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Archive for the ‘Inflammasome’ Category


LIVE 9/21 3:20PM to 6:40PM KINASE INHIBITORS FOR CANCER IMMUNOTHERAPY COMBINATIONS & KINASE INHIBITORS FOR AUTOIMMUNE AND INFLAMMATORY DISEASES at CHI’s 14th  Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

#BostonDOT16

@BostonDOT

 

KINASE INHIBITORS FOR CANCER IMMUNOTHERAPY COMBINATIONS

3:20 Chairperson’s Opening Remarks

Guido J.R. Zaman, Ph.D., Managing Director & Head of Biology, Netherlands Translational Research Center B.V. (NTRC)

3:25 FEATURED PRESENTATION: Inhibition of PI3K and Tubulin

Doriano_Fabbro

Doriano Fabbro, Ph.D., CSO, PIQUR Therapeutics

The PI3K signaling pathway is frequently activated in tumors. PQR309 is a selective dual inhibitor of PI3K and mTOR (currently in Phase I) in cancer patients. The preclinical pharmacology and toxicology of PQR309 is presented, including its activity in lymphoma preclinical models. In addition, we elucidate structural factors defining the PI3K inhibitory activity and tubulin-binding of PQR309 derivatives.

  • PQR309 & GDC0941 arrest cells i G1/S (typical for PI3K/mTOR Inhibitor)
  • What drives Antiproliferative Activity of BKM120: PI3K or MT or both?
  • BKM120 Binds to beta-Tubulin/alpha -Tubulin Interfere
  • T2R-TTL complex
  • Orientation of BKM120 in PI3K
  • PQR309 – is a brain penetrating, PK and BAV by PO, good metabolic stability
  • PQR309 ANti-proliferative in Lymphoma
  • Clinical efficacy – Now in Phase II

4:05 Design and Development of a Novel PI3K-p110β/δ Inhibitor, KA2237 with Combined Tumor Immunotherapeutic, Growth Inhibition and Anti-Metastatic Activity

Stephen_Shuttleworth

Stephen Shuttleworth, Ph.D., FRSC, CChem, CSO, Karus Therapeutics Ltd.

The design and development of KA2237, a novel and selective inhibitor of PI3K-p110β/δ, will be described. This molecule has clinical potential in the treatment of solid and hematological malignancies, through its direct inhibition of tumor growth and metastatic spread, and through immunotherapeutic mechanisms. Phase I studies for KA2237 are scheduled to commence in Q2 2016 at the MD Anderson Cancer Center.

  • Design & Development of Novel, Oral, selective PI3K enzyme family: CLass I,II, III, IV based upon:
  • Class I IA IB
  • KA2237: DUal PI3K – p110beta/delta-selective inhibitor: CTL, Treg, p1 106 T sell response
  • Molecular signature in the tumor
  • WT p110delta, WT 1 10beta+, Mutant p1 10Beta+, PTEN-null, Ibrutinib-resistance, Growth inhibition; suppression of metastesis (p110beta
  • small molecule combination agents: potential aided by selectivity over p110
  • KA2237: clinical Pi3K-p110beta/delta Inhibitor- ATP -comtetitive
  • Doxorubicin -cytotoxic control
  • KA2237 superior activity to Idelasib
  • KA2237 – suppression of micro-metastasis in 4T1 synergenic model
  • Tumor Growth inhibition Pre-Surgery
  • Tumor Re-Growth Inhibition Post-Surgery
  • metastasis post surgery
  • Tumor-free mice post-surgery
  • CHemistry: IHC -pAKT; IHC – FOxp3+
  • KA2237 inhibits HGF-stimulated 4T1 tumor
  • 2004 – Preclinical develpemnt PI3K is reported
  • 2006 First PI#K is enter Clinical Trials
  • Targeting p1110Beta (PIKeCB) mutations in cancer with KA2237
  • DIscovery of the mutations lead drug discovery
  • KA@@#&: Potential in treatment of B-Cell Lymphom AS IN TARGETING IBRUTINIB RESISTENCE
  • GROWTH INHIBITION IN HEMATOLOGICAL CANCERS TUMOE CELL LINE PANEL
  • KA2237 – differentiated from competing Pi3K is Superior efficacy cf. p110delta
  • Combination: Not histone deacetylase but a tubulin deacetylase – Hsp90 ans Hsp70
  • T cell exhausion: Tumor growth inhibition vs Suppression of lung metastasis
  • Tumor BiologyRationale vs Clinical Agents
  • Oncogenic mutants, solid tumor supression magrophage, combination PD-1, CTLA$
  • FDA -approved kinase inhibitors

Summary

  1. phase I clinical study commenced in pathients with B cell Lymphoma
  2. Potential for treatment of solid and hematological malignancies

4:35 InCELL Pulse: A Novel Cellular Target Engagement Assay Platform for Drug Discovery

Treiber_Daniel

Daniel Treiber, Ph.D., Vice President, KINOMEscan, DiscoverX Corporation

InCELL Pulse is a quantitative and rapid method for measuring cellular target engagement potencies for small molecule inhibitors. InCELL Pulse capitalizes on two novel DiscoverX technologies, Enzyme Fragment Complementation (EFC) and Pulse Denaturation, which overcome the limitations of related target engagement methods. Examples across multiple target classes will be described.

  • InCELL Pulse – cellular Target ENgagement Assays
  • cellular thermal stabilization-based approach
  • simple, rapid and generig cellular alternative to CETSa
  • Thermal melting Curves vs Isothermal Inhibitor EC50 curves
  • Pulse Denaturation compound binding, or not binding
  • ABL1 Tyrosine Kinase – dose response curve – allosteric Inhibitor
  • MTH1 Hydrolase: InCELL Pulseassay validated for multiple substrate-competitive inhibitors
  • Validated InCELL Pulse Assays for Diverse Kinases
  • Kinase targets; BRAF, MEC1

Summary

  1. validation across proteins

TTP Labtech4:50 Potential Application of Fluorescence Lifetime Assays to Enable Robust, Rapid Protein Binding Assays

Wylie_Paul

Paul Wylie, Ph.D., Head, Applications, TTP Labtech

Current methods to screen protein binding interactions often have limitations due to the reliance on antibodies, but also interference from fluorescent molecules. Fluorescence lifetime has the potential to overcome these problems through directly labelled proteins and lifetime measurements that are independent of total fluorescence intensity.

  • Protein binding as a target class
  • protein-protein interactions (PPIs)
  1. FRET/HTRF
  2. FP
  3. AlphaScreen

What new in FLT?

  • long lifetime fluorophores, economical reagent platform
  • directly labelled reagents – no antibodies
  • independent of total intensity – reduced interference
  • robustness screen vs nuisance screen – caspase-3
  • productive; reduction false positives: FRET
  • protein-binding assays & FLT formats:
  1. protein – small molecule binding – CECR2
  2. protein – peptide binding: long and sholt lifetime
  3. Site-specific labelling vs Non-selective labelling
  4. Toolbox for PoC
  5. Detection reagents
  6. Further develop technology

5:05 Refreshment Break in the Exhibit Hall with Poster Viewing

 

6:40 End of Day

 

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Anti-tumor necrosis factor drugs (TNF inhibitors) is the treatment for otulipenia, a new inflammatory disease discovered by NIH researchers using NGS – Inflammation

Reporter: Aviva Lev-Ari, PhD, RN

NIH researchers discover otulipenia, a new inflammatory disease – Rare and sometimes lethal disease affects young children

An international network of scientists studying inflammatory diseases identified four children from Pakistani and Turkish families with unexplained skin rashes and inflamed joints. NIH scientists then searched for disease-causing genes using next-generation DNA sequencing, technology that allows researchers to sequence DNA quickly and economically.

Once they found that the OTULIN gene was abnormal in the sick children, they studied the immune pathway in order to understand the mechanisms of disease and to improve treatment of these patients. They discovered a problem in the processing of a small protein, ubiquitin, which is critical to the regulation of many other proteins in the body, including immune molecules. In the affected children, the inability to remove the ubiquitin proteins from various molecules resulted in an increased production of chemical messengers that lead to inflammation (inflammatory cytokines).

The researchers determined that the children with otulipenia might respond to drugs that turned off tumor necrosis factor, a chemical messenger involved in systemic inflammation. Inflammation subsided in the children who had been treated with anti-tumor necrosis factor drugs (TNF inhibitors). TNF inhibitors are also used to treat chronic inflammatory diseases such as rheumatoid arthritis.

SOURCE

https://www.nih.gov/news-events/news-releases/nih-researchers-discover-otulipenia-new-inflammatory-disease

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Targeted Liposome Based Delivery System to Present HLA Class I Antigens to Tumor Cells: Two papers

Reporter: Stephen J. Williams, Ph.D.

 

Abstract

Cell-mediated immunotherapies have potential as stand-alone and adjuvant therapies for cancer. However, most current protocols suffer from one or more of three major issues: cost, safety, or efficacy. Here we present a nanoparticle delivery system that facilitates presentation of an immunogenic measles antigen specifically in cancer cells. The delivery system does not contain viral particles, toxins, or biologically derived material. Treatment with this system facilitates activation of a secondary immune response against cancer cells, bypassing the need to identify tumor-associated antigens or educate the immune system through a primary immune response. The delivery system consists of a stealth liposome displaying a cancer-specific targeting peptide, named H1299.3, on its exterior surface and encapsulating H250, an immunogenic human leukocyte antigen class 1 restricted peptide. This targeted-nanoparticle facilitates presentation of the H250 peptide in major histocompatibility complex class I molecules. Activation is dependent on the targeting peptide, previous antigen exposure, and utilizes a novel autophagy-mediated mechanism to facilitate presentation. Treatment with this liposome results in a significant reduction of tumor growth using an aggressive LLC1 model in vaccinated C57BL/6 mice. These data provide proof-of-principle for a novel cell-mediated immunotherapy that is scalable, contains no biologically derived material, and is an efficacious cancer therapy.

