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Posts Tagged ‘T cells’


Cancer Drugs Shed Light on Rheumatism

Reporter: Irina Robu, PhD

The human body is often described as being ‘at war’. By this, it is meant that the body is constantly under attack from things that are trying to do it harm. These include toxins, bacteria, fungi, parasites and viruses. The human immune system is one of the most effective defense mechanisms known to nature and can sometimes can be overwhelmed by disease. Yet, on occasions our immune systems turn on our own tissue and attack it which can trigger conditions such as type I diabetes, rheumatoid arthritis and lupus.

In the case of rheumatoid arthritis, immune cells start to attack tissues in the joins which causes them to become painful, stiff and swollen. It is known that one third of those who develop rheumatoid arthritis, feel the horrible effects of the disease within two years of its onset. Immunologist Adrian Hayday, which is a researcher at Francis Crick Institute of London says that the current treatment for rheumatoid arthritis require patients to take the drugs for the rest of their lives. But, researchers such as Hayday found an unexpected ally in the battle against autoimmune disease, cancer.

However, there is a positive consequence to the discovery that cancer immunotherapies have the effect of triggering autoimmune diseases and for the first-time rheumatoid arthritis can be detected at the earliest stages. At present, people are not diagnosed with the condition until symptoms have already made their lives so unpleasant, they have gone to see their doctors. As a result, research backed by Cancer Research UK and Arthritis Research UK, has been launched with the aim of uncovering the roots of autoimmune disease from research on cancer patients.

The scientists mentioned stress that their work is only now start and warn that it will still take several years of research to get substantial results. Nevertheless, uncovering the first stages of an autoimmune disease emerging in a person’s body should give researchers a vital lead in ultimately developing treatments that will prevent or halt a range of conditions that currently cause a great deal of misery and require constant medication.

Our immune defenses consist of a range of cells and proteins that notice invading micro-organisms and attack them. The first line of defense, yet, consists of simple physical barriers similar to skin, which blocks invaders from entering your body. When this defense is penetrated, they are attacked by a number of agents. The key cells, leukocytes seek out and destroy disease-causing organisms. Neutrophils rush to the site of an infection and attack invading bacteria. Helper T-cells give instructions to other cells while killer T-cells punch holes in infected cells so that their contents ooze out. After these macrophages clean up the mess left behind.

Another significant agent is the B-cell, which produces antibodies that lock on to sites on the surface of bacteria or viruses and immobilize them until macrophages consume them. These cells can live a long time and can answer quickly following a second exposure to the same infections. In conclusion, suppressor T-cells act when an infection has been distributed with and the immune system needs to be reassured, the killer cells may keep on attacking, as they do in autoimmune diseases. By slowing down the immune system, regulatory T-cells prevent damage to “good” cells.

SOURCE

https://www.theguardian.com/science/2018/mar/03/immunotherapy-cancer-patients-rheumatoid-arthritis-robin-mckie

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells.

 

CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.

 

Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells. Using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), an average insertion or deletion (indel) efficiency greater than 80% was achieved. This high efficiency of insertion or deletion generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs.

 

The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. These results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. As hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

 

CRISPR-based editing of T cells to treat cancer, as scientists at the University of Pennsylvania are studying in a clinical trial, should also not have a p53 problem. Nor should any therapy developed with CRISPR base editing, which does not make the double-stranded breaks that trigger p53. But, there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

 

References:

 

https://techonomy.com/2018/06/new-cancer-concerns-shake-crispr-prognosis/

 

https://www.statnews.com/2018/06/11/crispr-hurdle-edited-cells-might-cause-cancer/

 

https://www.biorxiv.org/content/early/2017/07/26/168443

 

https://www.nature.com/articles/s41591-018-0049-z.epdf?referrer_access_token=s92jDP_yPBmDmi-USafzK9RgN0jAjWel9jnR3ZoTv0MRjuB3dEnTctGtoy16n3DDbmISsvbln9SCISHVDd73tdQRNS7LB8qBlX1vpbLE0nK_CwKThDGcf344KR6RAm9k3wZiwyu-Kb1f2Dl7pArs5yYSiSLSdgeH7gst7lOBEh9qIc6kDpsytWLHqX_tyggu&tracking_referrer=www.statnews.com

 

https://www.nature.com/articles/s41591-018-0050-6.epdf?referrer_access_token=2KJ0L-tmvjtQdzqlkVXWVNRgN0jAjWel9jnR3ZoTv0Phq6GCpDlJx7lIwhCzBRjHJv0mv4zO0wzJJCeuxJjzoUWLeemH8T4I3i61ftUBkYkETi6qnweELRYMj4v0kLk7naHF-ujuz4WUf75mXsIRJ3HH0kQGq1TNYg7tk3kamoelcgGp4M7UTiTmG8j0oog_&tracking_referrer=www.statnews.com

 

https://www.biorxiv.org/content/early/2018/01/05/243345

 

https://www.nature.com/articles/nmeth.4293.epdf

 

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Signaling through the T Cell Receptor (TCR) Complex and the Co-stimulatory Receptor CD28

Curator: Larry H. Bernstein, MD, FCAP

 

 

New connections: T cell actin dynamics

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.

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Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

 

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T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex at the site of the T cell receptors (TCRs). Srikanth et al. found that the lymphocyte-specific large guanosine triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human CD4+ T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T cell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium and kinase signaling.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

 

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New insights in cancer, cancer immunogenesis and circulating cancer cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Revised 4/20/2016

 

Circulating Tumor Cells Traverse Tiny Vasculature

Clusters of tumor-derived cells can pass through narrow channels that mimic human capillaries, scientists show in vitro and in zebrafish.

By Tanya Lewis | April 18, 2016

http://www.the-scientist.com/?articles.view/articleNo/45883/title/Circulating-Tumor-Cells-Traverse-Tiny-Vasculature

http://www.the-scientist.com/images/News/April2016/tumorclusters1.gif

A stained cluster of cancer cells passes through a 7-μm channel in vitro. PNAS, AU ET AL

Clusters of circulating tumor cells (CTCs) may play a larger role in cancer metastasis than previously thought. Researchers at Massachusetts General Hospital have now shown that these clusters can squeeze through microfluidic channels just 7 microns (μm) in diameter. The team’s findings were published today (April 18) in PNAS.

“There’s a common belief in the field that even single CTCs traversing through capillary beds would destroy the majority of them through physical shearing,” Edward Cho of Spectrum Genomics wrote in an email toThe Scientist. This study “demonstrates new evidence that clusters of CTCs may have a mechanism to prevent shearing as they traverse through small capillaries, and thus may have greater metastatic potential than previously thought,” added Cho, who was not involved in the work.

Most cancer deaths are caused by tumors metastasizing to different organs. Traditionally, clusters of these cells were thought to be too large to pass through capillaries, instead getting stuck and forming blood clots. Yet, more recently, these clusters have been detected in blood drawn from cancer patients. “If they’re so big, how can we find them in blood collection in the arm?” study coauthor Mehmet Toner of Massachusetts General Hospital told The Scientist.

To investigate, Toner and his colleagues created 7-μm-wide microfluidic channels designed to mimic the mechanical properties of human capillaries, and filmed clusters of CTCs—derived from patient blood samples and from cell lines—as they moved through the channels.

As the videos revealed, clusters of 10 to 20 cells loosely disassembled as they entered the channels and moved through the tiny passageways, single-file—like a crowd of people holding hands as they squeeze through a narrow alleyway in a line formation. The cells were “squeezed beyond belief,” said Toner. Upon exiting the channel, the CTCs reassembled into nonlinear clusters.

Next, the researchers sought to validate their model in vivo. They injected human CTC clusters into the bloodstreams of 3-day-old transgenic zebrafish embryos. Zebrafish are a good model for humans because their capillaries are almost identical to those of humans in size and pressure, Toner explained. Again the researchers found that the CTC clusters could traverse the fish capillaries.

http://www.the-scientist.com/images/News/April2016/tumorclusters2.gif

A cluster of four tumor cells elongates and compresses as it travels through a 7-μm microfluidic capillary.PNAS, AU ET AL.

http://www.the-scientist.com/images/News/April2016/tumorclusters3.gif

Clusters of tumor cells (green) moving through a transgenic zebrafish blood vessel (arrows indicate direction of flow through dorsal aorta, caudal vein, and pivot point)PNAS, AU ET AL.

Finally, Toner’s team showed that these CTC clusters could be broken up with certain drugs. The researchers treated clusters with either FAK inhibitor 14, a molecule overproduced by many tumors that inhibits a protein involved in cell-cell adhesion, or the chemotherapy drug paclitaxel, which also weakens cell junctions. When the researchers injected the treated clusters into the microfluidic channels, the clusters broke up into smaller clumps or single CTCs, suggesting a possible avenue for treatment.

“It’s a very interesting paper,” Sanjiv Sam Gambhir of Stanford, a professor of radiology and nuclear medicine who did not take part in the study, told TheScientist. “It’s not known how these clusters of cells end up being the bad [guys] in terms of metastasis. This work very nicely—both through computational modeling, as well as microfluidic devices and zebrafish models—attempts to elucidate this finding.”

However, Gambhir added, the results are still based on models. “Unless you’re doing this in actual human capillaries, you still can’t prove this is what really goes on in humans,” he said.

However, Gambhir added, the results are still based on models. “Unless you’re doing this in actual human capillaries, you still can’t prove this is what really goes on in humans,” he said.

Missing from both the in vitro and zebrafish models was information on how CTC clusters behave in branching capillary beds like those seen in human capillaries, Cho noted. Cho’s team and others have previously shown that CTC clusters may also become stuck in veins, creating clots that can be fatal.

Further, developing treatments based on breaking up these clusters relies on the assumption that CTC clusters have greater metastatic potential than single CTCs, which is still up for debate.

“This study is a good first effort to help us understand how cells might transit through a simplified model of the circulatory system,” Cho wrote in an email, “but until we can model the true complexity of CTCs and CTC clusters traveling through the human circulatory system, we should be cautious not to extrapolate too much in terms of the potential therapeutic potential from the conclusions of studies like these.”

S.H. Au et al., “Clusters of circulating tumor cells traverse capillary-sized vessels,” PNAS,doi:10.1073/pnas.1524448113, 2016.

Clusters of circulating tumor cells traverse capillary-sized vessels

Sam H. Aua,bBrian D. StoreycJohn C. Moored,e,fQin Tangd,e,f, et al.    Sam H. Au,  http://dx.doi.org:/10.1073/pnas.1524448113

Metastasis is responsible for 90% of cancer-related deaths and is driven by tumor cells circulating in blood. However, it is believed that only individual tumor cells can reach distant organs because multicellular clusters are too large to pass through narrow capillaries. Here, we collected evidence by examining clusters in microscale devices, computational simulations, and animals, which suggest that this assumption is incorrect, and that clusters may transit through capillaries by unfolding into single-file chains. This previously unidentified cell behavior may explain why previous experiments reported that clusters were more efficient at seeding metastases than equal numbers of single tumor cells, and has led to a strategy that, if applied clinically, may reduce the incidence of metastasis in patients.

 

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.
Signal Loop Pulls Healthy Cells into Cancer’s Echo Chamber

http://www.genengnews.com/gen-news-highlights/signal-loop-pulls-healthy-cells-into-cancer-s-echo-chamber/81252618/

In the cellular media environment, some of the most pernicious messaging occurs within tumors, which form a kind of echo chamber that amplifies molecular interactions. These interactions, which support the growth and spread of cancer, occur not only between genetically diverse cancer cells, but also between cancer cells and healthy cells.

That healthy cells should participate in such distorted discourse is disappointing but undeniable, say scientists based at the Institute of Cancer Research (ICR). These scientists report that stromal cells are all too receptive to KRAS signals secreted by cancer cells. Under the influence of oncogenic KRAS, stromal cells secrete a message of their own, one that cancer cells cannot produce themselves, and the stromal cells’ messaging ends up reinforcing the cancer cells’ malignant behavior.

These findings appeared April 14 in the journal Cell, in an article entitled, “Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.” The article describes how the ICR researchers studied communication networks in cells from a type of pancreatic cancer called pancreatic ductal adenocarcinoma, one of the most deadly forms of cancer.

“By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells,” wrote the authors of the Cell article. “Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D.”

Normal KRAS makes occasional signals that tell a cell to divide; but when the gene is mutated, it becomes hyperactive and helps drive cancer cells’ rapid and uncontrolled growth. KRAS is mutated in more than 90% of pancreatic cancer, and in nearly 20% of all cancers.

The authors determined that, “…reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis.”

In other words, by monitoring reciprocal signaling, the ICR scientists discovered that healthy cells were responding with a totally new message, one propagated via IGF1R/AXL-AKT. This message doubled the capacity for KRAS to drive malignant behavior in cancer cells.

“We now know that tumors are a complex mix of genetically diverse cancer cells and multiple types of healthy cells, all communicating with each other via an intricate web of interactions,” noted Claus Jørgensen, Ph.D., the ICR scientist who led the study and is currently a junior group leader at the Cancer Research UK Manchester Institute. “Untangling this web, and decoding individual signals, is vital to identify which of the multitude of communications are most important for controlling tumor growth and spread.”

“We have identified a key role played by the most commonly mutated gene in cancer in communicating with healthy cells. Blocking its effects could be an effective cancer treatment.”

 

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation

Christopher J. Tape, Stephanie Ling, Maria Dimitriadi4,…., Douglas A. Lauffenburger, Claus Jørgensen
Cell Apr 2016    http://dx.doi.org/10.1016/j.cell.2016.03.029

In Brief – Cell-specific proteome labeling reveals that oncogenic KRAS stimulates stromal cells to initiate reciprocal signaling back to pancreatic tumor cells, thereby enabling signaling capacity beyond the traditionally studied cell-autonomous pathways.

Highlights

  1.  KRASG12D establishes a reciprocal signaling axis via heterotypic stromal cells
  2.  Reciprocal signaling further regulates tumor cell signaling downstream of KRASG12D
  3.  Reciprocal signaling regulates tumor cell behavior via AXL/ IGF1R-AKT
  4.  Heterocellularity expands tumor cell signaling beyond cellautonomous pathways

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2053320047/2060147607/fx1.jpg

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12Dproduces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer.

Solid cancers are heterocellular systems containing both tumor cells and stromal cells. Coercion of stromal cells by tumor cell oncogenes profoundly impacts cancer biology (Friedl and Alexander, 2011, Quail and Joyce, 2013) and aberrant tumor-stroma signaling regulates many hallmarks of cancer (Hanahan and Weinberg, 2011). While individual oncogene-driven regulators of tumor-stroma signaling have been identified, the propagation of oncogene-dependent signals throughout a heterocellular system is poorly understood. Consequently, our perspective of oncogenic signaling is biased toward how oncogenes regulate tumor cells in isolation (Kolch et al., 2015).

In a heterocellular cancer, tumor cell oncogenes drive aberrant signaling both within tumor cells (cell-autonomous signaling) and adjacent stromal cells (non-cell-autonomous signaling) (Croce, 2008, Egeblad et al., 2010). As different cell types process signals via distinct pathways (Miller-Jensen et al., 2007), heterocellular systems (containing different cell types) theoretically provide increased signal processing capacity over homocellular systems (containing a single cell type). By extension, oncogene-dependent signaling can theoretically engage additional signaling pathways in a heterocellular system when compared to a homocellular system. However, to what extent activated stromal cells reciprocally regulate tumor cells beyond cell-autonomous signaling is not well understood.

We hypothesized that the expanded signaling capacity provided by stromal heterocellularity allows oncogenes to establish a differential reciprocal signaling state in tumor cells. To test this hypothesis, we studied oncogenic KRAS (KRASG12D) signaling in pancreatic ductal adenocarcinoma (PDA). KRAS is one of the most frequently activated oncogenic drivers in cancer (Pylayeva-Gupta et al., 2011) and is mutated in >90% of PDA tumor cells (Almoguera et al., 1988). PDA is an extremely heterocellular malignancy—composed of mutated tumor cells, stromal fibroblasts, endothelial cells, and immune cells (Neesse et al., 2011). Crucially, the gross stromal pancreatic stellate cell (PSC) expansion observed in the PDA microenvironment is non-cell-autonomously controlled by tumor cell KRASG12D in vivo (Collins et al., 2012, Ying et al., 2012). As a result, understanding the heterocellular signaling consequences of KRASG12D is essential to comprehend PDA tumor biology.

Comprehensive analysis of tumor-stroma signaling requires concurrent measurement of cell-specific phosphorylation events. Recent advances in proteome labeling now permit cell-specific phosphoproteome analysis in heterocellular systems (Gauthier et al., 2013, Tape et al., 2014a). Furthermore, advances in proteomic multiplexing enable deep multivariate phospho-signaling analysis (McAlister et al., 2012, Tape et al., 2014b).

Here, we combine cell-specific proteome labeling, multivariate phosphoproteomics, and inducible oncogenic mutations to describe KRASG12Dcell-autonomous, non-cell-autonomous, and reciprocal signaling across a heterocellular system. This study reveals KRASG12D uniquely regulates tumor cells via heterotypic stromal cells. By exploiting heterocellularity, reciprocal signaling enables KRASG12D to engage oncogenic signaling pathways beyond those regulated in a cell-autonomous manner. Expansion of KRASG12D signaling via stromal reciprocation suggests oncogenic communication should be viewed as a heterocellular process.

….

Whether oncogenes regulate tumor cell signaling via stromal cells is a fundamental question in tumor biology. Using heterocellular multivariate phosphoproteomics, we demonstrate how oncogenic KRAS signals through local non-tumor cells to achieve a differential reciprocal signaling state in the inceptive tumor cells. In PDA, this reciprocal axis supplements oncogenic cell-autonomous signaling to control protein abundance, transcription, mitochondrial activity, proliferation, apoptosis, and colony formation. Reciprocal signaling is the exclusive product of heterocellularity and cannot be achieved by tumor cells alone. These observations imply oncogenes expand their capacity to deregulate cellular signaling via stromal heterocellularity (Figure 7).

Despite the well-established heterocellularity of cancer, our understanding of oncogenic signaling within tumor cells has largely excluded non-tumor cells. We observe that stromal cells approximately double the number of tumor cell signaling nodes regulated by oncogenic KRAS, suggesting both cell-autonomous (internal) and reciprocal (external) stimuli should be considered when defining aberrant oncogenic signaling states. For example, although KRAS is thought to cell-autonomously regulate AKT in PDA (Eser et al., 2014), we show that KRASG12D activates AKT, not cell-autonomously, but reciprocally. As PI3K signaling is essential for PDA formation in vivo (Baer et al., 2014, Eser et al., 2013, Wu et al., 2014) reciprocal signaling may control oncogene-dependent tumorigenesis. Our findings suggest future genetic studies should consider the heterocellular signaling consequences of oncogene/tumor-suppressor deregulation.

The observation that many oncogene-dependent tumor cell signaling nodes require reciprocal activation has important implications for identifying pharmacological inhibitors of oncogene signaling. For example, if PDA tumor cells were screened alone, one would expect MEK, MAPK, and CDK inhibitors to perturb KRASG12D signaling. However, when screened in conjunction with heterotypic stromal cells, our study additionally identified SHH, AKT, and IGF1R/AXL inhibitors as KRASG12D-dependent targets in tumor cells. Inhibitors of signaling specific to reciprocally engaged tumor cells, such as or AKT or IGF1R/AXL, block heterocellular phenotypes (e.g., protein expression, proliferation, mitochondrial performance, and anti-apoptosis), but have little effect on KRASG12D tumor cells alone. An appreciation of reciprocal nodes increases our molecular understanding of drug targets downstream of oncogenic drivers and highlights focal points where reciprocal signals converge (e.g., AKT). These trans-cellular observations reinforce the importance of understanding cancer as a heterocellular disease.

Previous work in PDA tumor cells under homocellular conditions demonstrated cell-autonomous KRASG12D shifts metabolism toward the non-oxidative pentose phosphate pathway (Ying et al., 2012), whereas KRASG12D-ablated cells depend on mitochondrial activity (Viale et al., 2014). Here, we show that heterocellular reciprocal signaling can restore the expression of mitochondrial proteins and subsequently re-establish both mitochondrial polarity and superoxide levels. This suggests KRASG12D regulates non-oxidative flux through cell-autonomous signaling and mitochondrial oxidative phosphorylation through reciprocal signaling. These results provide a unique example of context-dependent metabolic control by oncogenes and reinforce the emerging role of tumor-stroma communication in regulating cancer metabolism (Ghesquière et al., 2014).

In PDA, the stroma has dichotomous pro-tumor (Kraman et al., 2010, Sherman et al., 2014) and anti-tumor (Lee et al., 2014, Rhim et al., 2014) properties. It is becoming increasingly evident that non-cell-autonomously activated stromal cells vary within a tumor and can influence tumors in a non-obvious manner. For example, while vitamin D receptor normalization of stromal fibroblasts improves PDA therapeutic response (Sherman et al., 2014), total stromal ablation increases malignant behavior (Lee et al., 2014, Rhim et al., 2014). Thus, while stromal purging is unlikely to provide therapeutic benefit in PDA, “stromal reprogramming” toward an anti-tumor stroma is now desirable (Brock et al., 2015). Although we describe a largely pro-tumor reciprocal axis, both pro- and anti-tumor stromal phenotypes likely transduce across reciprocal signaling networks. Our work suggests future efforts to therapeutically reprogram the PDA stroma toward anti-tumor phenotypes will require an understanding of reciprocal signaling. In describing the first oncogenic reciprocal axis, this study provides a foundation to measure the cell-cell communication required for anti-tumor stromal reprogramming.

We demonstrate heterocellular multivariate phosphoproteomics can be used to observe reciprocal signaling in vitro. Unfortunately, cell-specific isotopic phosphoproteomics is not currently possible in vivo. To delineate reciprocal signaling in vivo, experimental systems must support manipulation of multiple cell-specific variables and provide cell-specific signaling readouts. Simple pharmacological perturbation of reciprocal nodes (e.g., IGF1R, AXL, AKT, etc.) in existing PDA GEMMs will in principle affect all cell types (e.g., tumor cells, PSCs, immune cells) and cannot provide axis-specific information in vivo. Future in vivo studies of reciprocal signaling will require parallel inducible genetic manipulation (e.g., oncogene activation in cancer cell and/or inhibition of reciprocal node in stromal cell), combined with cell-specific signaling data (e.g., using epithelial tissue mass-cytometry) (Simmons et al., 2015).

We describe KRASG12D reciprocal signaling between PDA tumor cells and PSCs. However, it is likely oncogenic reciprocal signaling occurs across multiple different cell types in the tumor microenvironment. For example, in PDA, FAP+stromal fibroblasts secrete SDF1 that binds tumor cells to suppress T cells (Feig et al., 2013). Our model predicts oncogene signaling expands across several cell types in the tumor microenvironment—including immune cells. Moreover, as oncogenes non-cell-autonomously regulate the stroma in many other tumor types (Croce, 2008), our model predicts oncogenic reciprocal signaling to be a broad phenomenon across all heterocellular cancers. The presented heterocellular multivariate phosphoproteomic workflow now enables future characterization of oncogenic reciprocal signaling in alternative cancer types.

As differentiated cells process signals in unique ways, heterocellularity provides increased signal processing space over homocellularity. We provide evidence that KRASG12D exploits heterocellularity via reciprocal signaling to expand tumor cell signaling space beyond cell-autonomous pathways. Given the frequent heterocellularity of solid tumors, we suspect reciprocal signaling to be a common—albeit under-studied—axis in oncogene-dependent signal transduction.

New Genomic Analysis of Immune Cell Infiltration in Colorectal Cancer

http://www.genengnews.com/gen-news-highlights/new-genomic-analysis-of-immune-cell-infiltration-in-colorectal-cancer/81252616/

Through whole-exome sequencing of colorectal tumors, researchers were able to identify additional driver genes that correlate high neoantigen load with increased lymphocytic infiltration and improved survival. [Giannakis et al., 2016, Cell Reports 15, 1–9]     http://www.genengnews.com/Media/images/GENHighlight/thumb_fx11248133146.jpg

The past several years have seen some exciting results for cancer immunotherapy. However, there remains a fundamental lack of understanding of immune system recognition in various cancers. Many large-scale sequencing efforts have added to our collective knowledge base, but too many of these studies have been deficient in comprehensive epidemiological and demographic information.

Now, researchers at the Dana-Farber Cancer Institute and the Broad Institute of MIT and Harvard report on their findings from a new study, which found that colorectal cancers festooned with tumor-related proteins called neoantigens were likely to be saturated with disease-fighting white blood cells, mainly lymphocytes.

Using several data sets from patients in two large health-tracking studies, the Nurses’ Health Study and the Health Professionals Follow-up Study, investigators performed whole-exome sequencing on colorectal tumor samples from 619 patients—itemizing each DNA base that specifies how cell proteins are to be constructed. This information was merged with data from tests of the immune system’s response to the tumors and with patient clinical data, including length of survival.

“We were looking for genetic features that predict how extensively a tumor is infiltrated by lymphocytes and which types of lymphocytes are present,” explained co-lead study author, Marios Giannakis, M.D., Ph.D., medical oncologist and clinical investigator at the Dana-Farber Gastrointestinal Cancer Treatment Center, and researcher at the Broad Institute of MIT and Harvard. “We found that tumors with a high ‘neoantigen load’—which carry large quantities of neoantigens—tended to be infiltrated by a large number of lymphocytes, including memory T cells, which provide protection against previously encountered infections and diseases. Patients whose tumors had high numbers of neoantigens also survived longer than those with lower neoantigen loads.”

The findings from this study were published recently in Cell Reports in an article entitled “Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma.”

Neoantigens are mutated forms of protein antigens, which are found on normal cells. Genetic mutations often cause cancer cells to produce abnormal proteins, some of which get trafficked to the cell surface, where they serve as a red flag to the immune system that something has gone awry with the cell.

“There can be hundreds or thousands of neoantigens on tumor cells,” noted Dr. Giannakis explained. “Only a few of these may actually provoke T cells to infiltrate a tumor. However, the more neoantigens on display, the greater the chance that some of them will spark an immune system response.”

Physicians often take advantage of therapies known as immune checkpoint inhibitors, which work by removing some of the barriers to an immune system attack on cancer. Although these agents have produced astonishing results in some cases, they’re effective only in patients whose immune system has already launched an immune response to cancer. This new study may help investigators identify which patients are most likely to benefit in new clinical trials of immune checkpoint inhibitors by showing that tumors with high antigen loads are apt to be laced with T cells—and therefore able to provoke an immune response.

Interestingly, this new analysis found several often-mutated genes that had not previously been strongly associated with the disease, including BCL9L, RBM10, CTCF, and KLF5, suggesting that they may be valuable targets for new therapies.

“Our study helps shed light on the overall development of colorectal cancer,” Dr. Giannakis remarked. “It also shows the insights that can be gained by integrating molecular research with findings from other areas such as epidemiology and immunology.”

“Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma”

 

Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival
Scott D. Brown,1,2 Rene L. Warren,1 Ewan A. Gibb,1,3 Spencer D. Martin,1,3,4 John J. Spinelli,5,6 Brad H. Nelson,3,4,7 and Robert A. Holt1,3,8,9
Genome Res. 2014 May; 24(5): 743–750.    doi:  10.1101/gr.165985.113

Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patient’s autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.

The accumulation of somatic mutations underlies the initiation and progression of most cancers by conferring upon tumor cells unrestricted proliferative capacity (Hanahan and Weinberg 2011). The analysis of cancer genomes has revealed that tumor mutational landscapes (Vogelstein et al. 2013) are extremely variable among patients, among different tumors from the same patient, and even among the different regions of a single tumor (Gerlinger et al. 2012). There is a need for personalized strategies for cancer therapy that are compatible with mutational heterogeneity, and in this regard, immune interventions that aim to initiate or enhance anti-tumor immune responses hold much promise. Therapeutic antibodies and chimeric antigen receptor (CAR) technologies have shown anti-cancer efficacy (Fox et al. 2011), but such antibody-based approaches are limited to cell surface target antigens (Slamon et al. 2001; Coiffier et al. 2002; Yang et al. 2003;Cunningham et al. 2004; Kalos et al. 2011). In contrast, most tumor mutations are point mutations in genes encoding intracellular proteins. Short peptide fragments of these proteins, after intracellular processing and presentation at the cell surface as MHC ligands, can elicit T cell immunoreactivity. Further, the presence of tumor infiltrating lymphocytes (TIL), in particular, CD8+ T cells, has been associated with increased survival (Sato et al. 2005; Nelson 2008; Oble et al. 2009; Yamada et al. 2010; Gooden et al. 2011; Hwang et al. 2012), suggesting that the adaptive immune system can mount protective anti-tumor responses in many cancer patients (Kim et al. 2007; Fox et al. 2011). The antigen specificities of tumor-infiltrating T cells remain almost completely undefined (Andersen et al. 2012), but there are numerous examples of cytotoxic T cells recognizing single amino acid coding changes originating from somatic tumor mutations (Lennerz et al. 2005;Matsushita et al. 2012; Heemskerk et al. 2013; Lu et al. 2013; Robbins et al. 2013;van Rooij et al. 2013; Wick et al. 2014). Thus, the notion that tumor mutations are reservoirs of exploitable neo-antigens remains compelling (Heemskerk et al. 2013). For a mutation to be recognized by CD8+ T cells, the mutant peptide must be presented by MHC I molecules on the surface of the tumor cell. The ability of a peptide to bind a given MHC I molecule with sufficient affinity for the peptide-MHC complex to be stabilized at the cell surface is the single most limiting step in antigen presentation and T cell activation (Yewdell and Bennink 1999). Recently, several algorithms have been developed that can predict which peptides will bind to given MHC molecules (Nielsen et al. 2003; Bui et al. 2005; Peters and Sette 2005; Vita et al. 2010; Lundegaard et al. 2011), thereby providing guidance into which mutations are immunogenic.

The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) is an initiative of the National Institutes of Health that has created a comprehensive catalog of somatic tumor mutations identified using deep sequencing. As a member of The Cancer Genome Atlas Research Network, our center has generated extensive tumor RNA-seq data. Here, we have used public TCGA RNA-seq data to explore the T cell immunoreactivity of somatic missense mutations across six tumor sites. This type of analysis is challenged not only by large numbers of mutations unique to individual patients, but also by the complexity of personalized antigen presentation by MHC arising from the extreme HLA allelic diversity in the outbred human population. Previous studies have explored the potential immunogenicity of tumor mutations (Segal et al. 2008; Warren and Holt 2010; Khalili et al. 2012), but these have been hampered by small sample size and the inability to specify autologous HLA restriction. Recently, we described a method of HLA calling from RNA-seq data that shows high sensitivity and specificity (Warren et al. 2012). Here, we have obtained matched tumor mutational profiles and HLA-A genotypes from TCGA subjects and used these data to predict patient-specific mutational epitope profiles. The evaluation of these data together with RNA-seq-derived markers of T cell infiltration and overall patient survival provides the first comprehensive view of the landscape of potentially immunogenic mutations in solid tumors.    …..

The results of the present study have clinical implications. We have shown that patients with tumors bearing missense mutations predicted to be immunogenic have a survival advantage (Fig. 3D). These tumors also show evidence of higher CD8+ TIL, which suggests that a number of these mutations might be immunoreactive. The existence of these mutations is encouraging because, in principle, they could be leveraged by personalized therapeutic vaccination strategies or adoptive transfer protocols to enhance anti-tumor immunoreactivity. Likewise, patients with tumors showing naturally immunogenic mutations and associated TIL are potential candidates for treatment with immune modulators such as CTLA4- or PDCD1-targeted antibodies. There is evidence that such therapies are most effective against tumors infiltrated by T cells (Moschos et al. 2006; Hamid et al. 2009). Our results indicate that tumors bearing predicted immunogenic mutations have not only elevated CD8A expression (Fig. 3C) but also elevated expression of CTLA4 and PDCD1 (Fig. 4), reinforcing the notion that these patients may be optimal candidates for immune modulation. Importantly, we observed that tumors with low levels of CD8+ TIL invariably have far fewer immunogenic mutations. Such patients would be better suited to conventional therapy or to immunotherapies (e.g., chimeric antigen receptor modified T cells) that target nonmutated antigens.

  1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229261/Apr 12, 2011 Keywords: tumor immune infiltrate, T-cells, cancer prognosis, colon … By conducting genomic and in situ immunostaining on resected tumors from ….. of tumor-infiltrating immune cells correlates with better overall survival.

  2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234325/May 27, 2011 Keywords: Colorectal cancer, Immune response, T lymphocytes, Microsatellite instability … In CRC, at least 3 distinct pathways of genomic instability have been …. The potential influence of these immunecell infiltrates in CRC on the … in controlling cytolytic activity of CD8+ T cells, inversely correlates to the …   

 

Cancer-Associated Immune Resistance and Evasion of Immune Surveillance in Colorectal Cancer

Gastroenterology Research and Practice
Volume 2016 (2016), Article ID 6261721, 8 pages
http://dx.doi.org/10.1155/2016/6261721


Data from molecular profiles of tumors and tumor associated cells provide a model in which cancer cells can acquire the capability of avoiding immune surveillance by expressing an immune-like phenotype. Recent works reveal that expression of immune antigens (PDL1, CD47, CD73, CD14, CD68, MAC387, CD163, DAP12, and CD15) by tumor cells “immune resistance,” combined with prometastatic function of nonmalignant infiltrating cells, may represent a strategy to overcome the rate-limiting steps of metastatic cascade through (a) enhanced interactions with protumorigenic myeloid cells and escape from T-dependent immune response mediated by CD8+ and natural killer (NK) cells; (b) production of immune mediators that establish a local and systemic tumor-supportive environment (premetastatic niche); (c) ability to survive either in the peripheral blood as circulating tumor cells (CTCs) or at the metastatic site forming a cooperative prometastatic loop with foreign “myeloid” cells, macrophages, and neutrophils, respectively. The development of cancer-specific “immune resistance” can be orchestrated either by cooperation with tumor microenvironment or by successive rounds of genetic/epigenetic changes. Recognition of the applicability of this model may provide effective therapeutic avenues for complete elimination of immune-resistant metastatic cells and for enhanced antitumor immunity as part of a combinatorial strategy.

1. Introduction

Metastasis remains the most significant cause of cancer-associated morbidity and mortality and specific targeting molecules have had limited success in reversing metastatic progression in the clinical setting [13]. Understanding the exact molecular and cellular basis of the events that facilitate cancer metastasis has been difficult so far. Over the past years, a well-accepted theory suggests that genomic alterations of the malignant cells accompanied by the so-called tumor microenvironment “nonmalignant cells” contribute to the metastatic cascade [4, 5]. As such, metastasis is frequently described as the sequential execution of multiple steps. To establish the metastatic tumor, cancer cells have to acquire the traits that enable them to efficiently cooperate with the host stroma and simultaneously avoid antitumor immune response [49]. At early stage of carcinogenesis, tumors appear to be vulnerable because mutant and thus potentially immunogenic tumor cells are being exposed to the immune system, which can recognize them and restrict their growth [10, 11]. This is the case of tumor-infiltrating immune cells particularly CD8+ T cells and NK cells which have the potential to restrict the tumor outgrowth or reject metastatic tumor cells [12, 13]. According to this notion, in most primary tumors, a strong Th1/cytotoxic T cells infiltration correlates with a longer patient’s survival [1214]. Unfortunately, tumor develops multiple mechanisms of evading immune responses, by forming a compromised microenvironment that allows the dissemination of malignant cells in a foreign microenvironment through molecular mechanisms still poorly characterized. A variety of stromal cells, particularly M2-phenotype macrophages and myeloid-derived suppressor cells (MDSCs), are recruited to primary tumors; these not only enhance growth of the primary cancer but also facilitate its metastatic dissemination to distant organs [13, 14]. Notably, cooperative “dialogue” between malignant cells and their microenvironment will go on in the systemic circulation and subsequently in the future metastatic site [1317]. In fact, recent studies have demonstrated that a high systemic inflammatory response, that is, blood neutrophil-lymphocyte ratio (NLR), predicts lower overall survival, higher tumor stage, and a greater incidence of metastasis in multiple tumor types [18, 19]. Therefore, a substantial amount of data suggests a novel dimension of the tumor biology and offers the opportunity to revisit the mechanisms describing evasion of cancer immunosurveillance during the metastatic process. The present review analyses recent studies that elucidate and reinforce the theory by which immune-phenotypic features or “immune resistance” by cancer cells may need to sustain the metastatic cascade and avoid antitumor immune response.

2. Tumor Antigens and Antitumor Immune Response by Effectors of Adaptive Immunity

A decade of studies has emphasized the nature of cancer as a systemic disease remarking a key role of host microenvironment as a critical hallmark. As a result, a new picture of cancer is emerging in particular due to unexpected cross-talk between malignant cells and the immune system [35]. Recent data have expanded the mechanisms of cancer-immune system interactions revealing that every known innate and adaptive immune effector component participates in tumor recognition and control [9, 10]. It is now recognized that in different individuals and with different cancers, at early stage of tumorigenesis, the few cancer cells are detected by NK cells through their encounter with specific ligands on tumor cells [5]. In turn, activation of macrophages and dendritic cells and particularly T and B cells expands production of additional cytokines and further promotes activation of tumor-specific T cells “CD8+ cytotoxic T cells” leading to the generation of immune memory to specific tumor components [1416]. However, in cases where the immune system is not able to eliminate the cancer, a state of equilibrium develops or eventually cancer cells can resist, avoid, or suppress the antitumor immune response, leading to the immune escape and a fully developed tumor (Figure 1) [915]. For example, investigations into the nature of cancer as a genetic disease have suggested two paradigmatic subtypes of colorectal cancer (CRC): chromosomal instability (CIN) and microsatellite instability (MSI), in which the expression of immune-checkpoint proteins can be differentially dysregulated to unleash the potential of the antitumor immune response [11]. In particular, tumors with mismatch-repair deficiency (dMMR) (10–20%) of advanced colorectal cancer tend to have 10 to 100 times more somatic mutations and higher amount of lymphocyte infiltrates than mismatch-repair-proficient colorectal cancers (pMMR), a finding consistent with a stronger antitumor immune response (Figure 1) [11, 20]. According to this notion, recent studies suggest that certain cancer subtypes dMMR CRC with high numbers of somatic mutations are more responsive to PD-1 blockade, a well-known immune-checkpoint inhibitor [20]. In particular, CD8-positive lymphoid infiltrate and membranous PDL1 expression on either tumor cells or tumor-infiltrating lymphocytes at the invasive fronts of the tumor are associated with an improved response to anti-PD-1 therapy in patients with mismatch-repair-deficient cancer [11, 20]. In addition, cancer subtypes with stronger antitumor immune responses (immunogenic) are characterized by surface-exposed calreticulin or heat shock protein 90 (HSP90), which serve as a powerful mobilizing signal to the immune system in the context of damage-associated molecular patterns (DAMPs) [17]. As danger signals, DAMPs accompanied by subversion MHC Class I and II antigens on the plasma membrane of cancer cells appear to be characteristic of stressed or injured cells and can act as adjuvant signals to enhance antitumor immunity mediated by the innate immune system [17]. As described in this review, unfortunately, the large majority of human tumors can suppress the immune system to enhance their survival, rendering them invisible to cytotoxic T lymphocytes through a variety of mechanisms. Furthermore, in most cases, tumor-infiltrating immune cells differentiate into cells that promote each step of the tumor progression supporting ability of cancer cells to invade and survive in foreign organs. In addition, the intricate network of malignant and immune components represents a prominent obstacle to the effects of therapeutic agents.

………..

High-resolution genomic analysis: the tumor-immune interface comes into focus

Jonathan J Havel1 and Timothy A Chan1,2*
Havel and Chan     Genome Biology (2015) 16:65      http://dx.doi.org:/10.1186/s13059-015-0631-3

A genomic analysis of heterogeneous colorectal tumor samples has uncovered interactions between immunophenotype and various aspects of tumor biology, with implications for informing the choice of immunotherapies for specific patients and guiding the design of personalized neoantigen-based vaccines.

Please see related article: http://dx.doi.org/10.1186/s13059-015-0620-6

Immunotherapy is a promising new approach for treating human malignancies. Approximately 20% of melanoma and lung cancer patients receiving immune checkpoint inhibitors show responses [1,2]. Current major challenges include identification of patients most likely to respond to specific therapies and elucidation of novel targets to treat those who do not. To address these problems, a detailed understanding of the dynamic interactions between tumors and the immune system is required. In a new study, Zlatko Trajanoski and colleagues [3] describe a powerful approach to dissecting these issues through high-resolution analysis of patient genomic data. This study represents a significant advance over previous work from this group, which defined 28 immune-cell-type gene expression signatures and identified specific cell types as prognostic indicators in colorectal cancer (CRC) patients [4]. Here, the authors [3] integrate genomic analyses of CRC tumor molecular phenotypes, predicted antigenicity (called the ‘antigenome’), and immune-cell infiltration derived from multiple independent cohorts to gain refined insights into tumor-immune system interactions.

Not all tumor-infiltrating lymphocytes are created equal

Past studies have used immune-staining techniques to determine associations between a limited set of infiltrating immune cells and patient survival [5] or tumor molecular phenotype [6]. Here, the authors [3] use gene set enrichment analysis (GSEA) of immune cell expression signatures to ascertain associations of 28 immune-cell populations with patient survival and tumor molecular phenotypes. Effector memory CD8+ and CD4+ T cells, natural killer cells, and activated dendritic cells are significantly associated with improved overall survival. Interestingly, although the authors’ previous work found no significant prognostic value of regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs) [4], negative associations of these cell types with overall survival are among the strongest relationships observed in the current study. It is possible that variations in sample collection and preparation may have contributed to this discrepancy. The conclusions, supported by the numerous animal studies demonstrating the importance of cell-mediated immunosuppression, are substantially strengthened by a much larger cohort size used in this study.

Another important observation is the association of specific immune cell subsets with CRC tumor stage and molecular phenotypes as classified by mutation rate, microsatellite instability, and methylation status. This knowledge will be crucial in determining which types of immunotherapy are most likely to benefit individual patients. Interestingly, although hypermutated microsatellite-unstable tumors show strong enrichment of adaptive immune cells, similar enrichment is notably lacking in the small population of hypermutated microsatellite-stable tumors. This raises an intriguing question of whether and how microsatellite instability/mismatch repair may independently shape immune responses. Furthermore, Trajanoski and colleagues [3] find that tumor-infiltrating lymphocytes transition from an adaptive to an innate immunophenotype with increasing tumor stage. This raises an interesting issue of whether immunotherapies that depend on the adaptive immune response can be effective in later stage CRC tumors.

Diversity of tumor antigens

In addition to characterizing immune components involved in tumor immune responses, it is equally important to identify and understand the tumor-associated antigens that elicit these responses, called the ‘antigenome’. The authors [3] analyze RNA-seq and genomic data to identify two types of tumor antigens in CRC – non-mutated cancer germline antigens that are aberrantly overexpressed, and neoantigens, which are generated from non-synonymous somatic mutations. Importantly, the authors [3] find that cancer-germline antigens are highly shared among patients and are independent of molecular and immune phenotype. In contrast, neoantigens are enriched in the hypermutated microsatellite-unstable phenotype tumors and rarely shared among patients. These results imply a heightened importance of neoantigens in comparison to cancer-germline antigens [7]. In addition, similar analytical methods have recently been applied to identify functional neoantigens in human melanoma and cholangiocarcinoma [810]. An emerging theme of these studies is that the in vitro validation rate for predicted neoantigens is relatively low; however, it is unclear whether this is due to limited sensitivity of functional assays or epigenetic silencing to circumvent immunoediting, or whether the number of immunogenic neoantigens is in fact small. Interestingly, Trajanoski and colleagues [3] find a modest yet significant decrease in neoantigen frequency with increasing tumor stage. Considering the concomitant decrease in adaptive immune cell infiltration, it is tempting to speculate that this phenomenon reflects immunoediting of critical neoepitopes during tumor progression. Furthermore, the authors find an association, albeit not statistically significant, between increased neoantigen burden and improved patient survival. This finding complements a recent report [9] showing that whereas neoantigen burden roughly predicts survival of anti-CTLA-4-treated melanoma patients, a collection of consensus neoepitope motifs is strongly associated with patient survival. It will be interesting to see if future studies can determine the effect of CRC neoantigen burden in the setting of immunotherapy, and answer the questions of whether an analogous signature of prognostic neoepitope motifs exists for CRC, and whether there are any similarities between substring signatures of different tumor types.

………

Not Your Average Circulating Tumor Cells   

Translational Scientists Profile Cancer Cells That Have Gone on the Lam

GEN Apr 15, 2016 (Vol. 36, No. 8)    http://www.genengnews.com/gen-articles/not-your-average-circulating-tumor-cells/5737/

 

  • For most malignant tumors, morbidity and mortality are, to a great extent, the result of metastatic dissemination, as opposed to the presence of the primary tumor.

    The existence of circulating tumor cells, which can be shed into the circulation by primary or metastatic malignancies, was first recognized almost 150 years ago, and their diagnostic and therapeutic values have been increasingly appreciated during the last few decades.

    One of the unique characteristics of circulating tumor cells is that they are in a fundamentally different environment from that established in either the primary tumor or the metastatic one. Although circulating tumor cells can be kept in place so that they can be assessed, the usual technique—immobilization to a solid surface—tends to yield distorted results. Free-floating cells are molecularly and functionally distinct from immobilized cells. For example, nonadhering breast cancer cells were shown to have tubulin-based microtentacles that shape their dynamic behavior, including their aggregation, retention in organs, or interaction with the endothelium.

    “These microtentacles are very hard to study because they depolymerize when cells bind either an endothelial cell or another tumor cell,” says Christopher M. Jewell, Ph.D., assistant professor of bioengineering  at the University of Maryland College Park. “Cells that form microtumors undergo massive mechanochemical and phenotypic changes as compared to when they are floating or circulating.”

    Characterizing circulating tumor cells, then, seems to amount to capturing the substance of freedom, a task that sounds self-defeatingly paradoxical—or at least fraught with difficulties. Overcoming difficulties, however, would likely be worth the effort. Two areas that immediately benefit from the characterization of circulating tumor cells are diagnostics and therapeutics.

    Capturing and analyzing circulating tumor cells opens not only the possibility of diagnosing patients earlier and more accurately, but also the potential for identifying new approaches to targeting malignancies. “Many groups are working on important technologies to capture circulating tumor cells,” informs Dr. Jewell. “We’re working on new technologies to analyze these populations.”

  • Floating in Place

    To address the existing gap in characterizing the biology of free-floating cancer cells, Dr. Jewell and collaborators in the University of Maryland laboratory of physiologist Stuart Martin, Ph.D., have designed an unusual  microfluidic device. It can spatially immobilize free-floating tumor cells while maintaining their free-floating characteristics.

    In this microfluidic device, polyelectrolyte multilayers inhibit the attachment of cells to multiwall plates, allowing their free-floating functional and morphological characteristics to be visualized and studied. Lipid tethers incorporated into the device interact with the cell membrane and allow cells to remain loosely attached and spatially localized, offering the possibility to perform applications such as real-time imaging and drug screening.

    “We are trying to understand what the signaling changes are in individual circulating tumors cells that are not nucleating into a tumor,” explains Dr. Jewell, “as compared to cells that contact enough cells and nucleate to form a tumor.”

    Surface tethering of circulating tumor cells also provides the opportunity to capture arrays of tumor cells; to introduce a perturbation such as a drug or a change in flow rate or mechanical properties; and then to collect the same individual cells that had already been imaged. In these cells, morphological changes can be correlated with genomic or proteomic information, providing an opportunity to dynamically understand how the mechanochemical properties of the cells change in response to external perturbations.

    “Our collaborators,” notes Dr. Jewell, “are also developing algorithms to quantify some of the features of microtentacles and convert visual information into quantitative metrics.”

  • Filterless Filters

    At the University of California, Los Angeles, Dino Di Carlo, Ph.D., and colleagues have developed High-Throughput Vortex Chip (Vortex-HT) technology, which uses parallel microfluidic vortex chambers to accumulate the larger circulating tumor cells from flowing blood. Vortex-HT reportedly generates less contamination with white blood cells than other technologies and isolates cells in a smaller output volume.

    Early techniques to capture circulating tumor cells have taken advantage of cell size differences, leading to the development of filtration-based approaches. This was followed, more recently, by the emergence of inertial microfluidic-based approaches, of which vortex technology is one example.

    “We think of vortex technology as a filterless filter,” says Dino Di Carlo, Ph.D., professor of bioengineering and director of the Cancer Nanotechnology Program at the Jonsson Comprehensive Cancer Center of the University of California, Los Angeles. “There aren’t any structures that are smaller than the cell types, but cells are still isolated based on size.”

    Dr. Di Carlo and colleagues recently developed the High-Throughput Vortex Chip (Vortex HT), an improved microfluidic technology that allows the label-free, size-based enrichment and concentration of rare cells. The strategy involves minimal pretreatment steps, reducing cell damage, and allows an approximately 8 mL vial of blood to be processed within 15–20 minutes.

    “With this approach,” asserts Dr. Di Carlo, “we can concentrate cells from any volume to about 100 µL.”

    Circulating tumor cells can then be used for subsequent steps, such as real-time imaging or immunostaining. The capture efficiency, up to 83%, is slightly lower than with Dean flow fractionation and CTC-iChip, but Vortex HT generates much less contamination with white blood cells than other technologies and isolates cells in a smaller output volume.

    Along with circulating tumor cells, another promising noninvasive biomarker is provided by circulating tumor DNA. Such DNA can be detected in the plasma or serum of many cancer patients as a result of the active or passive release of nucleic acid from apoptotic or necrotic tumor cells.

    While circulating tumor DNA can be used to dynamically collect information about specific mutations, and provides advantages for some applications, it is not powered to offer certain types of information that can be captured only from circulating tumor cells. For example, it cannot provide details about cellular morphology or protein expression and localization. Also, it cannot enable investigators to perform proteomic profiling in parallel with genomic profiling.

    These are not the only situations in which circulating DNA serves as a poor substitute for circulating tumor cells. “Another example,” notes Dr. Di Carlo, occurs with “applications that involve a drug screen that seeks to determine whether cells are sensitive or resistant to a particular compound.” Additionally, for certain cancers that have no dominant mutations, or for which mutations are not well known, circulating tumor DNA cannot provide the information that can be interrogated from profiling circulating tumor cells.

    • Insights beyond Counting

      “The field started off by enumerating circulating tumor cells as a potential biomarker,” says David T. Miyamoto, M.D., Ph.D., assistant professor of radiation oncology at Harvard Medical School and the Massachusetts General Hospital (MGH). “It is currently moving toward performing detailed molecular analyses of these circulating tumor cells and using them as a form of liquid biopsy that allows us to gain insights into the molecular biology of the tumor itself.”

      The Circulating Tumor Cell Center at MGH, led by Daniel Haber, M.D., Ph.D., and Mehmet Toner, Ph.D., has developed three generations of microfluidic technology. The technology of the first two generations captured circulating tumor cells on microfluidic surfaces. The technology of the third generation, known as CTC-iChip technology, introduces the unique capability—isolating cells in solution. Once the circulating tumor cells are captured or isolated, notes Dr. Miyamoto, they can be subjected to “a variety of sophisticated molecular analyses.”

      In a recent study using the CTC-iChip technology, Dr. Miyamoto and colleagues performed single-cell RNA sequencing. The investigators used 77 circulating tumor cells isolated by microfluidic enrichment from 13 patients.

      “The goal of this work was to use the circulating tumor cell technology to identify potential resistance mechanisms in metastatic castration-resistant prostate cancer,” explains Dr. Miyamoto. In patients who were undergoing therapy with an androgen receptor inhibitor, the retrospective analysis of their circulating tumor cells revealed that the noncanonical Wnt signaling pathway may play a role in resistance to therapy.

      “We need to validate the findings in larger patient cohorts,” concludes Dr. Miyamoto. “But this proof-of-concept study shows that detailed molecular analyses of liquid biopsy samples can be used to identify potentially clinically relevant mechanisms of resistance that can then be exploited to guide patient care.”

    • Variable to the Last

      Morphotek, a biopharmaceutical company that specializes in the development of protein and antibody products through the use of gene-evolution technology, has developed the ApoStream device, which uses continuous field-flow-assist and dielectrophoresis technology to isolate and recover circulating tumor cells from the blood of cancer patients. In a recent study in Biomarker Insights, Morphotek scientists described how they interrogated ApoStream-isolated circulating tumor cells by employing laser-scanning cytometry using highly selective antibodies. The scientists detected folate receptor alpha (FRα) expression in CK+/CD45 cells isolated from lung cancer, as indicated in these representative images.

      “Most of the work on circulating tumor cells has been done in late-stage cancers to direct therapeutic interventions,” says Daniel J. O’Shannessy, Ph.D., head of translational medicine and diagnostics at Morphotek. “Even in late-stage cancers, there is a great deal of variability with respect to the numbers of cells shed for a cancer type but especially between cancer types.”

      Many studies correlated the presence of circulating tumor cells with prognosis in several cancers, including breast, lung, and colorectal malignancies. However, one of the challenges associated with analyzing circulating tumor cells is that not every cancer releases them into the circulation. Also, even among cancers that do, not every cancer generates a lot of these cells.

      For example, as estimated using current techniques, ovarian cancers do not appear to shed as many circulating tumor cells as several other malignancies. “Another challenge is that existing technologies are often limited by sensitivity much more than by specificity,” cautions Dr. O’Shannessy. This limitation has the potential to make interpatient comparisons, and even the longitudinal follow-up of patients, particularly difficult.

      Previously, investigators at Morphotek described ApoStream®, a device that uses continuous field-flow-assist and dielectrophoresis technology to isolate and recover circulating tumor cells from the blood of cancer patients. In a recent study, Dr. O’Shannessy and colleagues used laser-scanning cytometry and highly selective antibodies to identify folate receptor alpha-positive cells from circulating tumor cells that had been isolated using the ApoStream technology.

      This proof-of-principle study was able to detect folate receptor alpha-positive cells in patients with breast cancer, ovarian cancer, and non-small cell lung adenocarcinoma, but not in patients with squamous cell lung cancer. These findings supporting previous findings that were made using the respective primary or metastatic tumors.

      The study demonstrated the utility of following the enrichment and identification of circulating tumor cells with immunofluorescence staining for a specific tumor marker. This combination of approaches emerges as a valuable noninvasive strategy for differentiating among tumor types. It can also be used to examine heterogeneous cell populations within tumors, particularly when tissue samples are not available.

    • Outliers among Outliers

      “We know that circulating tumor cells are present in cancer patients, but we have a limited understanding of the prognostic significance of their presence, or how to identify the ones that have more metastatic potential,” says Shana O. Kelley, Ph.D., professor of biochemistry at the University of Toronto. “These are questions we are trying to address to obtain functional information.”

      Recently, Dr. Kelley and colleagues described a new molecular approach based on a fluidic chip that captures circulating tumor cells using two-dimensional sorting. At a first stage, DNA aptamers specific to cell-surface markers bound to magnetic nanoparticles are used to capture circulating tumor cells. Subsequently, at a second stage, the corresponding antisense oligonucleotides are used to release the cells, enabling two-dimensional cell sorting.

      In a proof-of-concept experiment, Dr. Kelley and colleagues illustrated the strength of this approach in isolating cellular subpopulations that exhibit different phenotypes. Also, the investigators validated their results using an invasion assay.

      “Progress in working on the biology of circulating tumor cells motivates us to make devices to collect information about markers much more readily,” declares Dr. Kelley. “We hope this will provide information about outcomes and prognosis.”

 

 

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Programmed Cell Death and Cancer Therapy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Programmed Death: A Cat and Mouse Game

 http://www.cancernetwork.com/blog/programmed-death-cat-and-mouse-game

Prologue: The world of cancer care has been shaken up by the news that patients with hard-to-treat tumors benefit from a new type of immunotherapy, called checkpoint inhibition. A key receptor, called programmed death 1 (PD-1), is charged with suppressing the ability of activated T cells and other immune cells to destroy cancer cells, all in the name of preventing damage to normal tissue via autoimmunity. When PD-1 receptors on T cells bind with PD-L1 and PD-L2, complimentary receptors expressed on tumor cells, the immune response (call it the assassination of the cell) is checked and the tumor lives on. The anti PD-1 monoclonal antibodies nivolumab and pembrolizumab keep PD-L1 from turning off T cells, which has produced durable responses in several tumor types including melanoma, lung cancer, and renal cell carcinoma and represents a new hope for many.

Oncologists are excited to relay this news to patients, but is there a way to explain this without putting everyone in the room to sleep? Well, I like to use analogies to make seemingly complicated mechanisms easier to understand and the PD-1/PD-L1 relationship has inspired several colorful examples, to wit:

“Think of T cells as killers that use photographs to identify individual bad guys. Their weakness is that they will not act if the intended victim shakes their hand first. The bad guys used to be born without arms, but over time they evolved to grow arms and hands, thus avoiding elimination. The antibodies are boxing gloves that cover the hands of the T cells. Goodbye, bad guys.”

“Think of T cells as cats specially trained to eliminate mice wherever they hide. Their only weakness is if they smell catnip they will roll over and purr like idiots instead of doing their job. The mice then develop special glands that secrete catnip, thus pacifying the kitties. Solution: plug up the cats’ noses with nivolumab or pembrolizumab. Sayonara, Mr. Mouse.”

“Think of T cells as a fire sprinkler system designed to activate when a metal plug is heated to its melting point, releasing water from a pipe. The fire then emits a toxin that coats the fusible metal, keeping it below its melting point. By fitting a protective shield around the plug we block the toxic molecules and allow the plug to melt in a fire. The shield is the monoclonal antibody against PD-1 and thus the fire is successfully extinguished.”

This is getting exhausting, so I think I will stop, but don’t you agree that the concept of checkpoint inhibition lends itself to a plethora of metaphors? Now for the next lesson: how to explain chimeric antigen receptor T-cell therapy to patients. Hold on—I think I need to explain it to myself first.

 

As an clinical immunologist, i agree with the concept, looks pretty straightforward but definitely much more complex as our immune system work like a network. I would appreciate clinical trial data with statistical significance.

 

Much more confusing. Most people understand simplified concepts.

“Some cancer cells turn off your immune systems ability to recognise them. These drugs ramp up the immune system and prevent the cancer cells from hiding. This allows your cells to attack and kill cancer cells”

If i think patient seem to have better ability to understand I say “the drugs block the “off switch” that cancer cells use to escape their detection. This turns your immune systems ability to attack and kill cancer cells back on”

I haven’t had one patient that has looked confused since.

 

HDAC Inhibitors Enhance Immunotherapy Efficacy in Lung Cancer

http://www.oncotherapynetwork.com/lung-cancer-targets/hdac-inhibitors-enhance-immunotherapy-efficacy-lung-cancer

Histone deacetylase (HDAC) inhibitors like romidepsin might improve the efficacy of programmed cell death-1 (PD-1) blockade in lung cancer, suggest preclinical findings reported in the journal Clinical Cancer Research.

Most lung cancer patients’ tumors do not respond to immune checkpoint blockade agents like those that target PD-1. One possible mechanism underlying tumor resistance to PD-1 blockade is the failure of sufficient numbers of T cells to infiltrate tumor tissue.

Hypothesizing that upregulating T-cell chemokine expression and thereby T-cell infiltration of tumors would improve PD-1 blockade’s efficacy against lung tumors, the research team went hunting for FDA-approved oncology agents that induce chemokine expression. Screening 97 approved agents, they found one class that did: HDAC inhibitors.

The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor infiltration and impacted lung tumor growth in mouse models, the team reported—and when romidepsin was subsequently combined with PD-1 blockade in several lung tumor models, the combination showed greater antitumor activity than either agent on its own.

“These results suggest that combination of HDAC inhibitors with PD-1 blockade represent a promising strategy for lung cancer treatment,” said senior study author Amer A. Beg, PhD, of the Moffitt Cancer Center’s Immunology Program, in a news release.

Romidepsin and other HDAC inhibitors have already been approved by the FDA for use against lymphoma and other hematologic cancers, Dr. Beg noted.

The combination will next be tested in several clinical trials, including a study of patients diagnosed with non-small cell lung cancer (NSCLC) at Moffitt Cancer Center.

 

HDAC inhibitors enhance T cell chemokine expression and augment response to PD-1 immunotherapy in lung adenocarcinoma

Hong Zheng1,  weipeng zhao2Cihui Yan3Crystina C Watson4,…., Brian Ruffell13, and Amer A Beg4,*

Clin Cancer Res March 10, 2016; http://dx.doi.org:/10.1158/1078-0432.CCR-15-2584

Purpose: A significant limitation of checkpoint blockade immunotherapy is the relatively low response rate (e.g. ~20% with PD-1 blockade in lung cancer). In this study, we tested whether strategies which increase T cell infiltration to tumors can be efficacious in enhancing immunotherapy response. Experimental Design: We performed an unbiased screen to identify FDA-approved oncology agents with ability to enhance T cell chemokine expression with the goal of identifying agents capable of augmenting immunotherapy response. Identified agents were tested in multiple lung tumor models as single agents and in combination with PD-1 blockade. Additional molecular and cellular analysis of tumors was used to define underlying mechanisms. Results: We found that histone deacetylase (HDAC) inhibitors (HDACi) increased expression of multiple T cell chemokines in cancer cells, macrophages and T cells. Using the HDACi romidepsin in vivo, we observed increased chemokine expression, enhanced T cell infiltration, and T cell-dependent tumor regression. Importantly, romidepsin significantly enhanced the response to PD-1 blockade immunotherapy in multiple lung tumor models, including nearly complete rejection in two models. Combined romidepsin and PD-1 blockade also significantly enhanced activation of tumor-infiltrating T cells. Conclusions: These results provide evidence for a novel role of HDACs in modulating T cell chemokine expression in multiple cell types. In addition, our findings indicate that pharmacological induction of T cell chemokine expression represents a conceptually novel approach for enhancing immunotherapy response. Finally, these results suggest that combination of HDAC inhibitors with PD-1 blockade represents a promising strategy for lung cancer treatment.

 

Cancer Cell Survival Driven by Novel Metabolic Pathway

http://www.genengnews.com/gen-news-highlights/cancer-cell-survival-driven-by-novel-metabolic-pathway/81252584/

Researchers have identified a novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells. [National Cancer Institute, NIH] http://www.genengnews.com/Media/images/GENHighlight/thumb_28216_large1422477191.jpg

Being attached to the extracellular matrix (ECM) provides cells with numerous advantages for survival, for instance, receiving much needed growth stimuli. However, for malignant cells to function, they must overcome their anchorage-dependent growth—a scenario that is associated with increased production of reactive oxygen species (ROS) and altered glucose metabolism.

Now, researchers at the Children’s Medical Center Research Institute at UT Southwestern (CRI) believe they have uncovered a novel metabolic pathway that helps cancer cells thrive in conditions that would otherwise be lethal to healthy cells.

“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” explained senior study author Ralph DeBerardinis, M.D., Ph.D.,  associate professor, and director of CRI’s Genetic and Metabolic Disease Program. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”

This new study describes an alternate version of two well-known metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle, to defend against ROS that destroy cells via oxidative stress.

The findings from this study were published recently in Nature in an article entitled “Reductive Carboxylation Supports Redox Homeostasis During Anchorage-Independent Growth.”

Previous work from Dr. DeBerardinis’ laboratory found that the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.

Dr. DeBerardinis also noted that cells “are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival.” Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells. Yet, cancer cells seem to have evolved workaround.

A landmark study from 2009 elucidated that healthy cells were destroyed when detached from the ECM. Moreover, in the same study, investigators found that inserting an oncogene into a normal cell caused it to behave like a cancer cell and survive detachment.

“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.

Dr. DeBerardinis and his team worked from the premise that the two findings were pieces of the same puzzle and that a crucial part of the picture seemed to be missing.

It had been well known that the PPP was an important source of nicotine adenine dinucleotide phosphate (NADPH), which provides a source of reducing electrons to scavenge ROS; however, the PPP produces NADPH in the cytosol, whereas the ROS are generated primarily in another subcellular structure called the mitochondria.

“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis noted.  But how could NADPH from the PPP help deal with the stress of ROS produced in an entirely different part of the cell? “What we did was to discover how this happens.”

The CRI team was able to demonstrate that cancer cells use a “piggybacking” system to carry the reducing electron from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle.The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing electrons to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.

“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells. But we had no idea that they were linked in this unusual fashion,” Dr. DeBerardinis stated. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”

The researchers stressed that their findings were based on cultured cell models and more research will be necessary to test the role of the pathway in living organisms.

“We are particularly excited to test whether this pathway is required for metastasis because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” Dr. DeBerardinis concluded.

 

Reductive carboxylation supports redox homeostasis during anchorage-independent growth

Lei JiangAlexander A. ShestovPamela SwainChendong Yang, …., Brian P. DrankaBenjamin Schwartz & Ralph J. DeBerardinis

Nature(2016)      http://dx.doi.org:/10.1038/nature17393

Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM)1. Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells2. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism2. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

 

Liquid Biopsy Accurately Detects Mutations in Advanced NSCLC

http://www.oncotherapynetwork.com/lung-cancer/liquid-biopsy-accurately-detects-mutations-advanced-nsclc#sthash.bEPxRkAq.dpuf

Droplet digital polymerase chain reaction (ddPCR)-based plasma genotyping—referred to as liquid biopsy—exhibited perfect specificity in identifying EGFR and KRAS mutations in patients with advanced non–small-cell lung cancer (NSCLC), according to the results of a study published in JAMA Oncology.

“We see plasma genotyping as having enormous potential as a clinical test, or assay—a rapid, noninvasive way of screening a cancer for common genetic fingerprints, while avoiding the challenges of traditional invasive biopsies,” said senior author, Geoffrey Oxnard, MD, thoracic oncologist and lung cancer researcher at Dana-Farber and Brigham and Women’s Hospital, in a press release. “Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients.”

According to the press release, the test proved so reliable in the study that the Dana-Farber/Brigham and Women’s Cancer Center this week became the first medical facility in the country to offer it to all patients with NSCLC, either at the time of first diagnosis or of relapse following previous treatment.

Oxnard and colleagues enrolled 180 patients with advanced NSCLC. Patients were either newly diagnosed with the disease (n = 120) or had acquired resistance to prior EGFR kinase inhibitors (n = 60) and were planned for rebiopsy. Patients underwent initial blood sampling and immediate plasma ddPCR screening for EGFR exon 19 deletion, L858R, the EGFR T790M acquired resistance mutation, or KRAS G12X. In addition, patients underwent biopsy for tissue genotyping used to compare the accuracy of ddPCR.

Among the enrolled patients, 80 had EGFR exon19/L858R mutations, 35 had T790M mutations and 25 had KRAS G12X mutations. The median test turnaround time for liquid biopsy was 3 days. In comparison, the median turnaround time for tissue genotyping was 12 days for newly diagnosed patients and 27 days for patients with acquired EGFR inhibitor resistance.

“This long turnaround time is due largely to the practical reality that many patients with newly diagnosed NSCLC require a repeat biopsy to obtain tissue for genotyping, as do all patients with acquired resistance,” the researchers noted.

The liquid biopsy showed 100% positive predictive value for detecting EGFR 19 deletion, L858R, and KRAS mutations. However, it had only a positive predictive value of 79% for T790M mutations. The sensitivity of the test was lower. ddPCR had a sensitivity of 82% for EGFR 19 deletion, 74% for L858R, 77% for T790M, and 64% for KRAS.

The researchers pointed out that “a key limitation of plasma ddPCR is that although this method is adept at rapidly detecting specific targetable mutations, it cannot easily detect copy number alterations and rearrangements. The ddPCR panel assessed in this study thus cannot currently detect targetable alterations in either ALK or ROS1,” two other common mutations in NSCLC.

In an editorial that accompanied the article, P. Mickey Williams, PhD, of Frederick National Laboratory for Cancer Research, and Barbara A. Conley, MD, from the National Cancer Institute, questioned whether or not these results, and the rapid turnaround time for liquid biopsy, could be replicated widely by other institutions.

“However, if this performance were generally applicable, this would indeed be an advance in clinical care, reducing the proportion of patients requiring biopsy, at least in the resistance setting,” Williams and Conley wrote.

“This study is a step in the right direction of preparing needed clinical validation for the use of ctDNA for detection and serial monitoring of clinically relevant tumor mutations. Owing to low sensitivity and high positive predictive value and specificity, this approach is probably best suited for detection of resistance mutations and for serial plasma testing to assess treatment response, and should not replace tumor biopsy assessment for initial treatment decision-making,” they concluded.

 

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer

Adrian G. Sacher, MD1,2; Cloud Paweletz, PhD3; Suzanne E. Dahlberg, PhD4,5; Ryan S. Alden, BSc1; Allison O’Connell, BSc3; Nora Feeney, BSc3; Stacy L. Mach, BA1; Pasi A. Jänne, MD, PhD1,2,3; Geoffrey R. Oxnard, MD1,2

JAMA Oncol. Published online April 07, 2016.    http://dx.doi.org:/10.1001/jamaoncol.2016.0173

Importance  Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.

Objective  To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.

Design, Setting, and Participants  Patients with advanced nonsquamous non–small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute–designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.

Main Outcomes and Measures  Plasma ddPCR assay sensitivity, specificity, and TAT.

Results  Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRASG12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.

Conclusions and Relevance  Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

Plasma genotyping uses tumor-derived cell-free DNA (cfDNA) to allow for rapid noninvasive genotyping of tumors. This technology is currently poised to transition into a treatment decision-making tool in multiple cancer types. It is particularly relevant to the treatment of advanced non–small-cell lung cancer (NSCLC), in which therapy hinges on rapid and accurate detection of targetable epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and ROS1 alterations.1– 6Plasma genotyping is capable of circumventing many limitations of standard tissue genotyping including slow turnaround time (TAT), limited tissue for testing, and the potential for failed biopsies. It may be particularly useful in directing the rapid use of new targeted therapies for acquired resistance in advanced EGFR-mutant NSCLC, where the need for a repeat biopsy to test for resistance mechanisms has amplified the inherent limitations of traditional genotyping.7,8

The need to carefully validate the test characteristics of each of the myriad individual plasma genotyping assays before use in clinical decision making is paramount. We have previously reported the development of a quantitative droplet digital polymerase chain reaction (ddPCR)-based assay for the detection of EGFR kinase mutations andKRAS codon 12 mutations in plasma.9 The detection of these mutations has the potential to guide treatment by either facilitating targeted therapy with an EGFR tyrosine kinase inhibitor (TKI) or ruling out the presence of other potentially targetable alterations in the case of KRAS.5 Alternative platforms including Cobas, peptide nucleic acid–mediated PCR, multiplexed next-generation sequencing (NGS), high-performance liquid chromatography, and Scorpion–amplified refractory mutation system have also been examined in retrospective analyses of patient samples.10– 22 The test characteristics of these assays have been variable and may be attributable to differences in testing platforms, as well as the retrospective nature of these studies, their smaller size, and the timing of blood collection with respect to disease progression and therapy initiation. The absence of reliable prospective data on the use of specific plasma genotyping assays in advanced NSCLC has left key aspects of its utility largely undefined and slowed its uptake as a tool for clinical care in patients with both newly diagnosed NSCLC and EGFR acquired resistance.

To our knowledge, we have conducted the first prospective study of the use of ddPCR-based plasma genotyping for the detection of EGFR and KRAS mutations. This study was performed in the 2 settings where we anticipate clinical adoption of this assay: (1) patients with newly diagnosed advanced NSCLC and (2) those with acquired resistance to EGFR kinase inhibitors. The primary aim of this study was to prospectively evaluate the feasibility and accuracy of this assay for the detection ofEGFR/KRAS mutations in patients with newly diagnosed NSCLC and EGFR T790M in patients with acquired resistance in a clinical setting. Additional end points included test TAT and the effect of sample treatment conditions on test accuracy.

Key Points
  • Question What is the sensitivity, specificity, turnaround time, and robustness of droplet digital polymerase chain reaction (ddPCR)-based plasma genotyping for the rapid detection of targetable genomic alterations in patients with advanced non–small-cell lung cancer (NSCLC)?

  • Findings In this study of 180 patients with advanced NSCLC (120 newly diagnosed, 60 with acquired resistance to epidermal growth factor receptor [EGFR] kinase inhibitors), plasma genotyping exhibited perfect specificity (100%) and acceptable sensitivity (69%-80%) for the detection of EGFR-sensitizing mutations with rapid turnaround time (3 business days). Specificity was lower for EGFR T790M (63%), presumably secondary to tumor heterogeneity and false-negative tissue genotyping.

  • Meaning The use of ddPCR-based plasma genotyping can detect EGFR mutations with the rigor necessary to direct clinical care. This assay may obviate repeated biopsies in patients with positive plasma genotyping results.

CYP3A7*1C Allele Associated With Poor Outcomes in CLL, Breast, and Lung Cancer

 http://www.oncotherapynetwork.com/breast-cancer-targets/cyp3a71c-allele-associated-poor-outcomes-cll-breast-and-lung-cancer#sthash.7uiD8XFD.dpuf

Patients with the CYP3A7*1C allele suffer higher rates of cancer progression and mortality, possibly because of worse outcomes among patients treated with chemotherapy drugs that are broken down by the enzyme encoded by CYP3A7, according to authors of a retrospective study published in the journal Cancer Research.

“We found that individuals with breast cancer, lung cancer, or CLL [chronic lymphocytic leukemia] who carry one or more copy of the CYP3A7*1C allele tend to have worse outcomes,” said Olivia Fletcher, PhD, a senior investigator at the Breast Cancer Now Toby Robins Research Centre at the Institute of Cancer Research in London, England, in an American Association for Cancer Research (AACR) news release.

Approximately 8% of cancer patients harbor the CYP3A7*1C allele, the coauthors noted. For these patients, it is possible that standard chemotherapy with CYP3A substrates “may not be optimal,” they cautioned.

The team analyzed DNA samples from 1,008 patients with breast cancer, 1,128 patients with lung cancer, and 347 patients with CLL. They found that the CYP3A7*1C-associated single nucleotide polymorphism (SNP) rs45446698 is associated with increased breast cancer mortality (hazard ratio [HR] 1.74; P = .03), all-cause mortality among patients with lung cancer (HR 1.43; P = .009), and progression of CLL (HR 1.62; P = .03). The rs45446698 SNP is one of seven SNPs that form the CYP3A7*1C allele.

The CYP3A7*1C allele is expressed in adults, whereas other variants of CYP3A7 are expressed during fetal development. CYP3A7 encodes an enzyme that degrades estrogen and testosterone, and some anticancer drugs.

“We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (P = .06),” they noted.

“Even though we did not see a statistically-significant difference when stratifying patients by treatment with a CYP3A7 substrate, the fact that we see the same effect in three very different cancer types suggests to me that it is more likely to be something to do with treatment than the disease itself,” commented Dr. Fletcher. “However, we are looking at ways of replicating these results in additional cohorts of patients and types of cancer, as well as overcoming the limitations of this study.”

Limitations included the retrospective nature of the study and the absence of data on how quickly individual patients metabolized chemotherapeutic agents, she said.

 

Cytochrome P450 AlleleCYP3A7*1C Associates with Adverse Outcomes in Chronic Lymphocytic Leukemia, Breast, and Lung Cancer

Nichola Johnson1,2Paolo De Ieso3Gabriele Migliorini4,….., Gillian Ross12Richard S. Houlston, and Olivia Fletcher1,2,*

Cancer Res March 10, 2016; http://dx.doi.org:/10.1158/0008-5472.CAN-15-1410

CYP3A enzymes metabolize endogenous hormones and chemotherapeutic agents used to treat cancer, thereby potentially affecting drug effectiveness. Here, we refined the genetic basis underlying the functional effects of a CYP3A haplotype on urinary estrone glucuronide (E1G) levels and tested for an association betweenCYP3A genotype and outcome in patients with chronic lymphocytic leukemia (CLL), breast, or lung cancers. The most significantly associated SNP was rs45446698, an SNP that tags the CYP3A7*1Callele; this SNP was associated with a 54% decrease in urinary E1G levels. Genotyping this SNP in 1,008 breast cancer, 1,128 lung cancer, and 347 CLL patients, we found that rs45446698 was associated with breast cancer mortality (HR, 1.74; P = 0.03), all-cause mortality in lung cancer patients (HR, 1.43; P = 0.009), and CLL progression (HR, 1.62; P= 0.03). We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (Pinteraction = 0.06). The CYP3A7*1C allele, which results in adult expression of the fetal CYP3A7 gene, is likely to be the functional allele influencing levels of circulating endogenous sex hormones and outcome in these various malignancies. Further studies confirming these associations and determining the mechanism by which CYP3A7*1C influences outcome are required. One possibility is that standard chemotherapy regimens that include CYP3A substrates may not be optimal for the approximately 8% of cancer patients who are CYP3A7*1C carriers. Cancer Res; 76(6); 1–9. ©2016 AACR.

 

​Specific Form of CYP3A7 Gene Associated With Poor Outcomes for Patients With Several Cancer Types

3/10/2016

PHILADELPHIA — Among patients with breast cancer, lung cancer, or chronic lymphocytic leukemia (CLL), those who had a specific form of the CYP3A7 gene (CYP3A7*1C) had worse outcomes compared with those who did not have CYP3A7*1C, and this may be related to how the patients metabolize, or break down, the therapeutics used to treat them, according to a study published in Cancer Research, a journal of the American Association for Cancer Research.

“The CYP3A7 gene encodes an enzyme that breaks down all sorts of naturally occurring substances—such as sex steroids like estrogen and testosterone—as well as a wide range of drugs that are used in the treatment of cancer,” saidOlivia Fletcher, PhD, a senior investigator at the Breast Cancer Now Toby Robins Research Centre at The Institute of Cancer Research in London. “The CYP3A7 gene is normally turned on in an embryo and then turned off shortly after a baby is born, but individuals who have one or more copy of the CYP3A7*1C form of the gene [the CYP3A7*1C allele] turn on their CYP3A7 gene in adult life.

“We found that individuals with breast cancer, lung cancer, or CLL who carry one or more copy of the CYP3A7*1C allele tend to have worse outcomes,” continued Fletcher. “One possibility is that these patients break down the drugs that they are given to treat their cancer too fast. However, further independent studies that replicate our findings in larger numbers of patients and rule out biases are needed before we could recommend any changes to the treatment that cancer patients with the CYP3A7*1C allele receive.”

To find out whether the CYP3A7*1C allele was associated with outcome for patients with breast cancer, lung cancer, or CLL, Fletcher and colleagues analyzed DNA samples from 1,008 breast cancer patients, 1,142 patients with lung cancer, and 356 patients with CLL for the presence of a single nucleotide polymorphism (SNP), rs45446698. Fletcher explained that a SNP is a type of genetic variant and that because of the way that we inherit our genetic material from our parents, we tend to inherit clusters of genetic variants. She went on to say that rs45446698 is one of seven SNPs that cluster together, forming the CYP3A7*1C allele.

The researchers found that among the breast cancer patients, rs45446698 (and, therefore, the CYP3A7*1C allele) was associated with a 74 percent increased risk of breast cancer mortality. Among the lung cancer patients, it was associated with a 43 percent increased risk of death from any cause, and among the CLL patients, it was associated with a 62 percent increased risk of disease progression.

Patients who were treated with a chemotherapeutic broken down by CYP3A7 tended to have worse outcomes compared with those treated with other chemotherapeutics, but the difference was not statistically significant.

“Even though we did not see a statistically significant difference when stratifying patients by treatment with a CYP3A7 substrate, the fact that we see the same effect in three very different cancer types suggests to me that it is more likely to be something to do with treatment than the disease itself,” said Fletcher. “However, we are looking at ways of replicating these results in additional cohorts of patients and types of cancer, as well as overcoming the limitations of this study.”

Fletcher explained that the main limitation of the study is that the researchers used samples and clinical information collected for other studies. Therefore, they did not have the same clinical information for each patient, and the samples were collected at different time points and for patients treated with various chemotherapeutics. She also noted that the team were not able to determine how quickly the patients broke down the therapeutics they received as treatment.

The study was supported by Breast Cancer Now, Leukaemia and Lymphoma Research (now known as Bloodwise), Cancer Research UK, the Medical Research Council, the Cridlan Trust, the Helen Rollason Cancer Charity, and Sanofi-Aventis. Funding for the authors’ institutions was received from the National Health Service of the United Kingdom. Fletcher declares no conflicts of interest.