Introduction

Cell-mediated (CM) immunotherapies for cancer treatment are designed to activate the body’s adaptive immune responses against a malignant growth.1,2 Generally, the goal of a CM response is to activate a cytotoxic T-cell response against a tumor to eliminate cancer cells. The principle of these treatments is straightforward, yet current work studying the complexity of the tumor micro-environment2,3 as well as methods that attempt to directly activate T cells against tumor antigens4,5,6 demonstrate the difficulty associated generating an immune response against a tumor.

Several CM cancer immunotherapies exist today. Major examples include PD-1 inhibitors, injection of live virus or viral particles into tumors, and adoptive T-cell therapies.1,6,7,8 However, concerns regarding efficacy, safety, and/or cost have limited the use of many of these treatments. To address these concerns, we sought to develop a novel treatment based on developing a fully synthetic, minimal delivery system that facilitates presentation of human leukocyte antigen (HLA) class I restricted immunogenic peptides specifically on cancer cells without using live virus, viral subunits, or biologically derived material.

Based on these requirements, we developed a liposomal based agent consisting of a neutral, stealth liposome that encapsulates a synthetically manufactured immunogenic HLA class I restricted peptide derived from measles virus.1,2,9 In addition, the liposome has a targeting peptide on the external surface that both specifically accumulates in cancer cells and facilitates presentation of the immunogenic peptide in HLA class I molecules (Figure 1a). Thus, this treatment is designed to generate a secondary CM immune response specifically against the tumor if the patient was previously vaccinated against or infected with measles.

Figure 1

The minimal antigen delivery system consists of three components. (a) PEGylated stealth liposomes are loaded with an immunogenic human leukocyte antigen (HLA) class 1 restricted peptide derived from measles virus, named H250. The surface of the liposome

In this proof-of-concept study, we synthesized a liposome that encapsulates H250,1 an immunogenic HLA class 1 restricted peptide identified from measles hemagglutinin protein. The liposome is designed to specifically internalize in cancer cells by displaying the recently identified targeting peptide H1299.3 on the exterior surface (Figure 1b).10 H1299.3 is a 20mer, cancer-specific targeting peptide that was recently identified by our group. The peptide was identified using a novel phage display technique that allows for selection of cancer-specific targeting peptides that preferentially internalize in cancer cells via a defined mechanism of endocytosis. This peptide was dimerized on a lysine core and is fully functional outside the context of the phage particle. The H1299.3 peptide accumulates specifically in a panel of non-small cell lung cancer (NSCLC) cell lines compared to a normal bronchial epithelial cell control cell line via a clathrin-dependent mechanism of endocytosis. In this study, we demonstrate that H1299.3 facilitates functional presentation of an immunogenic antigen in both major histocompatibility complex (MHC) and HLA class I molecules as indicated by CD8+-specific interferon (IFN)γ secretion. In addition, H1299.3 facilitated presentation utilizes an autophagy-dependent mechanism. Finally, treatment with H1299.3 targeted liposomes containing H250 substantially reduces the growth rate of subcutaneous LLC1 tumors implanted in vaccinated C57BL/6 mice compared to treatment with vehicle control.

Result summarized:

  1. The H1299.3 targeting ligand specifically accumulates in cancer and facilitates HLA class I presentation: H250 is an immunogenic peptide identified from sequencing peptides present in HLA A*0201 molecules following measles infection. identified two donors that were HLA A*02 positive and had previously been vaccinated against measles virus (the human NSCLC cell line, H1993, which we determined to be HLA A*02 positive)
  2. identified three different cancer-specific targeting peptides that internalize into H1993 that have been previously published: H1299.2, H2009.1, and H1299.3. Each of these peptides specifically internalize in NSCLC cell lines compared to normal bronchial epithelial cells
  3. H1299.3 facilitated HLA class I presentation requires autophagy. H1299.3 peptide colocalizes with Lamp-1 which is a marker of both lysosomes and autolysosomes, therefore it was possible autophagy involved and shown that H1299.3 colocalizes with autophagosomes.  Chlorpromazine, which inhibits clathrin coated mediatated endocytosis, decreased the HLA1 presentation of H250.
  4. H1299.3-targeted liposomes encapsulating H250 reduce tumor burden in vivo. Mice were first vaccinated against H250.  The J1299.3 targeted liposome encapsulation H250 reduced tumor growth of LLC1 s.c. xenograpfts  by 50%.
J Transl Med. 2011 Mar 31;9:34. doi: 10.1186/1479-5876-9-34.

Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy.

Abstract

BACKGROUND:

Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge.

METHODS:

To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro.

RESULTS:

We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses.

CONCLUSION:

Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

[PubMed – indexed for MEDLINE]

Free PMC Article

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Immune System in Perspective

Curator: Larry H. Bernstein, MD, FCAP

LPBI

How regulatory T cells work
Vignali DAA, Collison LW & Workman CJ
Nature Reviews Immunology 8, 523-532 (July 2008) |   doi:10.1038/nri2343
http://www.nature.com/nri/journal/v8/n7/full/nri2343.html

Regulatory T (TReg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting antitumour immunity. Given that TReg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we describe the basic mechanisms used by TReg cells to mediate suppression and discuss whether one or many of these mechanisms are likely to be crucial for TReg-cell function. In addition, we propose the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate TReg-cell function.

How regulatory T cells work.

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Basic mechanisms used by Treg cells

This schematic depicts the various regulatory T (Treg)-cell mechanisms arranged into four groups centred around four basic modes of action. ‘Inhibitory cytokines’ include interleukin-10 (IL-10), interleukin-35 (IL-35) and transforming growth factor-β (TGF-β). ‘Cytolysis’ includes granzyme-A- and granzyme-B-dependent and perforin-dependent killing mechanisms. ‘Metabolic disruption’ includes high affinity IL-2 receptor α (CD25)-dependent cytokine-deprivation-mediated apoptosis, cyclic AMP (cAMP)-mediated inhibition, and CD39- and/or CD73-generated, adenosine–purinergic adenosine receptor (A2A)-mediated immunosuppression. ‘Targeting dendritic cells’ includes mechanisms that modulate DC maturation and/or function such as lymphocyte activation gene-3 (LAG3; also known as CD223)–MHC-class-II-mediated suppression of DC maturation, and cytotoxic T lymphocyte antigen-4 (CTLA4)–CD80/CD86-mediated induction of indoleamine 2,3-dioxygenase (IDO), which is an immunosuppressive molecule, by DCs.

An external file that holds a picture, illustration, etc. Object name is nihms-102080-f0002.jpg

Model for how effector T cells might boost Treg-cell function

This occurs in three stages. (a) Initial regulatory T (Treg)-cell activation induces production of regulatory factors such as interleukin-35 (IL-35). (b) Treg cells ‘sense’ the presence of recently activated effector T cells through a receptor–ligand interaction (cell surface or soluble). (c) This in turn boosts or potentiates Treg-cell function resulting in the enhanced production of regulatory mediators, such as IL-35, and perhaps the induction of new mediators.

 

Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity. Given that Treg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we discuss the basic mechanisms used by Treg cells to mediate suppression, and discuss whether one or many of these mechanisms are likely to be crucial for Tregcell function. In addition, we present the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate Treg-cell function.

Several sophisticated regulatory mechanisms are used to maintain immune homeostasis, prevent autoimmunity and moderate inflammation induced by pathogens and environmental insults. Chief amongst these are regulatory T (Treg) cells that are now widely regarded as the primary mediators of peripheral tolerance. Although Treg cells play a pivotal role in preventing autoimmune diseases, such as type 1 diabetes1,2, and limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease (IBD)3,4, they also block beneficial responses by preventing sterilizing immunity to certain pathogens5,6 and limiting anti-tumour immunity7. A seminal advance in the analysis of Treg cells came with the identification of a key transcription factor, forkhead box P3 (FOXP3), that is required for their development, maintenance and function8,9. Mice and patients that lack FOXP3 develop a profound autoimmune-like lymphoproliferative disease that graphically emphasizes the importance of Treg cells in maintaining peripheral tolerance10-12 (BOX 1). Although FOXP3 has been proposed as the master regulator of Treg cells that controls the expression of multiple genes that mediate their regulatory activity13,14, this has been recently challenged raising the possibility that other transcriptional events may operate upstream of and/or concurrently with FOXP3 to mediate Treg-cell development15.

While Foxp3 has proven to be an invaluable marker for murine Treg cells, its role in human Treg cells is less straightforward (see BOX 2 for a discussion of Treg-cell markers). Humans that lack FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), a severe autoimmune disease that presents early in infancy. Although FOXP3 appears to be required for human Treg-cell development and function, expression of FOXP3 alone is clearly not sufficient as a significant percentage of human activated T cells express FOXP3 and yet do not possess regulatory activity16-20. Furthermore, induction of FOXP3 in human T cells by transforming growth factor-β (TGFβ) does not confer a regulatory phenotype, in contrast to their murine counterparts20. Consequently, FOXP3 is not a good marker for human Treg cells (BOX 2). Whether this distinction is due to intrinsic differences between mouse and human FOXP3 and/or a requirement for an additional cofactor/ transcription factor is an important question that needs to be resolved.

Significant progress has been made over the last few years in delineating the molecules and mechanisms that Treg cells use to mediate suppression21,22. In this Review, we outline our current understanding of the mechanisms used by Treg cells to mediate suppression, and the challenges that lie ahead in defining their mode of action. We also discuss whether Treg cells are likely to depend on one, a few or many of these mechanisms. In addition, we propose that effector T cells may have a significant role in boosting and/or modulating Treg-cell function. Unless stated, we focus here primarily on the mechanisms that are used by thymus-derived natural CD4+CD25+ FOXP3+ Treg cells.

Basic mechanisms of Treg-cell function Defining the mechanisms of Treg-cell function is clearly of crucial importance. Not only would this provide insight into the control processes of peripheral tolerance but it would probably provide a number of potentially important therapeutic targets. Although this quest has been ongoing since interest in Treg cells was reignited in 199523, there has been significant progress in the last few years. From a functional perspective, the various potential suppression mechanisms of Treg cells can be grouped into four basic ‘modes of action’: suppression by inhibitory cytokines, suppression by cytolysis, suppression by metabolic disruption, and suppression by modulation of dendritic-cell (DC) maturation or function (FIG. 1).

Suppression by inhibitory cytokines Inhibitory cytokines, such as interleukin-10 (IL-10) and TGFβ, have been the focus of considerable attention as a mechanism of Treg-cell-mediated suppression. There has also been significant interest in their ability to generate induced (also known as adaptive) Treg-cell populations, either naturally in vivo or experimentally as a potential therapeutic modality (BOX 3). Although the general importance of IL-10 and TGFβ as suppressive mediators is undisputed, their contribution to the function of thymus-derived, natural Treg cells is still a matter of debate24. This is partly due to the general perception that Treg cells function in a contactdependent manner25,26. Indeed, in vitro studies using neutralizing antibodies or T cells that are unable to produce or respond to IL-10 and TGFβ suggested that these cytokines may not be essential for Treg-cell function25-28. However, this contrasts with data from in vivo studies29,30.

In allergy and asthma models, evidence suggests that both natural and antigen-specific Treg cells control disease in a manner that is, in part, dependent on IL-1029 and in some reports dependent on both IL-10 and TGFβ 31. Adoptive transfer of allergen-specific Treg cells induced significant IL-10 production by CD4+ effector T cells in the lung following allergen challenge and this Treg-cell-mediated control of disease was reversed by treatment with an IL-10- receptor-specific antibody32. However, suppression of allergic inflammation and airway hyper-reactivity, and increased production of IL-10 still occurred following transfer of IL-10- deficient Treg cells, suggesting that Treg cells can suppress the Th2-driven response to allergens in vivo through an IL-10-dependent mechanism, but that the production of IL-10 by Treg cells themselves is not required for the suppression observed. This contrasts with a recent study suggesting that the Treg-cell-specific ablation of IL-10 expression resulted in increased lung allergic inflammation and hyperreactivity33.

This scenario might occur in other disease models. For instance, the effects of IL-10 can only be partially attributed to Treg-cell-derived IL-10 in the immune response to hepatitis B virus34 and in the allograft tolerance response elicited by splenocytes exposed to non-inherited maternal antigens35. Recently, it was also shown that IL-10 is crucial for the control of various infections in which Treg cells have been reported to be involved including Mycobacterium tuberculosis36, Toxoplasma gondii37, Leishmania major38, and Trichinella spiralis39. However, Treg cells were not the source of IL-10 in all of these infection models.

By contrast, several studies have shown that IL-10 production by Treg cells is essential for the prevention of colitis in mouse models of IBD40. Moreover, it appears that the tumour microenvironment promotes the generation of FOXP3+ Treg cells that mediate IL-10- dependent, cell-contact independent, suppression41. Similarly, in UV-radiation-induced carcinogenesis, IL-10 production by Treg cells appears to be important for blocking anti-tumour immunity42. IL-10 produced by Treg cells also appears to be crucial for IL-10-mediated tolerance in a model of hepatitis induced by concanavalin A43 and tolerance to bacterial and viral superantigens44. In addition, recent papers suggest new roles for Treg-cell-derived IL-10 in the induction of feto-maternal tolerance45 and B-cell-enhanced recovery from experimental autoimmune encephalomyelitis46. Collectively, the picture that appears to be emerging is that the relative importance of Treg-cell-derived IL-10 is very dependent on the target organism or disease and on the experimental system. Furthermore, the Treg-cell-specific deletion of IL-10 did not result in the development of spontaneous systemic autoimmunity, but did result in enhanced pathology in the colon of older mice and in the lungs of mice with induced airway hypersensitivity, suggesting that the function of Treg-cell-derived IL-10 may be restricted to controlling inflammatory responses induced by pathogens or environmental insults33.

While some early in vitro studies using neutralizing antibodies to TGFβ or Treg cells lacking TGFβ 25,47 indicated that TGFβ was not required for natural Treg-cell function, other studies, both in vitro and in vivo suggested a critical role for Treg-cell surface bound TGFβ 48,49. Therefore, the importance of TGFβ for natural Treg-cell function has also been a controversial topic. Indeed, there has been considerably more focus recently on the importance of TGFβ in the development of induced Treg cells and perhaps in Treg-cell maintenance in general (BOX 3). However, there are studies that suggest that TGFβ produced by Treg cells may directly participate in effector T-cell suppression. For instance, effector T cells that are resistant to TGFβ-mediated suppression cannot be controlled by Treg cells in an IBD model50. In addition, TGFβ produced by Treg cells has been found to be important in the control of the host immune response to M. tuberculosis36, suppression of allergic responses31 and prevention of colitis in an IBD model51. Interestingly, TGFβ produced by Treg cells has also been implicated in limiting anti-tumour immunity in head and neck squamous-cell carcinoma52 and in follicular lymphoma53 by rendering T cells unresponsive to the tumour. TGFβ also appears to limit the anti-tumour activity of cytokine-induced killer cells54.

Membrane-tethered TGFβ can also mediate suppression by Treg cells in a cell-cell contactdependent manner48. Treg cells can control islet infiltration of CD8+ T cells and delay the progress of diabetes through membrane-tethered TGFβ 49. However, experiments using mice deficient in TGFβ-receptor (TGFβR) signalling in effector T cells or using TGFβ or TGFβR blocking reagents failed to show that membrane-tethered TGFβ is required for natural Treg cell development or function47. More recently, however, interest in membrane-tethered TGFβ has re-surfaced with the description of a previously unappreciated role for it in the tumour microenvironment. TGFβ associated with tumour exosome membranes appears to enhance the suppressive function of Treg cells and skew T cells away from their effector functions and towards regulatory functions55. Furthermore, ovalbumin-induced airway inflammation can be attenuated by heme oxygenase-1 through membrane-tethered TGFβ and IL-10 secretion by Treg cells56, a process that activates the Notch1–HES1 (hairy and enhancer of split 1) axis in target cells57. Thus, in light of the most current data, it now appears that soluble and/or membrane-tethered TGFβ may have a previously unappreciated role in natural Treg-cell function.

Recently, a new inhibitory cytokine, IL-35, has been described that is preferentially expressed by Treg cells and is required for their maximal suppressive activity58. IL-35 is a novel member of the IL-12 heterodimeric cytokine family and is formed by the pairing of Epstein–Barr virus induced gene 3 (Ebi3), which normally pairs with p28 to form IL-27, and p35 (also known as  Il12a), which normally pairs with p40 to form IL-12. Both Ebi3 and Il12a are preferentially expressed by murine Foxp3+ Treg cells58,59, but not resting or active effector T cells, and are significantly upregulated in actively suppressing Treg cells58. As predicted for a heterodimeric cytokine, both Ebi3−/− and Il12a−/− Treg cells had significantly reduced regulatory activity in vitro and failed to control homeostatic proliferation and cure IBD in vivo. This precise phenocopy suggested that IL-35 is required for the maximal suppressive activity of Treg cells. Importantly IL-35 was not only required but sufficient, as ectopic expression of IL-35 conferred regulatory activity on naive T cells and recombinant IL-35 suppressed T cell proliferation in vitro58. Although IL-35 is an exciting addition to the Treg-cell portfolio, there is clearly much that remains to be defined about this cytokine and its contribution to Treg-cell function. For instance, it remains to be determined if IL-35 suppresses the development and/or function of other cell types such as DCs and macrophages.

It is now clear that three inhibitory cytokines, IL-10, IL-35 and TGFβ, are key mediators of Treg-cell function. Although they are all inhibitory, the extent to which they are utilized in distinct pathogenic/homeostatic settings differs suggesting a non-overlapping function, which needs further refinement.

……….

How many mechanisms do Treg cells need? Although efforts to define the suppressive mechanisms used by Treg cells continue, an important question looms large. Is it likely that all these molecules and mechanisms will be crucial for Treg-cell function? There are three broad possibilities.

One, a single, overriding suppressive mechanism is required by all Treg cells Until the entire mechanistic panoply of Treg cells is defined, one cannot completely rule out this possibility. However, this possibility would seem unlikely as none of the molecules and/ or mechanisms that have been defined to date, when blocked or deleted, result in the complete absence of regulatory activity — a consequence that one might predict would result in a ‘Scurfy-like’ phenotype (BOX 1). So, although Treg cells that lack a single molecule, for instance IL-10, IL-35 or granzyme B, exhibit significantly reduced suppressor function, a scurfy phenotype does not ensue. Given that none of the current Treg-cell mechanisms can exclusively claim this distinction, it seems unlikely that any ‘unknown’ molecules or mechanisms could do so either.

Two, multiple, non-redundant mechanisms are required for maximal Treg-cell function In the studies conducted to date, Treg cells that lack various suppressive molecules have been shown to be functionally defective. This favours a scenario where there are multiple mechanisms that can be used by Treg cells but they are non-redundant, with each molecule contributing to the mechanistic whole. At present, this possibility would seem plausible. Indeed, this is supported by the recent analysis of mice possessing a Treg-cell-specific ablation of IL-10 expression, in which enhanced pathology was observed following environmental insult33. One would predict that at some point we should be able to generate knockout mice that lack a particular set of genes which results in a complete loss of Treg-cell activity. For this to be truly non-redundant, this list would probably be restricted and small (2–4 genes).

Three, multiple, redundant mechanisms are required for maximal Treg-cell function With the plethora of regulatory mechanisms described to date and the possibility of more yet to be identified, it is conceivable that there are multiple mechanisms that function redundantly. Such a redundant system would help to mitigate against effector T-cell escape from regulatory control. Also, given the very small size of the Treg-cell population, a sizable arsenal may be required at the height of an effector T-cell attack. Of course, it is possible that a semi-redundant scenario exists.