 

Liquid Biopsy for NSCLC

‘Liquid biopsy’ blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.

http://www.technologynetworks.com/Diagnostics/news.aspx?ID=190276

A simple blood test can rapidly and accurately detect mutations in two key genes in non-small cell lung tumors, researchers at Dana-Farber Cancer Institute and other institutions report in a new study – demonstrating the test’s potential as a clinical tool for identifying patients who can benefit from drugs targeting those mutations.

The test, known as a liquid biopsy, proved so reliable in the study that Dana-Farber/Brigham and Women’s Cancer Center (DF/BWCC) expects to offer it soon to all patients with non-small cell lung cancer (NSCLC), either at the time of first diagnosis or of relapse following previous treatment.

…….

“Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients. The trial was such a success that we are transitioning the assay into a clinical test for lung cancer patients at DF/BWCC.”

The study involved 180 patients with NSCLC, 120 of whom were newly diagnosed, and 60 of whom had become resistant to a previous treatment, allowing the disease to recur. Participants’ cell-free DNA was tested for mutations in the EGFR and KRAS genes, and for a separate mutation in EGFR that allows tumor cells to become resistant to front-line targeted drugs. The test was performed with a technique known as droplet digital polymerase chain reaction (ddPCR), which counts the individual letters of the genetic code in cell-free DNA to determine if specific mutations are present. Each participant also underwent a conventional tissue biopsy to test for the same mutations. The results of the liquid biopsies were then compared to those of the tissue biopsies.

The data showed that liquid biopsies returned results much more quickly. The median turnaround time for liquid biopsies was three days, compared to 12 days for tissue biopsies in newly diagnosed patients and 27 days in drug-resistant patients.

Liquid biopsy was also found to be highly accurate. In newly diagnosed patients, the “predictive value” of plasma ddPCR was 100 percent for the primary EGFR mutation and the KRAS mutation – meaning that a patient who tested positive for either mutation was certain to have that mutation in his or her tumor. For patients with the EGFR resistance mutation, the predictive value of the ddPCR test was 79 percent, suggesting the blood test was able to find additional cases with the mutation that were missed using standard biopsies.

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFRand KRAS Mutations in Advanced Lung Cancer

Adrian G. Sacher, MD1,2; Cloud Paweletz, PhD3; Suzanne E. Dahlberg, PhD, et al.       JAMA Oncol. Published online April 07, 2016.  http://dx.doi.org::/10.1001/jamaoncol.2016.0173

Importance  Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.

Objective  To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.

Design, Setting, and Participants  Patients with advanced nonsquamous non–small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute–designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.

Main Outcomes and Measures  Plasma ddPCR assay sensitivity, specificity, and TAT.

Results  Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRASG12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.

Conclusions and Relevance  Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

 

In this prospective study, we demonstrate the highly specific and rapid nature of plasma genotyping. No false-positive test results were seen for driver mutations inEGFR or KRAS, and TAT from when the specimen was obtained to result was a matter of days. This assay exhibited 100% positive predictive value for the detection of these mutations. Sensitivity was more modest and was directly correlated with both number of metastatic sites and the presence of liver or bone metastases. This newly demonstrated relationship is likely related to increased cfDNA shed in the setting of more extensive disease where tumor cfDNA shed is the chief driver of assay sensitivity and determines its upper limit. The characteristics of plasma ddPCR prospectively demonstrated in this study were similar or improved compared with previous retrospective reports of other cfDNA genotyping assays.10– 13,15,16,24,25 These retrospective studies are smaller, frequently examined a mix of tumor types and/or stages, and lack the careful prospective design needed to demonstrate the readiness of this technology to transition to a tool for selecting therapy. Studies that use retrospective samples from clinical trials that enrolled only EGFR-mutant patients are further limited by an inability to both blind laboratory investigators to tissue genotype and to generalize their assay test characteristics to a genetically heterogeneous real-world patient population.11 These differences and the multiple platforms examined previously have led to variable test characteristics and uncertainty regarding the clinical application of these technologies. This study is the first to prospectively demonstrate the ability of a ddPCR-based plasma genotyping assay to rapidly and accurately detect EGFR and KRAS mutations in a real-world clinical setting with the rigor necessary to support the assertion that use of this assay is capable of directing clinical care.

Even with a diagnostic sensitivity of less than 100%, such a rapid assay with 100% positive predictive value carries the potential for immense clinical utility. The 2- to 3-day TAT contrasts starkly with the 27-day TAT for tumor genotyping seen in patients needing a new tumor biopsy. This long TAT is due largely to the practical reality that many patients with newly diagnosed NSCLC require a repeat biopsy to obtain tissue for genotyping, as do all patients with acquired resistance. Consider the case of 1 study participant, an octogenarian with metastatic NSCLC who had developed acquired resistance to erlotinib with painful bone metastases (Figure 3). Due to the patient’s age and comorbidities, significant concerns existed about the risks of a biopsy and further systemic therapy. A plasma sample was obtained, and within 24 hours ddPCR demonstrated 806 copies/mL of EGFR T790M. A confirmatory lung biopsy was performed, which confirmed EGFR T790M. Treatment with a third-generation EGFR kinase inhibitor, osimertinib mesylate, was subsequently initiated and the patient had a partial response to therapy that was maintained for more than 1 year. The potential of this technology to obviate repeated biopsy in both patients with newly diagnosed NSCLC with insufficient tissue, as well as patients with acquired resistance, is considerable.

A key limitation of plasma ddPCR is that although this method is adept at rapidly detecting specific targetable mutations, it cannot easily detect copy number alterations and rearrangements. The ddPCR panel assessed in this study thus cannot currently detect targetable alterations in either ALK or ROS1. This limitation may potentially be addressed by using targeted NGS of cfDNA for broad, multiplexed detection of complex genomic alterations including ALK and ROS1 rearrangements, although this method is potentially slower than ddPCR-based methods and has been less thoroughly evaluated.23 The potential exists to use these technologies in tandem in advanced NSCLC to facilitate rapid initiation of therapy. Tissue genotyping and repeated biopsy would be specifically used to direct therapy in cases in which plasma genotyping was uninformative due to limitations of assay sensitivity. This approach would be particularly useful in cases of EGFR acquired resistance in which a repeated biopsy for T790M testing could be avoided entirely in many patients. Beyond detecting targetable alterations in order to drive therapy, the identification of nontargetable oncogenic drivers such as KRAS mutations that preclude the presence of other targetable alterations may guide a clinician to rapidly initiate alternative therapies such as chemotherapy or immunotherapy.5 The finding that assay sensitivity is highest in patients with more extensive metastatic disease suggests that those patients most in need of rapid treatment initiation would also be least likely to have false-negative results.

One surprising result of our study was evidence of recurrent false-positive results forEGFR T790M in patients with acquired resistance, despite no false-positive test results for other mutations studied. The sensitivity of the EGFR T790M assay was comparable to that of the EGFR sensitizing mutation assays and similarly related to both disease burden and the presence of liver or bone metastases, which are likely predictive of increased tumor cfDNA shed. We hypothesize that the lower assay specificity is due to the genomic heterogeneity whereby the T790M status of the biopsied site is not representative of all metastatic sites in a patient, a phenomenon supported by mounting evidence in the acquired resistance setting.26,27 This is consistent with the finding that a minority of patients with apparently EGFR T790M tissue-negative disease respond to therapy with third-generation EGFR kinase inhibitors.7,8,28 These observations raise questions regarding the fallibility of tissue-based genotyping as the reference standard for T790M status. The use of plasma genotyping to detect EGFR T790M thus has great potential to identify patients who would benefit from newly approved third-generation EGFR kinase inhibitors but would be unable to access them based on falsely negative tissue genotyping results. Indeed, plasma genotyping may allow more reliable assessment of both T790M status as well as the mechanisms of resistance across all sites of a heterogeneous cancer as opposed to a tissue biopsy and is likely to be an essential tool for future trials targeting drug resistance. The potential to avoid a repeat biopsy entirely in patients in whom plasma ddPCR detects T790M further strengthens the utility of this technology, although a repeat biopsy would still be needed in patients with uninformative plasma ddPCR due to limitations with respect to assay sensitivity.

This study also examined the potential of the quantitative nature of ddPCR-based plasma genotyping to allow for the early prediction of treatment response. Distinct patterns of change in mutant allele copy number were observed as early as 2 weeks after treatment and were similar to those reported in other tumor types.19,20 We hypothesize that these distinct patterns of change in this study will correlate with specific patterns of radiographic response and emergence of acquired resistance and plan to report these data once mature. The observed differences in treatment discontinuation rates observed in this study comparing patients with complete resolution of detectable mutant cfDNA with those with incomplete resolution support this hypothesis. The use of this technology to monitor disease status in real time has potential utility for both routine clinical care, as well as use as an integrated biomarker in early-phase clinical trials.10

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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This is a compilation of articles on Inflammatory Disorders that were published 

@ pharmaceuticalintelligence.com, since 4/2012 to date

There are published works that have not been included.  However, there is a substantial amount of material in the following categories:

  1. The systemic inflammatory response
    https://pharmaceuticalintelligence.com/2014/11/08/introduction-to-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/
    https://pharmaceuticalintelligence.com/2014/11/09/summary-and-perspectives-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/
    https://pharmaceuticalintelligence.com/2015/12/19/neutrophil-serine-proteases-in-disease-and-therapeutic-considerations/
    https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/
    https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/
    https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/
    https://pharmaceuticalintelligence.com/2012/07/08/zebrafish-provide-insights-into-causes-and-treatment-of-human-diseases/
    https://pharmaceuticalintelligence.com/2016/01/25/ibd-immunomodulatory-effect-of-retinoic-acid-il-23il-17a-axis-correlates-with-the-nitric-oxide-pathway/
    https://pharmaceuticalintelligence.com/2015/11/29/role-of-inflammation-in-disease/
    https://pharmaceuticalintelligence.com/2013/03/06/can-resolvins-suppress-acute-lung-injury/
    https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/
  2. sepsis
    https://pharmaceuticalintelligence.com/2012/10/20/nitric-oxide-and-sepsis-hemodynamic-collapse-and-the-search-for-therapeutic-options/
  3. vasculitis
    https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/
    https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/
    https://pharmaceuticalintelligence.com/2012/11/20/the-potential-for-nitric-oxide-donors-in-renal-function-disorders/
  4. neurodegenerative disease
    https://pharmaceuticalintelligence.com/2013/02/27/ustekinumab-new-drug-therapy-for-cognitive-decline-resulting-from-neuroinflammatory-cytokine-signaling-and-alzheimers-disease/
    https://pharmaceuticalintelligence.com/2016/01/26/amyloid-and-alzheimers-disease/
    https://pharmaceuticalintelligence.com/2016/02/15/alzheimers-disease-tau-art-thou-or-amyloid/
    https://pharmaceuticalintelligence.com/2016/01/26/beyond-tau-and-amyloid/
    https://pharmaceuticalintelligence.com/2015/12/10/remyelination-of-axon-requires-gli1-inhibition/
    https://pharmaceuticalintelligence.com/2015/11/28/neurovascular-pathways-to-neurodegeneration/
    https://pharmaceuticalintelligence.com/2015/11/13/new-alzheimers-protein-aicd-2/
    https://pharmaceuticalintelligence.com/2015/10/31/impairment-of-cognitive-function-and-neurogenesis/
    https://pharmaceuticalintelligence.com/2014/05/06/bwh-researchers-genetic-variations-can-influence-immune-cell-function-risk-factors-for-alzheimers-diseasedm-and-ms-later-in-life/
  5. cancer immunology
    https://pharmaceuticalintelligence.com/2013/04/12/innovations-in-tumor-immunology/
    https://pharmaceuticalintelligence.com/2016/01/09/signaling-of-immune-response-in-colon-cancer/
    https://pharmaceuticalintelligence.com/2015/05/12/vaccines-small-peptides-aptamers-and-immunotherapy-9/
    https://pharmaceuticalintelligence.com/2015/01/30/viruses-vaccines-and-immunotherapy/
    https://pharmaceuticalintelligence.com/2015/10/20/gene-expression-and-adaptive-immune-resistance-mechanisms-in-lymphoma/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
  6. autoimmune diseases: rheumatoid arthritis, colitis, ileitis, …
    https://pharmaceuticalintelligence.com/2016/02/11/intestinal-inflammatory-pharmaceutics/
    https://pharmaceuticalintelligence.com/2016/01/07/two-new-drugs-for-inflammatory-bowel-syndrome-are-giving-patients-hope/
    https://pharmaceuticalintelligence.com/2015/12/16/contribution-to-inflammatory-bowel-disease-ibd-of-bacterial-overgrowth-in-gut-on-a-chip/
    https://pharmaceuticalintelligence.com/2016/02/13/cytokines-in-ibd/
    https://pharmaceuticalintelligence.com/2016/01/23/autoimmune-inflammtory-bowl-diseases-crohns-disease-ulcerative-colitis-potential-roles-for-modulation-of-interleukins-17-and-23-signaling-for-therapeutics/
    https://pharmaceuticalintelligence.com/2014/10/14/autoimmune-disease-single-gene-eliminates-the-immune-protein-isg15-resulting-in-inability-to-resolve-inflammation-and-fight-infections-discovery-rockefeller-university/
    https://pharmaceuticalintelligence.com/2015/03/01/diarrheas-bacterial-and-nonbacterial/
    https://pharmaceuticalintelligence.com/2016/02/11/intestinal-inflammatory-pharmaceutics/
    https://pharmaceuticalintelligence.com/2014/01/28/biologics-for-autoimmune-diseases-cambridge-healthtech-institutes-inaugural-may-5-6-2014-seaport-world-trade-center-boston-ma/
    https://pharmaceuticalintelligence.com/2015/11/19/rheumatoid-arthritis-update/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
    https://pharmaceuticalintelligence.com/2012/09/13/tofacitinib-an-oral-janus-kinase-inhibitor-in-active-ulcerative-colitis/
    https://pharmaceuticalintelligence.com/2013/03/05/approach-to-controlling-pathogenic-inflammation-in-arthritis/
    https://pharmaceuticalintelligence.com/2013/03/05/rheumatoid-arthritis-risk/
    https://pharmaceuticalintelligence.com/2012/07/08/the-mechanism-of-action-of-the-drug-acthar-for-systemic-lupus-erythematosus-sle/
  7. T cells in immunity
    https://pharmaceuticalintelligence.com/2015/09/07/t-cell-mediated-immune-responses-signaling-pathways-activated-by-tlrs/
    https://pharmaceuticalintelligence.com/2015/05/14/allogeneic-stem-cell-transplantation-9-2/
    https://pharmaceuticalintelligence.com/2015/02/19/graft-versus-host-disease/
    https://pharmaceuticalintelligence.com/2014/10/14/autoimmune-disease-single-gene-eliminates-the-immune-protein-isg15-resulting-in-inability-to-resolve-inflammation-and-fight-infections-discovery-rockefeller-university/
    https://pharmaceuticalintelligence.com/2014/05/27/immunity-and-host-defense-a-bibliography-of-research-technion/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
    https://pharmaceuticalintelligence.com/2013/04/14/immune-regulation-news/

Proteomics, metabolomics and diabetes

https://pharmaceuticalintelligence.com/2015/11/16/reducing-obesity-related-inflammation/

https://pharmaceuticalintelligence.com/2015/10/25/the-relationship-of-stress-hypermetabolism-to-essential-protein-needs/

https://pharmaceuticalintelligence.com/2015/10/24/the-relationship-of-s-amino-acids-to-marasmic-and-kwashiorkor-pem/

https://pharmaceuticalintelligence.com/2015/10/24/the-significant-burden-of-childhood-malnutrition-and-stunting/

https://pharmaceuticalintelligence.com/2015/04/14/protein-binding-protein-protein-interactions-therapeutic-implications-7-3/

https://pharmaceuticalintelligence.com/2015/03/07/transthyretin-and-the-stressful-condition/

https://pharmaceuticalintelligence.com/2015/02/13/neural-activity-regulating-endocrine-response/

https://pharmaceuticalintelligence.com/2015/01/31/proteomics/

https://pharmaceuticalintelligence.com/2015/01/17/proteins-an-evolutionary-record-of-diversity-and-adaptation/

https://pharmaceuticalintelligence.com/2014/11/01/summary-of-signaling-and-signaling-pathways/

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

https://pharmaceuticalintelligence.com/2014/10/24/diabetes-mellitus/

https://pharmaceuticalintelligence.com/2014/10/16/metabolomics-summary-and-perspective/

https://pharmaceuticalintelligence.com/2014/10/14/metabolic-reactions-need-just-enough/

https://pharmaceuticalintelligence.com/2014/11/03/introduction-to-protein-synthesis-and-degradation/

https://pharmaceuticalintelligence.com/2015/09/25/proceedings-of-the-nyas/

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

https://pharmaceuticalintelligence.com/2013/03/05/irf-1-deficiency-skews-the-differentiation-of-dendritic-cells/

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

https://pharmaceuticalintelligence.com/2012/11/20/the-potential-for-nitric-oxide-donors-in-renal-function-disorders/

 

 

 

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Gene Expression and Adaptive Immune Resistance Mechanisms in Lymphoma

Larry H. Bernstein, MD, FCAP

 

Gene expression meta-analysis reveals immune response convergence on the IFNγ-STAT1-IRF1 axis and adaptive immune resistance mechanisms in lymphoma

Matthew A. Care1,2, David R. Westhead2 and Reuben M. Tooze1*
Care et al. Genome Medicine (2015) 7:96    http://dx.doi.org:/10.1186/s13073-015-0218-3

Background: Cancers adapt to immune-surveillance through evasion. Immune responses against carcinoma and melanoma converge on cytotoxic effectors and IFNγ-STAT1-IRF1 signalling. Local IFN-driven immune checkpoint expression can mediate feedback inhibition and adaptive immune resistance. Whether such coupled immune polarization and adaptive resistance is generalisable to lymphoid malignancies is incompletely defined. The host response in diffuse large B-cell lymphoma (DLBCL), the commonest aggressive lymphoid malignancy, provides an empirical model.

Methods: Using ten publicly available gene expression data sets encompassing 2030 cases we explore the nature of host response in DLBCL. Starting from the “cell of origin” paradigm for DLBCL classification, we use the consistency of differential expression to define polarized patterns of immune response genes in DLBCL, and derive a linear classifier of immune response gene expression. We validate and extend the results in an approach independent of “cell of origin” classification based on gene expression correlations across all data sets.

Results: T-cell and cytotoxic gene expression with polarization along the IFNγ-STAT1-IRF1 axis provides a defining feature of the immune response in DLBCL. This response is associated with improved outcome, particularly in the germinal centre B-cell subsets of DLBCL. Analysis of gene correlations across all data sets, independent of “cell of origin” class, demonstrates a consistent association with a hierarchy of immune-regulatory gene expression that places IDO1, LAG3 and FGL2 ahead of PD1-ligands CD274 and PDCD1LG2.

Conclusion: Immune responses in DLBCL converge onto the IFNγ-STAT1-IRF1 axis and link to diverse potential mediators of adaptive immune resistance identifying future therapeutic targets.

 

Emergence of clinically detectable malignant disease is associated with escape from tumour immune surveillance [1]. Two principal mechanisms may operate: on the one hand the immune systems loses the ability to detect the neoplastic population through changes in antigen presentation or editing of the antigen receptor repertoire; on the other hand initially effective immune responses may be rendered ineffective through development of an immune suppressive environment [2]. In the latter scenario, local expression of immune checkpoint components can be viewed as subversion of a physiological mechanism, which acts during chronic infections to balance effective immunity with immunemediated tissue damage [3].

In a range of cancers the density, location and functional polarization of tumour infiltrating lymphocytes are of prognostic value [4], providing evidence that the nature of immune evasion remains of importance after clinical detection. This is particularly relevant in the context of novel therapeutic strategies aimed at re-invigorating the “exhausted” anti-tumour immune response through immune checkpoint blockade [5, 6]. Gene expression analysis of bulk tumour tissue integrates expression profiles from multiple cellular sources, often allowing global assessment of the predominant vector of functional immune polarization. A paradigm has been proposed in which cancer-associated immune responses converge on a common “immunologic constant of rejection” characterized by a pattern of cytotoxic and T-cell immune responses and a dominant IFNγ-STAT1-IRF1 signalling axis [4, 7]. Linking the polarized pattern of interferon (IFN)γ-driven immune responses to the expression of immune checkpoints is the concept of “adaptive immune resistance” [5, 8]. In this model IFNγ signalling drives local feedback inhibition through the transcriptional regulation of ligands for the inhibitory receptor PD1 [5, 8]. The common association between cytotoxic responses and expression of IFN signatures and potential mediators of adaptive immune resistance has been further supported by analysis of solid tumour gene expression data from The Cancer Genome Atlas [9]. Importantly, such feedback may be mediated both at the immediate interface between tumour cell and cytotoxic lymphocyte, and by the establishment of a wider immune suppressive milieu in the tumour microenvironment.

The combination of convergent IFN-polarized immune responses [4, 7], coupled to IFN-driven adaptive immune resistance [5, 8], provides a powerful model with which to explain common pathologic associations in carcinoma and melanoma. The recent success of therapies targeting CTLA4 and PD1 immune checkpoints [10–12], combined with an extended range of other therapeutic options [6], means that evidence to support prioritization of therapeutic combinations in different tumour settings is required. Lymphoma, which comprises immune system malignancies, provides an instance in which these pathways are incompletely studied. Classical Hodgkin lymphoma is the archetype in which host response elements dominate to the point of obscuring the neoplastic B-cell clone [13], and in classical Hodgkin lymphoma PD1 pathway blockade has recently been described as a promising therapeutic approach [14]. Diffuse large B-cell lymphoma (DLBCL) is the commonest form of nodal lymphoma in the western world and represents an aggressive malignancy that frequently remains incurable. It is well established that this lymphoma type is associated with a varied extent of host response at diagnosis, which can include elements of IFN signalling [15]. Since several large data sets are publicly available [15–25], this malignancy represents an empirical human model in which to test the association between immune polarization and adaptive immune resistance mechanisms.

The “cell of origin” (COO) classification provides the dominant paradigm for our current understanding of DLBCL [24, 26]. This classification relates the gene expression profiles in DLBCL to those of germinal centre B cells (GCBs) or activated B cells (ABCs), the latter representing the initial stage of B-cell terminal differentiation to plasma cells. Although the COO classification allows the division of DLBCL based on expression of a restricted set of classifier genes into the two principal classes [24], a subset of cases show patterns of classifier gene expression that do not allow confident assignment to either GCB or ABC subsets. Such cases are referred to as “type 3” [24, 26], or “unclassified” [27, 28]. To avoid ambiguity we refer to these cases as COOunclassified DLBCL in the following. In a parallel “consensus cluster” classification developed by Monti et al. [15], it was shown that DLBCL could be divided into three categories characterized by preferential expression of genes linked to proliferation and B-cell receptor signalling, metabolic oxidative phosphorylation, or host response. The latter included multiple elements attributable to components of the immune system and supporting stromal cell types. It was noted that a greater proportion of COO-unclassified DLBCL belonged to the host/ immune response cluster, which had increased numbers of intra-tumoral T cells and macrophages and a relative decrease in neoplastic B cells [15]. We reasoned that the potential association of COOunclassified DLBCL with intense host responses provided a starting point for a meta-analysis of immune response elements in DLBCL. In originating from a prevailing paradigm this provided a wider biological and clinical context. Furthermore, by asking whether evidence supporting a common polarized immune response could be discovered from within the construct of the COO paradigm, we sought to avoid bias that might have arisen by focusing ab initio on components of the polarized immune response or immune checkpoints. With this approach we identify a distinct signature characterised by a pattern of cytotoxic T-cell and IFNγ-polarized immune response genes as a dominant pattern across ten DLBCL data sets encompassing 2030 cases. Using components of this polarized pattern we then explore the immune context of DLBCL in a fashion independent of COO class. We demonstrate the strong association with an IFNγSTAT1-IRF1 axis and an expression hierarchy of immune checkpoints/modulators, consistent with adaptive immune resistance as a common feature operating in DLBCL.
….

Normalisation and re-annotation of data For each data set the probes were re-annotated with the latest version of HUGO Gene Nomenclature Committee (HGNC)-approved symbols [30]. The complete HGNC list was downloaded (on 1 October 2014). Each probe was re-annotated to the latest approved symbol if an unambiguous mapping (i.e. single symbol mapping to approved symbol) could be determined, else the original gene name was maintained. Each data set was quantile normalised using the R Limma package [31]. The probes for each gene were merged by taking the median value for probe sets with a Pearson correlation ≥0.2 and the maximum value for those with a correlation <0.2 [15].

COO classifications We used the COO classifications assigned by the DLBCL automatic classifier (DAC) classifier in our previous work [32].

…..

Gene ontology analysis Meta-profile gene lists were assessed for gene ontology (GO) enrichment using the Cytoscape BiNGO tool [41]. GO and annotation files were downloaded from [42] (13 June 2014). The background reference was set to a nonredundant list of the genes present in the 11 data sets. The FDR rate (Benjamini and Hochberg) was set to ≤0.1.

Signature enrichment visualisation See Additional file 2 for an outline of the process for integrating and visualizing analysis of gene signature and ontology enrichments. The results from gene signature and gene ontology enrichment were used to create heatmap visualisations. For each meta-profile the top 100 most enriched signatures and 100 most enriched GO terms were used to construct a matrix of signatures against genes. This is a binary matrix with 1 s depicting an assigned signature/ GO annotation. Using Python a row-wise (gene correlation) and column-wise (signature correlation) phi coefficient was calculated. These were then hierarchical clustered using GENE-E [43] with complete linkage.

……

Results

Shared meta-profiles for COO-unclassified and COO-classified DLBCL Given the importance of the COO paradigm to both the biological and clinical assessment of DLBCL, we anchored our initial analysis on this classification. We previously developed a COO classifier implementation that allows the robust classification of multiple DLBCL data sets [32], which is currently in clinical usage in the context of a phase 3 clinical trial [44]. Applying this to the 11 largest publicly available DLBCL data sets (GSE10846 was split according to treatment into CHOP and R-CHOP components), encompassing 2030 cases [15–25], provided a resource for gene expression metaanalysis. To determine genes consistently linked to COO class we used both the consistency of differential expression between data sets as well as absolute level of differential expression to identify and rank genes associated with each class. We restricted the gene lists by applying a threshold of differential expression in 6 out of 11 data sets; we refer to these as meta-profiles. To explore the relationship of COO-unclassified DLBCL to each of the principal COO classes, we employed sequential pairwise comparisons (Additional file 1). From the initial comparison, we identified 127 genes associated with COO-unclassified DLBCL relative to both ABC- and GCB-DLBCL, while 209 genes were associated with both
COO classes relative to COO-unclassified DLBCL (Additional file 5; Fig. 1). The extent of overlap was highly significant (p=1.32E-157 and p=2.09E-200 for genes associated with COO-unclassified DLBCL or COO class, respectively). We subsequently refer to these sets of overlapping genes as COO-unclassified and COO-classified meta-profiles, respectively.

Figures not shown

Fig. 1 Consistent gene expression differences separate COO-unclassified DLBCL from either principal COO class. The overlap of genes consistently associated with either COO-classified DLBCL (left Venn diagram and Wordle) or COO-unclassified DLBC (right Venn diagram and Wordle) are shown. Left: the Venn diagram shows genes up-regulated in ABC (yellow) or GCB (blue) relative to COO-unclassified. Right: the Venn diagram shows genes up-regulated in COO-unclassified relative to ABC-DLBCL (brown) or GCB-DLBCL (turquoise). For the Wordles, word size is given by differential expression (between contrasts) to the power of median-fold change
COO-unclassified DLBCL is enriched for features of a polarized immune response To assess underlying biology in the COO-classified and COO-unclassified meta-profiles we developed an approach for integrated analysis of GO and gene signature enrichment (Additional file 2) which applies hierarchical clustering to reciprocally assess the relationships of enriched ontology and signature terms and associated genes contributing to enrichments (Additional file 6). The results are displayed as heatmaps of the hierarchically clustered correlations. In the COO-classified meta-profile a striking representation of genes linked to cell proliferation resulted in multiple distinct clusters of enriched terms reflecting a wide range of processes associated with cell proliferation (Fig. 2a; Additional file 7). In addition to this, distinct enrichment of signatures of the B-cell lineage was evident. From the gene perspective this was reflected in one main branch associated with cell cycle and cell proliferation, and the second including two principal subclusters associated on the one hand with RNA binding and processing, and on the other with core B-cellassociated genes (Fig. 2b; Additional file 8).

Fig. 2 Integrated gene signature and ontology enrichment analysis demonstrates association of the COO-classified meta-profile with cell proliferation and B-cell signatures. a The top gene signature and ontology terms enriched in the COO-classified meta-profile, clustered according to the correlation of signatures given their gene membership. b The corresponding clustering of genes contributing to signature and ontology term enrichments for the COO-classified meta-profile, clustered according to correlation of genes given their signature membership. To the right general categories corresponding to major correlation clusters are illustrated. Corresponding high resolution versions are available in Additional files 7 and 8

In contrast the COO-unclassified meta-profile was linked to terms related to T-cell populations, T-cell receptor signalling and T-cell activation. While the second principal branch of ontology/signature terms was linked to additional more diverse immune response elements (Fig. 3a; Additional file 9). Hierarchical clustering from the gene perspective (Fig. 3b; Additional file 10) generated a principal branch related to T cells composed of a cluster of genes representing core elements of the T-cell state (CD2, CD3D, CD3E, CD3G, CD28 and TRBC1) and another cluster of genes with T-cell associations, including BCL11B, GZMA, GZMK, MAF and STAT4. The second principal branch of the hierarchical tree included genes derived from monocytes and other immune/host response signatures. This also included a subcluster comprising IFNG, and interferon responsive genes GBP1 and IFITM1, as well as the chemokine receptors CCR5, CXCR3 and CXCR6, which are linked to Th1 polarized T-cell populations [45, 46]. We therefore conclude that COO-unclassified DLBCL is generally distinguished from COO-classified DLBCL by a predominant T-cell immune response with skewing toward IFNG gene expression. Furthermore the paucity of both proliferation and B-cell gene expression is indicative of a relatively low representation of neoplastic B cells.

Fig. 3 Integrated gene signature and ontology enrichment analysis demonstrates association of the COO-unclassified meta-profile with polarized immune response. a The top gene signature and ontology terms enriched in the COO-unclassified meta-profile, clustered according to the correlation of signatures given their gene membership. b The corresponding clustering of genes contributing to signature and ontology term enrichments for the COO-unclassified meta-profile, clustered according to correlation of genes given their signature membership. To the right general terms corresponding to major correlation clusters are illustrated (NOS not otherwise specified). Corresponding high resolution versions are available in Additional files 9 and 10

A cytotoxic and interferon polarized immune response as an independent molecular feature of DLBCL We next addressed to what extent the identified polarized pattern of immune response was selective for COO-unclassified DLBCL or whether equivalently intense expression of polarized immune response genes might be detectable amongst some DLBCL cases that could be assigned to a principal COO class. As noted above, the COO-unclassified meta-profile separated on hierarchical clustering from the gene perspective into two branches, one of which was more strongly linked to core T-cell and cytotoxic genes (Fig. 4). To examine the relative ranking of genes belonging to these two hierarchical clustering branches within the COOunclassified meta-profile we superimposed the cluster membership onto scatter plots of differential expression ranking. We first ranked and then plotted genes
belonging to the meta-profile by median fold differential expression in the comparison of COO-unclassified with ABC- or GCB-DLBCL. This demonstrated a significant overall correlation in the differential expression of COO-unclassified meta-profile genes relative to either principal COO class. Furthermore, genes belonging to the “T-cell cluster” (cluster 1) were significantly skewed toward most consistent association with COO-unclassified DLBCL (Additional file 11). To address whether the consistency of differential detection between data sets would alter this conclusion we ranked genes by a measure derived from both the number of data sets (consistency of differential expression) in which a gene was differentially expressed and the normalised median fold differential expression (Additional file 12). This again showed a significant overall correlation and a skewing of the T-cell cluster toward most consistent association with COO unclassified DLBCL (p=6.57E-06, hypergeometric test; Fig. 4). However, using either approach IFNG was identified as amongst the cluster 2 genes most consistently linked to COO-unclassified DLBCL.

Fig. 4 Genes most consistently associated with COO-unclassified DLBCL are related to a polarized immune response. The two principal branches of the gene-centred hierarchical clustering tree of the COO-unclassified meta-profile are illustrated on the left. Colour-coding identifies: red cluster 1, corresponding to the T-cell cluster; black cluster 2, IFN and monocyte/immune NOS (not otherwise specified). On the right the relative rank of differentially expressed genes contributing to the COO-unclassified meta-profile is plotted using a differential expression ranking, derived from the number of data sets with differential expression to the power of normalized median fold change; the x-axis indicates differential expression rank in the comparison COO-unclassified versus ABC-DLBCL; the y-axis indicates differential expression rank in the comparison COO-unclassified versus GCB-DLBCL. Cluster membership is superimposed on the scatter plot of differential expression rank according to the colour coding shown (red cluster 1, black cluster 2). The 16 genes most consistently separating COO-unclassified DLBCL from either ABC- or GCB-DLBCL are illustrated below with cluster membership and mean differential expression rank shown. See corresponding Additional file 11

To examine the contribution of polarized immune response genes associated with COO-unclassified DLBCL across all data sets on a case-by-case basis we developed a linear additive classifier. For this we employed the 16 genes most strongly linked to COO-unclassified DLBCL derived from analysis using both the consistency/data set number and median fold differential expression. Given the contribution of core T-cell elements, cytotoxic genes and IFNG, we consider this to represent an integrated assessment of a polarized immune response. We ranked all cases in each data set by this linear score and plotted the incidence of cases classified as ABC, GCB and unclassified on this ranking. Overall, individual COO-unclassified DLBCL cases showed a stronger association with the polarized immune response score relative to either ABC- or GCB-DLBCL (Fig. 5a; Additional file 13). This was particularly evident in the larger data sets GSE31312, GSE22470 and GSE10846. However, ABC- and GCBDLBCL cases with high levels of expression of the polarized immune response score were present in all data sets.

Fig. 5 The polarized immune response is a dominant feature across DLBCL, independent of COO class. a The incidences of individual cases across all data sets (note GSE10846 is subdivided into CHOP and R-CHOP treated components) ranked according to polarized immune response score. The top and bottom 25 cases for each data set are illustrated with colour coding for COO class shown in the top bar (yellow ABC, blue GCB, green unclassified), class confidence assigned during classification shown in the middle bar (blue low confidence to red high confidence), and polarized immune response score shown in the bottom bar (blue low polarized immune response score to red high polarized immune response score). b Complete results for data sets GSE10846 R-CHOP and GSE31312, showing all cases ranked by polarized immune response score. Each heatmap displays class assignment, classification confidence and polarized immune response score summary as in (a) followed by COO-classifier gene expression (yellow and blue bars), the 16 genes of the polarized immune response score (green bar), and the extended set of COO-unclassified meta-profile genes (black bar). A corresponding high-resolution figure comprising equivalent representation for all data sets is provided in Additional file 13

To assess whether the 16-gene score also reflected the expression of other genes associated with the immune response in COO-unclassified DLBCL we added further components of the meta-profile. Expression of these genes followed the overall pattern of expression of the 16-gene score across all DLBCL data sets (Fig. 5b; Additional file 13). Thus, the 16-gene score provides a tool with which to identify the overall pattern of this polarized immune response in DLBCL. Since some COO-unclassified DLBCL cases in all data sets showed low polarized immune response scores, we examined the pattern of T-cell gene expression further by hierarchical clustering within each COO class. This demonstrated, particularly in the larger data sets such as GSE31312 and GSE22470, that COO-unclassified DLBCL could be segregated into principal groups with a subset of cases characterized both by weak expression of COO-classifier genes and weak expression of polarized immune response genes (Fig. 6; Additional file 14). Within the ABC- and GCB-DLBCL subsets there was a common concordance between expression of core T-cell genes and components of the polarized immune response. Only a few cases, particularly in the GCBDLBCL subset, could be identified in which core T-cell genes were co-expressed in the absence of other elements of the polarized response. These cases were, however, too few to allow meaningful analysis (data not shown). Thus, across all DLBCL data sets the expression of core T-cell genes is paralleled by the expression of genes linked to functional polarization irrespective of COO class.

Fig. 6 The polarized immune response subdivides COO-unclassified DLBCL and identifies subsets of cases within ABC- and GCB-DLBCL classes. Heatmaps illustrate data for GSE10846 R-CHOP and GSE31312 hierarchically clustered according to all genes shown, and constrained by COO class assignment. Assigned COO class is shown above each heat map by the blue (GCB), green (COO-unclassified) and yellow (ABC) bars. To the right is shown the corresponding general category of genes: yellow ABC-classifier genes, blue GCB-classifier genes, green polarized immune response score genes, and black extended COO-unclassified meta-profile. A corresponding high-resolution figure comprising equivalent representation for all data sets is provided in Additional file 14

Polarised immune response and COO-unclassified DLBCL do not overlap significantly with signatures of primary mediastinal B-cell lymphoma COO-unclassified DLBCL cases lacking both polarized immune response and COO-classifier gene expression are distinct from the subset of cases in which the extent of the polarized immune response obscures the characterization of the neoplastic B-cell population. At least two principal explanations could be considered for this subgroup: on the one hand these might include cases in which gene expression was technically challenging with poor representation of tumour cell RNA; alternatively, they might include a subset of large B-cell lymphoma which fails to express COO-classifier genes at significant levels. Primary mediastinal B-cell lymphoma (PMBL) is a biologically distinct subgroup of large B-cell lymphoma, more common in women, with a mediastinal localization, distinct molecular genetics and possible derivation from a thymic B-cell population [47]. This lymphoma class can be associated with a pattern of gene expression distinct from either GCB- or ABC-DLBCL. While many PMBL cases would be excluded on the basis of diagnosis from conventional DLBCL gene expression data sets, it was possible that some PMBL cases might contribute to the COO-unclassified DLBCL cases, in particular those lacking a polarized immune response signature. To address this we used the 23-gene PMBL signature described by Rosenwald et al. [40], and first tested for enrichment within the COO-classified and COO-unclassified meta-profiles, but this showed no evidence of significant enrichment, nor was a signature separating PMBL from Hodgkin lymphoma enriched (Additional file 6). We next used the 23-gene PMBL signature in place of the extended immune response gene list to reanalyse the DLBCL data sets by hierarchical clustering (Additional file 15). We found no evidence of distinct clusters of cases identifiable with the 23-gene PMBL signature amongst COO-unclassified DLBCL, although a few elements of the 23-gene signature, most notably PDCD1LG2, CD274 and BATF3, do correlate with the polarized immune response. In contrast, in several data sets small clusters of cases were identifiable with coordinated high expression of the 23 genes of the PMBL signature, but such cases were classifiable as GCB-DLBCL, suggesting a greater overlap of PMBL signature gene expression amongst cases otherwise classifiable as GCB-DLBCL rather than ABC-DLCBL or COO-unclassified DLBCL. Thus, we found no gene expression-based evidence for a significant contribution of PMBL-like gene expression patterns amongst COOunclassified DLBCL in the data sets analysed. Inclusion of PMBL-like cases does not have a major impact on the detection of the polarized immune response signature, nor provide an explanation for the subset of COOunclassified DLBCL that lacks both COO-classifier and polarized immune response gene expression.