These possibilities have been discussed from the perspective of there being a single homogeneous Treg-cell population. However, as for helper T cell subsets it remains possible that a few or even many different Treg-cell subsets exist24. Each of these may rely on one or multiple regulatory mechanisms. Several recent studies have provided support for both phenotypic and functional heterogeneity amongst Treg cells. For instance, it has recently been shown that a small sub-population of Treg cells express the chemokine receptor CCR6, which is associated with T cells possessing an effector-memory phenotype102. CCR6+ Treg cells appeared to accumulate in the central nervous systems of mice with experimental autoimmune encephalomyelitis (EAE) suggesting that they may have a prevalent role in controlling responses in inflamed tissues. Heterogeneous expression of HLA-DR has also been suggested to mark different subpopulations of functionally distinct human Treg cells103. Indeed, HLADR positive Treg cells were found to be more suppressive than their DR negative counterparts. One might speculate that their enhanced inhibitory activity is due to DR-mediated ligation of the inhibitory molecule LAG3 expressed by activated effector T cells95,96.

So, if multiple suppressor mechanisms exist, how might these be integrated and used productively by Treg cells in vivo? We would propose the following possible models21. First, a ‘hierarchical’ model in which Treg cells possess many mechanisms that could be used but only one or two that are really crucial and consistently important in a variety of regulatory settings. Second, a ‘contextual’ model where different mechanisms become more or less important depending on the background or context in which the Treg cells reside and the type of target cell that they have to repress. For example, some cell types may be inhibited primarily by cytokines, whereas others are most effectively suppressed through lysis by Treg cells. Alternatively, different mechanisms may be more effective in different tissue compartments or in different disease settings. This notion is supported by the recent analysis of mice in which IL-10 expression was specifically ablated in Treg cells33. Whereas Treg-cell-derived IL-10 was not required for the systemic control of autoimmunity, it did seem to be required from the control of inflammatory events at mucosal interfaces such as the lungs and colon. As a clear picture of the available Treg-cell weaponry emerges, an important challenge will be to determine their relative importance and contribution to Treg-cell function in different disease models.

A hypothesis: effector T cells potentiate Treg-cell function? Most cellular interactions within the immune system are bidirectional, with molecular signals moving in both directions even though the interaction has broader unidirectional intentions (for example, CD4+ T-cell help). However, to date the general perception is that Treg cells suppress and effector T cells capitulate. We hypothesize that this is in fact an incomplete picture and that effector T cells have a very active role in their own functional demise. Three recent observations support this view. First, we have recently examined the molecular signature of activated Treg cells in the presence and absence of effector T cells and were surprised to find that it was strikingly different, with hundreds of genes differentially modulated as a consequence of the presence of effector T cells (C.J.W. and D.A.A.V., unpublished observations). Second, we have shown that Ebi3 and Il12a mRNA are markedly upregulated in Treg cells that were co-cultured with effector T cells, supporting the idea that effector T cells may provide signals which boost IL-35 production in trans58. Third, we found that Treg cells were able to mediate suppression of effector T cells across a permeable membrane when placed in direct contact with effector T cells in the upper chamber of a Transwell™ plate (L.W.C. and D.A.A.V., unpublished observations). Interestingly, this suppression was IL-35 dependent, as Ebi3−/− Treg cells were unable to mediate this ‘long-distance’ suppression. Collectively, these data suggest that it is the ‘induction’, rather than the ‘function’, of Treg-cell suppression that is contact-dependent and that effector T cells have an active role in potentiating Treg-cellmediated suppression. Therefore, we hypothesize that receptor–ligand interactions between the co-cultured CD4+ effector T cells and Treg cells initiate a signalling pathway that leads to enhanced IL-35 secretion and regulatory activity (FIG. 2). While the molecule that mediates this enhanced Treg-cell suppression is unknown, it is possible that IL-2 may serve this function104. Given the contrasting genetic profiles of activated Treg cells in the presence and absence of effector T cells, it seems possible that this interaction may boost the expression of other regulatory proteins. It may well be that effector T cells unwittingly perform the ultimate act of altruism.

Concluding remarks Although significant progress has been made over the last few years in defining the mechanisms that Treg cells use to mediate their suppressive function, there is clearly much that remains to be elucidated and many questions persist. First, are there more undiscovered mechanisms and/ or molecules that mediate Treg-cell suppression? What is clear is that the transcriptional landscape of Treg cells is very different from naive or activated effector T cells. There are literally thousands of genes that are upregulated (or downregulated) in Treg cells compared with effector T cells. Although it seems unlikely that all or many of these will be crucial for Treg-cell function, it is quite possible that a few undiscovered genes might be important. It should be noted that although we are discussing mechanisms here, it is clear that some of these molecules may perform key Treg-cell functions, such as Treg-cell homing and homeostasis, which are likely to indirectly influence their suppressive capacity in vivo but don’t directly contribute to their inhibitory activity. It is also possible that some of these unknown molecules may represent more specific markers for the characterization and isolation of Treg cells, a particularly important issue for the analysis and use of human Treg cells (BOX 2).

Second, which mechanisms are most important? An important but potentially complex challenge will be to determine if a few mechanisms are important in many Treg-cell settings or whether different mechanisms are required in different cellular scenarios. At present it is difficult to assess this objectively as these mechanisms have predominantly been elucidated in different labs using distinct experimental systems and thus none have really been compared in side-by-side experiments. Furthermore, only recently have conditional mutant mice been examined that have a regulatory component specifically deleted in Treg cells33.

It almost goes without saying that although defining the Treg-cell mode of action is of great academic importance, it is also essential in order to develop effective approaches for the clinical manipulation of Treg cells. Given the capacity of Treg cells to control inflammation and autoimmunity, and their implication in blocking effective anti-tumour immunity and preventing sterilizing immunity, it seems probable that a clear understanding of how Treg cells work will present definitive opportunities for therapeutic intervention.

Box 1 Scurfy mice: misplaced mechanistic expectations?

Mice that carry a spontaneous loss-of-function mutation (known as Scurfy mice) or a deletion of Foxp3 develop a fatal autoimmune-like disease with hyperresponsive CD4+ T cells9,12. More recently Foxp3:diptheria toxin receptor (DTR) knockin mice have allowed for the selective depletion of Treg cells following DT treatment105. These mice have been invaluable for dissecting the role of Foxp3 in Treg-cell function. Given the profound phenotype in these mice, there is a general expectation that genetic disruption of any key Treg-cell inhibitory molecule or mechanism would probably result in a Scurfy-like phenotype. Of course, it is also possible that deletion of a key Treg-cell gene may be more synonymous with DT-mediated Treg-cell depletion where Foxp3 may still serve to prevent expression of proinflammatory cytokines105. Nonetheless, this has lead to the notion that if mutant mice don’t have a Scurfy-like or a Treg-cell-depleted phenotype, then the disrupted gene probably isn’t important for Treg-cell function. This may not necessarily be correct. Indeed, it is possible that no mouse lacking a Treg-cell inhibitory effector molecule will ever be generated that develops a profound, spontaneous autoimmune disease21. It should be noted that mutant mice that are Helicobacter spp. and/or Citrobacter rodentium positive may have an exacerbated phenotype, as several studies have shown that opportunistic enteric bacteria can significant exacerbate gut pathology4. Ultimately, the occurrence of disease in knockout mice will depend on whether Treg cells rely on a single or multiple suppressive mechanisms. Given the number of genes induced or modulated by FOXP3, it is probable that a programme of intrinsic and extrinsic regulation is induced that involves multiple proteins9,13. Therefore, it would not be surprising if deletion of a single molecule does not provoke the profound Scurfy-like phenotype seen in mice that lack Foxp3.

Box 2. Treg-cell markers

Identifying discriminatory cell surface markers for the characterization and isolation of Treg cells has always been a critical goal. Although excellent markers exist for murine Treg cells, this goal has remained elusive for human Treg cells. Traditionally, murine and human Treg cells have been characterized as CD4+CD25+ (also known as interleukin-2 receptor α (IL-2Rα)). Indeed, murine Treg cells can be effectively isolated based on staining for CD4+CD25+CD45RBlow expression. However, the purity of isolated human Treg cells has always been an issue because T cells up-regulate CD25 upon activation106. Indeed, during the influenza or allergy season a substantial proportion of human CD4+ T cells can express CD25. Although the identification of forkhead box P3 (Foxp3) as a key regulator of Treg-cell development and function has facilitated their identification in the mouse8, many activated (non-regulatory) human T cells express FOXP3, precluding it as a useful marker for human Treg cells16-20. Consequently, the search for Treg-cell-specific cellsurface markers, particularly in humans, has continued in earnest with a growing number of candidates proposed (reviewed by Zhao and colleagues107). For instance, it was shown that the expression of CD127 (also known as IL-7R) is down-regulated on Treg cells and that this could be used to increase the purity of human Treg-cell isolation. Indeed, there is a 90% correlation between CD4+CD25+CD127low T cells and FOXP3 expression108, 109. In addition, it was recently found that Treg cells expressed a higher level of folate receptor 4 (FR4) compared with activated effector T cells110. It is also important to recognize that Treg cells, like their T helper cell counterparts, may be heterogeneous and thus a collection of cell surface markers could facilitate their isolation and functional characterization. Indeed, such heterogeneity has recently been described based on differential expression of HLA-DR or CCR6102,103. However, the general use of both markers remains to be fully established so it is quite probable that the search for better Treg-cell markers will continue for some time.

Box 3 Induced or adaptive Treg cells: development and mode of action

Naturally occurring FOXP3+CD4+CD25+ Treg cells develop in the thymus and display a diverse T-cell receptor (TCR) repertoire that is specific for self-antigens111,112. However, Treg cells can also be ‘induced’, ‘adapted’ or ‘converted’ from effector T cells during inflammatory processes in peripheral tissues, or experimentally generated as a possible therapeutic29,113,114. For instance, T regulatory 1 cells (Tr1) and T helper 3 cells (Th3) can be generated experimentally by, and mediate their suppressive activity through interleukin-10 (IL-10) and transforming growth factor-β (TGFβ), respectively114,115. Typically, these regulatory populations do not express FOXP3. In vivo, it has recently been suggested that stimulation of mouse effector T cells by CD103+ dendritic cells (DCs) in the presence of TGFβ and retinoic acid induces the generation of Foxp3+ T cells in the gutassociated lymphoid tissue (GALT)116-121. Furthermore, Treg cells can be preferentially induced in the periphery by exposure to αVβ8-integrin-expressing DCs122 or suppressor of cytokine signalling 3 (Socs3) −/− DCs123. Interestingly, independent of its role in generating induced Treg cells, TGFβ may also have an important role in helping to maintain Foxp3 expression in natural Treg cells124, a process that can be blocked by IL-4 or interferon-γ (IFNγ) 125. In contrast to mouse T cells, FOXP3 induction by TCR stimulation in the presence of TGFβ in human T cells does not confer a regulatory phenotype20. The mechanism of action of adaptive Treg cells may not necessarily be restricted to suppressive cytokines. Indeed, human adaptive Treg cells (CD4+CD45RA+ T cells stimulated with CD3- and CD46-specific antibodies) have also been shown to express granzyme B and killing target cells in a perforin-dependent manner126. Treg cells often have a restricted specificity for particular cell types, tumours or foreign antigens127. Therefore, induced Treg cells may be ideally suited to respond to infectious agents. This may also be of particular importance in the GALT and in the tumour microenvironment where TGFβ drives the conversion of induced Treg cells118,128. A significant challenge in deciphering data from in vivo experiments is to assess the contribution of natural Treg cells versus induced Treg cells, and to determine whether inhibitory molecules, such as IL-10 or TGFβ, were derived from the former or the latter (or elsewhere).

 

 

Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers.

Keisuke Kataoka, Yuichi Shiraishi, Yohei Takeda, Seiji Sakata, et al.
Nature may 23,2016; http://dx.doi.org:/10.1038/nature18294

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development1, 2, 3, 4, 5, 6. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma6, 7, 8, 9, 10. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3′ region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3′-untranslated region (UTR). Disruption of the Pd-l1 3′-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3′-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

 

Viruses are a dominant driver of protein adaptation in mammals.

David Enard, Le Cai, Carina Gwennap and Dmitri A Petrov.
eLife May 16, 2016; 5:e12469. http://dx.doi.org/10.7554/eLife.12469

Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes.

 

Purdue scientists use adaptors to advance CAR-T therapy

by Oliver Worsley | May 25, 2016

http://www.fiercebiotech.com/research/purdue-scientists-use-adaptors-to-advance-car-t-therapy

Chimeric antigen receptor (CAR) T cells, developed in the 1990s, are a genetically engineered type of T cell that can target a specific cancer. Now, scientists at Purdue University say they’ve made improvements in this strategy–overcoming the several limitations of traditional CAR-T therapy.

Purdue professor of chemistry Philip Low and his team presented their findings at the American Association for Cancer Research meeting in New Orleans last month.

T cells are a type of immune cell that recognizes and clears the body of invading cells or pathogens, like cancer. They are fine-tuned by the immune system in order to specifically target and kill these foreign invaders–but cancer cells may respond by jumping these safety barriers.

CAR-T therapy was therefore proposed and has been recently used for cancer treatment. It has been hailed for its promising remission rates after early stage clinical trials for acute lymphoblastic leukemia.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said in a university news release. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

They did this by teaming up with Endocyte ($ECYT) scientist Haiyan Chu and designing CAR T cells that require activation by a small molecule adaptor before proceeding. In this way, they can carefully control the amount of active CAR T cells in the circulation.

So far, they have only tried the novel therapy in animal models, but when they tested it in mice they observed antitumor activity only when both the CAR T cells and the correct adaptor molecules were present.

Low believes it will allow clinicians to target multiple cancer subtypes at once. “Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said in the release. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

– here’s the release

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Read More: CAR-T   Cancer

 

Purdue research may expand engineered T-cell cancer treatment

PURDUE UNIVERSITY

http://www.eurekalert.org/multimedia/pub/116141.php

A graphic depicting the activation and inactivation of CAR T cells through a small molecule adaptor is shown. Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery, and graduate student Yong Gu Lee led a team that designed new engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The system has the potential to control the engineered cells to overcome existing limitations in CAR T-cell therapy. CREDIT Purdue University image courtesy of Yong Gu Lee

Purdue University researchers may have figured out a way to call off a cancer cell assassin that sometimes goes rogue and assign it a larger tumor-specific “hit list.”

T cells are the immune system’s natural defense against cancer and other harmful entities in the human body. However, the cells must be activated and taught by the immune system to recognize cancer cells in order to seek out and destroy them. Unfortunately, many types of cancer manage to thwart this process.

 

In the 1990s scientists found a way to genetically engineer T cells to recognize a specific cancer. These engineered T cells, called chimeric antigen receptor, or CAR, T cells, have been recently used as treatment for cancer, said Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery who led the work.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

Low and Purdue graduate student Yong Gu Lee collaborated with Endocyte Inc. scientist Haiyan Chu to design genetically engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The technology has been tested in animal models but no human trials have been performed. A poster presentation describing the work was presented Tuesday (April 19, 2016) at the American Association for Cancer Research annual meeting in New Orleans.

“While the traditional CAR T cells could remain and replicate in the human body for many years, the adaptors we have created are expected to be excreted fairly quickly,” Lee said. “By controlling the level of adaptors in the system, we can control the numbers and potencies of active CAR T cells. Those that aren’t stimulated by an adaptor molecule are blind and do not recognize or target any cells. Eventually, if they remain inactive for a while, they should die and be eliminated from the body.”

A study in mice showed the anti-tumor activity was induced only when both the engineered CAR T cell and the correct adaptor molecules were present.

The system also offers the potential to treat multiple cancer subtypes at once, Low said.

“Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

The adaptor molecule serves as a bridge between the CAR T-cell and the cancer cell. It is made with a yellow dye called fluorescein isothiocyanate on one end, to which the engineered CAR T cells have been designed to bind, and a cancer-targeting molecule on the other.

Low’s research has focused on the design and synthesis of technologies for targeted delivery of therapeutic and imaging agents to treat cancer, inflammatory and autoimmune diseases, and infectious diseases.

He has developed molecules that target folate-receptors and prostate-specific membrane antigen on the surfaces of cancer cells. Approximately 85 percent of ovarian cancers; 80 percent of endometrial and lung cancers; and 50 percent of breast, kidney and colon cancers express folate receptors on their cellular surfaces. Prostate-specific membrane antigen receptors are found on nearly 90 percent of all prostate cancers. Other tumor-specific ligands developed by Low’s lab can target each of the other major human cancers, he said.

Each CAR T cell has thousands of receptors on its surface to which an adaptor molecule can bind. One CAR T cell could have a variety of adaptor molecules bound to its surface and the cancer cell it targets will depend on which of those adaptors first encounters a targeted cancer cell. Once the CAR T cell binds to a cancer cell, it begins the process of destroying it. When that process is complete, the CAR T cell is released and can bind to a new cancer cell, he said.

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

In addition to Low, Chu and Lee, members of the research group include Purdue postdoctoral research associates at the time of the study Srinivasarao Tenneti and Ananda Kumar Kanduluru.

Drug discovery is one of the priorities within Purdue Moves, an initiative designed to broaden the university’s global impact and enhance educational opportunities for its students. All of the moves fall into four broad categories: science, technology, engineering and math (STEM) leadership; world-changing research; transformative education; and affordability and accessibility.

The Purdue University Center for Drug Discovery supports more than 100 faculty in six colleges with research focused on several major disease categories: cancer; diabetes, obesity and cardiovascular; immune and infectious disease; and neurological disorders and trauma.

The center and drug discovery initiative builds upon Purdue’s strengths along all points of the drug discovery pipeline, including 14 core units to provide shared resources for analysis, screening, synthesis and testing of potential therapeutic compounds.

With more than 44 Purdue-developed compounds at various stages of preclinical development, and 16 in human clinical trials, Purdue is among the most productive universities in the world of drug discovery.

The center also is aligned with the university’s recently announced $250 million investment in the life sciences.

Endocyte Inc., a Purdue Research Park-based company that develops receptor-targeted therapeutics for the treatment of cancer and autoimmune diseases, funded the study, holds exclusive rights to the technology and assisted Purdue researchers in the development of the technology. Low is a founder and chief science officer of Endocyte Inc. and serves on the Endocyte board of directors.

AACR press release: http://www.aacr.org/Newsroom/Pages/News-Release-Detail.aspx?ItemID=874#.VxZFs2N8V0c

Endocyte press release: http://investor.endocyte.com/releasedetail.cfm?ReleaseID=965753

ABSTRACT

A Universal Remedy for CAR T Cell Limitations

Yong Gu Lee, Haiyan Chu, Srinivasarao Tenneti, Ananda Kumar Kanduluru, Philip S. Low

Chimeric antigen receptor (CAR) T cells show significant potential for treating cancer due to their tumor-specific activation and ability to focus their killing activity on cells that express a tumor antigen. Unfortunately, this promising therapeutic technology is still limited by: (1) an inability to control the rate of cytokine release and tumor lysis; (2) the absence of an “off switch” that can terminate cytotoxic activity when tumor eradication is complete; (3) a failure to eliminate tumor cells that do not express the targeted antigen; and (4) a requirement to generate a different CAR T cell for each unique tumor antigen. In order to address these limitations, we have exploited a low molecular weight bi-specific adaptor molecule that must bridge between the CAR T cell and its targeted tumor cell by simultaneously binding to the chimeric antigen receptor on the CAR T cell and the unique antigen on the tumor. Using this bispecific adaptor, one can control CAR T cell cytotoxicity by adjusting the concentration and rate of administration of the adaptor. Because the half life of the adapter is <20 minutes in vivo, termination of CAR T cell killing can be accomplished by cessation of adapter administration. Moreover, when heterogeneous tumors containing cells that express orthogonal antigens must be treated, the same CAR T cell can be targeted to multiple antigens by attachment of the same CAR ligand to the appropriate selection of tumor-specific ligands. Finally, when the targeted tumor antigen is also expressed at low levels on normal cells, tumor specificity can be achieved by adjusting the affinity of the tumor-specific ligand to enable CAR T cell engagement only when a highly multivalent interaction is possible. To experimentally demonstrate the aforementioned benefits of using low molecular weight bispecific adaptors, CAR T cells were constructed by fusing an anti-fluorescein isothiocyanate (FITC) scFv to a CD3 zeta chain containing the intracellular domain of CD137 (i.e. CAR4-1BBZ T cells). Then, to enable their tumor-specific cytotoxicity, a bispecific adaptor molecule comprised of fluorescein linked to a small organic ligand with high affinity and specificity for a tumor-specific antigen (FITC-SMC) was synthesized. For these studies, the tumor-specific ligands were: i) folate for recognition of the folate receptor that is over-expressed on ~1/3 of human cancers, ii) DUPA for binding to prostate specific membrane antigen that is over-expressed on prostate cancers, and iii) NK-1R ligand that is over-expressed on neuroendocrine tumors. The ability of the same clone of CAR4-1BBZ T cells to eliminate tumors expressing each of the above antigens was then demonstrated by administration of the desired FITC-SMC to mice injected with the CAR4-1BBZ T cells. Our data show that anti-tumor activity: i) is only induced when both CAR4-1BBZ T cells and the correct antigen-specific FITC-SMC are present, ii) anti-tumor activity and toxicity can be sensitively controlled by adjusting the dosing of FITC-SMC, and iii) treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired FITC-SMCs. Taken together, these data show that many of the limitations of CAR T cell technology can be addressed by use of a bi-specific adaptor molecule to mediate tumor cell recognition and killing.