A polarized immune response is associated with improved outcome in DLBCL Across several cancer types the extent of tumour infiltrating lymphocytes, and their polarization toward cytotoxic T/natural killer (NK) cell gene expression linked to an IFNγ-STAT1-IRF1 signalling axis has been identified as a feature associated with good prognosis [4]. We therefore asked whether the expression of the polarized immune response signature, alone or taken in conjunction with COO class, was associated with differences in overall survival. Currently DLBCL is treated with an immunochemotherapy regimen, R-CHOP, which combines the anti-CD20 therapeutic monoclonal antibody rituximab with cyclophosphamide, hydroxydaunorubicin, vincristine (Oncovin), and prednisolone. Based on the success of the R-CHOP regimen, current treatment and future therapeutic trials in DLBCL will be based on immunochemotherapeutic approaches encompassing rituximab or related therapeutic antibodies. Therefore, only those data sets (GSE10846, GSE31312 and GSE32918) encompassing R-CHOP-treated cases associated with appropriate survival data were considered. This analysis demonstrated a consistent trend toward a reduced hazard ratio of death with increasing polarized immune response score across all three R-CHOP-treated DLBCL data sets. This reached statistical significance when considered independently of COO class in data sets GSE32918 and GSE31312, the latter representing the largest data set of R-CHOP-treated DLBCL [23]. However, in these two data sets the polarized immune response score was also significantly associated with lower age. When considered according to COO classification a consistent trend toward better outcome with high polarized immune response score was observed across all three categories. This trend was most pronounced for GCB-DLBCL, and reached statistical significance for improved outcome associated with high polarized immune response score in the largest data set GSE31312 (Additional file 16; Fig. 7). We conclude, therefore, that the presence of a polarized and IFNγ-associated immune response shows an association with good outcome which is modified by consideration of COO class, such that in the context of current R-CHOP therapy a polarized immune response is most consistently linked to improved outcome in patients with GCB-DLBCL.

Fig. 7 A high polarized immune response score is associated with improved outcome in R-CHOP-treated GCB-DLBCL. The figure illustrates Kaplan– Meier plots of overall survival derived from R-CHOP-treated DLBCL cases from data sets GSE10846, GSE31312 and GSE32918. Illustrated is the overall survival for the top and bottom 25 % of cases divided by polarized immune response score. The left graphs illustrate results independent of COO class and the right graphs results for cases assigned to the GCB-DLBCL class. CI confidence interval, HR hazards ratio

Polarization along an IFNγ-STAT1-IRF1 axis is a defining feature of the DLBCL immune response While the above analysis pointed to a common convergence onto a cytotoxic and IFNγ-polarized immune response in DLBCL, not all components of the IFNγSTAT1-IRF1 axis were sufficiently differentially expressed between COO-classified and COO-unclassified DLBCL to be identified by this approach. In order to explore the
DLBCL-associated immune response in a fashion which was not constrained by the COO paradigm we reanalyzed the DLBCL data sets, assessing the consistency and degree of correlated gene expression across all data sets relative to a selected “focus gene” (Fig. 8a; Additional files 3 and 4). We followed this by applying the integrated signature and GO enrichment analysis (Additional file 17). As focus genes we selected two components of the 16gene polarized immune response signature, TRAT1 and FGL2, to reflect origin from the two branches of the COO-unclassified meta-profile (Fig. 8b; Additional files 18 and 19). TRAT1 was selected as the most highly correlated gene from cluster 1 (Fig. 4), while FGL2 was selected as the second most highly correlated gene in cluster 2, and of more established immunologic interest than TC2N and less overt connection to immune response polarization than IFNG, the other two genes derived from cluster 2 that contribute to the 16-gene polarized immune response classifier. Genes correlating with TRAT1 could be assigned to clusters of signatures and GO terms related to T-cell state, and T-cell signal transduction, cell motility and interferon response. For FGL2 as the focus gene a similar pattern emerged, including an expanded cluster of signature enrichments related to interferon responses, including some derived from models of viral infection, and an additional association with monocyte/macrophage-derived signatures. To examine the strength of correlation with IFN responsive genes we ranked genes by median correlation, plotted rank against median gene correlation for each focus gene context and assessed the distribution of selected IFN signature genes (derived from the previous analysis) on the resulting correlation curves. We applied this approach using TRAT1 and FGL2 as focus genes, but observed similar results with all 16 genes of the polarised immune response classifier (Fig. 9; Additional files 20). In either context IFN pathway genes were consistently present within the leading edge of most correlated genes, including IFNG, STAT1, IRF1, GBP1, GBP5 and IDO1. These genes were also consistently present within the leading edge when considering a more generic T/NK cell-associated gene, CD2, as focus gene. Components of the IFNγ-STAT1-IRF1 axis therefore emerge as a consistent and dominant feature of the DLBCL immune environment linked to expression of a wider complement of IFN-responsive genes.

Fig. 8 A focus gene analysis independent of COO class verifies the dominant polarized immune response in DLBCL. a An outline of the focus gene analysis (high resolution version in Additional file 3). Upper panel: the approach within each data set, with initial selection of the 80 % most variable genes, and subsequent generation of linked matrices of gene correlation values and associated p values. Middle panel: the merging of all data sets (11 data sets; data set GSE10846 subdivided by treatment type) is shown to give matrices of median correlations and p values. Lower panel: the selection of an individual focus gene for downstream analysis. b Results of integrated gene signature and ontology analysis for two focus genes (left panel TRAT1) and (right panel FGL2) displaying the clustering of enriched signature and GO terms. General terms corresponding to major correlation clusters are illustrated to the right of each heatmap. Corresponding high resolution versions are available in Additional files 18 and 19, which also include the corresponding heatmaps clustered from the gene perspective

Fig. 9 IFN-responsive genes and the IFNγ-STAT1-IRF1 axis are amongst the leading edge of highly correlated DLBCL immune response genes. Correlation curves were generated from the focus gene analysis by ranking genes according to median correlation, and then plotting the gene correlation rank (x-axis) against the corresponding median gene correlation (y-axis, median Rho). This illustrates both the relative strength of correlations for each focus gene and identifies a leading edge of genes with most significant correlations. The position of a set of IFN-associated genes was plotted for each focus gene context as indicated in the figure. Note only the top 2000 of 20,121 genes tested are illustrated. See corresponding Additional file 20
IFNγ-STAT1-IRF1 axis and adaptive immune regulatory pathways in DLBCL In the model of adaptive immune resistance IFNγ-driven expression of PD1 ligands CD274 and PDCD1LG2 on tumour cells and the microenvironment limits local T-cell responses [5, 8]. We reasoned that the hierarchy of gene expression correlations would allow a ranking of immune checkpoint/modulatory gene expression linked to the IFNγ-STAT1-IRF1 polarized response in DLBCL. In this pathway STAT1 and IRF1 encode the transcriptional regulators; we therefore selected these along with CD2 as a generic representative of the T/NK cell response for analysis (Fig. 10; Additional file 4). When considering immune modulatory/checkpoint genes a consistent cluster of three genes, LAG3, IDO1 and FGL2, emerged as most highly ranked and amongst the leading edge in all three focus gene contexts. In contrast, CD274 and PDCD1LG2 showed significantly weaker correlations with each focus gene, but nonetheless remained well correlated in comparison with all genes tested (rank <1000 out of 20,121 tested). To further confirm this pattern we extended the analysis to all 16 genes of the polarized immune response classifier, and observed similar patterns of gene correlation ranking (Additional file 21). Since the relative contribution of immune modulatory/checkpoint gene expression in tumour cells themselves relative to the wider microenvironment cannot be determined from these analyses, we conclude that, in addition to CD274 and PDCD1LG2,a wider complement of immune modulators provides a potentially high degree of redundancy in adaptive immune resistance in DLBCL. Amongst these components IDO1, FGL2 and LAG3 are particularly strongly correlated with IFNγ polarized immune responses.

Discussion

The common convergence of cancer immune responses onto patterns of cytotoxic and IFNγ-dominated pathways has been summarised in the concept of an “immune constant of rejection” [4, 7]. In parallel the model of adaptive immune resistance argues for the control of such immune responses via local feedback driven through IFN-mediated upregulation of immune checkpoints [5, 8]. Our analysis here provides extensive empirical evidence across currently available large DLBCL data sets that this combination of IFNγ polarisation and induction of adaptive immune resistance mechanisms is a feature of the immune response to DLBCL. Unbiased analysis of gene expression correlations moreover suggests a hierarchy of IFN-associated immune modulatory gene expression with LAG3, IDO1 and FGL2 as key elements in this context. Thus, adaptive immune resistance is likely to provide an important component of immune evasion in DLBCL.

Other mechanisms of immune evasion have been previously identified as playing an important role in the pathogenesis of DLBCL, including mutation and deletion of B2M and CD58, and amplification of genomic regions encompassing genes encoding PD1 ligands [48, 49]. Furthermore previous studies have demonstrated the presence of PD1 expression on infiltrating T-cell populations and PD-L1(CD274) on tumour cells and in the microenvironment of DLBCL and related neoplasms [50, 51]. In the context of gene expression profiling, morphologically defined T-cell and histiocyte-rich large B-cell lymphoma, which represents a relatively rare subcategory, has been characterized by evidence of an IFNassociated immune response, linked on the one hand with over-expression of PD1 (PDCD1) on infiltrating T cells when compared with classical Hodgkin lymphoma [52], or the expression of IDO1 when compared with nodular lymphocyte predominant Hodgkin lymphoma, another relatively rare lymphoma subtype [53]. Indeed, expression of IDO1 has been defined as a feature associated with poor outcome in generic DLBCL in one patient series [54]. Thus, the involvement of several pathways of immune modulation in large B-cell lymphomas is supported by prior studies.

Using the 16-gene polarized immune response score we have ranked DLBCL cases across multiple data sets, and demonstrate that a substantial fraction of cases regardless of COO class are linked to a polarized immune response. In the context of the COO classification, the dominance of this immune response at the expense of proliferating B cells provides the most common explanation for DLBCL cases that are “unclassifiable” as originally suggested by Monti et al. [15]. Equally important is the identification of a distinct group of DLBCL characterized by an absence of host response elements, which is consistent with “immunological ignorance”,  a feature which in other cancers is associated with poor response to immune checkpoint blockade [12]. These cases are also consistent with a model of host tissue “effacement” proposed by Scott and Gascoyne [49] as distinguishing subsets of aggressive lymphomas. Immune evasion in DLBCLs can be associated with loss of MHC class I expression consequent on mutation and/or deletion of B2M, which may be further accompanied by inactivation of CD58 [48], and a prediction might be that such cases would be enriched in the subset characterized by apparent immunological ignorance. However, analogous lesions affecting B2M were recently identified as recurrent events positively associated with cytotoxic gene signatures in solid tumours [9]. This suggests a model in which adaptive immune resistance mechanisms may be followed by somatic genetic alterations that further enhance tumour immune escape. Whether a similar positive association between cytotoxic response and B2M or CD58 mutation status exists in DLBCL is, to our knowledge, not established.

Across several cancer types the intensity of tumour infiltrating lymphocytes and their functional polarization has proved to be of prognostic significance in the absence of specific immune checkpoint blockade [4, 55–57]. Our analysis indicates that a trend toward an improved outcome in association with a more intense polarized immune response is generally maintained in the context of DLBCL treated with the current immunochemotherapy regimen, R-CHOP. However, this benefit is not equivalent across all DLBCL when considered in relation to COO class, and is most pronounced for GCB-DLBCL. Indeed, in the largest available data set of R-CHOP-treated DLBCL, GSE31312 [23], a substantial group of patients with both a GCB-DLBCL classification and a high polarized immune response score appeared curable with current therapy. As a statistically significant association is not consistently observed across all three data sets of DLBCL treated with R-CHOP, and there is a potentially confounding association with young age, the overall prognostic value of this association remains uncertain in the context of current therapy. Additional features of the host response, which did not emerge as principal discriminants between COO-classified versus COO-unclassified DLBCL, such as contributions from macrophage/monocyte lineage cells, may add value to immune response classifiers. These will need to be considered alongside the polarized immune response signature in future work. Nonetheless, the analysis presented here demonstrates a graded pattern of immune response in DLBCL, with one extreme characterized by minimal cytotoxic immune response signature and tendency to poor outcome, and another extreme characterized by intense polarized immune response and a tendency toward better outcome which is modified by COO class. In other settings the pattern of pre-existing immune response prior to immune checkpoint therapy has proved to be of predictive value [11, 12, 58, 59]. Based on this evidence, it is the subset of DLBCL cases with preexisting polarized immune response which is most likely to benefit from immune checkpoint/modulatory therapy, and is readily identifiable in a quantitative fashion from gene expression data.

Immune checkpoint inhibitors are already under evaluation in the context of large cell lymphomas [60, 61]. Recent clinical trials with PD1 pathway blockade have shown substantial promise in Hodgkin lymphoma [14], as in other tumour types [11, 12, 62]. Combining immune checkpoint inhibitors may hold particular promise, and both LAG3 and IDO1 are therapeutic targets with novel agents in current clinical evaluation. Our analyses support these as high priority candidates for therapeutic evaluation in DLBCL alongside PD1 blockade. In addition to direct interventions specifically targeting immune checkpoints, signalling pathways that mediate survival of neoplastic B cells, and are the targets of novel therapeutic agents in lymphoma, overlap with pathways controlling T-cell responses. Such agents have the potential to de-repress cytotoxic T-cell populations and promote anti-tumour immunity [63]. Thus, companion biomarkers evaluating the potential association between pre-existing immune response at diagnosis and treatment response should arguably also be included in the setting of lymphoma clinical trials where agents targeting lymphocyte signalling pathways are being evaluated.

A notable element of the DLBCL immune response is the consistent association with FGL2 expression. This encodes fibrinogen-like 2 prothrombinase, a protein that has dual roles as a pro-coagulant and immune modulator. FGL2 has been shown to act as an immune responsive coagulant in settings such as foetal loss driven by Th1 polarized immune responses [64] and fulminant hepatitis [65]. Subsequently, FGL2 has been implicated as a repressor of T-cell activation both in the ability of recombinant FGL2 to block graft rejection [66] and in the context of Fgl2 knockout mice developing autoimmune glomerulonephritis [67]. In several experimental models FGL2 has been associated with suppression of cytotoxic and Th1-polarized immune responses [67–69]. FGL2 effects in DLBCL could relate to both pro-coagulant and immune modulatory functions. In DLBCL FGL2 expression correlates with multiple elements of the IFNγSTAT1-IRF1 axis; supporting direct regulation, FGL2 expression has previously been shown to be responsive to IFNγ in Tcells [70, 71], and was shown to act downstream of IRF1 in Th1-driven foetal loss [64]. Thus, the relationships in DLBCL suggest that FGL2 may provide an additional element of negative feedback and adaptive immune resistance, which is potentially suitable for therapeutic targeting [72, 73].

We note that some DLBCL cases with a prominent immune response may be associated with Epstein-Barr virus (EBV) infection and oncogenic drive. In the metaanalysis approach taken here the contribution of EBV cannot be systematically assessed from available data since EBV status is incompletely annotated, and not necessarily assessed using both immunohistochemistry for EBV LMP1 and RNA-FISH for EBERs. Immune surveillance is known to contribute to the control of EBVmediated tumours [74], and the presence of high EBV loads can contribute to the establishment of an exhausted cytotoxic response [75]. Indeed, there are significant overlaps between the gene expression profiles of the immune response in EBV-associated large cell lymphomas occurring in the post-transplant setting [76] and the polarized IFNγ-associated gene expression that is evident from our DLBCL meta-analysis. However, while the frequency of EBV infection in generically diagnosed DLBCL has been established at close to 10 % [77], significant expression of genes linked to the polarized immune response is more frequent across DLBCL data sets. An overlap of gene expression profiles between the immune response targeting EBV-driven and EBV-independent lymphomas is consistent with the model of convergent patterns of “immune rejection” across diverse immune contexts [4, 7]. It is arguable that the principal predictive factor of response to immune checkpoint inhibition will be the presence of a pre-existing polarized immune response and the mechanisms controlling its chronic activation/exhaustion rather than the nature of the initial triggering antigens whether viral or cancer-associated.
Conclusions The analysis presented here supports the central importance of convergent patterns of immune response linked to the IFNγ-STAT1-IRF1 axis, coupled to IFN-driven feedback pathways in DLBCL. This argues for the generalisable nature of these interconnected mechanisms, and implicates a hierarchy of immune modulators, known to promote the establishment of an immunosuppressive microenvironment [2], in the process of IFNγ-driven adaptive immune resistance.

Additional files
Additional file 1: Figure S1. Outline of meta-profile generation using COO classification. Upper panel: the data sets used. Please note one data set, GSE10846, is divided into two component parts reflecting underlying differences in treatment (CHOP versus R-CHOP), giving a total of 11 separate data set components. Illustrated are all cases for each data set, subdivided by COO classification established using the DAC classifier [32] and ranked by classification confidence, with classifier genes illustrated on the right (yellow bars ABC classifier genes and cases, blue bars GCB classifier genes and cases, green bars COO-unclassified cases). Middle panels: the pairwise comparisons of differentially expressed genes between three classes, and the integration of differentially expressed genes across data sets. Bottom panels: the resulting meta-profiles of differentially expressed genes for both components of the three possible pairwise comparisons are shown as Wordles for illustrative purposes (complete details provided in Additional file 5). (PDF 2024 kb)

Additional file 2: Figure S2. Outline of the process for integrating and visualizing analysis of gene signature and ontology enrichments. The flow diagram illustrates the process for integrating gene signature and ontology enrichments. The initial assessment of overlap between meta-profiles derived from the comparison of ABC-DLBCL versus COO-unclassified (CU) DLBCL and GCB-DLBCL versus COO-unclassified (CU) DLBCL is shown at the top of the figure, followed by the parallel analysis of gene ontology (BiNGO) and hypergeometric testing of signature enrichments. Next a matrix is illustrated showing the occurrence of genes versus enriched signatures (green fill), followed by analysis of correlations (Phi coefficient) by column (signature/ontology terms) or by row (genes) and hierarchical clustering. (PDF 1658 kb)

Additional file 3: Figure S11. An outline of the focus gene approach, and a high resolution image to accompany Fig. 8a. Upper panel: the approach within each data set with initial selection of the 80 % most variable genes, and subsequent generation of linked matrices of gene correlation values and associated p values. Middle panel: merging of all data sets (11 data sets; data set GSE10846 subdivided by treatment type) to give gene by gene matrices of median correlations and p values. Lower panel: the selection of an individual focus gene for downstream analysis. (PDF 1375 kb)

Additional file 4: Table S5. Lists of correlated genes for selected focus genes. (XLSX 16708 kb)

Additional file 5: Table S1. Lists of Meta-profile genes differentially expressed between DLBCL COO classes. (XLSX 2318 kb)

Additional file 6: Table S2. Lists of enriched gene signature and gene ontology terms for COO-classified and COO-unclassified meta-profiles. (XLSX 2848 kb)

Additional file 7: Figure S3. High resolution image corresponding to Fig. 2a. Integrated gene signature and ontology enrichments for COOclassified meta-profile clusters from signature and ontology term perspectives. The figure represents the hierarchical clustering of enriched gene signature and ontology terms related to the COO-classified meta-profile. Correlations are illustrated in heat maps on a blue (least) to red (most) scale as indicated at the top of the figure. Along the edges of the heatmap the signature terms are provided (and correspond to terms listed in Additional file 6). The FDRcorrected p value for enrichment of the signature is illustrated as a bar on either side of the heatmap, along with an indication of the type of term (signature versus ontology) and the origin of the terms as indicated in the figure. (PDF 283 kb)

………   Additional file 21: Figure S15.

 

Fig. 10 Immune-modulatory and checkpoint gene expression is strongly correlated with elements of the IFNγ-STAT1-IRF1 axis in DLBCL. IRF1 and STAT1 along with CD2 were analysed as focus genes, and resultant correlation curves are illustrated. Genes were plotted according to correlation rank (x-axis) against median gene correlation (y-axis, median Rho). The position of immune checkpoint/modulatory genes on the resulting curves was plotted for each focus gene as indicated in the figure. Note only the top 2000 of 20,121 genes tested are illustrated. See corresponding Additional file 21

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20. Salaverria I, Philipp C, Oschlies I, Kohler CW, Kreuz M, Szczepanowski M, et al. Translocations activating IRF4 identify a subtype of germinal centerderived B-cell lymphoma affecting predominantly children and young adults. Blood. 2011;118:139–47. http://dx.doi.org:/10.1182/blood-2011-01-330795.

 

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Gluten-free Diets

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

 

Clinical, Serologic, and Histologic Features of Gluten Sensitivity in Children

Ruggiero Francavilla, Fernanda Cristofori, Stefania Castellaneta, et al.
J Pediatr 2014; 164:463-7.
http://dx.doi.org/10.1016/j.jpeds.2013.10.007

Until a few years ago, the spectrum of gluten-related disorders included only celiac disease (CD) and wheat allergy (WA).  Recent data, however, suggest the existence of another form of gluten intolerance, known as nonceliac gluten sensitivity, or simply gluten sensitivity (GS). Some individuals experience distress after eating gluten-containing products and show improvement after institution of a gluten-free diet (GFD). Although the gastrointestinal symptoms may resemble those seen in CD, patients with CD do not have positive celiac-related antibodies or intestinal damage. This entity was described more than 30 years ago in 8 adult females suffering from abdominal pain and chronic diarrhea who experienced relief from a GFD and a return of symptoms on a gluten challenge.

GS is diagnosed in patients with symptoms that respond to removal of gluten from the diet, after CD and WA are excluded. Currently, it is a clinical diagnosis based on response to the GFD and relapse after gluten challenge; no specific blood test is available for GS. Sapone et al, aiming to elucidate the underlying pathophysiological mechanisms of GS, found that GS, as opposed to CD, is a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.

Recently, the existence of GS was confirmed by Biesiekierski et al in a double-blind, randomized, placebo-controlled challenge trial performed in a selected group of patients with irritable bowel syndrome who were symptomatically controlled on a GFD. Patients with irritable bowel syndrome-GS frequently demonstrate serum IgG class native anti-gliadin antibodies (AGA) as a possible marker of immune activation to gluten.

Objective To describe the clinical, serologic, and histologic characteristics of children with gluten sensitivity (GS). Study design We studied 15 children (10 males and 5 females; mean age, 9.6 + 3.9 years) with GS who were diagnosed based on a clear-cut relationship between wheat consumption and development of symptoms, after excluding celiac disease (CD) and wheat allergy, along with 15 children with active CD (5 males and 10 females; mean age, 9.1 + 3.1 years) and 15 controls with a functional gastrointestinal disorder (6males and 9 females; mean age, 8.6 + 2.7 years). Method: All children underwent CD panel testing (native anti-gliadin antibodies IgG and IgA, anti-tissue transglutaminase antibody IgA and IgG, and anti-endomysial antibody IgA), hematologic assessment (hemoglobin, iron, ferritin, aspartate aminotransferase, erythrocyte sedimentation rate), HLA typing, and small intestinal biopsy (on a voluntary basis in the children with GS).
Results Abdominal pain was the most prevalent symptomin the children with GS (80%), followed by chronic diarrhea in (73%), tiredness (33%), bloating (26%), limb pain, vomiting, constipation, headache (20%), and failure to thrive (13%). Native antigliadin antibodies IgG was positive in 66% of the children with GS. No differences in nutritional, biochemical, or inflammatory markers were found between the children with GS and controls. HLA-DQ2 was found in 7 children with GS. Histology revealed normal to mildly inflamed mucosa (Marsh stage 0-1) in the children with GS. Conclusion Our findings support the existence of GS in children across all ages with clinical, serologic, genetic, and histologic features similar to those of adults. (J Pediatr 2014;164:463-7).

 

Coeliac disease

C Leivers, G Martin, M Gasparetto, H Shelley, M Valente
Paediatrics and Child Health  2014; 24(11):481-84

Celiac disease is an immune-mediated systemic disorder, which is triggered by dietary gluten in genetically susceptible individuals. It is characterised by the presence of HLA-DQ2 or HLADQ8 genetic haplotypes, gluten-dependent signs and symptoms, celiac-specific antibodies and enteropathy.

The pathogenesis of celiac disease is complex and involves both genetic and environmental factors. Genetics is important: there is a high concordance in monozygotic twins (between 70 and 86%) and the HLA haplotype of DQ2/DQ8 is the principal genetic factor described. In the Caucasian population, between 30 and 35% will be carriers of these markers, but only 2-5% will go on to develop celiac disease. In patients with coeliac disease, 95% have HLA-DQ2 and 5-10% will carry HLA-DQ8.

Non-celiac gluten sensitivity

This occurs in those who have had a diagnosis of celiac disease excluded, but whereby there is a clear adverse response associated with gluten ingestion. Non-IgE mediated food allergy is a potential cause.

Differential diagnoses/causes of villous atrophy

  • Coeliac disease
  • Food protein hypersensitivity (particularly cow’s milk and/or soya proteins)
  • Eosinophilic gastroenteritis
  • Hypogammaglobulinemia
  • Whipple diseases
  • Abetalipoproteinaemia (Bassen-Kornzweig syndrome)
  • Intestinal lymphoma
  • Crohn’s disease
  • Infectious diseases (e.g. tuberculosis, giardiasis, parasitic infestations, infectious enteritis)
  • Small bowel bacterial overgrowth
  • Severe malnutrition
  • Small bowel ischemia
  • Radiotherapy
  • Autoimmune enteropathy
  • Cytotoxic drugs

Gluten challenge

Routine gluten challenge is not recommended. However, the process is advised when the initial diagnosis was not secure. Challenges should be undertaken at age 6-7 years or when pubertal growth is complete. Prior to a challenge HLA-DQ2/8 haplotype should be determined; if absent, celiac disease is unlikely. At least 4-6 weeks (ideally three months) of a normal gluten-containing diet (2-3 meals per day containing at least 5 g of gluten) is recommended prior to testing. Celiac serology and symptoms should be closely monitored to decide on the timing of biopsies. A pediatric dietitian is useful to support families and clinicians through this process.

Serological testing

Blood tests including tTG-IgA and full blood count are undertaken and growth is assessed. tTG-IgA levels are used to assess recovery and dietary adherence, particularly in asymptomatic patients. The high sensitivity and specificity of tTG-IgA in the diagnosis of celiac disease, has been extensively validated for diagnostic and follow up purposes.

Dietetic assessment and interview

A dietary assessment includes a review of the gluten free diet; the child and family’s level of adherence and its overall nutritional adequacy. In particular, the child’s intake of calcium and iron is assessed and if required, the family is advised on how to increase the intake of these nutrients up to the Reference Nutrient Intake (RNI).

Practice points

  1. The incidence of coeliac disease remains high, currently estimated to be around 1% of the UK population, although only 10-20% of these are diagnosed
  2. Prior to confirming a diagnosis, it is important to ensure the child is on a gluten-containing diet
  3. The diagnostic process in children has changed and depends on whether the child is symptomatic or asymptomatic, and on the level of their tTG
  4. Duodenal biopsy may be avoided in symptomatic children who meet strict criteria upon further laboratory testing
  5. A lifelong gluten free diet is currently the only treatment for celiac disease
  6. The management of coeliac disease involves examination, repeat serology and dietetic interview and support
  7. Prolonged, untreated coeliac disease has associated morbidity and mortality

 

Controlled Trial of Gluten-Free Diet in Patients with Irritable Bowel Syndrome-Diarrhea: Effects on Bowel Frequency and Intestinal Function

Maria I. Vazquez–Roque, Michael Camilleri, Thomas Smyrk, et al.
Gastroenterology 2013;144:903–911
http://dx.doi.org/10.1053/j.gastro.2013.01.049

Background & Aims: Patients with diarrhea-predominant irritable bowel syndrome (IBS-D) could benefit from a gluten-free diet (GFD).
Methods: We performed a randomized controlled 4-week trial of a gluten-containing  diet (GCD) or GFD in 45 patients with IBS-D; genotype analysis was performed for HLA-DQ2 and HLA-DQ8. Twenty-two patients were placed on the GCD (11 HLA-DQ2/8 negative and 11 HLA-DQ2/8 positive) and 23 patients were placed on the GFD (12 HLA-DQ2/8 negative and 11 HLADQ2/8 positive). We measured bowel function daily, small bowel (SB) and colonic transit, mucosal permeability (by lactulose and mannitol excretion), and cytokine production by peripheral blood mononuclear cells after exposure to gluten and rice. We collected rectosigmoid biopsy specimens from 28 patients, analyzed levels of messenger RNAs encoding tight junction proteins, and performed H&E staining and immune-histochemical analyses. Analysis of covariance models was used to compare data from the GCD and GFD groups.
Results: Subjects on the GCD had more bowel movements per day (P < .04); the GCD had a greater effect on bowel movements per day of HLA-DQ2/8–positive than HLA-DQ2/8–negative patients (P < .019). The GCD was associated with higher SB permeability (based on 0-2 h levels of mannitol and the lactulose/
mannitol ratio); SB permeability was greater in HLA-DQ2/8–positive than HLADQ2/8–negative patients (P < .018). No significant differences in colonic permeability were observed. Patients on the GCD had a small decrease in expression of zonula occludens 1 in SB mucosa and significant decreases in expression of zonula occludens 1, claudin-1, and occludin in rectosigmoid mucosa; the effects of the GCD on expression were significantly greater in HLA-DQ2/8–positive patients. The GCD vs the GFD had no significant effects on transit or histology. Peripheral blood mononuclear cells produced higher levels of interleukin-10, granulocyte colony-stimulating factor, and transforming growth factor-α in response to gluten than rice (unrelated to HLA genotype). Conclusions: Gluten alters bowel barrier functions in patients with IBS-D, particularly in HLA-DQ2/8–positive patients. These findings reveal a reversible mechanism for the disorder. Clinical trials.gov NCT01094041.

Our data convincingly showed effects of gluten on the increased mRNA expression of all the measured TJ proteins in colonic tissue relative to GFD. One limitation of the study was the inability to document alterations in colonic permeability using the 2-sugar excretion profile from 8 to 24 hours. We perceive that this may represent a lack of sensitivity of the lactulose and mannitol excretion test, for example, because of the metabolism of both sugars by colonic bacteria. There are advantages to measuring both tissue and in vivo markers of barrier function. Another limitation was that the mechanism for improvement in stool frequency on a GFD in the absence of changes in colonic transit was not elucidated by our studies. Because it is unclear whether gluten or its metabolic products induce specific secretory mechanisms, the current hypothesis is that change in stool frequency may reflect change in fluid secretion from increased mucosal permeability. Our current studies did not evaluate effects of gluten on the microbiome, afferent functions, or cytokine expression in the mucosal biopsy specimens from patients before and after the interventions. These would be interesting parameters to include in future studies. Finally, our study did not specifically address the effects of gluten protein per se, and it is possible that other proteins in wheat flour may be responsible for the changes observed.

Overall, our data provide mechanistic explanations for the observation that gluten withdrawal may improve patient symptoms in IBS. The data also explain, in part, the observation of the relationship of HLA genotype to beneficial effects of gluten withdrawal in view of our results showing that biological effects of gluten were associated with HLA-DQ2 or HLA-DQ8 genotype. The data suggest that the relationship of dietary factors, innate and adaptive immune responses, and mucosal interactions in IBS-D deserve further study, and they support the need for further clinical intervention studies to evaluate the clinical effects of gluten withdrawal in patients with IBS-D.

 

Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides

Melinda Y. Hardy, Jason A. Tye-Din, Jessica A. Stewart, et al.
Journal of Autoimmunity 56 (2015) 56-65
http://dx.doi.org/10.1016/j.jaut.2014.10.003

Celiac disease (CD) is a common CD4+ T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin specific T cell response in vivo. We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLADQ2.5+ CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye. Avenin-specific responses were observed in 6/73 HLA-DQ2.5+ CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability. Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.

 

Diagnosis and classification of celiac disease and gluten sensitivity

Elio Tonutti, Nicola Bizzaro
Autoimmunity Reviews 13 (2014) 472–476
http://dx.doi.org/10.1016/j.autrev.2014.01.043
Celiac disease is a complex disorder, the development of which is controlled by a combination of genetic (HLA alleles) and environmental (gluten ingestion) factors. New diagnostic guidelines developed by ESPGHAN emphasize the crucial role of serological tests in the diagnostic process of symptomatic subjects, and of the detection of HLA DQ2/DQ8 alleles in defining a diagnosis in asymptomatic subjects belonging to at-risk groups. The serological diagnosis of CD is based on the detection of class IgA anti-tissue transglutaminase (anti-tTG) and anti-endomysial antibodies. In patients with IgA deficiency, anti-tTG or anti-deamidated gliadin peptide antibody assays of the IgG class are used. When anti-tTG antibody levels are very high, antibody specificity is absolute and CD can be diagnosed without performing a duodenum biopsy. Non-celiac gluten sensitivity is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out. Diagnostic criteria include the presence of symptoms similar to those of celiac or allergic patients; negative allergological tests and absence of anti-tTG and EMA antibodies; normal duodenal histology; evidence of disappearance of the symptoms with a gluten-free diet; relapse of the symptoms when gluten is reintroduced.

Celiac disease (CD) is a chronic, immune-mediated, gluten-induced gut disorder that manifests itself with a range of clinical symptoms in genetically susceptible subjects. Immune reaction to wheat, barley and rye gliadin fractions and glutenins triggers an inflammatory state of the duodenal mucosa: the result is reduced intestinal villus height and hyperplastic cryptae that may lead to complete villus atrophy. The critical role played by gluten is demonstrated by the fact that in CD patients on a gluten free diet (GFD) clinical symptoms disappear, anti-transglutaminase 2 antibodies (anti-tTG2, the serological markers of the disorder) normalize, and villus atrophy recedes. As to the role
of genetic factors, CD development has been demonstrated to be closely associated with MHC class II HLA-DQ2 and HLA-DQ8 molecules; in fact, virtually all CD patients express at least one of these HLA molecules compared to the general population in which about 30–35% have either DQ2 or DQ8.

A new gluten-associated clinical condition, named ‘non-celiac gluten sensitivity’ (NCGS) [4], also described in literature as gluten hypersensitivity or gluten intolerance, has been recently identified. NCGS is characterized by gastrointestinal or extra-intestinal symptoms comparable, in many cases, to those of CD patients; however, to date no specific immunological mechanisms or serological markers have been identified for this disorder. The diagnosis is made by exclusion of CD or IgE-mediated allergy to wheat, and is based on the direct association between gluten ingestion and symptom onset.

The development of highly sensitive immunological methods for identifying diagnostic antibodies (e.g. anti-tTG autoantibodies and anti-DGP antibodies) has enabled an increasing number of CD patients with vague or asymptomatic clinical presentations to be identified. Population-based studies now indicate that approximately 0.5–1% of the Western European and Northern American populations suffer from CD. In a recent paper, Abadie and coworkers correlate gluten consumption with HLA DQ2 and DQ8 haplotype frequency in the populations of the different world countries. The authors found a significant correlation between CD prevalence and wheat consumption, and between CD prevalence and DQ2–DQ8 frequency in most countries. However, outlier countries have been observed: Finland and Russia, for example, have similar wheat consumption levels and comparable HLA haplotype frequencies, but the prevalence of CD in Finland is 1–2.4% whereas in the adjacent Russian republic of Karelia the prevalence of CD is considerably lower (0.2%). In the Maghreb area, wheat and barley are the major staple foods. Despite similar frequencies of the DR3–DQ2 and DR4–DQ8 haplotypes, the prevalence of CD in Algeria (5.6%) is by far the highest reported worldwide, whereas CD prevalence in Tunisia (0.28%) remains one of the lowest. These observations suggest that similar levels of wheat consumption and predisposing HLA expression can be associated with strikingly different levels of CD prevalence.

CD is characterized by multiple clinical expressions. An ESPGHAN (European Society for Paediatric Gastroenterology, Hepatology and Nutrition) working group has recently developed new guidelines for the diagnosis of CD based on scientific and technical developments using an evidence-based approach. The ESPGHAN working group decided to revise the classification, also taking into consideration signs and symptoms that had not been considered in the previous classification. In particular, it was deemed advisable to eliminate the distinction between classic and atypical CD based on symptoms, as atypical signs and symptoms (e.g. anemia, neuropathy, reduced bone density) may be considerably more common than classic symptoms (e.g. chronic diarrhea).

Patients suffering from certain disorders (especially Hashimoto’s thyroiditis, type I diabetes, IgA deficiency and Down’s syndrome) have a higher risk of developing CD than the normal population. In these patients it is advisable to perform HLA DQ2/DQ8 and serological tests for CD even in the absence of symptoms.

CD and NGCS cannot be distinguished clinically, since the symptoms experienced by NGCS patients are often seen in CD. The definition of NGCS is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out (diagnosis by exclusion criteria).

Specifically: symptoms similar to those of celiac or allergic patients must be present; in vivo and in vitro wheat allergy tests (prick test and specific IgE), as well as anti-tTG and EMA antibodies must be negative; duodenal histology must be normal; the patients must also experience a disappearance of the symptoms when on a GFD and their reappearance after the reintroduction of gluten. The most frequent symptoms in NGCS patients are abdominal pain, eczema or rash, headache, blurred vision, fatigue, diarrhea, depression, anemia, numbness in the legs, arms or fingers, and joint pain.

Signs and symptoms of patients with non-celiac gluten sensitivity (NCGS)

Abdominal pain
Abdominal distension/bloating
Diarrhea
Eczema
Rash
Headache
Foggy mind
Fatigue
Depression
Anemia
Numbness in the legs, arms
Joint pain


An important aspect, confirmed by numerous studies, is the correlation between anti-tTG count and histological damage. One of the latest studies assessed retrospectively 412 consecutive anti-tTG and EMA patients who received a biopsy for suspected CD: the subjects whose levels of anti-tTG were greater than 7-fold the cut-off value had a 99.7% positive predictive value for histological damage (with Marsh score N2). To date, there are no specific laboratory markers for NCGS; a recent study by Volta found that 78 patients with NCGS were AGA IgG positive in 56.4% of the cases and AGA IgA positive in 7.7% of the cases. All patients were negative for anti-DGP IgG and IgA, as well as for anti-tTG and EMA.

Analysis of multiple biopsies is important: patchiness of the lesion has been reported and recent work suggests that different degrees of severity may be present, even in the same bioptic fragment. The biopsies should be taken from the second/third portion of the duodenum and at least one biopsy should be taken from the duodenal bulb. Patients with NCGS do not exhibit significant alterations of the duodenal mucosa; histological negativity is an essential parameter for a diagnosis of NCGS.

The diagnostic criteria proposed by ESPGHAN in 1990 envisaged the performance of gastro-duodenoscopy and histological confirmation of mucosal damage as the conclusive phase of the diagnostic process. These criteria did not indicate which serological tests should be positive, were not applicable to children aged below 2 years, and in any case required other clinical conditions to be ruled out. Therefore, in 2010, the ESPGHAN working group deemed appropriate to set out new criteria based on new knowledge and diagnostic tools developed in the last few years.

The new CD guidelines are revolutionary in two major respects: the crucial role of serological tests in the diagnostic process of symptomatic subjects, and the detection of HLA DQ2/DQ8 in diagnosing asymptomatic subjects belonging to groups at risk of CD.

Concerning the diagnosis of children and adolescents with signs and symptoms suggestive of CD, the ESPGHAN guidelines recommend, as the initial approach to symptomatic patients, testing for anti-tTG IgA antibodies as well as for total serum IgA to exclude IgA deficiency. As an alternative to total serum IgA, direct testing for IgG anti-DGP antibodies can be performed. The decision to perform IgA anti-tTG as the initial test in this population is based on the high sensitivity and specificity of the test, its widespread availability, and low costs compared with the EMA IgA test.

A fundamental aspect of the new guidelines concerns the possibility of not necessarily performing an intestinal biopsy if the anti-tTG antibody levels are very high, as in these cases the specificity of the antibody is absolute. Indeed, pediatric gastroenterologists should discuss with the parents and the patient who is positive for anti-tTG antibody levels N10 times ULN (as appropriate for age) the option of omitting the biopsies and the implications of doing so. If the parents (patient) accept this option, then blood should be drawn for HLA and EMA testing.

Patients with positive anti-tTG antibody levels lower than 10 times the upper limit for the normal population (ULN) given by the manufacturer of this particular test should undergo upper endoscopy with multiple biopsies.

As far as diagnosis methods in asymptomatic pediatric patients belonging to
at-risk groups are concerned, the ESPGHAN guidelines suggest a different procedure. In these patients, HLA-DQ2 and HLA-DQ8 testing as the initial action is probably cost-effective since a significant proportion of the patients can be excluded from further studies because they do not harbor DQ2 or DQ8. In individuals with DQ2 or DQ8 positivity, IgA anti-tTG and total serum IgA determination should be performed. If IgA anti-tTG is negative and IgA deficiency is excluded, then CD is unlikely; however, the disease may still develop later in life. Therefore, serological testing should be repeated at regular intervals. If anti-tTG antibodies are positive, then signs related to CD should be searched for (e.g. anemia, elevated liver enzymes).

 

Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation

Veronica Montserrat, Maaike J. Bruins, Luppo Edens, Frits Koning
Food Chemistry 174 (2015) 440–445
http://dx.doi.org/10.1016/j.foodchem.2014.11.053

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.

 

No Effects of Gluten in Patients with Self-Reported Non-Celiac Gluten Sensitivity after Dietary Reduction of Fermentable, Poorly Absorbed, Short-Chain Carbohydrates

Jessica R. Biesiekierski, Simone L. Peters, Evan D. Newnham, et al.
Gastroenterology 2013;145:320–328
http://dx.doi.org/10.1053/j.gastro.2013.04.051

Background & Aims: Patients with non-celiac gluten sensitivity (NCGS) do not have celiac disease but their symptoms improve when they are placed on gluten-free diets. We investigated the specific effects of gluten after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates (fermentable, oligo-, di-, monosaccharides, and polyols [FODMAPs]) in subjects believed to have NCGS. Methods: We performed a double-blind crossover trial of 37 subjects (aged 2461 y, 6 men) with NCGS and irritable bowel syndrome (based on Rome III criteria), but not celiac disease. Participants were randomly assigned to groups given a 2-week diet of reduced FODMAPs, and were then placed on high-gluten (16 g gluten/d), low-gluten (2 g gluten/d and 14 g whey protein/d), or control (16 g whey protein/d) diets for 1 week, followed by a washout period of at least 2 weeks. We assessed serum and fecal markers of intestinal inflammation/injury and immune activation, and indices of fatigue. Twenty-two participants then crossed over to groups given gluten (16 g/d), whey (16 g/d), or control (no additional protein) diets for 3 days. Symptoms were evaluated by visual analogue scales. Results: In all participants, gastrointestinal symptoms consistently and significantly improved during reduced FODMAP intake, but significantly worsened to a similar degree when their diets included gluten or whey protein. Gluten-specific effects were observed in only 8% of participants. There were no diet-specific changes in any biomarker. During the 3-day rechallenge, participants’ symptoms increased by similar levels among groups. Gluten-specific gastrointestinal effects were not reproduced. An order effect was observed. Conclusions: In a placebo controlled, cross-over rechallenge study, we found no evidence of specific or dose-dependent effects of gluten in patients with NCGS placed diets low in FODMAPs. www.anzctr.org.au.ACTRN12610000524099

 

Gluten Sensitivity: Not Celiac and Not Certain

See “No effects of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction of fermentable, poorlyabsorbed, short-chain carbohydrates,” by Biesiekierski JR, Peters SL, Newnham ED, et al.

Rohini Vanga, Daniel A. Leffler
http://dx.doi.org/10.1053/j.gastro.2013.06.027

The past few years have seen a flurry of clinical and basic research studies targeting NCGS, the results of which seem determined to thwart any attempt to come to broad consensus regarding what NCGS is or is not, what causes it, and who it might affect.

Some studies suggest that NCGS generally belongs on the spectrum of functional bowel disorders. Other studies are more suggestive that NCGS may actually fit better within the spectrum of celiac disease. For example, in contrast with the studies by Biesiekierski et al. and Saponi et al., a number of studies have reported that nonceliac individuals with gluten-responsive symptoms are more likely to carry human leukocyte antigen (HLA)-DQ2/8. Taking a somewhat different tack, Carroccio et al. reported that NCGS patients with negative wheat IgE allergy testing developed greater symptoms with wheat exposure compared with placebo (P < .0001). The presence of anemia, weight loss, self-reported wheat intolerance, history of food allergy in infancy, and coexistent atopic diseases were more frequent in wheat-sensitive patients than in non–gluten-responsive IBS controls. There was also a higher frequency of positive serum assays for IgG/IgA anti-gliadin and greater association with DQ2 or DQ8 haplotype than controls.

In this issue of Gastroenterology, Biesiekierski et al. return with another double-blind, randomized, controlled trial on NCGS. Although in many ways this work seems to have been designed as a more thorough follow-on study to their prior work, the most significant variation from the prior study was the recommendation that participants restrict to low-fermentable, poorly absorbed, short-chain carbohydrates (FODMAPs) throughout the study. With the changing patterns of food intake and dietary behaviors over the last 20 years so-called westernization, FODMAPs have constituted significant proportion in food consumption. FODMAPs have been identified as important triggers for functional gut symptoms in people with visceral hypersensitivity or abnormal motility responses, largely by inducing luminal distension via a combination of osmotic effects and gas production related to their rapid fermentation by bacteria in the small and proximal large intestines. This seems to have been the rationale behind the addition of a low FODMAP diet in the current study, limiting alternate dietary triggers that could confound results. In the current study,  subjects with NCGS defined as “IBS fulfilling Rome III criteria that self-reportedly improved with a GFD” after exclusion of celiac disease were enrolled into the trial.

This study calls into question the very existence of NCGS as a discrete entity and suggests that FODMAPs, rather than gluten or other wheat proteins, might be the mediator by which low-gluten diets improve gastrointestinal symptoms. As noted, there are many potential ways in which FODMAPs may elicit gastrointestinal symptoms in predisposed individuals; however, limited as our understanding of NCGS is, investigation into FODMAPs in gastrointestinal disease has been nearly nonexistent outside of a few small studies published by this same group. The other clear possibility is that NCGS is a real entity but confounded by a low FODMAP diet by an unclear mechanism. In either case, it is tempting to say that everything seems to be at a standstill and therefore NCGS remains a controversial topic. Overall, these studies have highlighted the great potential of specific dietary interventions in gastrointestinal disorders outside of celiac disease. Although few facets of NCGS are clear, it is apparent that only the combination of larger, high-quality clinical trials on the role of specific diets in patients with chronic gastrointestinal symptoms, and translational studies evaluating mechanisms and potential biomarkers of NCGS and other food sensitivities, will allow us to make advances on this elusive entity.

 

Predictors of dietary gluten avoidance in adults without a prior diagnosis of celiac disease

Pornthep Tanpowpong , S Broder-Fingert, AJ. Katz, CA. Camargo Jr.
Nutrition 31 (2015) 236–238
http://dx.doi.org/10.1016/j.nut.2014.07.001

Objective: Prior studies have shown that dietary gluten avoidance (DGA) is relatively common in children without previously diagnosed celiac disease (CD), and several clinical predictors of DGA have been found. However, available data on predictors of DGA in adults without diagnosed CD are limited. The aim of this study was to determine the independent predictors of DGA in this population. Methods: We performed a structured medical record review of 376 patients, ages 20 y, who had never been formally diagnosed with CD, presenting for an initial CD evaluation (ICD-9-CM 579.0) between January 2000 and December 2010 at two large Boston teaching hospitals. We collected data including demographic characteristics, medical history, history of CD serology before referral, and self-reported DGA. Predictors of DGA were determined using multivariable logistic regression. Results: Mean age was 47 (SD ¼ 17) years. We found that 41 patients (10.9%; 95% confidence interval [CI], 7.9–14.5) had avoided gluten at some time in their lives. Most patients had subjective abdominal complaints or bowel movement changes. History of CD seropositivity before referral was noted in 14%. Independent predictors of DGA (P < 0.05) were lactose intolerance (odds ratio [OR], 2.8; 95% CI, 1.1–7.5), food allergy (OR, 3.8; 95% CI, 1.04–13.7), and history of positive serology of less-specific CD markers before the referral (OR, 3.2; 95% CI, 1.3–7.9). Conclusions: Gluten avoidance is common in a clinic population of adults without prior CD diagnosis. The recognized predictors suggest that DGA may associate with conditions presenting with nonspecific gastrointestinal complaints and perhaps with the perceived benefits of DGA among patients with prior history of positive CD serology.

Solubilization of gliadins for use as a source of nitrogen in the selection of bacteria with gliadinase activity

Patricia Alvarez-Sieiro, Begoña Redruello, Victor Ladero, Elena Cañedo, et al.
Food Chemistry 168 (2015) 439–444
http://dx.doi.org/10.1016/j.foodchem.2014.07.085

For patients with celiac disease, gliadin detoxification via the use of gliadinases may provide an alternative to a gluten-free diet. A culture medium, in which gliadins were the sole source of nitrogen, was developed for screening for microorganisms with gliadinase activity. The problem of gliadin insolubility was solved by mild acid treatment, which renders an acid-hydrolysed gliadin/peptide mixture (AHG). This medium provided a sensitive and reliable means of detecting proteases, compared to the classical spectrophotometric method involving azocasein. When a sample of fermented wheat (a source of bacteria) was plated on an AHG-based culture medium, strains with gliadinase activity were isolated. These strains’ gliadinase profiles were determined using an AHG-based substrate in zymographic analyses.

 

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells

Charles J.M. Bell, Yongliang Sun, Urszula M. Nowak, Jan Clark, et al.
Journal of Autoimmunity 56 (2015) 66e80
http://dx.doi.org/10.1016/j.jaut.2014.10.002

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRg binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, supra-threshold pSTAT5α induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

Over the last 20 years we have progressed from discovering that IL-2 and IL-2RA are genetically associated with autoimmune diabetes and the functional state of Tregs to seeing dramatic clinical success with IL-2 in chronic GVHD. The central role of IL-2 in the maintenance of self-tolerance and Treg function is now immunological canon and many attempts are being made to harness Tregs to combat a variety of autoimmune and inflammatory diseases. The recent clinical successes with Proleukin are noteworthy since pharmacologically it is a drug with limitations: its short half-life requires daily or every other day injection and the doses used to date stimulate CD4+ T effector cells, NK cells and eosinophils in addition to Tregs. Our goal was to develop and characterize IL-2 molecules with improved pharmacologic profiles that could be delivered less frequently and at lower doses than Proleukin and selectively expand Tregs that maintained their epigenetic profiles at FOXP3 and CTLA4.

Increasing the in vivo half-life of IL-2 by fusion to IgG1, i.e. IgG-IL-2, results in a molecule that can induce a 4-fold increase in Tregs after a single dose in cynomolgus, a response that multiple-dose, but no single dose, of Proleukin can achieve. Increasing the stoichiometry and hence the avidity, i.e. IgG-(IL-2)2, increases the potency and stimulates a similar increase in Tregs albeit at a 5-fold lower dose than IgG-IL-2. A detailed characterization of the in vivo dose responses for Proleukin and IgG-(IL-2)2 highlights that the magnitude and duration of Treg expansion, defined by its AUC, correlates with the magnitude and duration of pSTAT5α upregulation, also defined by its AUC. Single doses of Proleukin that increase pSTAT5α for one day have a minimal AUC and as a consequence little impact on Treg numbers; whereas single dose IgG-IL-2 and IgG-(IL-2)2 or multiple-dose Proleukin stimulate pSTAT5α that is  sustained for 4 days resulting in 3-4-fold larger pSTAT5α AUCs and corresponding increases in Tregs and the AUCs of Treg/mm3. Intermediate levels and duration of pSTAT5α induction result in moderate increases in Tregs. Following in vivo activation with Proleukin and IgG-(IL-2)2, Treg cell surface CD25 as well as intracellular FOXP3 and Ki-67, increased in a dose-dependent manner and persisted longer than the corresponding pSTAT5α responses; the effects of IgG-(IL-2)2 were >10-fold more potent and persisted longer than those induced by Proleukin. Of particular significance, the cynomolgus Tregs present after IgG-IL-2 and IgG-(IL-2)2-induced in vivo expansion retain their fully demethylated FOXP3 and CTLA4 epigenetic signatures indicating a functional suppressive phenotype.

The ability of cynomolgus to respond and differentiate amongst different forms and doses of IL-2 with varying degrees of activation and increases in Tregs speaks to their utility as a translational preclinical species. In fact, single doses of IgG-IL-2 and IgG-(IL-2)2 replicated the increased number of Tregs seen in GVHD patients given daily Proleukin. Furthermore, Proleukin given to cynomolgus following the same multiple-dose protocol at the human equivalent dose achieved the same increases in Tregs and eosinophils as patients with type 1 diabetes.

The long half-lives of IgG-IL-2 and IgG-(IL-2)2 enable the detection of receptor-mediated clearance of IL-2 in vivo; the half-lives of the fusion proteins are five times longer in mice in the absence of the high affinity IL-2 receptor. The competition for injected IL-2 by different cell populations and the upregulation of IL-2 receptors in response to injections of the cytokine are important considerations when interpreting IL-2 doses required for preferential Treg expansion.  The failure of low-dose IL-2 to expand cynomolgus NK cells in vivo means that this aspect of IL-2 immunotherapy using novel, long-lived molecules will need to be addressed in future human studies. Despite these differences, the pharmacokinetic and pharmacodynamic analyses in this cynomolgus study strongly support the hypothesis that increasing the half-life of IL-2 allows for lower doses of IL-2 to be delivered far less frequently thereby favoring prolonged Treg-specific cell expansion.

 

T cell subsets and their signature cytokines in autoimmune and inflammatory diseases

Itay Raphael, Saisha Nalawade, Todd N. Eagar, Thomas G. Forsthuber
Cytokine xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.cyto.2014.09.011

CD4+ T helper (Th) cells are critical for proper immune cell homeostasis and host defense, but are also major contributors to pathology of autoimmune and inflammatory diseases. Since the discovery of the Th1/Th2 dichotomy, many additional Th subsets were discovered, each with a unique cytokine profile, functional properties, and presumed role in autoimmune tissue pathology. This includes Th1, Th2, Th17, Th22, Th9, and Treg cells which are characterized by specific cytokine profiles. Cytokines produced by these Th subsets play a critical role in immune cell differentiation, effector subset commitment, and in directing the effector response. Cytokines are often categorized into proinflammatory and anti-inflammatory cytokines and linked to Th subsets expressing them. This article reviews the different Th subsets in terms of cytokine profiles, how these cytokines influence and shape the immune response, and their relative roles in promoting pathology in autoimmune and inflammatory diseases. Furthermore, we will discuss whether Th cell pathogenicity can be defined solely based on their cytokine profiles and whether rigid definition of a Th cell subset by its cytokine profile is helpful.

T helper cell subsets differentiate and express their protective and pathogenic roles of their lineage-signature cytokines. The signature cytokines for each subset are as follows: IL-12 induces the expression of T-bet and differentiation into the Th1 subset which produces IFN-c and TNF; Th2 differentiation and GATA3 expression is induced by IL-4, leading to the production of IL-4, IL-5 and IL-13, whereas TGF- T helper-cell subset differentiation and the protective and pathogenic roles of their lineage-signature cytokines. The signature cytokines for each subset is as follows:  IL-12 induces the expression of T-β and differentiation into the Th1 subset which produces IFN-c and TNF; Th2 differentiation and GATA3 expression is induced by IL-4, leading to the production of IL-4, IL-5 and IL-13, whereas TGF-β and IL-4 induce PU.1 expression. This causes differentiation into the Th9 subset and leads to the production of IL-9. TGF-β induces the expression of Foxp3, which leads to differentiation into the Treg lineage; Th17 differentiation is a result of RORct expression induced by TGF-β, IL-6 and IL-23, leading to the production of IL-17, IL-22, IL-21, IL-25 and IL-26 (human); IL-6 and TNF induce AHR and differentiation into the Th22 subset and production of IL-22. STAT: Signal transducer and activator of transcription; RORc: RAR related orphan receptor gamma, AHR: Aryl hydrocarbon receptor, Foxp3: forkhead box P3 and IL-4 induce PU.1 expression which causes differentiation into the Th9 subset leading to the production of IL-9. TGF-β induces the expression of Foxp3, which leads to differentiation into the Treg lineage; Th17 differentiation is a result of RORct expression induced by TGF-β, IL-6 and IL-23, leading to the production of IL-17, IL-22, IL-21, IL-25 and IL-26 (human); IL-6 and TNF induce AHR and differentiation into the Th22 subset and production of IL-22. STAT: Signal transducer and activator of transcription; RORc: RAR related orphan receptor gamma, AHR: Aryl hydrocarbon receptor, Foxp3: forkhead box P3.

In autoimmune diseases, Th2 cells were initially described as anti-inflammatory based on their ability to suppress cell-mediated or Th1 models of disease. Th2 cells have been described in lesions of MS patients, and IL-4 and IL-4R expression has been reported in several cell types in close proximity to active demyelinating lesions. Over the years, however, a number of reports established a role for Th2 cells in tissue inflammation and implicated their cytokines in immunopathology.  Genain et al. reported that in marmoset monkeys with EAE the cytokine production was shifted from a Th1 to a Th2 pattern, and titers of autoantibodies to myelin oligodendrocyte glycoprotein (MOG) were enhanced. They concluded that induction of Th2 responses may exacerbate autoimmunity by enhancing production of pathogenic autoantibodies.

The involvement of Th2 cells and pathogenic antibodies contrast the prevailing models of murine EAE which are considered to be Th1 and Th17-effector T cell-mediated diseases. However, pathogenic roles for Th2 cells have also been reported in murine EAE. Lafaille et al. showed that adoptive transfer of Th2-polarized MBP-specific T effector cells elicited EAE in immunocompromised recipient mice (RAG-1 or TCRα deficient), but not immune-sufficient hosts. When compared with other T effector subsets, mice receiving Th2 cells developed EAE with delayed onset and milder symptoms. Jager et al. have also reported that 2D2 MOG-specific Th2 cells can induce EAE with delayed onset and low severity. Taken together, these reports support that Th2 cells can promote pathogenicity, but ensuing disease may be less severe. Alternatively, but not mutually exclusive, development of EAE may not have been mediated by ‘‘Th2’’ cytokines, but might have been due to the switch of Th2 cells to a Th1-like phenotype and secretion of proinflammatory cytokines such as IFN-c. Th2 cytokines are associated with the pathogenesis of antibody-mediated autoimmune diseases.

The expression of one signature cytokine, such as IL-17, may not tell the full story about Th subset commitment, since the stability of its expression may be influenced by different factors as mentioned above. Along these lines, IL-17 is enhanced by IL-23, which promotes the pathogenic potential of Th17 cells and enhances the expression of IL-17 by these cells. Thus, adoptive transfer of IL-23-induced Th17 cells results in severe EAE, and in the absence of IL-23 signaling the mice are resistant to EAE. However, the disease resistance seen in the absence of IL-23 signals was not due to the lack of expression of IL-17 or IL-22 by Th17 cells, but rather by the failure of these cells to produce GM-CSF, a cytokine that was initially believed to be produced by encephalitogenic, IFN-c producing Th1 cells. Indeed both Th1 cells and Th17 cells can produce GM-CSF. Interestingly, induction of GM-CSF expression by human Th cells is constrained by the IL-23/ROR-ct/Th17 cell axis but promoted by the IL-12/T-bet/Th1 cell axis. Thus the enigma remains as to why IL-23-induced Th17 cells are indispensable for the induction of EAE. As it turns out, IL-23-induced Th17 cells not only produce GM-CSF, but are also producing IFN-c. The observation of IFN-c producing Th17 cells lead to the realization that IL-17 and IFN-c double-producing cells, belonging to the Th17 subset, developed under the influence of IL-23 and converted into IL-17 producing Th1-like cells, and later to ‘‘exTh17’’ cells, while discontinuing the production of IL-17.

The concept of a specialized subset of T lymphocytes with suppressive function has been around since the early 1970s. In the mid-1990s a novel subset of Th cells with ‘‘regulatory’’ function was identified and designated Tregs. Tregs were later found to express the signature Foxp3 transcription factor, which is critical for their development, lineage commitment, and regulatory functions. Foxp3 expressing Treg subsets include thymically derived or natural Tregs (nTregs) and Tregs that are induced via post-thymic maturation (iTregs). Later, iTregs were further discriminated into Foxp3+ cells (Th3) and Foxp3 cells (Tr1). Numerous studies have identified Tregs as important immunoregulators in many inflammatory and autoimmune disease conditions including asthma, MS, and type-I diabetes.

Several mechanisms of Treg-mediated immune suppression have been identified, including: the secretion of anti-inflammatory  cytokines, expression of inhibitory receptors, and cytokine deprivation. For the purpose of this review we will focus on regulatory cytokine production. The two cytokines mostly associated with Tregs are IL-10 and TGF-β. Importantly, Tregs can themselves secrete these cytokines and use them to carry out their suppressive function. TGF-β is produced by both nTreg and Th3 cells, however other cells including B cells, macrophages, DCs, and many other non-immune cells, can also produce this cytokine. TGF-β is required for the generation of iTregs by inducing the expression of Foxp3 in a paracrine feedback loop that will convert naïve T cells (Th0) to differentiate into iTregs. The positive feedback loop between TGF-β and Foxp3 plays a critical role in maintaining peripheral tolerance and is key to the generation and maintenance of Tregs. In vivo, TGF-β producing Tregs have been shown to suppress EAE by inhibiting autoimmune T cell responses in the CNS of EAE mice.

Not shown.  A proposed model reveals an immune switch point from pathogenic Th17 cells to suppressive ex-Th17 cells in EAE. TGF-β, IL-6 and IL-23 induce the differentiation of Th17 cells in the immune periphery. In the CNS, signaling by IL-23 induces the expression of GM-CSF and IFN-c in Th17 cells, thereby rendering these cells pathogenic. In an autocrine signaling loop, IFN-c suppresses the expression of RORct and the production of GM-CSF (as well as IL-17) by pathogenic Th17 cells, thereby inducing a switch to ‘‘suppressive’’ exTh17 cells.

ExTh17 cells are expressing the transcription factor T-bet and as a result IFN-c, in an IL-23 dependent manner, which is important for the pathogenic potential of exTh17 cells. Furthermore, IFN-c acts as a potent negative regulator of ROR-ct, the master regulator of the Th17 subset that drives the production of GM-CSF. Similar observations were made in other inflammatory and autoimmune conditions, illustrating the transition of Th17 cells into Th1-like cells. These observations further support the view of a switch point at which anti-inflammatory pathways are activated by the same Th subsets that initially promoted pathogenicity. In this scenario, IFN-c inhibits GM-CSF production by Th17 cells in the target tissues. We propose a possible model for a switch point for GM-CSF production by ‘‘pathogenic’’ Th-17 cells which is mediated by IL-23 and IFN-c in EAE.

Taken together, the one cytokine, one pathogenic Th cell, does not fit the bill anymore. The discovery of Th1-like Th17 cells, exTh17 cells, etc. complicates the question as to whether targeting a single cytokine or pathogenic T cell subset will ever result in the cure for autoimmune diseases.

The immune system seems to favor a balance between pathogenic and protective Th cells via dual roles for ‘‘subset-specific’’, or ‘‘signature cytokines’’, as well as allowing plasticity for subset differentiation and expression of ‘‘signature’’ cytokine(s) by other Th subsets. The observation that many Th subsets can convert into IFN-c secreting Th1-like cells illustrates this fact since IFN-c can be both pathogenic and protective. Targeting cytokines as therapy for autoimmune and/or inflammatory disorders remains a conceptual challenge more than ever. Clearly, cytokine therapy proved successful in some cases, such as anti-TNF therapy of RA, with the caveat that surprising adverse effects were observed in some patients indicative of the additional roles of this cytokine.

 

Regulatory T-cells in autoimmune diseases: Challenges, controversies and—yet—unanswered question

Charlotte R. Grant, R Liberal, G Mieli-Vergani, D Vergani, MS Longhi
Autoimmunity Reviews 14 (2015) 105–116
http://dx.doi.org/10.1016/j.autrev.2014.10.012

Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.

There are four basic mechanisms that Tregs use to suppress immune responses:

  1. the modulation of antigen presenting cell (APC) maturation and function,
  2. the killing of target cells,
  3. the disruption of metabolic pathways and
  4. the production of anti-inflammatory cytokines

Fig not shown. A) Regulatory T cellmechanisms of suppression. Regulatory T cell (Treg) can suppress by four basicmechanisms. The interaction between cytotoxic T lymphocyte antigen-4 (CTLA4) and CD80/CD86, expressed by antigen presenting cells (APCs), leads to CD80/CD86 down-regulation. Removal of these co-stimulatory molecules modulates APC function, limiting the initiation of an adaptive immune response. Tregs induce effector T cell (Teff) apoptosis by the interaction between Galectin-9 (Gal-9) and the T cell immunoglobulin and mucin domain-3 (TIM-3), and by the release of granzymes which enter Teffs via perforin pores. Tregs release the anti-inflammatory cytokines TGFβ, IL10 and IL35. Treg expression of the ecto-enzymes CD39 and CD73 enables the hydrolysis of pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine (ADO). B) Regulatory T cell defects in autoimmunity. In health, Tregs maintain tolerance by exerting suppression of effector T cells. In organ specific autoimmune disease, Tregs fail to suppress autoreactive effector T cells, therefore leading to target cell death. Reported reasons for this include inadequate numbers of Tregs, impaired suppressive ability, Treg conversion into effector cells and resistance of effector T cells to Treg-mediated suppression.

In the following sections, studies investigating the frequency and suppressive function of Tregs in the archetypal non-organ specific autoimmune disease SLE, and the organ specific autoimmune diseases MS, T1D, RA, autoimmune thyroid disease, psoriasis and IBD will be discussed.

Treg defects are frequently reported in autoimmune disease. There are, however, often discrepancies in the literature, which can be accounted for by the choice of study participants and the techniques used to study this challenging population of cells. The search for new markers that could unequivocally identify bona fide human Tregs—for the purposes of both phenotypic and functional analysis—will greatly facilitate our understanding of the role of Tregs in autoimmune disease. Studies suggest that the nature of the Treg impairment differs according to the autoimmune disease under investigation. There are reports of numerical and functional Treg impairments, of resistance of effector T cells to Treg suppression and of conversion of Tregs to effector cells. It is, therefore, important to consider numerical, phenotypic and functional defects affecting a range of Treg subsets. Moreover, current evidence strongly implies that systemic or regional factors can confine Treg impairments to the target organ. Treg studies would, therefore, benefit from more thorough investigation of the inflammatory site.

Take-home message

  • Tregs are central to tolerance maintenance and tissue homeostasis.
  • Treg impairment has been reported in several autoimmune diseases.
  • Systemic or regional factors can confine Treg impairment to the target organ.
  • Challenges remain when defining and investigating Tregs in autoimmune diseases.

 

 

 

 

 

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Graft-versus-Host Disease

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

This piece is a follow up to the article on allogeneic transfusion reactions, which extends into transplantation and transplantation outcomes for hematological diseases, both malignant and nonmalignant. The safety of transfusions in Western countries has improved substantially, and the causes for transfusion mishaps has been reduced to unexpected infectious sources, uncommon immune incompatibilities, and errors in processing the blood products.  The greatest risk is incurred in platelet transfusions because of the short shelf-life of the product, and the time needed for testing prior to release.  This portion of the review is concerned with Graft-versus-Host Disease, which is unique to transfusion and transplanting of blood. In other transplantation, there is graft failure because of host versus graft incompatibility or complications.  The reverse order applies to blood.  In this case, on the contrary, the transfused or grafted donor tissue becomes a pursuer after the recipients hematopoietic cells.

Peter Brian Medawar: Father of Transplantation

Thomas E. Starzl, M.D., PH.D., F.A.C.S.
J Am Coll Surg. 1995 Mar; 180(3): 332–336

Most of the surgical specialities can be tracked to the creative vision of a surgeon. Transplantation is an exception. Here, the father of the field is succinctly defined in the dictionary as: “Peter Brian Medawar: a Brazilian born British Zoologist who at the age of 45 shared a 1960 Nobel Prize for his work on acquired immunologic tolerance”. Medawar was mysteriously overwhelming to many colleagues and observers, even when he was young. He was the son of a Lebanese father and an English mother—tall, athletic, abnormally handsome, hypnotically articulate in public, and politely cordial in his personal relations. In September 1969, at the age of 54, he had the first of a series of strokes. These crippled him physically but not in spirit. Although I saw Medawar often professionally and privately over a 22 year period, before and after he was disabled, this sporadic exposure was not enough to understand him. My sense is that no one did, except perhaps Jean, his wife for nearly 50 years.

Medawar’s dazzling personality before and great courage after his strokes was inspirational, but his fame was based on the unique achievement in 1953 captured by the terse dictionary mention of “acquired immunologic tolerance.” The roots leading to this accomplishment had fed on the blood of war. More than 12 years earlier, the recently wed zoologist Medawar—24 years of age and fresh from graduate studies at Oxford University—was assigned to
the service of the British surgeon, Dr. Thomas Gibson, to determine if skin allografts could be used to treat casualties from the Battle of Britain. First,
in human studies with Gibson, and then with simple and logical rabbit experiments, Medawar showed that rejection of the skin was an immunologic phenomenon. This later was shown  to be analogous to the cell-mediated delayed hypersensitivity that confers immunity to diseases such as tuberculosis. The principal evidence in the early studies was that repetitive grafts from the same donor were rejected more rapidly with each successive attempt —the sensitization and donor specificity confirming an earlier clinical observations by Emil Holman of Stanford in skin-grafted burn victims. Once it was established that rejection was an immune reaction, strategies began to evolve to weaken the recipient immune system. By 1953, total body irradiation and adrenal cortical steroids had been shown to delay skin rejection. However, this immunosuppressive effect was either minor if the animals survived, or lethal to the recipient if the grafts were spared.

Bombshell

In the resulting atmosphere of nihilism about clinical applications, a three and one-half page article by Billingham, Brent, and Medawar in the October 3, 1953 issue of Nature describing acquired tolerance, came as a blinding beacon of hope. The three men had learned that donor splenocytes could be engrafted by their intravenous infusion into immunologically immature mice in utero or perinatally. When these inoculated recipients matured, they could accept skin and other tissues from the donor (but from no other) mouse strain. The immune system of the recipients had been populated by the immunocytes of the donor, meaning that they were now chimeras. The race was on to convert this principle to humans. However, the dark side of their accomplishment soon was revealed by the two younger members of Medawar’s team, Billingham and Brent and by the Dane, Simonsen. The engrafted donor cells could turn the tables and reject the defenseless recipient unless the tissue match was a good one. This was the dreaded graft versus host disease (GVHD) in which transplanted donor cells attacked the recipient skin, gastrointestinal tract, lungs, liver, and the bone marrow itself. Medawar’s dream of 1953 was suddenly a nightmare. Or was it?

On the contrary, the work took a straight line to clinical application, after the demonstration by Prehn and Main that similar tolerance could be induced in adult mice rendered immunologically defenseless by total body irradiation before splenocyte (or later bone marrow) infusion. The recipient conditioning, known as cytoablation, also could be accomplished with myelotoxic drugs. However, as Billingham, Brent, and Medawar had predicted, donor specific tolerance could be induced in humans without GVHD only if there was a good tissue (HLA) match. In 1968, 15 years after the epic Billingham, Brent and Medawar publication, Robert Good and Fritz Bach reported the first two successful human bone marrow transplants. Both recipients of well matched bone marrow from blood relatives are still alive. This was a triumph in which the principal clinicians were internists, as summarized 25 years later in the acceptance speech by the 1990 Nobel Laureate Donnall Thomas.

The growth of bone marrow and whole organ transplantation

The growth of bone marrow and whole organ transplantation

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681237/bin/nihms-87975-f0001.gif

The growth of bone marrow (right) and whole organ transplantation (left) from the seed planted by Peter Medawar during World War II. GVHD, Graft versus host disease.

Immunological Tolerance: Medawar Nobel Acceptance Lecture

“Immunological tolerance” may be described as a state of indifference or non-reactivity towards a substance that would normally be expected to excite an immunological response. The term first came to be used in the context of tissue transplantation immunity, i.e. of the form of immunity that usually prohibits the grafting of tissues between individuals of different genetic make-up; and it was used to refer only to a non-reactivity caused by exposing animals to antigenic stimuli before they were old enough to undertake an immunological response. For example, if living cells from a mouse of strain CBA are injected into an adult mouse of strain A, the CBA cells will be destroyed by an immunological process, and the A-line mouse that received them will destroy any later graft of the same origin with the speed to be expected of an animal immunologically forearmed. But if the CBA cells are injected into a foetal or newborn A-line mouse, they are accepted; more than that, the A-line mouse, when it grows up, will accept any later graft from a CBA donor as if it were its own. I shall begin by using the term “immunological tolerance” in the rather restricted sense that is illustrated by this experiment, and shall discuss its more general usage later on.

The experiment I have just described can be thought of as an artificial reproduction of an astonishing natural curiosity, the phenomenon of red-cell chimerism in certain dizygotic twins. The blood systems of twin cattle before birth are not sharply distinct from each other, as they are in most other twins; instead, the blood systems make anastomoses with each other, with the effect that the twins can indulge in a prolonged exchange of blood before birth. In 1945, R.D. Owen2 made the remarkable discovery that most twin cattle are born with, and may retain throughout life, a stable mixture – not necessarily a fifty-fifty mixture – of each other’s red cells; it followed, then, that the twin cattle must have exchanged red-cell precursors and not merely red cells in their mutual transfusion before birth. This is the first example of the phenomenon we came to call immunological tolerance; the red cells could not have “adapted” themselves to their strange environment, because they were in fact identified as native or foreign by those very antigenie properties which, had an adaptation occurred, must necessarily have been transformed. A few years later R.E. Billingham and I3, with the help of three members of the scientific staff of the Agricultural Research Council, showed that most dizygotic cattle twins would accept skin grafts from each other, and that this mutual tolerance was specific, for skin transplanted from third parties was cast off in the expected fashion.

Some properties of the tolerant state

The main points that emerged from our analysis of the tolerant state were these. In the first place, tolerance must be due to an alteration of the host, not to an antigenic adaptation of the grafted cells, for grafts newly transplanted in adult life have no opportunity to adapt themselves, and the descendants of the cells injected into foetal or newborn animals can be shown by N.A. Mitcbison’s methods to retain their antigenic power10. Once established, the state of tolerance is systemic; if one part of the body will tolerate a foreign graft, so will another; we found no evidence that a tolerated graft builds up a privileged position for itself within its own lymphatic territory. The stimulus that is responsible for instating tolerance is an antigenic stimulus – one which, had it been applied to older animals, would have caused them to become sensitive or immune. A plural stimulus can induce plural tolerance; the donor will usually contain several important antigens that are lacking in the recipient, and long-lasting tolerance must imply tolerance of them all. The state of tolerance is specific in the sense that it will discriminate between one individual and another, for an animal made tolerant of grafts from one individual will not accept grafts from a second individual unrelated to the first; but it will not discriminate between one tissue and another from the same donor.

Tolerance and auto-immunity: 50 years after Burnet.

Martini A1, Burgio GR
Eur J Pediatr. 1999 Oct;158(10):769-75.

Fifty years ago Sir F. Macfarlane Burnet published his first fundamental contribution to the theory of immune tolerance he perfected 10 years later. Since then an impressive amount of new information on the function of the immune system has been gathered. As any original meaningful theory, Burnet’s hypothesis on the development of immune tolerance has undergone extensive modifications to take into account all these new findings. An improved understanding of the mechanisms of tolerance has led to new possibilities for the treatment of auto-immune diseases.

Clonal Selection
http://en.wikipedia.org/wiki/Clonal_selection

Clonal selection theory is a scientific theory in immunology that explains the functions of cells (lymphocytes) of the immune system in response to specific antigens invading the body. The concept was introduced by an Australian doctor Frank Macfarlane Burnet in 1957 in an attempt to explain the formation of a diversity of antibodies during initiation of the immune response. The theory has become a widely accepted model for how the immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens.

The theory states that in a pre-existing group of lymphocytes (specifically B cells), a specific antigen only activates (i.e. selection) its counter-specific cell so that particular cell is induced to multiply (producing its clones) for antibody production. In short the theory is an explanation of the mechanism for the generation of diversity of antibody specificity. The first experimental evidence came in 1958, when Gustav Nossal and Joshua Lederberg showed that one B cell always produces only one antibody. The idea turned out to be the foundation of molecular immunology, especially in adaptive immunity.

The fundamental contribution of Robert A. Good to the discovery of the crucial role of thymus in mammalian immunity

Domenico Ribatti
Immunology. 2006 Nov; 119(3): 291–295.
http://dx.doi.org:/10.1111/j.1365-2567.2006.02484.x

Robert Alan Good was a pioneer in the field of immunodeficiency diseases. He and his colleagues defined the cellular basis and functional consequences of many of the inherited immunodeficiency diseases. His was one of the groups that discovered the pivotal role of the thymus in the immune system development and defined the separate development of the thymus-dependent and bursa-dependent lymphoid cell lineages and their responsibilities in cell-mediated and humoral immunity.  Keywords: bursa of Fabricius, history of medicine, immunology, thymus

Robert Alan Good (May 21, 1922 – June 13, 2003) was an American physician who performed the first successful human bone marrow transplant

Robert A. Good began his intellectual and experimental queries related to the thymus in 1952 at the University of Minnesota, initially with pediatric patients. However, his interest in the plasma cell, antibodies and the immune response began in 1944, while still in Medical School at the University of Minnesota in Minneapolis, with his first publication appearing in 1945.

Idiopathic Acquired Agammaglobulinemia Associated with Thymoma (1953)

  • a markedly deficient ability to produce antibodies and significant deficits of all or most of the cell-mediated immunities
  • in no instance did removal of the thymic tumour restore immunological function or correct the protein deficit

Good syndrome: thymoma with immunodeficiency

  • increased susceptibility to bacterial infections by encapsulated organisms and opportunistic viral and fungal infections
  • immunodeficiencies, leukopenia, lymphopenia and eosinophylopenia
  • severely hypogammaglobulinemic rather than agammaglobulinemic

Good and others found that the patients lacked all of the subsequently described immunoglobulins. These patients were found not to have plasma cells or germinal centers in their hematopoietic and lymphoid tissues. They possessed circulating lymphocytes in normal numbers.

Speculation on the reason for immunological failure following neonatal thymectomy has centered on the thymus as a source of cells or humoral factors essential to normal lymphoid development and immunological maturation.

The bursa of Fabricius and the thymus are ‘central lymphoid organs’ in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Studies by Papermaster and co-workers in Good’s laboratory34,35 indicated that bursectomy in the newly hatched chicks did not completely abolish immunological potential in the adult animal but rather produced a striking quantitative reduction insufficient to eliminate the homograft reaction. The failure of thymectomy in newly hatched chicks to alter the immunological potential of the maturing animal probably only reflected the participation of the bursa of Fabricius in the development of full immunological capacity.

Bursectomized and irradiated birds were completely devoid of germinal centers, plasma cells and the capacity to make antibodies yet they had perfectly normal development of thymocytes and lymphocytes elsewhere in the body that mediated cellular immune reactions. On the other hand, thymectomized and irradiated animals were deficient in lymphocytes that mediated cellular immunity as assessed by skin graft rejection, delayed-type hypersensitivity and graft versus host assays, but they still produced germinal centers, plasma cells and circulating immunoglobulins.

 

Graft vs Host Disease

Graft-versus-host disease (GVHD) is a complication that can occur after a stem cell or bone marrow transplant. With GVHD, the newly transplanted donor cells attack the transplant recipient’s body.

Graft-versus-host disease (GVHD) is a common complication following an allogeneic tissue transplant. It is commonly associated with stem cell or bone marrow transplant but the term also applies to other forms of tissue graft. Immune cells (white blood cells) in the tissue (the graft) recognize the recipient (the host) as “foreign“. The transplanted immune cells then attack the host’s body cells. GVHD can also occur after a blood transfusion if the blood products used have not been irradiated or treated with an approved pathogen reduction system.

http://en.wikipedia.org/wiki/Graft-versus-host_disease

Causes

GVHD may occur after a bone marrow or stem cell transplant in which someone receives bone marrow tissue or cells from a donor. This type of transplant is called allogeneic. The new, transplanted cells regard the recipient’s body as foreign. When this happens, the newly transplanted cells attack the recipient’s body.

GVHD does not occur when someone receives his or her own cells during a transplant. This type of transplant is called autologous.

Before a transplant, tissue and cells from possible donors are checked to see how closely they match the person having the transplant. GVHD is less likely to occur, or symptoms will be milder, when the match is close. The chance of GVHD is:

  • Around 30 – 40% when the donor and recipient are related
  • Around 60 – 80% when the donor and recipient are not related

There are two types of GVHD: acute and chronic. Symptoms in both acute and chronic GVHD range from mild to severe.