 

 

CTLA-4 found in dendritic cells suggests New cancer treatment possibilities

Matthew Halpert, et al. Dendritic Cell Secreted CTLA-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Stem Cells and Development, 2016; http://dx.doi.org:/10.1089/scd.2016.0009

Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the “general” leading T cells “soldiers” to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.

  Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Halpert MM1, Konduri V1, Liang D1, Chen Y1, Wing JB2, Paust S3,4, Levitt JM1,5, Decker WK1,6.  Stem Cells Dev. 2016 May 15;25(10):774-87. doi: 10.1089/scd.2016.0009. Epub 2016 May 2.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

 

Non-invasive strategy to guide personalized cancer immunotherapy

Cancer immunotherapy is the rising hope to offer ultimate solutions for cancer. Neoantigens, derived from products of mutated genes in tumor cells, are found to be closely related to the efficacy of cancer immunotherapies. A non-invasive approach to identify unique, patient-specific neoantigens has been advanced by Dr. Steven Rosenberg’s group. A recent article published in Nature Medicine reported that a small population of circulating CD8+PD-1+ tumor-reactive T lymphocytes can be used to identify neoantigens, in addition to tumor-infiltrating T cells. The study paves the way for designing personalized cancer immunotherapy with a novel non-invasive approach.

Gros, A. et al.
Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.
Nat. Med. (2016)   http://dx. doi.org:/10.1038/nm.4501

Detection of lymphocytes that target tumor-specific mutant neoantigens-derived from products encoded by mutated genes in the tumor-is mostly limited to tumor-resident lymphocytes, but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(-), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8+ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8+ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8+lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8+ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8+PD-1+ compared with CD8+PD-1 TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8+ and the CD8+PD-1+ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8+ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

Cancer immunotherapy has experienced major progress in the last decade. Adoptive transfer of ex vivo–expanded tumor-infiltrating lymphocytes (TILs) can cause substantial regression of metastatic melanoma (1, 2). Blockade of the interaction of cytotoxic T lymphocyte antigen 4 (CTLA-4; also known as CD152) or programmed cell death 1 receptor (PD-1; also known as CD279) with their ligands using blocking antibodies alone or in combination have been shown to unleash an otherwise-ineffective immune response against melanoma (37), renal cell carcinoma (3), and non–small cell lung cancer (3). The antitumor responses observed in these clinical trials support the presence of naturally occurring tumor-reactive CD8+ T cells and their immunotherapeutic potential. In the particular case of TIL therapy, persistence of transferred tumor-specific T cell clones is associated with tumor regression (8). Moreover, retrospective clinical studies have shown an association of autologous tumor recognition by TILs and clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. However, the identification of the diverse repertoire of tumor-reactive cells limits the ability to study these cells, enhance clinical efficacy, and extend this therapy to other malignancies.

Melanoma TILs represent a heterogeneous population that can target a variety of antigens, including melanocyte differentiation antigens, cancer germline antigens, self-antigens overexpressed by the tumor, and mutated tumor neoantigens (11). The latter appear to be of critical importance for the antitumor responses observed after transfer of TILs, given the substantial regression of metastatic melanoma in up to 72% of patients in phase 2 clinical trials, in the absence of any autoimmune side effects in the great majority of patients (2). This contrasts with the modest antitumor activity but high prevalence of severe autoimmune manifestations observed after transfer of peripheral blood gene-engineered T cells expressing TCRs targeting shared melanocyte differentiation antigens MART1 and gp100 (12,13). Furthermore, T cells targeting mutated neoepitopes are not subject to negative selection in the thymus and may constitute the predominant naturally occurring tumor-reactive population in cancer patients. In support of this notion, a recent study reported the frequent detection and dominance of T cell populations targeting mutated epitopes in melanoma-derived TILs (14). Conversely, T cells targeting shared melanocyte differentiation antigens and cancer germline antigens in bulk melanoma TILs were represented at a strikingly low frequency (15). These findings have shifted our interest from the more accessible and commonly studied T cells targeting melanocyte differentiation antigens to T cells targeting unique patient-specific mutations. However, the often rare availability of autologous tumor cell lines necessary to study these reactivities, and the hurdles associated with the identification of the unique mutations targeted, have thus far hindered immunobiological studies of these T cell populations in the tumor.

Naturally occurring tumor-reactive cells are exposed to their antigen at the tumor site. Thus, the immunobiological characterization of T cells infiltrating tumors represents a unique opportunity to study their function and to identify the patient-specific repertoire of tumor-reactive cells. TCR stimulation triggers simultaneous upregulation of both costimulatory and coinhibitory receptors, which can either promote or inhibit T cell activation and function. Expression of the inhibitory receptors PD-1, CTLA-4, lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) is regulated in response to activation and throughout differentiation (16, 17). Chronic antigen stimulation has been shown to induce coexpression of inhibitory receptors and is associated with T cell hyporesponsiveness, termed exhaustion (18). Exhaustion in response to persistent exposure to antigen was first delineated in a murine model of chronic lymphocytic choriomeningitis virus (19), but has been observed in multiple human chronic viral infections (2022) as well as in tumor-reactive MART1-specific TILs (23, 24), and has provided the rationale for restoring immune function using immune checkpoint blockade. Conversely, 4-1BB (also known as CD137) is a costimulatory member of the TNF receptor family that has emerged as an important mediator of survival and proliferation, particularly in CD8+ T cells (2527). 4-1BB is transiently expressed upon TCR stimulation, and its expression has been used to enrich for antigen-specific T cells in response to acute antigen stimulation (28). However, expression of this marker has not been extensively explored in CD8+ lymphocytes infiltrating human tumors. In addition to changes in the expression of cosignaling receptors on the surface of T cells, antigen-specific stimulation typically results in clonal expansion. TCR sequence immunoprofiling can be used to monitor T cell responses to a given immune challenge even without a priori knowledge of the specific epitope targeted, through determination of the abundance of specific clonotypes (29, 30). However, there is limited knowledge regarding the TCR repertoire and the frequency of tumor-reactive clonotypes infiltrating human tumors.

We hypothesized that the assessment of unique phenotypic traits expressed by CD8+ TILs and TCR β chain (TCRβ; encoded by TRB) clonotypic immunoprofiling of lymphocytes infiltrating the tumor could provide a powerful platform to study antitumor T cell responses and evaluated their usefulness in identifying the diverse repertoire of tumor-reactive cells. Despite the accepted negative regulatory role of PD-1 in T cells, our findings establish that expression of PD-1 on CD8+ melanoma TILs accurately identifies the repertoire of clonally expanded tumor-reactive, mutation-specific lymphocytes and suggest that cells derived from this population play a critical role in tumor regression after TIL administration.

PD-1 was initially described to be expressed on a T cell hybridoma undergoing cell death (37). Its negative effect on T cell responses was first delineated in PD-1 knockout mice (38, 39). Since then, PD-1 expression and coexpression of other inhibitory receptors such as CTLA-4, TIM-3, BTLA, CD160, LAG-3, and 2B4 have become a hallmark of chronically stimulated T cells during chronic infection or in the tumor microenvironment. This altered phenotype, and the interaction of these receptors with their corresponding ligands on target cells, is associated with impaired proliferation and effector function frequently referred to as exhaustion (18, 24, 40). Expression of PD-1 in patients with chronic viral infections correlates with disease progression (22, 41). Additionally, CD8+ lymphocytes targeting melanoma differentiation antigens in the tumor express PD-1, CTLA-4, TIM-3, and LAG-3 and exhibit impaired IFN-γ and IL-2 secretion (23, 24), supporting a negative regulatory role of PD-1 and inhibitory receptors in naturally occurring T cell responses to cancer and providing a rationale for the treatment of cancer with immune checkpoint inhibitors.