  • Acute GVHD usually happens within the first 6 months after a transplant.
  • Chronic GVHD usually starts more than 3 months after a transplant, and can last a lifetime.

Bone marrow transplant

A bone marrow transplant is a procedure to replace damaged or destroyed bone marrow with healthy bone marrow stem cells.  Stem cells are immature cells in the bone marrow that give rise to all of your blood cells.

There are three kinds of bone marrow transplants:

  • Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.
  • Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.
  • Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

  • Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.
  • Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

Histocompatibility antigen:

  • A histocompatibility antigen blood test looks at proteins called human leukocyte antigens (HLAs). These are found on the surface of almost all cells in the human body. HLAs are found in large amounts on the surface of white blood cells. They help the immune system tell the difference between body tissue and substances that are not from your own body.

http://www.nlm.nih.gov/medlineplus/ency/article/001309.htm

Induction of transplantation tolerance in haploidenical transplantation under reduced intensity conditioning: The role of ex-vivo generated donor CD8+ T cells with central memory phenotype

Eran Ophir, Y Eidelstein, E Bachar-Lustig, D Hagin, N Or-Geva, A Lask, , Y Reisner
Best Practice & Research Clinical Haematology 24 (2011) 393–401
http://dx.doi.org:/10.1016/j.beha.2011.05.007

Haploidentical hematopoietic stem cell transplantation (HSCT) offers the advantage of readily available family member donors for nearly all patients. A ‘megadose’ of purified CD34þ hematopoietic stem cells is used to overcome the host’s residual immunity surviving the myeloablative conditioning, while avoiding severe GVHD. However, the number of CD34+ cells that can be harvested is insufficient for overcoming the large numbers of host T cells remaining after reduced intensity conditioning (RIC). Therefore, combining a ‘megadose’ of CD34+ HSCT with other tolerizing cells could potentially support and promote successful engraftment of haploidentical purified stem cell transplantation under a safer RIC. One approach to address this challenge
could be afforded by using Donor CD8 T cells directed against 3rd-party stimulators, bearing an ex-vivo induced central memory phenotype (Tcm). These Tcm cells, depleted of GVH reactivity, were shown to be highly
efficient in overcoming host T cells mediated rejection and in promoting
fully mismatched bone-marrow (BM) engraftment, in HSCT murine models.
This is likely due to the marked lymph node homing of the Tcm, their strong proliferative capacity and prolonged persistence in BM transplant recipients. Thus, combining anti 3rd-party Tcm cell therapy with a ‘megadose’ of purified CD34+ stem cells, could offer a safer RIC protocol for attaining hematopoietic chimerism in patients with hematological diseases and as a platform for organ transplantation or cell therapy in cancer patients.

Induction of tolerance in organ recipients by hematopoietic stem cell transplantation

Eran Ophir, Yair Reisner
International Immunopharmacology 9 (2009) 694–700
http://dx.doi.org:/10.1016/j.intimp.2008.12.009

The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+linhematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism.

Intrinsic unresponsiveness of Mertk/B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression

Wen-Hai Shao, Y Zhen, FD Finkelman, RA Eisenberg, PL Cohen
Journal of Autoimmunity 39 (2012) 412e419
http://dx.doi.org/10.1016/j.jaut.2012.07.001

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk-/- mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk-/-mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk-/-  mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk-/- mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk-/- mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis

Kazuki Sakai, Eri Kawata, Eishi Ashihara, Yoko Nakagawa, et al.
Eur. J. Immunol. 2011. 41: 67–75 http://dx.doi.org:/10.1002/eji.200939931

Galectins comprise a family of animal lectins that differ in their affinity for β-galactosides. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently shown to function as a ligand for T-cell immunoglobin domain and mucin domain-3 (Tim-3) expressed on terminally differentiated CD41 Th1 cells. Gal-9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal-9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal-9 inhibit MLR in a dose-dependent manner, involving both Ca21 influx and apoptosis in T cells. Next, we revealed that recombinant human Gal-9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal-9 ameliorates aGVHD, possibly by inducing T-cell apoptosis, suggesting that gal-9 may be an attractive candidate for the treatment of aGVHD.

 

GVHD Prevention: An Ounce Is Better Than a Pound

Pavan Reddy, Gerard Socie, Corey Cutler, Daniel Weisdorf
Biol Blood Marrow Transplant 18:S17-S26, 2012  http://dx.doi.org:/10.1016/j.bbmt.2011.10.034

The pathophysiology of acute graft-versus-host disease (aGVHD) is known to involve donor T cells responding to host histoincompatible allo-antigens presented by the host antigen presenting cells (APCs) and the subsequent induction of pro-inflammatory cytokines and cellular effectors that cause target organ damage. In a more general sense, GVHD can be considered as an immune response against foreign antigens that has gone awry. Similar to all immune responses, GVHD, can be understood as a process that consists of (A) triggers, (B) sensors, (C) mediators, and (D) effectors of GVHD.

Like all immune responses, certain triggers are critical for induction of acute graft-versus-host disease (aGVHD). These include: (1) Disparities between histocompatibility antigens: antigen disparity can be at the level of major histocompatibility complex (MHC), that is, MHC mismatched or at the level of minor histocompatibility antigens (miHA), that is, MHC matched but miHA mismatched. The severity of aGVHD is directly related to the degree of M HC mismatch. In bone marrow transplants (BMT) that are MHC matched but miHA disparate, donor T cells still recognize MHC-peptide derived from the products of recipient polymorphic genes, the miHAs.

Damage induced by conditioning regimens and underlying diseases: under most circumstances, the initiation of an adaptive immune response is triggered by the innate immune response. The innate immune system is triggered by certain exogenous and endogenous molecules. This is likely the case in the induction of aGVHD. Pattern recognition receptors such as Toll-like receptors (TLR), nucleotide-binding oligomerization domain containing 2 (NOD2) play an essential role in innate immunity and in initiating the cellular signaling pathways that activate cytokine secretion, such as NF-kB. Some of their ligands, such as lipopolysaccharide, CpG, and MDP2, which is recognized by TLR-4, TLR-9, and NOD2, respectively, are released by the preparative regimens and contribute to the induction and enhancement of allo-T cell responses. In this way, the conditioning regimens amplify the secretion of proinflammatory cytokines like interleukin (IL)-1, tumor necrosis factor (TNF)-α,  IL-6, and other interferon family members in a process described as a ‘‘cytokine storm.’’

The triggers that initiate an immune response have to be sensed and presented. APCs might be considered the sensors for aGVHD. The APCs sense the DAMPs, present the MHC disparate or miHA disparate protein, and provide the critical secondary (costimulatory) and tertiary (cytokine) signals for activation of the alloreactive T cells, the mediators of aGVHD. APCs sense allo-disparity through MHC and peptide complexes. Dendritic cells (DCs) are the most potent APCs and the primary sensors of allo-disparity.

APCs provide the critical costimulation signals for turning on the aGVHD process. The interaction between the MHC/allopeptide complex on APCs and the T cell receptor of donor T cells along with the signal via T cell costimulatory molecules and their ligands on APCs is required to achieve T cell activation, proliferation, differentiation, and survival and the in vivo blockade of positive costimulatory molecules (such as CD28, ICOS, CD40, CD30, etc.), or inhibitory signals (such as PD-1 and CTLA-4) mitigate or exacerbate aGVHD, respectively.

Evidence suggests that alloreactive donor T cells consist of several subsets with different stimuli responsiveness, activation thresholds, and effector functions.

The allo-antigen composition of the host determines which donor T cells subsets differentiate and proliferate. As mentioned previously, in the majority of HLA-matched HCT, aGVHD may be induced by either or both CD41 and CD81 subsets responses to miHAs. The repertoire and immunodominance of the GVHD-associated peptides presented by MHC class I and class II molecules has not been defined. Donor naive CD62L1 T cells are the primary alloreactive T cells that drive the GVHD reaction while the donor effector memory CD62L2 T cells do not. Interestingly, donor regulatory T cells (Tregs) expressing CD62L are also critical to the regulation of GVHD. We now know that it is possible to modulate the alloreactivity of na€ıve T cells by inducing anergy with costimulation blockade, deletion via cytokine modulation, or mixed chimerism. Donor effector memory T cells that are nonalloreactive do not induce GVHD, yet are able to transfer functional memory. In contrast, memory T cells that are alloreactive can cause severe GVHD.

The effector phase that leads to GVHD target organ damage is a complex cascade that involves cytolytic cellular effectors such as CD8 cytotoxic T lymphocytes (CTLs), CD4 T cells, natural killer cells, and inflammatory molecules such as IL-1β, TNF-α, IFN-ϒ, IL-6, and reactive oxygen species. The cellular effectors require cell-cell contact to kill the cells of the target tissues via activation of perforin granzyme, Fas-FasL (CD95-CD95L), or TNFR TRAIL pathways. Other CTLs killing mechanisms such as TWEAK, and LTβ/LIGHT pathways have also been implicated in GVHD. It is important to note that
CTL pathways are essential for GVL effects as well.

All of the above aspects of the biology of aGVHD have been summarized in the mold of a normal immune response. Although this allows for accessing the biology of GVHD, it is important to note that GVHD is a complicated systemic process with as yet still many unknowns and is not a simplified, linear, or cyclical process.

Kinetics of CD4+ and CD8+ T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii

Yasuhiro Shibasakia, H Todaa, Isao kobayashib, T Moritomoa, T Nakanishia
Developmental and Comparative Immunology 34 (2010) 1075–1081
http://dx.doi.org:/10.1016/j.dci.2010.05.009

We have previously demonstrated the presence of graft-versus-host reaction (GVHR) in fish employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. To elucidate the role of CD8+ T cells in the induction of GVHR, we investigate the kinetics of CD4+ and CD8+ T-cell subsets in GVHR along with the pathological changes associated with GVH disease (GVHD) in ginbuna. GVHR was not induced with a leukocyte fraction lacking CD8+ T cells separated by magnetic cell sorting. Ploidy and immunofluorescence analysis revealed that CD4+ and CD8+  T cells from sensitized donors greatly

increased in the host trunk kidney, constituting more than 80% of total cells 1–2 weeks after donor cell injection, while those from non-sensitized donors constituted less than 50% of cells present. The increase of CD4+ T cells was greater and more rapid than that of CD8+ T cells. The number of donor CD4+ and CD8+ T cells was highest in trunk kidney followed by spleen. Increases in donor CD4+ and CD8+ T cells were also found in liver and PBL, although the percentages were not as high. Pathologic changes similar to those in human and murine acute GVHD were observed in the lymphoid organs as well as target organs such as skin, liver and intestine, including the destruction of cells and tissues and massive leukocyte infiltration. The pathologic changes became more severe with the increase of CD8+ T cells. These results suggest that donor-derived CD8+ T cells play essential roles for the induction of acute GVHR/D in teleosts as in mammals.

Fludarabine and Exposure-Targeted Busulfan Compares Favorably with Busulfan/Cyclophosphamide-Based Regimens in Pediatric Hematopoietic
Cell Transplantation: Maintaining Efficacy with Less Toxicity

I.H. Bartelink, E.M.L. van Reij, C.E. Gerhardt, E.M. van Maarseveen, et al
Biol Blood Marrow Transplant 20 (2014) 345e353
http://dx.doi.org/10.1016/j.bbmt.2013.11.027

Busulfan (Bu) is used as a myeloablative agent in conditioning regimens before allogeneic hematopoietic cell transplantation (allo-HCT). In line with strategies explored in adults, patient outcomes may be optimized by replacing cyclophosphamide (Cy) with or without melphalan (Mel) with fludarabine (Flu). We compared outcomes in 2 consecutive cohorts of HCT recipients with a nonmalignant HCT indication, a myeloid malignancy, or a lymphoid malignancy with a contraindication for total body irradiation (TBI). Between 2009 and 2012, 64 children received Flu + Bu at a target dose of 80-95 mg-h/L, and between 2005 and 2008, 50 children received Bu targeted to 74-80 mg-h/L þ Cy. In the latter group, Mel was added for patients with myeloid malignancy (n = 12). Possible confounding effects of calendar time were studied in 69 patients receiving a myeloablative dose of TBI between 2005 and 2012. Estimated 2-year survival and event-free survival were 82% and 78%, respectively, in the FluBu arm and 78% and 72%, respectively, in the BuCy (Mel) arm (P,  not significant). Compared with the BuCy (Mel) arm, less toxicity was noted in the FluBu arm, with lower rates of acute (noninfectious) lung injury (16% versus 36%; P < .007), veno-occlusive disease (3% versus 28%; P < .003), chronic graft-versus-host disease (9% versus 26%; P < .047), adenovirus infection (3% versus 32%; P < .001), and human herpesvirus 6 infection reactivation (21% versus 44%; P < .005). Furthermore, the median duration of neutropenia was shorter in the FluBu arm (11 days versus 22 days; P < .001), and the patients in this arm required fewer transfusions. Our data indicate that Flu (160 mg/m2) with targeted myeloablative Bu (90 mg-h/L) is less toxic than and equally effective
as BuCy (Mel) in patients with similar indications for allo-HCT.

Fibrotic and Sclerotic Manifestations of Chronic Graft-versus-Host Disease

Carrie L. Kitko, Eric S. White, Kristin Baird
Biol Blood Marrow Transplant 18:S46-S52, 2012
http://dx.doi.org:/10.1016/j.bbmt.2011.10.021

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity
and mortality following allogeneic stem cell transplantation (HCT), with approximately 50% to 60% of long-term HCT survivors developing one or more manifestations of the disorder. Although acute GVHD is typically limited to skin, liver, and gastrointestinal involvement, virtually every organ is at risk for the development of cGVHD. Although the pathophysiology of cGVHD remains poorly understood, some of the most severe organ manifestations are linked by end-organ fibrosis. In particular, fibrotic cutaneous and bronchiolar changes, resulting in scleroderma-like changes and bronchiolitis obliterans syndrome (BOS), respectively, are two of the most devastating outcomes for these patients. Both sclerotic GVHD (ScGVHD) and BOS have been reported in 5% to 15% of patients with cGVHD.

Many of the manifestations of cGVHD share clinical characteristics seen in nontransplant conditions, including systemic sclerosis or pulmonary fibrosis. Thus, understanding the pathophysiology underlying these related conditions may help identify potential mechanisms and ultimately new therapeutic options for patients with cGVHD.

Tyrosine kinase inhibitors (TKIs) have been shown to inhibit two different profibrotic pathways (transforming growth factor β [TGF-β] and platelet-derived growth factor [PDGF]) in various mouse models of fibrotic disease and offer a possible novel treatment approach for cGVHD patients suffering from severe sclerosis. Likewise, overexpression of TNF-α has been shown to induce fibrogenesis in experimental hepatocellular disease and has been linked with human scleroderma-associated interstitial pulmonary fibrosis and profibrotic responses in human osteoarthritic hip joint fibroblasts. The use of TNF antagonists has been examined in some clinical situations associated with fibrosis, suggesting they may also be of some benefit to patients with cGVHD; however, this must first be prospectively tested.

Table. Proposed Modifications to NIH BOS Clinical Definition

  • Absence of infection (no change)
  • Another cGVHD manifestation in another organ (no change)
  • FEV1 <75% predicted (no change) or >10% decline from pre-HCT value (modification)
  • Signs of Obstruction
  • FEV1/SVC ratio <0.7 (modification), or
  • RV >120% predicted (no change), or
  • RV/TLC >120% (modification), and
  • HRCT with evidence of air trapping (no change)

SVC indicates slow vital capacity; RV, residual volume; TLC, total lung capacity; HRCT, high-resolution computed tomography

Figure (not shown)
Effect of etanercept on survival in post-HCT patients with subacute lung injury. (A) Overall 5-year survival by pulmonary function testing defect. Patients with an obstructive defect (solid line) had a 5-year survival of 67% compared with 44% in those with a restrictive lung defect (dashed line) (P 5 .19). (B) Overall 5-year survival by response to therapy. Patients who responded to etanercept therapy (solid line) had a 5-year survival of 90% compared with 55% in patients who failed to respond (dashed line) (P 5.07). (Figures reprinted with permission, Biol Blood and Marrow Trans).

Extensive, sclerotic skin changes with superficial or deep subcutaneous or fascial involvement are seen in approximately 4% to 13% of patients with cGVHD and can be a life-threatening manifestation. ScGVHD of the skin includes several cutaneous presentations characterized by inflammation and progressive fibrosis of the dermis and subcutaneous tissues. These changes can resemble morphea, systemic sclerosis, or eosinophilic fasciitis and may or may not occur in the setting of concurrent overlying epidermal GVHD. When severe, ScGVHD can result in contractures, severe wasting, and chest wall restriction.

Development of clinical trials for patients with cGVHD is difficult because of the complexity and heterogeneity of disease, variable approaches to treatment, and the lack of standardized assessments of disease. In particular, the study of ScGVHD lacks universally accepted measures of disease burden and response. Investigators have used several measures to assess ScGVHD involvement including body surface area, magnetic resonance imaging, ultrasound, and range-of-motion measurements. Additionally, investigators have tried to apply the Rodnan score, the standardmeasure for skin involvement in scleroderma. Thus far, none of these measures has proven
to be completely reliable in the setting of ScGVHD, and it is likely that multiple measures will need to be integrated into the assessment of ScGVHD.

Imatinib mesylate (Gleevec in the US; Glivec in Europe, Australia, and Latin America, marketed by Novartis) is a TKI that has biological activity against both PDGF and TGF-β signaling pathways. Both cytokines have been implicated in the pathogenesis of several fibrosing diseases, including hepatic, renal, and lung, as well as in scleroderma, a disease that closely resembles ScGVHD. In addition, stimulatory antibodies specific for the PDGF receptor (PDGFR) were identified in a series of 39 patients with extensive cGVHD with higher levels detected in those patients with skin involvement. Similar stimulatory antibodies targeting PDGFR have been reported in patients with scleroderma, suggesting an important therapeutic target for these fibrosing conditions. Imatinib mesylate has particularly potent activity against PDGF and is FDA approved in the United States for the treatment of several disorders associated with aberrant PDGFR signaling. The side effect profile of the drug is well established in non-HCT patients, which is helpful in the setting of a therapy for allogenic HCT patients, many of whom have multiorgan system symptoms and possible dysfunction and who will require ongoing immunosuppressive therapy.

Through the efforts of the Chronic GVHD Consortium, led by Stephanie Lee at the Fred Hutchinson Cancer Research Center, there is a multicenter, ongoing prospective evaluation of the NIH diagnostic and assessment tools. This effort has already resulted in several publications that have further refined essential criteria for cGVHD evaluation, including organ-specific manifestations such as BOS and ScGVHD. Currently, the Consortium is conducting a multicenter prospective clinical trial of fluticasone propionate, azithromycin, and montelukast for the treatment of BOS (ClinicalTrials.gov NCT01307462); a separate trial of imatinib versus rituximab for treatment of ScGVHD is also enrolling subjects (ClinicalTrials.gov NCT01309997).

Although cGVHD remains a significant problem for many long-term survivors of HCT, critical advances in cGVHD research and treatment can be achieved by cooperative group efforts such as those put forth by the Chronic GVHD Consortium and the Clinical Trials Network.

Hematopoietic stem cell transplantation (HSCT): An approach to autoimmunity

Carmen Alaez, Mariana Loyola, Andrea Murguıa, Hilario Flores, et al.
Autoimmunity Reviews 5 (2006) 167– 179
http://dx.doi.org:/10.1016/j.autrev.2005.06.003

HSCT provides the opportunity to replace a damaged tissue. It is the most important treatment for high risk hematologic malignant and nonmalignant disorders. An important challenge in the identification of matched donors/patients is the HLA diversity. The Mexican Bone Marrow Registry (DONORMO) has nowadays N5000 donors. The prevalent alleles are Amerindian, Mediterranean (Semitic and Spanish genes) and African. In theory, it is possible to find 11% of 6/6 A–B–DR low resolution matches for 70% of patients with Mexican ancestry. We contributed with 39 unrelated, cord blood and autologous HSCT for patients with malignant, genetic and autoimmune disorders. Overall disease survival was 50% (2–7 years) depending on the initial diagnosis, conditioning, disease evolution or other factors. Clinical studies using autologous and unrelated HSC are performed on patients with refractory autoimmune diseases producing mixed results: mainly, T1D, RA, MS, SLE. Improvement has been observed in skin damage and quality of life in SLE and systemic sclerosis. Disease stabilization in 2/3 of MS patients. However, in RA and T1D, initial benefits have been followed by eventual relapse. With growing clinical experience and protocol improvement, treatment-related mortality is decreasing. Proof efficacy will be achieved by comparing HSCT with standard therapy in autoimmunity.

Monoclonal Antibody-Mediated Targeting of CD123, IL-3 Receptor α Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells

Liqing Jin, Erwin M. Lee, Hayley S. Ramshaw, Samantha J. Busfield, et al.
Cell: Stem Cell 5, 31–42, July 2, 2009
http://dx.doi.org:/10.1016/j.stem.2009.04.018

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor α chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing
to bone marrow (BM) and activating innate immunity of nonobese diabetic/ severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival.
Mice with preestablished disease showed reduced AML burden in the BM
and periphery and impaired secondary transplantation upon treatment, establishing that AMLLSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34+ CD38[1] cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Many Days at Home during Neutropenia after Allogeneic Hematopoietic Stem Cell Transplantation Correlates with Low Incidence of Acute Graft-versus-Host Disease

Olle Ringdén, Mats Remberger, Katarina Holmberg, Charlotta Edeskog, et al.
Biol Blood Marrow Transplant 19 (2013) 314e320
http://dx.doi.org/10.1016/j.bbmt.2012.10.011

Patients are isolated in the hospital during the neutropenic phase after allogeneic hematopoietic stem cell transplantation. We challenged this by allowing patients to be treated at home. A nurse from the unit visited and checked the patient. One hundred forty-six patients treated at home were compared with matched hospital control subjects. Oral intake was intensified from September 2006 and improved (P < .002). We compared 4 groups: home care and control subjects before and after September 2006. The cumulative incidence of acute graft-versus-host disease (GVHD) of grades II to IV was 15% in the “old” home care group, which was significantly lower than that of 32% to 44% in the other groups (P <.03). Transplantation-related mortality, chronic GVHD, and relapse were similar in the groups. The “new” home care patients spent fewer days at home (P < .002). In multivariate analysis, GVHD of grades 0 to I was associated with home care (hazard ratio [HR], 2.46; P <.02) and with days spent at home (HR, .92; P < .005) but not with oral nutrition (HR, .98; P = .13). Five year survival was 61% in the home care group as compared with 49% in the control subjects (P < .07). Home care is safe. Home care and many days spent at home were correlated with a low risk of acute GVHD.

Impact on Outcomes of Human Leukocyte Antigen Matching by Allele-Level Typing in Adults with Acute Myeloid Leukemia Undergoing Umbilical Cord Blood Transplantation

Jaime Sanz, Francisco J. Jaramillo, Dolores Planelles, Pau Montesinos, et al.
Biol Blood Marrow Transplant 20 (2014) 106e110
http://dx.doi.org/10.1016/j.bbmt.2013.10.016

This retrospective study analyzed the impact of directional donor-recipient human leukocyte antigen (HLA) disparity using allele-level typing at HLA-A, -B, -C, and -DRB1 in 79 adults with acute myeloid leukemia (AML) who received single-unit umbilical cord blood (UCB) transplant at a single institution. With extended high resolution HLA typing, the donor-recipient compatibility ranged from 2/8 to 8/8. HLA disparity showed no negative impact on nonrelapse mortality (NRM), graft-versus-host (GVH) disease or engraftment. Considering disparities in the GVH direction, the 5-year cumulative incidence of relapse was 44% and 22% for patients receiving an UCB unit matched > 6/8 and < 6/8, respectively (P <.04). In multivariable analysis, a higher HLA disparity in the GVH direction using extended high-resolution typing (Risk ratio [RR] 2.8; 95% confidence interval [CI], 1.5 to 5.1; P ¼.0009) and first complete remission at time of transplantation (RR 2.1; 95% CI, 1.2 to 3.8; P < .01) were the only variables significantly associated with an improved disease-free survival. In conclusion, we found that in adults with AML undergoing single-unit UCBT, an increased number of HLA disparities at allele-level typing improved disease-free survival by decreasing the relapse rate without a negative effect on NRM.

HLA mismatch direction in cord blood transplantation: impact on outcome and implications for cord blood unit selection
Cladd E. Stevens, C Carrier, C Carpenter, D Sung, and A Scaradavou

Blood. 2011; 118(14):3969-3978
http://dx.doi.org:/10.1182/blood-2010-11-317271

Donor-recipient human leukocyte antigen mismatch level affects the outcome of unrelated cord blood (CB) transplantation. To identify possible “permissive” mismatches, we examined the relationship between  direction of human leukocyte antigen mismatch (“vector”) and transplantation outcomes in 1202 recipients of single CB units from the New York Blood Center National Cord Blood Program treated in United States Centers from 1993-2006. Altogether, 98 donor/patient pairs had only unidirectional mismatches: 58 in the graft-versus-host (GVH) direction only (GVH-O) and 40 in the host-versus-graft or rejection direction only (R-O). Engraftment was faster in patients with GVH-O mismatches compared with those with 1 bidirectional mismatch (hazard ratio [HR] = 1.6, P < .003). In addition, patients with hematologic malignancies given GVH-O grafts had lower transplantation-related mortality (HR = 0.5, P < .062), overall mortality (HR = 0.5, P < .019), and treatment failure (HR = 0.5, P < .016), resulting in outcomes similar to those of matched CB grafts. In contrast, R-O mismatches had slower engraftment, higher graft failure, and higher relapse rates (HR = 2.4, P < .010). Based on our findings, CB search algorithms should be modified to identify unidirectional mismatches. We recommend that transplant centers give priority to GVH-O-mismatched units over other mismatches and avoid selecting R-O mismatches, if possible.

Mutation of the NPM1 gene contributes to the development of donor cell–derived acute myeloid leukemia after unrelated cord blood transplantation
for acute lymphoblastic leukemia

G Rodríguez-Macías, C Martínez-Laperche, J Gayoso, V Noriega, .., Ismael Buño
Human Pathology (2013) 44, 1696–1699
http://dx.doi.org/10.1016/j.humpath.2013.01.001

Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

Graft-versus-leukemia in the bone marrow
Blood, 23 JAN 2014; 123(4)
http://imagebank.hematology.org.

63-year-old female with relapsed acute myeloid leukemia (AML) after allogeneic stem cell transplantation reached CR2 after re-induction therapy followed by consolidation with donor lymphocyte infusions: 3 x 107/kg and 3 x 108/kg after 1 and 2.5 months, respectively. No signs of graft-versus-host disease were observed at this time. At 5 months follow-up, her blood count deteriorated: hemoglobin: 6.9 mmol/L, thrombocytes: 58 x 109/L and leukocytes: 1.37 x 109/L. Bone marrow aspirate was not evaluable. Bone marrow trephine biopsy showed relapse AML with hypercellularity in the H&E staining (340 objective lens, panel A) and 20% CD341 blast cells without any signs of maturation (panel B). Also, a high number of CD3 positive T cells (panel C) was noted, intermingling with the CD34 positive blasts, both staining positively with CD43 (panel D). Only supportive care was given. However, normalization of the blood count was observed in the following months and she developed graft-versus-host disease of the lung, which was treated with ciclosporin and prednisone. A bone marrow aspirate performed 3 months after relapse showed a third remission with 0.8% myeloid blasts. In retrospect, one could therefore consider the picture of the bone marrow trephine biopsy at the second relapse as graft-versus-leukemia in the bone marrow.

GVL- panel A

GVL- panel A

GVL - panel B

GVL – panel B

GVL - panel C

GVL – panel C

GVL - panel D

Long-Term Outcomes of Alemtuzumab-Based Reduced-Intensity Conditioned Hematopoietic Stem Cell Transplantation for Myelodysplastic Syndrome and Acute Myelogenous Leukemia Secondary to Myelodysplastic Syndrome

Victoria T. Potter, Pramila Krishnamurthy, Linda D. Barber, ZiYi Lim, et al.
Biol Blood Marrow Transplant 20 (2014) 111e117
http://dx.doi.org/10.1016/j.bbmt.2013.10.021

Allogeneic hematopoietic stem cell transplantation (HSCT) with reduced-intensity conditioning (RIC) offers a potential cure for patients with myelodysplastic syndrome (MDS) who are ineligible for standard-intensity regimens. Previously published data from our institution suggest excellent outcomes at 1 yr using a uniform fludarabine, busulfan, and alemtuzumab-based regimen. Here we report long-term follow-up of 192 patients with MDS and acute myelogenous leukemia (AML) secondary to MDS (MDS-AML) transplanted with this protocol, using sibling (n = 45) or matched unrelated (n = 147) donors. The median age of the cohort was 57 yr (range, 21 to 72 yr), and median follow-up was 4.5 yr (range, 0.1 to 10.6 yr). The 5-yr overall survival (OS), event-free survival, and nonrelapse mortality were 44%, 33%, and 26% respectively. The incidence of de novo chronic graft-versus-host disease (GVHD) was low at 19%, illustrating the efficacy of alemtuzumab for GVHD prophylaxis. Conversely, the 5-yr relapse rate was 51%. For younger patients (age <50 yr), the 5-yr OS and relapse rates were 58% and 39%, respectively. On multivariate analysis, advanced age predicted significantly worse outcomes, with patients age >60 yr having a 5-yr OS of 15% and relapse rate of 66%. Patients receiving preemptive donor lymphocyte infusions had an impressive 5-yr OS of 67%, suggesting that this protocol may lend itself to the incorporation of immunotherapeutic strategies. Overall, these data demonstrate good 5-yr OS for patients with MDS and MDS-AML undergoing alemtuzumab-based RIC-HSCT. The low rate of chronic GVHD is encouraging, and comparative studies with other RIC protocols are warranted.

Natural killer cell activity influences outcome after T cell depleted stem cell transplantation from matched unrelated and haploidentical donors

Peter Lang, Matthias Pfeiffer,  Heiko-Manuel Teltschik, Patrick Schlegel, et al.
Best Practice & Research Clinical Haematology 24 (2011) 403–411
http://dx.doi.org:/10.1016/j.beha.2011.04.009

Lytic activity and recovery of natural killer (NK) cells was monitored in pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n = 18) and mismatched related (haploidentical, n = 29) donors. CD34+ selection with magnetic microbeads resulted in 8 x 103/kg residual T cells. No post-transplant immune suppression was given. NK cells recovered rapidly after transplantation (300 CD56+/mL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/mL at day 90). NK activity was measured as specific lysis of K 562 targets several times (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 2 years, p < 0.05). The subgroup of patients with ALL showed similar results (0.75 vs 0.14 at 2 years, p < 0.05). Speed of T cell recovery had no influence. These data suggest that both achieving and maintaining a high level of NK activity may contribute to prevent relapse. Since NK activity could be markedly increased by in vitro stimulation with Interleukin 2 (IL-2), in vivo administration should be considered.

Graft-versus-host disease: Pathogenesis and clinical manifestations of graft-versus-host disease

Sharon R. Hymes, Amin M. Alousi,  and Edward W. Cowen
J Am Acad Dermatol  2012; 66: 515.e1-18.

  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality in patients who undergo allogeneic hematopoietic cell transplantation.
  • Acute graft-versus-host disease manifests as a skin exanthem, liver dysfunction, and gastrointestinal involvement.
  • Chronic graft-versus-host disease of the skin is remarkably variable in its clinical presentation.
  • Chronic graft-versus-host disease is a multisystem disorder that may affect nearly any organ; the most common sites are the skin, oral mucosa, and eyes.

Key points

  • Allogeneic transplantation is in widespread use for hematologic malignancies, but is also increasingly used for marrow failure syndromes, immunodeficiencies, and other life-threatening conditions
  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality after allogeneic hematopoietic cell transplantation
  • Minimizing graft-versus-host disease without losing the graft-versus-tumor effect is an area of active research
  • The skin is the most common organ affected in patients with graft-versus-host disease

Outcomes of Thalassemia Patients Undergoing Hematopoietic Stem Cell Transplantation by Using a Standard Myeloablative versus a Novel Reduced-Toxicity Conditioning Regimen According to a New Risk Stratification

Usanarat Anurathapan, S Pakakasama, P Mekjaruskul, N Sirachainan, et al.
Biol Blood Marrow Transplant 20 (2014) 2056e2075
http://dx.doi.org/10.1016/j.bbmt.2014.07.016

Improving outcomes among class 3 thalassemia patients receiving allogeneic hematopoietic stem cell transplantations (HSCT) remains a challenge. Before HSCT, patients who were > 7 years old and had a liver size > 5 cm constitute what the Center for International Blood and Marrow Transplant Research defined as a very high risk subset of a conventional high-risk class 3 group (here referred to as class 3 HR). We performed HSCT in 98 patients with related and unrelated donor stem cells. Seventy-six of the patients with age < 10 years received the more conventional myeloablative conditioning (MAC) regimen (cyclophos-phamide, busulfan,  + fludarabine); the remaining 22 patients with age > 10 years and hepatomegaly (class 3 HR), and in several instances additional comorbidity problems, underwent HSCT with a novel reduced-toxicity conditioning (RTC) regimen (fludarabine and busulfan). We then compared the outcomes between these 2 groups (MAC versus RTC). Event-free survival (86% versus 90%) and overall survival (95% versus 90%) were not significantly different between the respective groups; however, there was a higher incidence of serious treatment-related complications in the MAC group, and although we experienced 6 graft failures in the MAC group (8%), there were none in the RTC group. Based on these results, we suggest that (1) class 3HRthalassemia patients can safely receive HSCT with our novel RTC regimen and achieve the same excellent outcome as low/standard-risk thalassemia patients who received the standard MAC regimen, and further, (2) that this novel RTC approach should be tested in the low/standard-risk patient population.

Pharmacological Immunosuppression Reduces But Does Not Eliminate the Need for Total-Body Irradiation in Nonmyeloablative Conditioning Regimens for Hematopoietic Cell Transplantation

Marco Mielcarek, Beverly Torok-Storb, Rainer Storb
Biol Blood Marrow Transplant 17: 1255-1260 (2011)
http://dx.doi.org:/10.1016/j.bbmt.2011.01.003

In the dog leukocyte antigen (DLA)-identical hematopoietic cell transplantation (HCT) model, stable marrow engraftment can be achieved with total-body irradiation (TBI) of 200 cGy when used in combination with postgrafting immunosuppression. The TBI dose can be reduced to 100 cGy without compromising engraftment rates if granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) are infused with the marrow. T cell-depleting the G-PBMC product abrogates this effect. These results were interpreted to suggest that the additional T cells provided with G-PBMC facilitated engraftment by overcoming host resistance.We therefore hypothesized that the TBI dose may be further reduced to 50 cGy by augmenting immunosupression either by (1) tolerizing or killing recipient T cells, or (2) enhancing the graft-versus-host (GVH) activity of donor T cells. To test the first hypothesis, recipient T cells were activated before HCT by repetitive donor-specific PBMC infusions followed by administration of methotrexate (MTX) (n 5 5), CTLA4-Ig (n = 4), denileukin diftitox (Ontak; n = 4), CTLA4-Ig 1 MTX (n = 8), or 5c8 antibody (anti-CD154) 1 MTX (n = 3). To test the second hypothesis, recipient dendritic cells were expanded in vivo by infusion of Flt3 ligand given either pre-HCT (n = 4) or pre- and post-HCT (n = 5) to augment GVH reactions. Although all dogs showed initial allogeneic engraftment, sustained engraftment was seen in only 6 of 42 dogs (14% of all dogs treated in 9 experimental groups). Hence, unless more innovative pharmacotherapy can be developed that more forcefully shifts the immunologic balance in favor of the donor, noncytotoxic immunosuppressive drug therapy as the sole component of HCT preparative regimens may not suffice to ensure sustained engraftment.

Pretransplant Immunosuppression followed by Reduced-Toxicity Conditioning and Stem Cell Transplantation in High-Risk Thalassemia: A Safe Approach to Disease Control

Usanarat Anurathapan, S Pakakasama, P Rujkijyanont, N Sirachainan, et al.
Biol Blood Marrow Transplant 19 (2013) 1254e1270
http://dx.doi.org/10.1016/j.bbmt.2013.04.023

Patients with class 3 thalassemia with high-risk features for adverse events after high-dose chemotherapy with hematopoietic stem cell transplantation (HSCT) are difficult to treat, tending to either suffer serious toxicity or fail to establish stable graft function. We performed HSCT in 18 such patients age 7 years and hepatomegaly using a novel approach with pretransplant immunosuppression followed by a myeloablative reduced-toxicity conditioning regimen (fludarabine and i.v. busulfan [Flu-IV Bu]) and then HSCT. The median patient age was 14 years (range, 10 to 18 years). Before the Flu-IV Bu þ antithymocyte globulin conditioning regimen, all patients received 1 to 2 cycles of pretransplant immunosuppression with fludarabine and dexamethasone. Thirteen patients received a related donor graft, and 5 received an unrelated donor graft. An initial prompt engraftment of donor cells with full donor chimerism was observed in all 18 patients, but 2 patients developed secondary mixed chimerism that necessitated withdrawal of immunosuppression to achieve full donor chimerism. Two patients (11%) had acute grade III-IV graft-versus-host disease, and 5 patients had limited chronic graft-versus-host disease. The only treatment-related mortality was from infection, and with a median follow-up of 42 months (range, 4 to 75), the 5-year overall survival and thalassemia-free survival were 89%. We conclude that this novel sequential immunoablative pretransplant-ation conditioning program is safe and effective for patients with high-risk class 3 thalassemia exhibiting additional comorbidities.

Profiling antibodies to class II HLA in transplant patient sera

Curtis McMurtrey, D Lowe, R Buchli, S Daga, D Royer, A Humphrey, et al.
Human Immunology 75 (2014) 261–270
http://dx.doi.org/10.1016/j.humimm.2013.11.015

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3 lg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to patient antibody concentra-tions did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

Reduced-intensity conditioning and HLA-matched hemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicenter study

Tayfun Güngör, P Teira, M Slatter, G Stussi, P Stepensky, D Moshous, et al.
Lancet 2014; 383: 436–48
http://dx.doi.org/10.1016/S0140-6736(13)62069-3

Background In chronic granulomatous disease allogeneic hemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients.       Methods This prospective study was done at 16 centers in ten countries worldwide. Patients aged 0–40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centers. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m² [infants <9 kg 1∙2 mg/kg]; one dose per day on days –8 to –3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days –4 to –1; or thymoglobulin 2·5 mg/kg, one dose per day on days –5 to –3]; or low-dose alemtuzumab [<1 mg/kg on days –8 to –6]), and low-dose (50–72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45–65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days –5 to –3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centers (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. Findings 56 patients (median age 12∙7 years; IQR 6·8–17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14–39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16–22) for neutrophils and 21 days (IQR 16–25) for platelets. At median follow-up of 21 months (IQR 13–35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86∙46–99∙09) and of EFS was 91% (79∙78–96∙17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III–IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. Interpretation This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease.

Refinement of the Definition of Permissible HLA-DPB1 Mismatches with Predicted Indirectly ReCognizable HLA-DPB1 Epitopes

Kirsten A. Thus, MTA Ruizendaal, TA de Hoop, Eric Borst, et al.
Biol Blood Marrow Transplant 20 (2014) 1705e1710
http://dx.doi.org/10.1016/j.bbmt.2014.06.026

Hematopoietic stem cell transplantation with HLA-DPB1emismatched donors leads to an increased risk of acute graft-versus-host disease (GVHD). Studies have indicated a prognostic value for classifying HLA-DPB1 mismatches based on T cell epitope (TCE) groups. The aim of this study was to determine the contribution of indirect recognition of HLA-DPe derived epitopes, as determined with the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) method. We therefore conducted a retrospective single-center analysis on 80 patients transplanted with a 10/10 matched unrelated donor that was HLA-DPB1 mismatched. HLADPB1 mismatches that were classified as GVH nonpermissive by the TCE algorithm correlated to higher numbers of HLA class I as well as HLA class II presented PIRCHE (PIRCHE-I and -II) compared with permissive or host-versus-graft nonpermissive mismatches. Patients with acute GVHD grades II to IV presented significantly higher numbers of PIRCHE-I compared with patients without acute GVHD (P < .05). Patients were divided into 2 groups based on the presence or absence of PIRCHE. Patients with PIRCHE-I or -II have an increased hazard of acute GVHD when compared with patients without PIRCHE-I or -II (hazard ratio [HR], 3.19; 95% confidence interval [CI], 1.10 to 9.19; P <.05; and HR, 4.07; 95% CI, .97 to 17.19; P < .06, respectively). Patients classified as having an HLA-DPB1 permissive mismatch by the TCE model had an increased risk of acute GVHD when comparing presence of PIRCHE-I with absence of PIRCHE-I (HR, 2.96; 95% CI, .84 to 10.39; P < .09). We therefore conclude that the data presented in this study describe an attractive and feasible possibility to better select permissible HLA-DPB1 mismatches by including both a direct and an indirect recognition model.