In the present study, we found that expression of PD-1 on CD8+ melanoma TILs captured the diverse repertoire of clonally expanded tumor-reactive lymphocytes. TCRβ sequencing revealed that tumor-reactive and mutation-specific clonotypes were highly expanded in the CD8+ population and preferentially expanded in the PD-1+ population. This is consistent with the TCR stimulation-driven expression of this receptor on T cells (42). The inhibitory receptors TIM-3 and LAG-3 and the costimulatory receptor 4-1BB were also expressed on CD8+PD-1+ TILs and could also be used to enrich for tumor-reactive cells. PD-1 was consistently expressed at a higher frequency and was found to be more comprehensive at identifying the diverse repertoire of tumor-reactive cells infiltrating melanoma tumors, although the less frequent PD-1/TIM-3+ and PD-1/LAG-3+ subpopulations could also represent tumor-reactive cells (Supplemental Figure 4 and Supplemental Table 6). Additionally, previous studies from our laboratory showing coexpression of PD-1 and CTLA-4 (23), and our preliminary data supporting coexpression of PD-1 and ICOS (Supplemental Figure 5), suggest that other receptors may also be used to distinguish tumor-reactive cells. Our present results further support immunotherapeutic intervention using immune checkpoint blockade using PD-1, TIM-3, and LAG-3 blocking antibodies or 4-1BB agonistic antibody to restore the function of tumor-reactive lymphocytes, which is currently being actively pursued in the clinic (3, 4, 6, 7, 43). The potential cooperative mechanisms of inhibition of these receptors when engaged with their ligands (44, 45) suggests that the combined targeting of different inhibitory receptors can further enhance antitumor efficacy, as already shown with the combination of anti–PD-1 and anti–CTLA-4 (5). Our present results demonstrate that PD-1 identifies the clonally expanded CD8+ tumor-reactive population and suggest that expression of PD-1 on CD8+TILs could function as a potential predictive biomarker of antitumor efficacy using immune checkpoint inhibitors.

Naturally occurring tumor-reactive cells play a pivotal role in mediating antitumor responses after TIL transfer. Currently, expansion of TILs for patient treatment involves nonspecific growth of TILs from tumor fragments in IL-2, and the diversity and frequency of antitumor T cells present in the final T cell product used for treatment remains largely uncharacterized. Prospective clinical studies have reported that in vitro recognition of autologous tumor by TILs is associated with a higher probability of clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. This is consistent with the idea that both tumor-reactive and non–tumor-reactive cells may compete for cytokines in vivo, especially in the absence of vaccination. However, the isolation of the patient-specific repertoire of tumor-reactive cells is not possible with current technologies (14, 28, 4650). Our findings established that expression of PD-1, TIM-3, LAG-3, and 4-1BB in CD8+ TILs can be used to enrich for tumor-reactive cells, regardless of the specific antigen targeted. One potential concern with isolating T cells expressing inhibitory receptors for therapy is that these cells may be exhausted or functionally impaired (23, 24, 44, 51, 52). However, we found that PD-1+, TIM-3+, and LAG-3+ CD8+ cells expanded in IL-2 were capable of secreting IFN-γ and lyse tumor in vitro. This supports the notion that immune dysfunction associated with coexpression of inhibitory receptors on CD8+ TILs can be reversed (21, 41, 51, 53), and may enable the reproducible enrichment of tumor-reactive cells for patient treatment. Notably, in a preliminary experiment (n= 8 nonresponders; 14 responders), there was no association between the frequency of expression of any of the markers studied in the CD8+ TILs in the fresh tumor and the clinical response to TILs derived from these tumor samples. However, the fresh tumors included in this study belonged to patients treated in several TIL protocols over the course of 10 years, and TILs were generated from these tumors using different methods, which makes these data difficult to interpret. In addition, the frequency of cells initially expressing PD-1 in the tumor may not reflect the frequency of the PD-1 derived cells in the infusion bag. For example, a low frequency of PD-1+ cells may be highly enriched during the process of TIL culture as a result of the presence of tumor cells. Although in vivo antitumor activity of tumor-isolated TILs based on PD-1 expression requires testing in a clinical trial, the observation that the overwhelming majority of tumor-reactive cells were derived from cells expressing PD-1 suggests that cells expressing PD-1 and inhibitory receptors in the tumor play a critical role in tumor regression after TIL administration.

The functional implications of selecting PD-1–, LAG-3–, TIM-3–, or 4-1BB–expressing T cells to enrich for tumor-reactive cells for patient treatment remain unclear. Although previous studies have reported differential expression of PD-1, LAG-3, and TIM-3 throughout differentiation (17), or preferential expression of TIM-3 in IFN-γ–secreting cells (54), our preliminary results have failed to show consistent phenotypic or functional differences between PD-1+, LAG-3+, TIM-3+, and 4-1BB+ selected TILs, including cytokine secretion, proliferation, and susceptibility to apoptosis (data not shown). We found that PD-1 expression was almost completely lost in the PD-1+ derived populations upon in vitro culture in IL-2. Conversely, TIM-3 and LAG-3 expression increased in the TIM-3 and LAG-3 populations after expansion. Overall, there were no differences in the expression of PD-1, TIM-3, or LAG-3 between any the populations after expansion. Thus, in agreement with previous reports (55, 56), we conclude that expansion in IL-2 alters the expression of these markers and compromises the potential use of inhibitory receptors to select for tumor-reactive cells after in vitro expansion. Recent work in animal models suggests that chronic antigen stimulation (5759) or a tolerizing microenvironment (60) may lead to permanent epigenetic changes in T cells, raising the possibility that the restoration of function observed in previously exhausted or tolerized cells in presence of cytokines may only be transient. These results have not yet been corroborated in human tumor-specific cells. However, given that the overwhelming majority of tumor-reactive cells appear to derive from cells expressing PD-1 in the tumor, studying permanent versus transient reversion of exhaustion may have important implications for adoptive cell transfer of TILs.

Tumor-reactive cells can also be found infiltrating other tumor malignancies, such as renal cell carcinoma (61) or ovarian (62), cervical (63), or gastrointestinal tract cancers (64), albeit at lower frequencies. Our findings provide alternatives to enrich and study tumor-reactive CD8+ TILs through selection of cells expressing the cell surface receptors PD-1, LAG-3, TIM-3, and 4-1BB, a hypothesis that we are actively investigating. Additionally, our present findings showed that the frequency of a specific clonotype in the CD8+ and PD-1+ populations can be used to predict its ability to recognize tumor and isolate tumor-specific TCRs, thus providing means to overcome potential irreversible functional impairments of TILs (52).

2 reports with opposing results have generated controversy regarding which may be the optimal marker for the identification of the tumor-reactive repertoire, PD-1 or 4-1BB. In one report studying PD-1 expression in the tumor, the authors showed promising although inconsistent ability to enrich for shared melanoma-reactive cells (55). In a more recent article studying the role of 4-1BB in fresh ovarian TILs, Ye et al. concluded that expression of 4-1BB, but not PD-1, on lymphocytes defines the population of tumor-reactive cells in the tumor (65). The results of Ye et al. appear to contradict our present findings, showing that expression of PD-1 rather than 4-1BB more comprehensively identifies the repertoire of tumor-reactive cells in the tumor. However, these inconsistencies can be explained by different experimental approaches undertaken to study the immunobiology of TILs. First, Ye et al. found that expression of 4-1BB in fresh ovarian TILs and tumor-associated lymphocytes was low, and thus exposed the tumor to IL-7 and IL-15 (65). In the 1 patient sample in which the authors enriched for tumor-reactive cells from fresh ovarian TILs or tumor-associated lymphocytes exposed to IL-7 and IL-15, expression of 4-1BB was dependent on in vitro activation, but no longer represented the natural expression of 4-1BB in the fresh tumor. Second, with the exception of the 1 experiment described above, the enrichment experiments reported were carried out with melanoma or ovarian TIL lines expanded in IL-2 and cocultured with tumor cell lines in vitro. It is well known that IL-2 can change the activation status and also the expression of inhibitory receptors on T cells (data not shown and ref. 56). Thus, the experiment comparing expression of PD-1 and 4-1BB performed by Ye et al. (65) addressed the significance of these receptors after in vitro coculture of a highly activated melanoma TIL line with a tumor cell line, rather than the role of PD-1 and 4-1BB expression in CD8+ lymphocytes in the fresh tumor. Finally, both Inozume et al. and Ye et al. used matched HLA-A2 cell lines to assess tumor reactivity (55, 65). However, the use of HLA-matched tumor cell lines does not enable the assessment of reactivities against unique mutations that are present only in the autologous tumor cell line. In our current study, we used fresh melanoma tumors for all our experiments, and these were rested in the absence of cytokines to preserve the phenotype of TILs. Moreover, we used autologous tumor cell lines to assess tumor recognition. We believe that our experimental approach overcomes the limitations described above, enabling us to conclude that tumor-reactive cells can be detected in both the PD-1+/4-1BB+ and PD-1+/4-1BB CD8+ TIL populations.

In summary, expression of PD-1 in CD8+ TILs in the fresh tumor identified and selected for the diverse patient-specific repertoire of tumor-reactive cells, including mutation-specific cells. In addition, analysis of the CD8+ TIL TCRβ repertoire in 2 melanomas showed that the frequency of a specific TCRβ clonotype in the CD8+ and PD-1+ populations could be used to predict its ability to recognize the autologous tumor. The use of inhibitory receptors and the frequency of individual TCRs to prospectively identify and select the diverse repertoire of tumor-reactive cells holds promise for the personalized treatment of cancer with T cell therapies, but may also facilitate the dissection and understanding of the immune response in human cancer patients.

Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?

Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.

Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:

  • LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
  • TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
  • VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice

In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets

  • OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
  • 4-1BB – Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
  • Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.

Sharma P and Allison JP.
Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential.
Cell. April 2015;161:205-214

 

Targeting single immune-checkpoint proteins has proven to be clinically effective at treating specific tumor types; can targeting two different proteins synergize effects?

Despite the success of targeting immune-checkpoint proteins, such as CTLA-4, PD-1, LAG-3, TIM-3 among others, percentages of patient response vary and rarely exceed 50%. It is highly tempting to speculate a strategy of dual-targeting of these checkpoint proteins. A recent presentation at the Keystone Symposium for Tumor Immunology: Multidisciplinary Science Driving Combination Therapy detailed findings of dual-targeting two immune-checkpoint proteins in mouse tumor models. Their key findings are summarized below:

  • Dual-targeting PD-1 and LAG-3 demonstrates superior efficacy over blocking either target alone
  • In addition to previous reported data on superior dual-targeting efficacy against fibrosarcoma (Sa1N) and colorectal adenosarcoma (MC38) tumor types1, anti-tumor activity against myeloma (SC J558L) and B-cell lymphoma (A20) hematological tumor types were also reported to be effacious.2

These exciting pre-clinical findings may result in further exploration of dual-targeting antibodies in the clinic, either as combination of existing antibody therapies, or as a new bi-specific antibody therapeutic.