Treosulfan-Thiotepa-FludarabineeBased Conditioning Regimen for
Allogeneic Transplantation in Patients with Thalassemia Major: A
Single-Center Experience from North India

Dharma Choudhary, SK Sharma, N Gupta,…, Satyendra Katewa
Biol Blood Marrow Transplant 19 (2013) 492e503
http://dx.doi.org/10.1016/j.bbmt.2012.11.007

Hematopoietic stem cell transplantation (HSCT) is the definite treatment
for patients with thalassemia major. A busulfan (Bu) and cyclophosphamide
(Cy)ebased regimen has been the standard myeloablative chemotherapy,
but it is associated with higher treatment-related toxicity, particularly in
patients classified as high risk by the Pesaro criteria. Treosulfan-based
conditioning regimens have been found to be equally effective and less
toxic. Consequently, we analyzed the safety and efficacy of treosulfan/
thiotepa/fludarabine (treo/thio/flu)-based conditioning regimens for
allogeneic HSCT in patients with thalassemia major between February
2010 and September 2012. We compared those results retrospectively
with results in patients who underwent previous HSCT with a Bu/Cy/
antithymocyte globulin (ATG)ebased conditioning regimen. A treo/thio/
flu-based conditioning regimen was used in 28 consecutive patients with
thalassemia major. The median patient age was 9.7 years (range, 2-18
years), and the mean CD34+ stem cell dose was 6.18 x 106/kg. Neutrophil
and platelet engraftment occurred at a median of 15 days (range, 12-23
days) and 21 days (range, 14-34 days), respectively. Three patients
developed veno-occlusive disease, 4 patients developed acute graft
versus-host disease (GVHD), and 2 patients had chronic GVHD. Treatment-
related mortality (TRM) was 21.4%. Two patients experienced secondary
graft rejection. We compared these results with results in patients who
underwent previous HSCT using a Bu/Cy/ATG-based conditioning regimen.
Twelve patients were treated with this protocol, at a median age of 7.2
years (range, 2-11 years). One patient had moderate veno-occlusive disease,
2 patients developed acute GVHD, 2 patients had chronic GVHD, and 2
patients experienced graft rejection. There was no TRM in this group. We
found no significant differences between the 2 groups (treo/thio/flu vs Bu/
Cy/ATG) in terms of the incidence of acute GVHD, chronic GVHD, TRM,
and graft failure, although a trend toward higher TRM was seen with the
treo/thio/flu regimen.

Graft-versus-Host Disease
James L.M. Ferrara, John E. Levine, Pavan Reddy, and Ernst Holler
Lancet. 2009 May 2; 373(9674): 1550–1561
http:dx.doi.org:/10.1016/S0140-6736(09)60237-3

The number of allogeneic hematopoietic cell transplantations (HCT)
continues to increase with more than 25,000 allogeneic transplantations
performed annually. The graft-versus leukemia/ tumor (GVL) effect during
allogeneic HCT effectively eradicates many hematological malignancies.
The development of novel strategies that use donor leukocyte infusions,
non-myeloablative conditioning and umbilical cord blood (UCB)
transplantation have helped expand the indications for allogeneic HCT
over the last several years, especially among older patients. Improvements
in infectious prophylaxis, immunosuppressive medications, supportive care
and DNA-based tissue typing have also contributed to improved outcomes
after allogeneic HCT. Yet the major complication of allogeneic HCT, graft-
versus-host disease (GVHD), remains lethal and limits the use of this
important therapy. Given current trends, the number of transplants from
unrelated donors is expected to double within the next five years,
significantly increasing the population of patients with GVHD. In this
seminar we review advances made in identifying the genetic risk
factors and pathophysiology of this major HCT complication, as well
as its prevention, diagnosis and treatment.

Non-HLA Genetics—Despite HLA identity between a patient and donor,
approximately 40% of patients receiving HLA-identical grafts develop
acute GVHD due to genetic differences that lie outside the HLA loci,
or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY
and HA-3, are expressed on all tissues and are targets for both GVHD
and GVL. Other minor HAs, such as HA-1 and HA-2, are expressed most
abundantly on hematopoietic cells (including leukemic cells) and may
therefore induce a greater GVL effect with less GVHD. Polymorphisms
in both donors and recipients for cytokines that are involved in the
classical `cytokine storm’ of GVHD have been implicated as risk factors
for GVHD. Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10),
Interferon-γ (IFNγ) variants have correlated with GVHD in some, but
not all, studies. Genetic polymorphisms of proteins involved in innate
immunity, such as nucleotide oligomerization domain 2 and Keratin 18
receptors, have also been associated with GVHD.

Future strategies to identify the best possible transplant donor will
probably incorporate both HLA and non-HLA genetic factors. Skin is most
commonly affected and is usually the first organ involved, often coinciding
with engraftment of donor cells. The characteristic maculopapular rash is
pruritic and can spread throughout the body, sparing the scalp. In severe
cases the skin may blister and ulcerate. Apoptosis at the base of epidermal
rete pegs is a characteristic pathologic finding. Other features include
dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent
to dyskeratotic epidermal keratinocytes, and a perivascular lymphocytic
infiltration in the dermis.

Gastrointestinal tract involvement of acute GVHD usually presents as
diarrhea but may also include vomiting, anorexia, and/or abdominal pain
when severe. The diarrhea of GVHD is secretory and often voluminous
(greater than two liters per day). Bleeding, which carries a poor prognosis,
occurs as a result of mucosal ulceration but patchy involvement of the
mucosa often leads to a normal appearance on endoscopy.

The incidence of the severity of acute GVHD is determined by the extent
of involvement of  three principal target organs. The overall grades are
classified as I (mild), II (moderate), III (severe) and IV (very severe). Severe
GVHD carries a poor prognosis, with 25% long term survival for grade III and
5% for grade IV. The incidence of acute GVHD is directly related to the
degree of mismatch between HLA proteins and ranges from 35-45% in
recipients of full matched sibling donor grafts to 60-80% in recipients of
one-antigen HLA mismatched unrelated donor grafts. The same degree
of mismatch causes less GVHD using UCB grafts and incidence of acute
GVHD is lower following the transplant of partially matched UCB units
and ranges from 35-65%.

Two important principles are important to consider regarding the
pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated
but normal inflammatory mechanisms mediated by donor lymphocytes infused
into the recipient where they function appropriately, given the foreign
environment they encounter. Second, the recipient tissues that stimulate
donor lymphocytes have usually been damaged by underlying disease,
prior infections, and the transplant conditioning regimen. As
a result, these tissues produce molecules (sometimes referred to as
“danger” signals) that promote the activation and proliferation of donor
immune cells.  Based largely on experimental models, the development
of acute GVHD can be conceptualized in three sequential steps or phases:
(1) activation of the APCs; (2) donor T cell activation, proliferation,
differentiation and migration; and (3) target tissue destruction.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein
expressed on a broad spectrum of leukocytes including lymphocytes,
monocytes, and dendritic cells. Its use in GVHD prophylaxis in a
Phase II trial decreased the incidence of acute and chronic GVHD
following reduced intensity transplant.98 In two prospective studies,
patients who received alemtuzumab rather than methotrexate showed
significantly lower rates of acute and chronic GVHD, but experienced
more infectious complications and higher rates of relapse, so that there
was no overall survival benefit. Alemtuzumab may also contribute to
graft failure when used with minimal intensity conditioning regimens.

An alternative strategy to TCD attempted to induce anergy in donor
T cells by ex vivo antibody blockade of co-stimulatory pathways prior
to transplantation. A small study using this approach in haploidentical
HCT recipients was quite encouraging, but has not yet been replicated.
Thus the focus of most prevention strategies remains  pharmacological
manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis
has also been extensively tested. The best studied drugs are anti-
thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations.
These sera, which have high titers of polyclonal antibodies, are made
by immunizing animals (horses or rabbits) to thymocytes or lymphocytes,
respectively. A complicating factor in determining the role of these
polyclonal sera in transplantation is the observation that even different
brands of the same class of sera exert different biologic effects. However,
the side effects of ATG/ALG infusions are common across different
preparations and include fever, chills, headache, thrombocytopenia
(from cross-reactivity to platelets), and, infrequently, anaphylaxis. In
retrospective studies, rabbit ATG reduced the incidence of GVHD in
related donor HSCT recipients without appearing to improve survival.
In recipients of unrelated donor HSCT, addition of ALG to standard
GVHD prophylaxis effectively prevented severe GVHD, but did not
result in improved survival because of increased infections. In a long
term follow-up study, however, pretransplant ATG provided significant
protection against extensive chronic GVHD and chronic lung dysfunction.

As allogeneic transplantation becomes an increasingly attractive therapeutic
option, the need for novel approaches to GVHD has accelerated. The
number of patients receiving transplants from unrelated donors is
expected to double in the next five years, significantly increasing
the population of patients with GVHD. The advent of RIC regimens
has reduced transplant-related mortality and lengthened the period
during which acute GVHD may develop (many new cases present up
to day 200) and the need for close monitoring of patients in this period
has increased. Patients have often returned to the care of their primary
hematologists by this time, increasing the need for these physicians to
collaborate with transplant specialists in the management of GVHD and
its complications.

Identification of biomarkers for GVHD with diagnostic (and possibly
prognostic) significance may eventually make the treatment of GVHD
preemptive rather than prophylactic. The use of cellular component therapy,
such as regulatory T cells that have been expanded ex vivo. will also
enter clinical trials in the near future, but the extensive infrastructure
required for such cellular approaches will likely limit their use initially.

Immunomodulatory Effects of Palifermin (Recombinant Human
Keratinocyte Growth Factor) in 
an SLE-Like Model of Chronic
Graft-Versus-Host Disease

C. A. Ellison, Y. V. Lissitsyn, I. Gheorghiu & J. G. Gartner
Scandinavian Journal of Immunology 2011; 75, 69–76
http://dx.doi.org:/10.1111/j.1365-3083.2011.02628.x

Keratinocyte growth factor (KGF) promotes epithelial cell proliferation
and survival. Recombinant human KGF, also known as palifermin, protects
epithelial cells from injury induced by chemicals, irradiation and acute murine
graft versus-host disease (GVHD). Findings from our studies and others
have shown that palifermin also has immunomodulatory properties. In a
model of acute GVHD, we showed that it shifts the immune response
from one in which Th1 cytokines dominate to mixed Th1 and Th2 cytokine
profile. Using the DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-hybrid model of chronic,
systemic lupus erythematosus-like GVHD, we showed that palifermin
treatment is associated with higher levels of Th2 cytokines, the production
of anti-nuclear antibodies, cryoglobulinemia and the development of more
severe pathological changes in the kidney. The aim of our current study
was to gain a better understanding of the immunobiology of KGF by
further characterizing the palifermin-mediated effects in this model of
chronic GVHD. Because the pathological changes we observed resemble
those seen in thymic stromal lymphopoietin (TSLP) transgenic mice, we
had originally hypothesized that palifermin might augment TSLP levels.
Surprisingly, we did not observe an increase in thymic

TSLP mRNA expression in palifermin-treated recipients. We did, however,
observe some differences in the percentages of CD4+CD25+Foxp3+
regulatory T cells in the spleen at some time points in palifermin-treated
recipients. Most importantly, we found that TGFβ levels were higher in
palifermin-treated recipients early in the GVH reaction, raising the
possibility that KGF might indirectly induce the development of fibrosis
and glomerulonephritis through a pathway involving TGFβ.

Keratinocyte growth factor (KGF) is an epithelial cell growth factor that is
produced by both mesenchymal cells and intraepithelial cdT cells. It is
also known as fibroblast growth factor 7. Its receptor, (KGFR⁄FGF7R), an
alternatively spliced form of FGFR2 ⁄ bek, is found on epithelial cells in
the intestine, mammary glands, ovaries and urinary tract, and on
hepatocytes, keratinocytes and alveolar type II cells. Previously, it
was shown that recombinant human KGF, also known as palifermin,
can protect the lung, bladder or intestine from chemical- or irradiation-
induced injury. This has been attributed to the ability of KGF to reduce
oxidative damage and enhance DNA repair.

Our own studies have provided a better understanding of the immuno-
biological properties of KGF in pathologically distinct models of systemic
disease driven by intense immunological and inflammatory responses.
The acute GVHD that develops in the C57BL ⁄ 6 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model is characterized by the activation of alloreactive donor T cells,
the production of Th1 cytokines and tissue injury in the skin, gastrointestinal
tract, liver, thymus and lung, where epithelia are present. Injury to the
intestinal mucosa permits the translocation of endotoxin into the system,
which, if untreated, leads to the development of endotoxemic shock. We
showed that palifermin treatment protects recipients from epithelial
cell injury, endotoxemia and morbidity in GVH mice. Palifermin also
shifts the immune response away from one that is predominated by Th1
cytokines towards a profile of mixed Th1 and Th2 cytokines, with a
preponderance of Th2 cytokines. The DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model of chronic GVHD is characterized by pathological changes
resembling those seen in systemic lupus erythematosus (SLE). Using this
model, we showed that palifermin treatment augments the production of Th2
cytokines such as IL-4, IL-5 and IL-13 and obviates IFN-c production. Both
untreated and palifermin-treated recipients developed pathological changes
in the kidney, but these changes were more severe in palifermin-treated
recipients. Some of the changes that developed in the palifermin-treated
recipients resemble those seen in thymic stromal lymphopoietin (TSLP)
transgenic mice. These similarities include the presence of ANA in the
sera, the development of cryoglobulinemia and the development of
glomerulonephritis featuring the deposition of immune complexes
consisting of IgG, IgA, IgM and C3 in the mesangium and the glomerular
capillaries. This led us to hypothesize that treating the recipient mice with
palifermin might induce TSLP expression in this model.

In this study, we were interested in determining whether palifermin
treatment was indeed associated with increased TSLP expression.
We were also interested in knowing whether palifermin treatment
changes the percentage of CD4+CD25+FoxP3+ cells in the spleen,
because palifermin treatment has been associated with increased
percentages of CD4+CD25+FoxP3+ cells in other studies including
our own. Lastly, we wished to study the effect of palifermin treatment
on TGFb levels, because this cytokine is known to play a pivotal role
in the development of glomerulonephritis.

We studied the histopathological changes to confirm that the pathological
changes seen in the kidney in this study were the same as those reported
by us previously.We examined kidney sections from both untreated and
palifermin-treated recipients. In these experiments, we were able to
reproduce findings from an earlier study that showed that palifermin-
treated recipients mice in this model of chronic GVHD develop a severe,
extracapillary proliferative glomerular nephritis characterized by epithelial
crescents and hyaline thrombi. These changes were associated with higher
levels of protein in the urine and the development of ascites, presumably
related to the development of nephrotic syndrome, as a consequence
of glomerular injury.

Pathological changes in the kidney

Pathological changes in the kidney. (A) shows a section from a BDF1-hybrid control
mouse that did not receive a graft. (B) shows increased epithelial cellularity within a
glomerulus from an untreated recipient with chronic graft-versus-host disease, on
day 50. No crescents were observed in sections from this group of recipients.
(C and D) show examples of pathological changes observed in kidneys from
palifermin-treated recipients on day 50. Arrows indicate examples of crescentic
glomerulonephritis and the development of protein casts within tubular lumena.
(E and F) show examples of the hyaline thrombi (arrows) seen in the glomeruli
in kidney sections from palifermin-treated recipients on day 50. All sections
were stained with haematoxylin and eosin except for that shown in (F), which
was stained with Masson Trichrome. The concentration of protein measured in
the urine is shown in the lower left corner of each photomicrograph. Original
magnification: ·200 (B–E) and ·400 (A and F).

TGFβ is a highly pleiotropic cytokine with three isoforms, TGFβ1, TGFβ2 and
TGFβ3 . Nearly, all cells have receptors for at least one of these isoforms,
but cells of the immune system primarily express TGFβ1. This cytokine
was implicated in the development of experimental glomerulonephritis in
experiments in which rats were treated with antiserum directed against
TGFβ1. The ability of palifermin to induce TGFβ release and reverse
limited airflow was demonstrated in a mouse model of emphysema. The
authors further showed that palifermin induced the release of TGFβ1
from primary cultures of mouse alveolar type 2 cells. Our results show
that palifermin treatment is associated with a rise in splenic TGFβ levels
during the first month of the GVH reaction. It is possible that by inducing
TGFβ production shortly after transplantation, palifermin treatment is able
to promote the development of the severe, crescentic glomerulonephritis
that we observed at later time points. As such, our findings raise the
possibility that endogenous KGF might play a role in the development
of glomerulonephritis and ⁄ or other autoimmune phenomena associated
with chronic GVHD and ⁄ or SLE.

T cells, murine chronic graft-versus-host disease and autoimmunity

Robert A. Eisenberg, Charles S. Via
Journal of Autoimmunity 39 (2012) 240e247
http://dx.doi.org:/10.1016/j.jaut.2012.05.017

The chronic graft-versus-host disease (cGVHD) in mice is characterized by
the production of autoantibodies and immunopathology characteristic of
systemic lupus erythematosus (lupus). The basic pathogenesis involves
the cognate recognition of foreign MHC class II of host B cells by alloreactive
CD4 T cells from the donor. CD4 T cells of the host are also necessary for
the full maturation of host B cells before the transfer of donor T cells.
CD8 T cells play critical roles as well. Donor CD8 T cells that are highly
cytotoxic can ablate or prevent the lupus syndrome, in part by killing
recipient B cells. Host CD8 T cells can reciprocally downregulate donor
CD8 T cells, and thus prevent them from suppressing the autoimmune
process. Thus, when the donor inoculum contains both CD4 T cells and
CD8 T cells, the resultant syndrome depends on the balance of activities
of these various cell populations. For example, in one cGVHD model
(DBA/2 (C57BL/6xDBA/2)F1, the disease is more severe in females, as
it is in several of the spontaneous mouse models of lupus, as well as in
human disease. The mechanism of this female skewing of disease
appears to depend on the relative inability of CD8 cells of the female host
to downregulate the donor CD4 T cells that drive the autoantibody response.
In general, then, the abnormal CD4 T cell help and the modulating roles
of CD8 T cells seen in cGVHD parallel the participation of T cells in
genetic lupus in mice and human lupus, although these spontaneous
syndromes are presumably not driven by overt alloreactivity.

Systemic lupus erythematosus (SLE) is characterized by a spectrum of auto-
antibodies that targets multiple normal cellular components, particularly
nucleic acids or proteins that are physiologically bound to nucleic acids.
Although SLE is highly diverse in its manifestations, a common theme
is the loss of B cell tolerance to these cellular autoantigens. More than
for any other human condition, several spontaneously arising mouse
models for SLE have been described, beginning with the New Zealand
strains in 1959. These models are largely genetic. In some cases, an
individual gene such as fas or Yaa plays a major role in driving the loss
of tolerance. However, in general the genetic contribution is complex and
involves multiple loci, which are not yet fully defined.

Despite extensive investigations, the failures in immunoregulation that
underlie the genetic SLE models remain poorly understood. It is not known
for sure which B cell tolerance checkpoints are breached in a given model,
and why. The autoantibody response to DNA, Sm, and other autoantigens
resembles the normal response to exogenous antigens: it involves clonal
expansion, somatic mutation, and a pattern of isotype use characteristic of
a T-cell dependent immunization. Thus the cellular dynamics of the response
may be basically normal. Yet the B-cell repertoire is abnormally autoreactive.

In this review we wish to focus more on the role of the T cell in SLE. As
stated above, the loss of B cell tolerance in SLE does appear in general
to require the participation of T cells. Multiple T cells abnormalities have
been described in human and in murine SLE, although in most cases it is
not clear if these are primary or secondary manifestations. Nevertheless,
it is striking how difficult it has been to demonstrate definitively the specificity
of the T cells that provide help for autoantibody production.

The key cellular mechanism in the cGVHD that results in the loss of B cell
tolerance and the production of the autoantibodies typical of SLE is the
cognate interaction of CD4 T cells with an MHC class II determinant on
the B cell surface. A variety of protocols have achieved this interaction.
In general, either the donor/recipient strains are paired in such away
that they only differ at the MHC class II loci, or the CD4 cells are isolated
free of CD8 cells that would recognize MHC class I. If the allorecognition
involves both CD4 T cell interaction with MHC II and CD8 interaction with
MHC I, an acute GVHD occurs, which is immunosuppressive, rather than
immunostimulatory. The DBA/2 (C57BL/6 DBA/2)F1 (B6D2F1) and the
BALB/c (BALB/c A/J)F1 models are exceptions to this rule. The former
has been investigated extensively for a deficiency in CD8 cytotoxic
lymphocytes.

The MHC class II recognition may be at either the I-A or the I-E locus.
However, the autoantibody specificities seen and the degree of immuno-
pathology differ depending on the locus targeted. In one set of experiments,
F1 mice were bred between either B6 or coisogenic bm12 mice and
B10.A(2R) or B10.A(4R) MHC recombinant congenics. The MHC class II
of B6 is I-Ab, while that of bm12 is I-Abm12. These two alleles differ by
only three amino acids, which is sufficient for a full strength MLR (mixed
lymphocyte reaction) between the two strains. Otherwise B6 and bm12
are identical. B10.A(2R) and B10.A(4R) differ only by the expression of
I-E in the former strain, but not in the latter strain. Thus, donor/recipient
combinations could be employed that provided for allogeneic differs only
at I-A, only at I-E, or at both loci.

Results from Busser et al. delineate requirements for this MHC class II
recognition. Utilizing several transgenic mouse strains that express a
more or less constricted CD4 autoreactive repertoire, they showed that
a diverse repertoire was essential to the production of SLE autoantibodies
by MHC II recognition. On the other hand, the non-specific, early polyclonal
B cell activation phase of cGVHD occurred even with a limited CD4 repertoire.

Figure not shown. Chronic GVHD in bm12 C57BL/6 mice. The MHC of the
bm12 donor differs from the MHC of the C57BL/6 recipient just in three
amino acids in the I-A class II molecule. Thus donor CD4 T cells recognize
MHC IIþ B cells as foreign. Donor CD8 T cells see only self MHC I. All T
cells do not express MHC II. Polyclonal activation and specific lupus
autoantibody responses ensue..

Lupus can result from unchecked CD4 T cell cognate help to a polyclonal
population of B cells. CD8 T cells can downregulate this CD4 driven B-cell
hyperactivity through CD8 CTL effectors and can maintain remission,
possibly through memory CD8 T cells. Whether CD8 CTL actually prevent
lupus in normals and fail in lupus prone individuals is not known; however,
data from the P F1 model suggest that therapeutic induction of CD8 CTL
and possibly long term memory cells may be beneficial in preventing or
limiting disease expression. The potential major role played by either
IFNa and IL-21 in both lupus expression and CD8 CTL function remains
to be further defined, but already these cytokines are being targeted in
human or murine lupus.

It is not surprising that the T cells have been shown to have diverse roles in
the autoimmune cGVHD in mice. Donor CD4 T cells drive the host B cell
activation, while host CD4 T cells are required to mature these B cells prior
to their encounter with donor T cells. Donor CD4 T cells also help activate
donor CD8 T cells, which in turn can downregulate or even ablate the
autoimmune response. Donor CD4 T cells license host DC cells, which in
turn can interact with donor CD8 T cells. Host CD8 T cells can suppress
the activity of donor CD8 T cells, and thereby favor the development of
the lupus syndrome. Although the precise mechanisms of T cell participation
in spontaneous lupus are still being defined, it seems reasonable to probe
these syndromes in humans and in mice for T cell mechanism that have
been shown to participate in cGVHD, CD4-B cell interactions almost
certainly are central to the pathogenesis of spontaneous lupus, and they
have been a target of investigation for several decades. If we understood
the peptide specificity of the alloreactive CD4 T cells that drive the formation
of the characteristic lupus autoantibodies, we would have a much clearer
idea where to look for such epitopes in spontaneous disease. Much less
is known about the other T cell activities defined in cGVHD, particularly
those that involve CD8 T cells. This area should invite further detailed
investigation. For example, the striking role of CD8 T cells in the stronger
female disease in the DBA BDF1 model clearly demands that similar
mechanisms be sought for in spontaneous disease.

Understanding Chronic GVHD from Different Angles

Bruce Blazar, Eric S. White, Daniel Couriel
Biol Blood Marrow Transplant 18:S184-S188, 2012
http://dx.doi.org:/10.1016/j.bbmt.2011.10.025

Whereas acute graft-versus-host disease (aGVHD) rates have decreased
with more intensive GVHD preventive agents and use of single and double
umbilical cord blood units as a source of donor cells in adult recipients,
significant chronic GVHD (cGVHD) rates unexpectedly have remained high.
Moreover, granulocyte colony stimulating factor mobilized peripheral blood
stem cell grafts have been associated with an increased overall risk of
cGVHD. As such, cGVHD has emerged as a primary cause of morbidity
and mortality following allogeneic hematopoietic stem cell transplantation.
Progress in developing cGVHD interventional strategies has been hampered
by variable onset and clinical and pathological manifestations of cGVHD, now
better defined by the National Institutes of Health (NIH) consensus conference,
and a dearth of preclinical models that closely mimic the conditions in which
cGVHD is generated and manifested. Although the exact causes of cGVHD
remain unknown, higher antibody levels have been associated with auto-
immunity and implicated in cGVHD. Newly diagnosed patients with
extensive cGVHD had elevated soluble B cell activating factor levels and
anti-double-strand DNA antibodies were found, which was associated with
higher circulating levels of pregerminal center (GC) B cells and post-GC
plasmablasts. B cells from cGVHD patients were hyperresponsive to Toll-like
receptor-9 signaling and have up-regulated CD86 levels.

By using a Cy and low doses of donor T cells, aGVHD was avoided and
cGVHD with BO favored. Histologic changes were similar to the findings in
human cGVHD with peribronchiolar and perivascular cuffing and infiltration
of the airway epithelium. The liver had inflammation and lymphocytic
infiltration, along with collagen deposition. The parotid and submandibular
salivary glands displayed lymphocytic infiltrates in both the bone marrow
and cGVHD groups, likely because of transplantation conditioning.

Treatment of steroid refractory cGVHD patients with rituximab, a B cell–
depleting anti-CD20 monoclonal antibody, has shown a beneficial role in
resolution of the autoimmune disorders such as systemic lupus erythmatosus
and rheumatoid arthritis, andcGVHD, with overall response rates of 29%
to 36% for oral, hepatic, gastrointestinal, and lung cGVHD, and 60% for
cutaneous cGVHD in aggregate data from multiple trials. Thus, we recently
undertook studies to identify the presence of CD41 T helper cells and B2201
B cells in the airways of mice that had BO, tissue-specific antibodies from sera,
and alloantibody deposition in the lung and liver of cGVHD recipients. cGVHD
development was associated with IgG2c deposition in the lung and liver,
abrogated if the donor bone marrow was deficient in mature B cells or
incapable of producing antihost reactive IgG. Robust GC formation was
seen in mice with cGVHD. Alleviation of symptoms in mice that received
B cell–deficient bone marrow confirms the requirement of B cells for lung
dysfunction and inflammation and fibrosis in the lung and liver.

Given a role for IgG antibodies, allo- or auto-Ab binding to the cGVHD organs
could enable tissue destruction or the pathology could be defined by the
specific function of these secreted antibodies. Pathogenic antibody production
therefore is likely to be an important inducer of cGVHD, and targeting this
specific function of the B cells is an attractive strategy for cGVHD. Because
GC B cells display lower susceptibility to rituximab-mediated clearance, probably
because they reside in a nonoptimal environment for antibody-based depletion,
our observation that GC B cells are critical to the development of cGVHD
suggests that agents that are more effective at disrupting the GC might be
more clinically useful. Treatment with LTbR-Ig, a fusion protein that blocks
interactions between LTbR and its ligands, had a direct effect on the
symptoms of cGVHD, at least in part by blocking GC formation and suggest
that LTbR-Ig could be a potential clinical interventional strategy for prevention
and therapy of cGVHD.

Fibrosis is the end result of a number of inflammatory and other injurious events,
resulting in replacement of normal tissue with a dense extracellular matrix (ECM)
scar composed primarily of collagens. While some degree of tissue fibrosis is
considered protective (e.g. in the setting of cutaneous wound healing),
exaggerated or unrelenting ECM deposition with replacement of the normal
tissue architecture is considered pathologic. Fibroproliferative disorders as
a class involving multiple organs (e.g. cGVHD following hematopoietic stem
cell transplant [affecting up to 30% of recipients surviving more than 100 days,
scleroderma [estimated to affect 70,000 in the US], idiopathic pulmonary fibrosis
[estimated to affect 200,000 in the US], hepatic cirrhosis [estimated to affect
up to 400,000 in the US], and renal fibrosis due to diabetic nephropathy and
other causes [estimated to affect over 400,000 in the US]) are a major cause
of morbidity and mortality. Combined, these disorders alone are conservatively
estimated to affect approximately 1 in 300 persons in the United States. When
coupled with a host of other disorders in which tissue fibrosis contributes to
morbidity (e.g. fibroproliferative acute respiratory distress syndrome,
hypersensitivity pneumonitis, solid organ transplant rejection), that estimate
is likely to be much greater.

Wound healing occurs by a highly orchestrated, complex process that has
been well defined. In general, wound repair occurs in 4 stages which overlap
considerably: clotting/coagulation, inflammation, fibroproliferation, and tissue
remodeling. The initial injury leads to a local disruption of epithelial and
endothelial barriers resulting in the elaboration of inflammatory mediators and
extravasation of cells and plasma proteins that serve to achieve hemostasis
and provide a provisional fibrin-rich matrix for the influx of inflammatory and
other reparative cells. Simultaneously, platelet degranulation provides a local
“boost” of vasodilators, growth factors, and ECM proteins that aid in the wound
healing response. Inflammatory cell influx occurs next, with polymorphonuclear
leukocytes (PMNs) arriving first. Following PMN degranulation, mononuclear
cells (macrophages and lymphocytes) arrive next and, along with PMN derived
products, sterilize and remove foreign materials from the wound. This process
also results in the elaboration of cytokines and chemokines designed to
augment the inflammatory response, to promote angiogenesis (allowing for
enhanced nutrient and oxygen delivery to the wound bed), and to recruit
fibroblasts to the wound bed. Fibroblast recruitment and transdifferentiation to
myofibroblasts (or recruitment of already-differentiated myofibroblasts or
fibroblast precursors; this point is still controversial) marks the fibroproliferative
stage, with the result being the elaboration of ECM proteins (collagens,
fibronectins) to repair the tissue defect.

Vorinostat plus tacrolimus and mycophenolate to prevent graft-versus-host
disease after related-donor reduced-intensity conditioning allogeneic
hemopoietic 
stem-cell transplantation: a phase 1/2 trial

Sung Won Choi, T Braun, L Chang, JLM Ferrara, A Pawarode, et al.
Lancet Oncol 2014; 15: 87–95
http://dx.doi.org/10.1016/S1470-2045(13)70512-6

Background Acute graft-versus-host disease (GVHD) remains a barrier to more
widespread application of allogeneic hemopoietic stem-cell transplantation.
Vorinostat is an inhibitor of histone deacetylases and was shown to attenuate
GVHD in preclinical models. We aimed to study the safety and activity of
vorinostat, in combination with standard immunoprophylaxis, for prevention of
GVHD in patients undergoing related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation. Methods Between March 31, 2009,
and Feb 8, 2013, we did a prospective, single-arm, phase 1/2 study at two
centers in the USA. We recruited adults (aged ≥18 years) with high-risk
hematological malignant diseases who were candidates for reduced-intensity
conditioning hemopoietic stem-cell transplantation and had an available 8/8
or 7/8 HLA matched related donor. All patients received a conditioning regimen
of fl udarabine (40 mg/m² daily for 4 days) and busulfan (3·2 mg/kg daily for
2 days) and GVHD immunoprophylaxis of mycophenolate mofetil (1 g three
times a day, days 0–28) and tacrolimus (0·03 mg/kg a day, titrated to a goal
level of 8–12 ng/mL, starting day –3 until day 180). Vorinostat (either 100 mg
or 200 mg, twice a day) was initiated 10 days before haemopoietic stem-cell
transplantation until day 100. The primary endpoint was the cumulative
incidence of grade 2–4 acute GVHD by day 100. This trial is registered with
ClinicalTrials.gov, number NCT00810602.
Findings 50 patients were assessable for both toxic effects and response;
eight additional patients were included in the analysis of toxic effects. All
patients engrafted neutrophils and platelets at expected times after
hemopoietic stem-cell transplantation. The cumulative incidence of grade
2–4 acute GVHD by day 100 was 22% (95% CI 13–36). The most common
non-hematological adverse events included electrolyte disturbances (n=15),
hyperglycemia (11), infections (six), mucositis (four), and increased activity
of liver enzymes (three). Non-symptomatic thrombocytopenia after
engraftment was the most common hematological grade 3–4 adverse
event (nine) but was transient and all cases resolved swiftly.
Interpretation Administration of vorinostat in combination with standard
GVHD prophylaxis after related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation is safe and is associated with a
lower than expected incidence of severe acute GVHD. Future studies
are needed to assess the effect of vorinostat for prevention of GVHD in
broader settings of hemopoietic stem-cell transplantation.

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Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Author: Larry H. Bernstein, MD

Author: Stephen Williams, PhD

and

Curator: Aviva Lev-Ari, PhD, RN

This article is Part II in a series of articles on Calcium and its role in Cell motility

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

 

This article, constitute, Part II, it is a broad, but not complete review of the emerging discoveries of the critical role of calcium signaling on cell motility and by extension, embryonic development, cancer metastasis, changes in vascular compliance at the junction between the endothelium and the underlying interstitial layer.  The effect of calcium signaling on the heart in arrhtmogenesis and heart failure will be a third in this series, while the binding of calcium to troponin C in the synchronous contraction of the myocardium had been discussed by Dr. Lev-Ari in Part I.

Universal MOTIFs essential to skeletal muscle, smooth muscle, cardiac syncytial muscle, endothelium, neovascularization, atherosclerosis and hypertension, cell division, embryogenesis, and cancer metastasis. The discussion will be presented in several parts:
1.  Biochemical and signaling cascades in cell motility
2.  Extracellular matrix and cell-ECM adhesions
3.  Actin dynamics in cell-cell adhesion
4.  Effect of intracellular Ca++ action on cell motility
5.  Regulation of the cytoskeleton
6.  Role of thymosin in actin-sequestration
7.  T-lymphocyte signaling and the actin cytoskeleton

Part 1.  Biochemical and Signaling Cascades in Cell Motility

BIOCHEMISTRY AND BIOMECHANICS OF CELL MOTILITY

Song Li, Jun-Lin Guan, and Shu Chien
Annu. Rev. Biomed. Eng. 2005. 7:105–50   [doi:10.1146/annurev.bioeng.7.060804.100340]
Cell motility or migration is an essential cellular process for a variety of biological events. In embryonic development, cells migrate to appropriate locations for the morphogenesis of tissues and organs. Cells need to migrate to heal the wound in repairing damaged tissue. Vascular endothelial cells (ECs) migrate to form new capillaries during angiogenesis. White blood cells migrate to the sites of inflammation to kill bacteria. Cancer cell metastasis involves their migration through the blood vessel wall to invade surrounding tissues.

Variety of important roles for cell migration:

1. Embryogenesis
2. Wound healing (secondary extension)
3. Inflammatory infiltrate (chemotaxis)
4. Angiogenesis
5. Cancer metastasis
6. Arterial compliance
7. Myocardial and skeletal muscle contraction
8. Cell division

Portrait of Cell in Migration:

1. protrusion of leading edge
2. Formation of new adhesions at front
3. Cell contraction
4. Release of adhesions at rear
Microenvironmental factor:
1. Concentration gradient of chemoattractants
2. Gradient of immobilized ECM proteins
3. Gradient of matrix rigidity
4. Mechanotaxis
Extracellular signals are sensed by receptors or mechanosensors on cell surface or in cell interior to initiate migration. Actin polymerization is the key event leading to protrusion at the leading edge and new focal adhesions anchor the actin filaments and the cell to the underlying surface.  This is followed by contraction of the actin filaments.  The contraction of actomyosin filaments pulls the elongate body forward and at the same time the tail retracts.

Part 2.  Cell-ECM Adhesions

Cytoskeleton and cell-ECM adhesions are two major molecular machineries involved in mechano-chemical signal transduction during cell migration. Although all three types of cytoskeleton (actin microfilaments, microtubules, and intermediate filaments) contribute to cell motility, actin cytoskeleton plays the central role. The polymerization of actin filaments provides the driving force for the protrusion of the leading edge as lamellipodia (sheet-like protrusions) or filopodia (spike-like protrusions), and actomyosin contraction generates the traction force at (focal adhesions) FAs and induces the retraction at the rear. It is generally accepted that actin filaments interact with the double-headed myosin to generate the force for cell motility and that actomyosin contraction/relaxation involves the modulation of myosin light chain (MLC) phosphorylation.  Rho family GTPases, including Cdc42, Rac, and Rho, are the key regulators of actin polymerization, actomyosin contraction, and cell motility.  Cdc42 activation induces the formation of filopodia; Rac activation induces lamellipodia; and Rho activation increases actin polymerization, stress fiber formation, and actomyosin contractility. All three types of Rho GTPases stimulate new FA formation.
Integrins are the major receptors for ECM proteins. The integrin family includes more than 20  transmembrane heterodimers composed of α and β subunits with noncovalent association. The extracellular domain of integrin binds to specific ligands, e.g., ECM proteins such as fibronectin (FN), vitronectin, collagen, and laminin. The cytoplasmic domain interacts with cytoskeletal proteins (e.g., paxillin, talin, vinculin, and actin) and signaling molecules in the focal adhesion (FA) sites. The unique structural features of integrins enable them to mediate outside-in signaling, in which extracellular stimuli induce the intracellular signaling cascade via integrin activation, and inside-out signaling, in which intracellular signals modulate integrin activation and force generation through FAs.

Part 3. Actin Dynamics in Cell-cell Adhesion

Actin filaments are linked to the focal adhesions (Fas) between cell and ECM through a protein complex that includes talin, vinculin, α-actinin, and filamin. Such a complex couples the actomyosin contractile apparatus to FAs, and plays an important role in the force transmission between ECM and the cell.

3a. Actin dynamics and cell–cell adhesion in epithelia

Valeri Vasioukhin and Elaine Fuchs
Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL
Current Opinion in Cell Biology 2001, 13:76–84
Recent advances in the field of intercellular adhesion highlight the importance of adherens junction association with the underlying actin cytoskeleton. In skin epithelial cells a dynamic feature of adherens junction formation involves filopodia, which physically project into the membrane of adjacent cells, catalyzing the clustering of adherens junction protein complexes at their tips. In turn, actin polymerization is stimulated at the cytoplasmic interface of these complexes. Although the mechanism remains unclear, the VASP/Mena family of proteins seems to be involved in organizing actin polymerization at these sites. In vivo, adherens junction formation appears to rely upon filopodia in processes where epithelial sheets must be physically moved closer to form stable intercellular connections, for example, in ventral closure in embryonic development or wound healing in the postnatal animal.
Located at cell–cell borders, adherens junctions are electron dense transmembrane structures that associate with the actin cytoskeleton. In their absence, the formation of other cell–cell adhesion structures is dramatically reduced. The transmembrane core of adherens junctions consists of cadherins, of which E-cadherin is the epithelial prototype. Its extracellular domain is responsible for homotypic, calcium-dependent, adhesive interactions with E-cadherins on the surface of opposing cells. Its cytoplasmic domain is important for associations with other intracellular proteins involved in the clustering of surface cadherins to form a junctional structure.
The extracellular domain of the transmembrane E-cadherin dimerizes and interacts in a calcium-dependent manner with similar molecules on neighboring cells. The intracellular juxtamembrane part of E-cadherin binds to p120ctn, an armadillo repeat protein capable of modulating E-cadherin clustering. The distal segment of E-cadherin’s cytoplasmic domain can interact with β-catenin or plakoglobin, armadillo repeat proteins which in turn bind to α-catenin. The carboxyl end of α-catenin binds directly to f-actin, and, through a direct mechanism, α-catenin can link the membrane-bound cadherin–catenin complex to the actin cytoskeleton. Additionally, α-catenin can bind to either vinculin or ZO1, and it is required for junctional localization of zyxin. Vinculin and zyxin can recruit VASP (and related family members), which in turn can associate with the actin cytoskeleton, providing the indirect mechanism to link the actin cytoskeleton to adherens junctions. ZO1 is also a member of tight junctions family, providing a means to link these junctions with adherens junctions.
Through a site near its transmembrane domain, cadherins bind directly to the catenin p120ctn, and through a more central site within the cytoplasmic domain, cadherins bind preferentially to β-catenin. Cell migration appears to be promoted by p120ctn through recruiting and activating small GTPases. β-catenin is normally involved in adherens junction formation through its ability to bind to β-catenin and link cadherins to the actin cytoskeleton. However, β-catenin leads a dual life in that it can also act as a transcriptional cofactor when stimulated by the Wnt signal transduction pathway

α-Catenin: More than just a Bridge between Adherens Junctions and the Actin Cytoskeleton

α-catenin was initially discovered as a member of the E-cadherin–catenin complex.  It is related to vinculin, an actin-binding protein that is found at integrin-based focal contacts. The amino-terminal domain of α-catenin is involved in α-catenin/plakoglobin binding and is also important for dimerization. Its central segment can bind to α-actinin and to vinculin, and it partially encompasses the region of the protein necessary for cell adhesion (which is the adhesion-modulation domain; amino acids 509–643). The carboxy-terminal domain of both vinculin and α-catenin is involved in filamentous actin (f-actin) binding, and for α-catenin, this domain is also involved in binding to ZO1.  VH1, VH2 and VH3 are three regions sharing homology to vinculin. The percentage amino acid identity and the numbers correspond to the amino acid residues of the α-catenin polypeptide.
α-catenin is the only catenin that can directly bind to actin filaments , and E-cadherin–catenin complexes do not associate with the actin cytoskeleton after α-catenin is removed by extraction with detergent. Cancer cell lines lacking α-catenin still express E-cadherin and β-catenin, but do not show proper cell–cell adhesion unless the wild-type gene is reintroduced into the cancer cell. This provides strong evidence that clustering of the E-cadherin–catenin complex and cell–cell adhesion requires the presence of α-catenin.
Although intercellular adhesion is dependent upon association of the E-cadherin–β-catenin protein complex with α-catenin and the actin cytoskeleton, it is unclear whether α-catenin’s role goes beyond linking the two structures. Fusion of a nonfunctional tailless E-cadherin (E C71) with α-catenin resulted in a chimeric protein able to confer cell–cell adhesion on mouse fibroblasts in vitro, and generation of additional chimeric proteins enabled delineation of the region of α-catenin that is important for cell aggregation. Not surprisingly, the essential domain of α-catenin was its carboxy-terminal domain (~amino acids 510–906), containing the actin-binding site, which encompasses residues 630–906 of this domain.
The binding of α-catenin to the actin cytoskeleton is required for cell–cell adhesion,  but α-catenin appears to have additional function(s) beyond its ability to link E-cadherin–β-catenin complexes to actin filaments.  The domain encompassing residues 509–643 of α-catenin has been referred to as an adhesion-modulation domain to reflect this added, and as yet unidentified, function.  Besides its association with β-catenin and f-actin, α-catenin binds to a number of additional proteins, some of which are actin binding proteins themselves.  Additionally, the localization of vinculin to cell–cell borders is dependent upon the presence of α-catenin. α-catenin can also bind to the MAGUK (membrane-associated guanylate kinase) family members ZO1 and ZO2.  Thus, the role for α-catenin might not simply be to link E-cadherin–catenin complexes to the actin cytoskeleton but rather to organize a multiprotein complex with multiple actin-binding, bundling and polymerization activities.
The decisive requirement for α-catenin’s actin-binding domain in adherens junction formation underscores the importance of the actin cytoskeleton in intercellular adhesion. Thus, it is perhaps not surprising that the majority of f-actin in epithelial cells localizes to cell–cell junctions.  When epidermal cells are incubated in vitro in culture media with calcium concentrations below 0.08 mM they are unable to form adherens junctions. However, when the calcium concentrations are raised to the levels naturally occurring in skin (1.5–1.8 mM), intercellular adhesion is initiated.
This switch in part promotes a calcium-dependent conformational change in the extracellular domain of E-cadherin that is necessary for homotypic interactions to take place.  It appears that the actin cytoskeleton has a role in facilitating the process that brings opposing membranes together and stabilizing them once junction formation has been initiated. In this regard, the formation of cell–cell adhesion can be divided into two categories:
  • active adhesion, a process that utilizes the actin cytoskeleton to generate the force necessary to bring opposing membranes together, and
  • passive adhesion, a process which may not require actin if the membranes are already closely juxtaposed and stabilized by the deposition of cadherin–catenin complexes.
Upon a switch from low to high calcium, cadherin-mediated intercellular adhesion is activated. Passive adhesion: in cells whose actin cytoskeleton has been largely disrupted by cytochalasin D, cadherin–catenin complexes occur at sites where membranes of neighboring cells directly contact each other. Active adhesion: neighboring cells with functional actin cytoskeletons can draw their membranes together, forming a continuous epithelial sheet.  Upon initial membrane contact, E-cadherin forms punctate aggregates or puncta along regions where opposing membranes are in contact with one another. Each of these puncta is contacted by a bundle of actin filaments that branch off from the cortical belt of actin filaments underlying the cell membrane. At later stages in the process, those segments of the circumferential actin cables that reside along the zone of cell–cell contacts disappear, and the resulting semi-circles of cortical actin align to form a seemingly single circumferential cable around the perimeter of the two cells. At the edges of the zone of cell–cell contact, plaques of E-cadherin–catenin complexes connect the cortical belt of actin to the line of adhesion. At the center of the developing zone of adhesion, E-cadherin puncta associate with small bundles of actin filaments oriented perpendicular to the zone.
Multiple E-cadherin-containing puncta that form along the developing contact rapidly associate with small bundles of actin filaments. As the contact between cells lengthens, puncta continue to develop at a constant average density, with new puncta at the edges of the contact. The segment of the circumferential actin cable that underlies the developing contact gradually ‘dissolves’, and merges into a large cable, encompassing both cells. This is made possible through cable-mediated connections to the E-cadherin plaques at the edges of the contact. As contact propagates, E-cadherin is deposited along the junction as a continuous line. The actin cytoskeleton reorganizes and is now oriented along the cell–cell contact. In primary keratinocytes, two neighboring cells send out filopodia, which, upon contact, slide along each other and project into the opposing cell’s membrane. Filopodia are rich in f-actin. Embedded tips of filopodia are stabilized by puncta, which are transmembrane clusters of adherens junction proteins.
This process draws regions of the two cell surfaces together, which are then clamped by desmosomes. Radial actin fibers reorganize at filopodia tips in a zyxin-, vinculin-, VASP-, and Mena-dependent fashion.  Actin polymerization is initiated at stabilized puncta, creating the directed reverse force needed to push and merge puncta into a single line as new puncta form at the edges. The actin-based movement physically brings remaining regions of opposing membranes together and seals them into epithelial sheets. As filopodia contain actin rather than keratin intermediate filaments, they become natural zones of adherens junctions, whereas the cell surface flanking filopodia becomes fertile ground for desmosome formation, alternating adherens junctions and desmosomes.

Possible Roles of Myosin in Cell–cell Adhesion.

[a] A hypothetical ‘purse string’ model for myosin-driven epithelial sheet closure at a large circular wound site in the cornea of an adult mouse. At the edge of wound site epithelial cables of actin appear to extend from cell to cell, forming a ring around the wound circumference. Contraction of actin cables  driven by myosin can lead to wound closure.
[b] Inside out ‘purse string’ model for contact propagation (compaction) in MDCK cells. During contact formation in MDCK cells, circumferential actin cables contact cadherin–catenin plaques at the edges of the contact. Contraction of actin cables driven by myosin can lead to the contact expansion.

What Regulates the Actin Dynamics that are Important for Cell–cell Adhesion?

The answer to this remains uncertain, but the small GTPases of the Rho family seem to be likely candidates, given that Rho, Rac1 and Cdc42 promote stress fiber, lamellipodia and filopodia formation, respectively.
In vivo mutagenesis studies in Drosophila reveal a role for Rac1 and Rho in dorsal closure and/or in head involution, processes that involve complex and well orchestrated rearrangements of cells. In contrast, Cdc42 appears to be involved in regulating polarized cell shape changes. In vitro, keratinocytes microinjected with dominant negative Rac1 or with C3 toxin, a specific inhibitor of Rho, are unable to form cadherin-based cell–cell contacts.  Similarly, overexpression of a constitutively active form of Rac1 or Cdc42 in MDCK cells increases junctional localization of E-cadherin–catenin complexes, whereas the dominant negative forms of Rac1 and Cdc42, or C3 microinjection, have the opposite effect. The finding that Tiam1, a guanine nucleotide exchange factor for Rac1, increases E-cadherin mediated cell–cell adhesion, inhibits hepatocyte growth-factor-induced cell scattering and reverses the loss of adhesion in Ras-transformed cells is consistent with the above.  Together, these findings provide compelling evidence that activation of the Rho family of small GTPases plays a key role in the actin dynamics that are necessary for adherens junction formation.
We found that E-cadherin–catenin-enriched puncta, which assemble during the first stages of epithelial sheet formation, are sites of de novo actin polymerization. This led us to postulate that actin polymerization might provide the force that is subsequently necessary to merge the double role of puncta into a single row and ultimately into an epithelial sheet. Knowledge of how actin polymerization might generate movement comes largely from studies of the mechanism by which the pathogen Listeria monocytogenes pirates actin polymerization and utilizes it for intracellular propulsion. For this endeavor, these bacteria recruit two types of cellular components, the VASP family of proteins and the Arp2/3 complex. The Arp2/3 protein complex is required for de novo nucleation of actin filament polymerization, whereas VASP appears to accelerate bacterial movement by about 10 fold.
Although most studies have revealed positive roles for VASP and its cousins in actin reorganization/ polymerization, recent experiments have shown that in certain instances these proteins act negatively in directing cell movement. A further complication is the finding that VASP family proteins can be phosphorylated, thereby inhibiting their actin nucleation and f-actin binding ability. A  role for VASP may be in the actin polymerization necessary for filopodia  extensions. In this regard, VASP family proteins localize to the tips of filopodia during neural growth and in calcium-stimulated keratinocytes. VASP family proteins in this process might provide directionality to the process of actin polymerization, reshaping f-actin into parallel bundles to produce and extend filopodia-like structures from branched lamellipodial networks.

The Might of Myosins

Although actin polymerization seems to be important in generating the cellular movement necessary for intercellular adhesion, this does not rule out the possibility that the myosin family of actin motor proteins may also play a role.  It is known, for instance, that cells can use myosin–actin contractile forces to alter cell shape, and myosin II is a ubiquitously expressed protein involved in such diverse processes as cell spreading, cytokinesis, cell migration, generation of tension within actin stress fiber networks and retrograde flow of actin filaments at the leading edge of moving cells. Interestingly, mouse corneal cells at a wound edge assemble cables of actin filaments anchored to E-cadherin–catenin complexes. The cells surrounding the wound site display myosin-II-associated actin filaments that are aligned in a structure resembling a purse string. It has been postulated that closure of the wound may be achieved through myosin-directed contraction of the actin filaments, in a mechanism similar to that of pulling on a purse string.
Overall, through guilt by association, myosins have been implicated in cell–cell adhesion and in adherens junction formation and although the models proposed are attractive, direct experimental evidence is still lacking. BDM (2,3-butanedione monoxime), a general inhibitor of myosin function, had no obvious effect on intercellular junction formation in our keratinocyte adhesion assays (V Vasioukhin, E Fuchs, unpublished data). However, the role of myosins clearly deserves a more detailed investigation, and this awaits the development of new and improved inhibitors and activators of myosin action.

 Key references:

1. Imamura Y, Itoh M, Maeno Y, Tsukita S, Nagafuchi A: Functional  domains of α-catenin required for the strong state of cadherin based cell adhesion. J Cell Biol 1999, 144:1311-1322.
Three distinct functional domains for α-catenin were identified: a vinculin binding domain, a ZO-1-binding domain and an adhesion modulation domain. Both ZO1-binding (also actin binding) and adhesion modulation domains are necessary for strong adhesion.
2. Vasioukhin V, Bauer C, Yin M, Fuchs E: Directed actin polymerization is the driving force for epithelial cell–cell adhesion. Cell 2000, 100:209-219.
A dynamic filopodia-driven process of cell–cell adhesion is described in primary mouse keratinocyte cultures. Newly forming adherens junctions were identified as sites of actin polymerization and/or reorganization, involving VASP/Mena family members.
3. Raich WB, Agbunag C, Hardin J: Rapid epithelial-sheet sealing in the Caenorhabditis elegans embryo requires cadherin-dependent filopodial priming. Curr Biol 1999, 9:1139-1146.
An elegant in vivo analysis of filopodia-based cell–cell junction formation during epithelial-sheet closure in embryonic development of C. elegans.
4. Loisel TP, Boujemaa R, Pantaloni D, Carlier MF: Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.  Nature 1999, 401:613-616.
Using an in vitro reconstitution approach, the authors show that Arp2/3, actin, cofilin and capping proteins are required for motility of Listeria, in contrast VASP seems to act by increasing the speed of movement by about 10 fold.

3b.  Role for Gelsolin in Actuating Epidermal Growth Factor Receptor-mediated Cell Motility

Philip Chen,  Joanne E. Murphy-Ullrich, and Alan Wells
Department of Pathology, University of Alabama at Birmingham, AL
J Cell Biology Aug 1996; 134(3): 689-698
Phospholipase C-~/(PLC~/) is required for EGF-induced motility (Chen, P., H. Xie, M.C. Sekar, K.B. Gupta, and A. Wells. J. Cell Biol. 1994. 127:847-857); however, the molecular basis of how PLC~/modulates the actin filament network underlying cell motility remains undetermined. One connection to the actin cytoskeleton may be direct hydrolysis of PIP 2 with subsequent mobilization of membrane-associated actin modifying proteins. We used signaling restricted EGFR mutants expressed in receptor-devoid NR6 fibroblast cells to investigate whether EGFR activation of PLC causes gelsolin mobilization from the cell membrane in vivo and whether this translocation facilitates cell movement. Gelsolin anti-sense  oligonucleotide (20 p,M) treatment of NR6 ceils expressing the motogenic full-length (WT) and  truncated c’ 1000 EGFR decreased endogenous gelsolin by 30–60%; this resulted in preferential reduction of EGF (25 nM)-induced cell movement by >50% with little effect on the basal motility. As 14 h of EGF stimulation of cells did not increase total cell gelsolin content, we determined whether EGF induced redistribution of gelsolin from the membrane fraction. EGF treatment decreased the gelsolin mass associated with the membrane fraction in motogenic WT and c’1000 EGFR NR6 cells but not in cells expressing the fully mitogenic, but nonmotogenic c’973 EGFR. Blocking PLC activity with the pharmacologic agent U73122 (1 ~M) diminished both this mobilization of gelsolin and EGF-induced motility, suggesting that gelsolin mobilization is downstream of PLC. Concomitantly observed was reorganization of submembranous actin filaments correlating directly with PLC activation and gelsolin mobilization. In vivo expression of a peptide that is reported to compete in vitro with gelsolin in binding to PIP2 dramatically increased basal cell motility in NR6 cells expressing either motogenic (WT and c’1000) or nonmotogenic (c’973) EGFR; EGF did not further augment cell motility and gelsolin mobilization. Cells expressing this peptide demonstrated actin reorganization similar to that observed in EGF-treated control cells; the peptide-induced changes were unaffected by U73122. These data suggest that much of the EGF induced motility and cytoskeletal alterations can be reproduced by displacement of select actin-modifying proteins from a PIP2-bound state. This provides a signaling mechanism for translating cell surface receptor mediated biochemical reactions to the cell movement machinery.

3c.  Actomyosin Contraction at the Cell Rear Drives Nuclear Translocation in Migrating Cortical Interneurons

Francisco J. Martini and Miguel Valdeolmillos
Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez, Alacant, Spain
Journal of Neuroscience 2010 • 30(25):8660–8670
Neuronal migration is a complex process requiring the coordinated interaction of cytoskeletal components and regulated by calcium signaling among other factors. Migratory neurons are polarized cells in which the largest intracellular organelle, the nucleus, has to move repeatedly. Current views support a central role for pulling forces that drive nuclear movement. By analyzing interneurons migrating in cortical slices of mouse brains, we have found that nucleokinesis is associated with a precise pattern of actin dynamics characterized by the initial formation of a cup-like actin structure at the rear nuclear pole. Time-lapse experiments show that progressive actomyosin contraction drives the nucleus forward. Nucleokinesis concludes with the complete contraction of the cup-like structure, resulting in an actin spot at the base of the retracting trailing process. Our results demonstrate that this actin remodeling requires a threshold calcium level provided by low-frequency spontaneous fast intracellular calcium transients. Microtubule stabilization with taxol treatment prevents actin remodeling and nucleokinesis, whereas cells with a collapsed microtubule cytoskeleton induced by nocodazole treatment, display nearly normal actin dynamics and nucleokinesis. In summary, the results presented here demonstrate that actomyosin forces acting at the rear side of the nucleus drives nucleokinesis in tangentially migrating interneurons in a process that requires calcium and a dynamic cytoskeleton of microtubules.

3d. Migration of Zebrafish Primordial Germ Cells: A Role for Myosin Contraction and Cytoplasmic Flow

H Blaser, M Reichman-Fried, I Castanon, K Dumstrei, F L Marlow, et al.
Max Planck Institute, Gottingen & Dresden, Germany;  Vanderbilt University, Nashville, Tenn; National Institute of Genetics, Shizuoka, Japan
Developmental Cell 2006; 11: 613–627 [DOI 10.1016/j.devcel.2006.09.023]
The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.

Part 4.  Calcium Signaling

4a. Indirect Association of Ezrin with F-Actin: Isoform Specificity and Calcium Sensitivity

Charles B. Shuster and Ira M. Herman
Tufts University Health Science Schools, Boston, MA
J Cell Biology Mar 1995; 128(5): 837-848
Muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains,  but the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Era) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KC1 step gradient.  A polypeptide of interest, which binds specifically to/3-actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-/3 interactions in vitro by passing extracts (Era) over isoactin affinity matrices in the presence of Ca2+-containing versus Ca2+-free buffers, with or without cytochalasin D. Ezrin binds and can be released from/3-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaC1 (at 4°C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaC12 or 2/~M cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to β-actin in the presence of calcium. Biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis shows that anti-ezrin and anti-β-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and β-actin interact in vivo. To test the hypothesis that ezrin binds directly to β-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either α- or β-actin. Results of these experiments reveal that purified ezrin does not directly bind to β-actin filaments. We mapped cellular free calcium in endothelial monolayers crawling in response to injury. Confocal imaging of fluo-3 fluorescence followed by simultaneous double antibody staining reveals a transient rise of free calcium within ezrin-/3-actin-enriched domains in the majority of motile cells bordering the wound edge. These results support the notion that calcium and calpain I modulate ezrin and β-actin interactions during forward protrusion formation.

4b.  Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons

Massimiliano Cristofanilli and Abram Akopian
New York University School of Medicine, New York, NY
J Physiol 575.2 (2006) pp 543–554
Intracellular Ca2+ regulates a variety of neuronal functions, including neurotransmitter release, protein phosphorylation, gene expression and synaptic plasticity. In a variety of cell types, including neurons, Ca2+ is involved in actin reorganization, resulting in either actin polymerization or depolymerization. Very little, however, is known about the relationship between Ca2+ and the actin cytoskeleton organization in retinal neurons. We studied the effect of high-K+-induced depolarization on F-actin organization in salamander retina and found that Ca2+ influx through voltage-gated L-type channels causes F-actin disruption, as assessed by 53±5% (n=23, P <0.001) reduction in the intensity of staining with Alexa-Fluor488-phalloidin, a compound that permits visualization and quantification of polymerized actin. Calcium-induced F-actin depolymerization was attenuated in the presence of protein kinase C antagonists, chelerythrine or bis-indolylmaleimide hydrochloride (GF 109203X). In addition, phorbol 12-myristate 13-acetate (PMA), but not 4α-PMA, mimicked the effect of Ca2+ influx on F-actin. Activation of ionotropic AMPA and NMDA glutamate receptors also caused a reduction in F-actin. No effect on F-actin was exerted by caffeine or thapsigargin, agents that stimulate Ca2+ release from internal stores. In whole-cell recording from a slice preparation, light-evoked ‘off’ but not ‘on’ EPSCs in ‘on–off’ ganglion cells were reduced by 60±8% (n=8, P <0.01) by cytochalasin D. These data suggest that elevation of intracellular Ca2+ during excitatory synaptic activity initiates a cascade for activity-dependent  actin remodelling, which in turn may serve as a feedback mechanism to attenuate excite-toxic Ca2+ accumulation induced by synaptic depolarization.

4c.  Electric Field-directed Cell Shape Changes, Displacement, and Cytoskeletal Reorganization Are Calcium Dependent

Edward K. Onuma and Sek-Wen Hui
Roswell Park Memorial Institute, Buffalo, New York
J Cell Biology 1988; 106: 2067-2075

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field induced disorganization of the stress fiber pattern, which was reduced when stimulation was conducted in calcium-depleted buffer or in buffer containing calcium antagonist CoC12, calcium channel blocker D-600, or calmodulin antagonist trifluoperazine. Treatment with calcium ionophore A23187 had similar effects, except that the presence of D-600 did not reduce the stress fiber disruption. The calcium-sensitive photoprotein aequorin was used to monitor changes in intracellular-free calcium. Electric stimulation caused an increase of calcium to the micromolar range. This increase was inhibited by calcium-depleted buffer or by CoC12, and was reduced by D-600. A calcium-dependent mechanism is proposed to explain the observed field-directed cell shape changes, preferential orientation, and displacement.

4d. Local Calcium Elevation and Cell Elongation Initiate Guided Motility in Electrically Stimulated osteoblast-Like Cells

N Ozkucur, TK Monsees, S Perike, H Quynh Do, RHW Funk.
Carl Gustav Carus, TU-Dresden, Dresden, Germany; University of the Western Cape, SAfrica.
Plos ONE 2009; 4 (7): e6131

Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration.
We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10–15 V/cm) and weak (#5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation. Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.

4e. TRPM4 Regulates Migration of Mast Cells in Mice

T Shimizua, G Owsianik, M Freichelb, V Flockerzi, et al.
Laboratory of Ion Channel Research, KU Leuven, Leuven, Belgium; Universität des Saarlandes, Homburg, Germany; National Institute for Physiological Sciences,Okazaki, Japan
Cell Calcium 2008; xxx–xxx

We demonstrate here that the transient receptor potential melastatin subfamily channel, TRPM4, controls migration of bone marrow-derived mast cells (BMMCs), triggered by dinitrophenylated human serum albumin (DNP-HSA) or stem cell factor (SCF). Wild-type BMMCs migrate after stimulation with DNPHSA or SCF whereas both stimuli do not induce migration in BMMCs derived from TRPM4 knockout mice (trpm4−/−). Mast cell migration is a Ca2+-dependent process, and TRPM4 likely controls this process by setting the intracellular Ca2+ level upon cell stimulation. Cell migration depends on filamentous actin (F-actin) rearrangement, since pretreatment with cytochalasin B, an inhibitor of F-actin formation, prevented both DNP-HSA- and SCF-induced migration in wild-type BMMC. Immunocytochemical experiments using fluorescence-conjugated phalloidin demonstrate a reduced level of F-actin formation in DNP-HSA-stimulated BMMCs from trpm4−/− mice. Thus, our results suggest that TRPM4 is critically involved in migration of BMMCs by regulation of Ca2+-dependent actin cytoskeleton rearrangements.
4f. Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells
G FAURY, Y USSON, M ROBERT-NICOUD, L ROBERT, AND J VERDETTI.
Institut Albert Bonniot, Universite´ J. Fourier, Grenoble, Fr; and Universite´ Paris, Paris, Fr
PNAS: Cell Biology 1998; 95: pp. 2967–2972.

The extracellular matrix protein ‘‘elastin’’ is the major component of elastic fibers present in the arterial wall. Physiological degradation of elastic fibers, enhanced in vascular pathologies, leads to the presence of circulating elastin peptides (EP). EP have been demonstrated to influence cell migration and proliferation. EP also induce, at circulating pathophysiological concentrations (and not below), an endothelium-and NO- dependent vasorelaxation mediated by the 67-kDa subunit of the elastin-laminin receptor. Here, by using the techniques of patch-clamp, spectrofluorimetry and confocal microscopy, we demonstrate that circulating concentrations of EP activate low specificity calcium channels on human umbilical venous endothelial cells, resulting in increase in cytoplasmic and nuclear free calcium concentrations. This action is independent of phosphoinositide metabolism. Furthermore, these effects are inhibited by lactose, an antagonist of the elastin-laminin receptor, and by cytochalasin D, an actin microfilament depolymerizer. These observations suggest that EP-induced signal transduction is mediated by the elastin-laminin receptor via coupling of cytoskeletal actin microfilaments to membrane channels and to the nucleus. Because vascular remodeling and carcinogenesis are accompanied by extracellular matrix modifications involving elastin, the processes here described could play a role in the elastin-laminin receptor-mediated cellular migration, differentiation, proliferation, as in atherogenesis, and metastasis formation.

Part 5. Regulation of the Cytoskeleton

5a Regulation of the Actin Cytoskeleton by PIP2 in Cytokinesis

MR Logan and CA Mandato
McGill University, Montreal, Ca
Biol. Cell (2006) 98, 377–388 [doi:10.1042/BC20050081]

Cytokinesis is a sequential process that occurs in three phases:

  • assembly of the cytokinetic apparatus, 
  • furrow progression and 
  • fission (abscission) of the newly formed daughter cells.

The ingression of the cleavage furrow is dependent on the constriction of an equatorial actomyosin ring in many cell types. Recent studies have demonstrated that this structure is highly dynamic and undergoes active polymerization and depolymerization throughout the furrowing process. Despite much progress in the identification of contractile ring components, little is known regarding the mechanism of its assembly and structural rearrangements. PIP2 (phosphatidylinositol 4,5-bisphosphate) is a critical regulator of actin dynamics and plays an essential role in cell motility and adhesion. Recent studies have indicated that an elevation of PIP2 at the cleavage furrow is a critical event for furrow stability. We discuss the role of PIP2-mediated signaling in the structural maintenance of the contractile ring and furrow progression. In addition, we address the role of other phosphoinositides, PI(4)P (phosphatidylinositol-4-phosphate) and PIP3 (phosphatidylinositol 3,4,5-triphosphate) in these processes.

Regulation of the actin cytoskeleton by PIPKs (phosphatidylinositol phosphate kinases) and PIP2 (phosphatidylinositol 4,5-bisphosphate)

PIP2 is generated by the activity of type I (PIPKIs) or type II (PIPKII) kinase isoforms (α, β, γ) which utilize PI(4)P (phosphatidylinositol 4-phosphate) and PI(5)P (phosphatidylinositol 5-phosphate) as substrates respectively. PIPKIs are localized to the plasma membrane and are thought to account for the majority of PIP2 synthesis, whereas PIPKIIs are predominantly localized to intracellular sites. PIP2 plays a key role in re-structuring the actin cytoskeleton in several ways. In general, high levels of PIP2 are associated with actin polymerization, whereas low levels block assembly or promote actin severing activity. PIP2 facilitates actin polymerization in multiple ways such as:

(i) activating N-WASp (neuronal Wiskott–Aldrich syndrome protein)- and Arp2/3 (actin-related protein 2/3)-mediated actin branching, 
(ii) binding and impairing the activity of actin-severing proteins, such as gelsolin and cofilin/ADF (actin depolymerizing factor); and
(iii) uncapping actin filaments for the addition on new actin monomers

This polymerization signal is counteracted by the generation of IP3 (inositol 1,4,5-triphosphate) and DAG (diacylglycerol), following PLC (phospholipase C)-mediated hydrolysis of PIP2. IP3-mediated activation of Ca2+/CaM (calmodulin) promotes the activation of severing proteins such as gelsolins and cofilin, which lead to solubilization of the actin network (Figure 1). In addition to influencing actin polymerization, PIP2 modulates the function of several actin cross-linking and regulatory proteins which are critical for the assembly of stress fibres, gel meshworks and membrane attachment. For example, PIP2 negatively regulates cross-linking mediated by filamin and the actin-bundling activity of α-actinin. In contrast, PIP2 induces conformational changes in vinculin, talin and ERM (ezrin/radixin/moesin) family proteins to promote anchoring of the actin cytoskeleton to the plasma membrane. PLC-mediated hydrolysis of PIP2 and the downstream activation of Ca2+/CaM and PKC (protein kinase C) also influences actin-myosin based contractility. Ca2+/CaM activates MLCK (myosin regulatory light chain kinase), leading to phosphorylation of the MLC (myosin regulatory light chain). Similarly, PKC has been shown to phosphorylate and activate MLC (Figure 1).

Figure 1 Summary of PIP2-mediated regulation of the actin cytoskeleton

Role of PIP2-mediated signaling in cell division

Prior to cell division cells undergo a global cell rounding which is a prerequisite step for the initiation of the cleavage furrow. In frog, sea urchin and newt eggs these shape changes correlate with an increase in cortical tension that precedes or occurs near the onset of the cleavage furrow.  Precise mapping of the changes in cortical tension have shown that peaks of tension are propagated in waves that occur in front of and at the same time as furrow initiation. These tension waves are generated by actomyosin-based contractility and subside after the furrow has passed. Experiments in Xenopus eggs, zebrafish and  Xenopus embryos indicated that site-specific Ca2+ waves were generated within the cleavage furrow that would be predicted to coincide with peaks of cortical tension. The injection of heparin, a competitive inhibitor of IP3 receptors, or Ca2+ chelators were both demonstrated to significantly delay or arrest furrowing , and a similar inhibitory effect was observed of microinjected PIP2 antibodies that caused a depletion of the intracellular pool of DAG and Ca2+ in Xenopus blastomeres. In addition, the increase in cortical contractility of Xenopus oocytes has been shown to occur via a PKC-dependent pathway. Together, these studies demonstrate a role for PIP2-mediated signaling at the early stages of cytokinesis.
Recent studies have supported that PIP2-mediated signaling also plays a critical role in ingression of the cleavage furrow, although significant differences have been shown in the localization of PIP2 and the role of PLC. Lithium and the PLC inhibitor, U73122, caused a rapid (within minutes) regression of cleavage furrows in crane fly spermatocytes, but did not block their initial formation. PIP2 may become concentrated within the cleavage furrow and could facilitate anchoring of the plasma membrane to structural components of the actomyosin ring. A PIPKI homologue, its3, and PIP2 were reported at the septum of dividing fission yeast, Schizosaccharomyces pombe. A temperature sensitive mutant of its3 exhibited disrupted actin patches, following a shift to the restrictive temperature, and also impaired cytokinesis. Although a contractile ring was still evident in these cells, abnormalities, such as an extra ring, were found. Two recent studies demonstrated an increase in PIP2-specific GFP-labeled PH domains within the cleavage furrow of mammalian cells. Both of these reports suggested de novo synthesis of PIP2 occurs within the furrow. Another study found that endogenous and over-expressed PIPKIβ, but not PIPKIγ, concentrated in the cleavage furrow of CHO (Chinese hamster ovary) cells. The expression of a kinase-dead mutant of this isoform and microinjection of PIP2-specific antibodies both caused a significant increase in the number of multinucleated cells. A multinucleated phenotype was, similarly, observed in multiple cell lines (CHO, HeLa, NIH 3T3 and 293T) transfected with high levels of PIP2-specific PH domains, synaptojanin [which dephosphorylates PIP2 to PI(4)P], or a kinase-dead mutant of PIPKIα. In addition, a small percentage of CHO and HeLa cells expressing high levels of PIP2-specific PH domains or synaptojanin showed signs of F-actin dissociation from the plasma membrane.  CHO cells transfected with PIP2 PH domains, but not PH domains specific for PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PIP3, also exhibited impaired furrow expansion induced by the application of hypotonic buffer. This suggests one of the primary roles of PIP2 is to promote cytoskeleton–membrane anchoring at the furrow.
Role of PI3Ks (phosphoinositide 3-kinases) and PI4Ks (phosphoinositide 4-kinases) in cytokinesis PI4Ks generate the PIPKI substrate, PI(4)P, and play a critical role in PIP2 generation.  Studies in lower organisms support  the requirement of PI4Ks for cytokinesis. In Saccharomyces cerevisiae two PI4Ks, STT4 and PIK1, have non-overlapping functions in Golgi-tomembrane trafficking and cell-wall integrity respectively.  Both genes are also required for cell division. Conditional mutants of Pik1p exhibited a cytokinesis defect: cells arrest with large buds and fully divided nuclei. In addition, STT4 was identified as a gene implicated in reorientation of the mitotic spindle prior to cytokinesis.  Spermatocytes derived from fwd mutant males had unstable furrows that failed to ingress and abnormal contractile rings with dissociated myosin II and F-actin, fwd has homology with yeast PIK1 and human PI4KIIIβ. Although PIK1 is an essential gene in yeast, the deletion of fwd was not lethal and female flies were fertile.  A study in fission yeast suggests that PI4Ks may be recruited to the furrow, as reported for PIPKs. Desautels et al. (2001) identified a PI4K as a binding partner of Cdc4p, a contractile ring protein with homology to the myosin essential light chain. A Cdc4p mutant, G107S, abolished the interaction with PI4K and induced the formation of multinucleated cells with defects in septum formation. This finding suggests that, at least for fission yeast, anchoring of PI4K to the contractile ring may concentrate PI(4)P substrate within the furrow for subsequent PIP2 generation.
An increased synthesis of PIP2 by PIPKIs at the cleavage furrow is anticipated to promote both actin polymerization and structural support to the contractile ring. Structural proteins of the contractile ring regulated by PIP2 include anillin, septin and ERM proteins. The concentration of PIP2 at the cleavage furrow is postulated to be a critical molecule in the recruitment of these proteins and their integration with the actomyosin ring. Anillin exhibits actin-bundling activity and is required at the terminal stages of cytokinesis in Drosophila and human cells.  The depletion of anillin in Drosophila and human cells causes cytokinesis failure, which is correlated with uncoordinated actomyosin contraction of the medial ring. Anillin also functions as a cofactor to promote the recruitment of septins to actin bundles. Mutations within the PH domain of anillin were recently demonstrated to impair septin localization to both the furrow canal and the contractile ring in Drosophila cells, blocking cellularization and furrow progression. Septins have also been shown to bind to phosphoinositides and this interaction regulates their subcellular localization. The mammalian septin, H5, bound PIP2 and PIP3 liposomes at its N-terminal basic region, which is conserved in most septin proteins. The over-expression of synaptojanin and treatment with neomycin (which depletes cellular PIP2) both caused disruption of actin stress fibres and dissociation of H5 from filamentous structures in Swiss 3T3 cells. Septins are co-localized with actin at the cleavage furrow and form ring structures that are postulated to structurally support  the contractile ring.
Studies suggest that PLC-mediated hydrolysis of PIP2 and the subsequent release of intracellular Ca2+ stores is a necessary event for furrow stability and ingression.  A role for Ca2+ is similarly supported by previous findings that Ca2+ waves were localized to the cleavage furrow in frog embryos, eggs and fish. PLC second messengers have also been implicated in cytokinesis. For example, CaM was localized to mitotic spindles of HeLa cells and the inhibition of its activity was reported to cause cytokinesis defects. A recent RNAi (RNA interference) screen also identified PI4Ks and PIPKs, but not PLC genes, as critical proteins for cytokinesis in Drosophila.  This may indicate PLC is required for completion of furrowing, rather than its initiation.
It is hypothesized that PLC activity may promote actin filament severing through the activation of Ca2+-dependent actin-severing proteins, such as gelsolin and cofilin. Depending on the localization of PLC, this could either drive disassembly of actin filaments of the medial ring or the cortical actin network. Furthermore, the activation of PKC and CaM would activate actomyosin contraction via the phosphorylation of MLCK. At the furrow, PKC and CaM could act in concert with the Rho effectors ROCK and Citron kinase, which also phosphorylate and activate MLC.
The activation of CaM and/or PKC may also provide positive feedback for the recruitment of PIP2 effectors and regulate GTPase-mediated actin polymerization. Both PKC and CaM have been shown to promote the dissociation of MARCKS (myristoylated alanine-rich C kinase substrates) family proteins from PIP2. MARCKS are postulated to play a major regulatory role in phosphoinositide signalling by sequestering PIP2 at the membrane. Thus the activation of PKC and CaM promotes PIP2 availability for the recruitment of PH-domain-containing effector proteins. Studies in yeast and mammalian cells have supported that CaM and PKC can mediate positive feedback for PIP2 synthesis by activating PIPKs.

Signaling Crosstalk: Role of GTPases and Phosphoinositides

On the basis of the present available data, PIP2 has been shown to be a critical molecule for structural integrity of the contractile ring and furrow stability. However, the observation that furrows are initiated in cells treated with agents that either sequester PIP2 or prevent its hydrolysis suggests PIP2 does not provide the originating signal for furrow formation. Recent studies suggest that the recruitment and activation of RhoA may provide this early signal.

Figure 2 Proposed model of PIP2 and GTPase signaling at the cleavage furrow

Ect2, is recruited to the cleavage furrow via its interaction withMgcRacGAP at the central spindle. Ect2 and MgcRacGAP regulate the activities of Rho GTPases (RhoA, Cdc42 and Rac) and are functionally implicated in the assembly of the contractile ring. Active RhoA and Cdc42 are increased at the furrow, whereas Rac is suppressed (grey). Furrow-recruited GTPases (RhoA, ARF6 and Cdc42) are predicted to activate PIPKI, leading to the generation of PIP2. PI3K activity is suppressed at the furrow (grey), which may be due to MgcRacGAP-mediated inhibition of Rac and/or the activity of the PIP3 phosphatase, PTEN. Cycles of PIP2 synthesis and hydrolysis by PLC are thought to play a critical role in re-structuring the contractile ring throughout the duration of furrowing. PIP2-mediated activation of anillin, septins and ERM proteins promotes cross-linking and membrane anchoring of the contractile ring. PLC-mediated activation of PKC and CaM can facilitate the contraction of the actomyosin ring, similar to RhoA effectors, ROCK and Citron kinase. CaM may also regulate IQGAP–Cdc42 interactions, and thereby modulate actin organization. It is hypothesized that Cdc42-mediated actin polymerization via effectors, such as N-WASp (neuronalWiskott–Aldrich syndrome protein) and Arp2/3 (actin-related protein 2/3), may reduce membrane tension outside the inner region of RhoA-mediated contractility.
Actin core bundle fimbrin

Actin core bundle fimbrin (Photo credit: Wikipedia)

English: Diagram showing Actin-Myosin filament...

English: Diagram showing Actin-Myosin filaments in Smooth muscle. The actin fibers attach to the cell wall and to dense bodies in the cytoplasm. When activated the slide over the myosin bundles causing shortening of the cell walls (Photo credit: Wikipedia)

English: Figure 2: The matrix can play into ot...

English: Figure 2: The matrix can play into other pathways inside the cell even through just its physical state. Matrix immobilization inhibits the formation of fibrillar adhesions and matrix reorganization. Likewise, players of other signaling pathways inside the cell can affect the structure of the cytoskeleton and thereby the cell’s interaction with the ECM. (Photo credit: Wikipedia)

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