Camelid single domain antibodies are a novel bi-specific antibody platform that may be used to develop a new generation of dual-targeting antibodies against multiple immune-checkpoint proteins.

1Woo SR et al.
Immune Inhibitory Molecules Lag-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape.
Cancer Res. Feb 2012. 15(4):917-927.

2Lewis KE et al.
Dual Targeting of PD-1 and LAG-3 demonstrates Superior Efficacy to Blocking Either PD-1 or LAG-3 Alone in Pre-Clinical Solid and Hematological Tumor Models.
Abstract J7 2033. Keystone Symposia: Tumor Immunology: Multidisciplinary Science Driving Combination Therapy. February 8-13, 2015. Banff, Alberta, Canada.

 

New insight behind the success of fighting cancer by targeting immune checkpoint proteins

Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.

In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.

In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.

Gubin MM. et al.
Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.
Nature. Nov 2014. 515:577-584

 

 

Myeloid-derived-suppressor cells as regulators of the immune system
Dmitry I. Gabrilovich and Srinivas Nagaraj  Nat Rev Immunol. 2009 March ; 9(3): 162–174. http://dx.doi.org:/10.1038/nri2506

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expands during cancer, inflammation and infection, and that has a remarkable ability to suppress T-cell responses. These cells constitute a unique component of the immune system that regulates immune responses in healthy individuals and in the context of various diseases. In this Review, we discuss the origin, mechanisms of expansion and suppressive functions of MDSCs, as well as the potential to target these cells for therapeutic benefit.

The first observations of suppressive myeloid cells were described more than 20 years ago in patients with cancer1-3. However, the functional importance of these cells in the immune system has only recently been appreciated due to accumulating evidence that has demonstrated their contribution to the negative regulation of immune responses during cancer and other diseases. It is now becoming increasingly clear that this activity is contained within a population known as myeloid-derived suppressor cells (MDSCs). Features common to all MDSCs are their myeloid origin, immature state and a remarkable ability to suppress T-cell responses (Box 1). In addition to their suppressive effects on adaptive immune responses, MDSCs have also been reported to regulate innate immune responses by modulating the cytokine production of macrophages4. Non-immunological functions of MDSC have also been described, such as the promotion of tumour angiogenesis, tumour-cell invasion and metastasis. However, as a discussion of these aspects of MDSC biology is beyond the scope of this article, the reader is referred to another recent Review on this topic5.

MDSCs represent an intrinsic part of the myeloid-cell lineage and are a heterogeneous population that is comprised of myeloid-cell progenitors and precursors of myeloid cells. In healthy individuals, immature myeloid cells (IMCs) generated in bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). In pathological conditions such as cancer, various infectious diseases, sepsis, trauma, bone marrow transplantation or some autoimmune disorders, a partial block in the differentiation of IMCs into mature myeloid cells results in an expansion of this population. Importantly, the activation of these cells in a pathological context results in the upregulated expression of immune suppressive factors such as arginase (encoded by ARG1) and inducible nitric oxide synthase (iNOS; also known as NOS2) and an increase in the production of NO (nitric oxide) and reactive oxygen species (ROS). Together, this results in the expansion of an IMC population that has immune suppressive activity; these cells are now collectively known as MDSCs. In this

Origin and subsets of MDSCs It is important to note that MDSCs that are expanded in pathological conditions (see later) are not a defined subset of myeloid cells but rather a heterogeneous population of activated IMCs that have been prevented from fully differentiating into mature cells. MDSCs lack the expression of cell-surface markers that are specific for monocytes, macrophages or DCs and are comprised of a mixture of myeloid cells with granulocytic and monocytic morphology6. Early studies showed that 1–5% of MDSCs are able to form myeloid-cell colonies7-9 and that about one third of this population can differentiate into mature macrophages and DCs in the presence of appropriate cytokines in vitro and in vivo7-9. In mice, MDSCs are characterized by the co-expression of the myeloid lineage differentiation antigen Gr1 (also known as Ly6G) and CD11b (also known as αM-integrin)10. Normal bone marrow contains 20–30% of cells with this phenotype, but these cells make up only a small proportion (2–4%) of spleen cells and are absent from the lymph nodes in mice (Fig. 1). In humans, MDSCs are most commonly defined as CD14-CD11b+ cells or, more narrowly, as cells that express the common myeloid marker CD33 but lack the expression of markers of mature myeloid and lymphoid cells and the MHC-class-II molecule HLA-DR11, 12. MDSCs have also been identified within a CD15+ population in human peripheral blood13. In healthy individuals, immature myeloid cells with described above phenotype comprise ∼0.5% of peripheral blood mononuclear cells.
Recently, the morphological heterogeneity of these cells has been defined more precisely in part based on their expression of Gr1. Notably, Gr1-specific antibodies bind to both Ly6G and Ly6C,  which are encoded by separate genes. However, these epitopes are recognized by different antibodies specific for each individual epitopes: anti-Ly6C and anti-Ly6G. Granulocytic MDSCs have a CD11b+Ly6G+Ly6Clow phenotype, whereas MDSCs with monocytic morphology are CD11b+Ly6G-Ly6Chigh 6,14. Importantly, evidence indicates that these two subpopulations may have different functions in cancer and infectious and autoimmune diseases15-17. During the analysis of ten different experimental tumour models, we found that both of these subsets of MDSCs were expanded. In most cases, however, the expansion of the granulocytic MDSC population was much greater than that of the monocytic subset6 and, interestingly, the two subpopulations used different mechanisms to suppress Tcell function (see later). In addition, the ability to differentiate into mature DCs and macrophages in vitro has been shown to be restricted to monocytic MDSCs6.
In recent years, several other surface molecules have been used to identify additional subsets of suppressive MDSCs, including CD80 (also known as B7.1)18, CD115 (the macrophage colony-stimulating factor receptor)19, 20 and CD124 (the IL-4 receptor α-chain)20. In our own studies, we observed that many MDSCs in tumour-bearing mice co-express CD115 and CD1246; however, direct comparison of MDSCs from tumour-bearing mice and Gr1+CD11b+ cells from naive mice showed that they expressed similar levels of CD115 and CD124. In addition, sorted CD115+ or CD124+ MDSCs from EL-4 tumour-bearing mice had the same ability to suppress T-cell proliferation on a per cell basis as did CD115- or CD124-MDSCs. This suggests that, although these molecules are associated with MDSCs, they might not be involved in the immunosuppressive function of these cells in all tumour models.

Overall, current data suggest that MDSCs are not a defined subset of cells but rather a group of phenotypically heterogeneous myeloid cells that have common biological activity.

MDSCs in pathological conditions MDSCs were first characterized in tumour-bearing mice or in patients with cancer. Inoculation of mice with transplantable tumour cells, or the spontaneous development of tumours in transgenic mice with tissue-restricted oncogene expression, results in a marked systemic expansion of these cells (Fig. 1 and Table 1). In addition, up to a tenfold increase in MDSC numbers was detected in the blood of patients with different types of cancer11, 12, 21, 22. In many mouse tumour models, as many as 20–40% of nucleated splenocytes are represented by MDSCs (in contrast to the 2-4% seen in normal mice). In addition, these cells are found in tumour tissues and in the lymph nodes of tumour-bearing mice.
Although initial observations and most of the current information regarding the role of MDSCs in immune responses has come from studies in the cancer field, accumulating evidence has shown that MDSCs also regulate immune responses in bacterial and parasitic infections, acute and chronic inflammation, traumatic stress, surgical sepsis and transplantation. A systemic expansion of both the granulocytic and monocytic subset of MDSCs was observed in mice primed with Mycobacterium tuberculosis as part of complete Freund’s adjuvant (CFA). Acute Trypanosoma cruzi infection, which induces T-cell activation and increased production of interferon-γ (IFNγ), also leads to the expansion of MDSCs23, 24. A similar expansion of MDSCs has been reported during acute toxoplasmosis25, polymicrobial sepsis26, acute infection with Listeria monocytogenes or chronic infection with Leishmania major27 and infection with helminths28,29, 30, Candida albicans31 or Porphyromonas gingivalis32.

MDSC expansion is also associated with autoimmunity and inflammation. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, an increase in CD11b+Ly6ChiLy6G− MDSCs was observed in the spleen and blood and these cells were found to enter the central nervous system during the inflammatory phase of the disease16. A significant increase in the number of MDSCs was also detected in experimental autoimmune uveoretinitis, an animal model of human intraocular inflammatory disease33, in the skin and spleens of mice that were repeatedly treated with a contact sensitizer to induce an inflammatory response34 and in inflammatory bowel diseases35. MDSCs were also found to infiltrate the spleen and suppress T-cell function in a model of traumatic stress36. Finally, a significant transient increase in MDSC numbers was also demonstrated in normal mice following immunization with different antigens such as ovalbumin or peptide together with CFA, a recombinant vaccinia virus expressing interleukin-2 (IL-2) or staphylococcal enterotoxin A 8, 37, 38. Therefore, current information clearly indicates that the expansion of an immunosuppressive MDSC population is frequently observed in many pathological conditions.

Expansion and activation of MDSCs Studies have demonstrated that the MDSC population is influenced by several different factors (Table 1), which can be divided into two main groups. The first group includes factors that are produced mainly by tumour cells and promote the expansion of MDSC through stimulation of myelopoiesis and inhibiting of the differentiation of mature myeloid cells. The second group of factors is produced mainly by activated T cells and tumour stroma, and is involved in directly activating MDSCs.
Mechanisms of MDSC expansion—Factors that induce MDSC expansion can include cyclooxygenase-2 (COX2), prostaglandins 39-41, stem-cell factor (SCF)39, macrophage colony-stimulating factor (M-CSF), IL-642, granulocyte/macrophage colony-stimulating factor (GM-CSF)41 and vascular endothelial growth factor (VEGF) 43 (Table 1). The signalling pathways in MDSCs that are triggered by most of these factors converge on Janus kinase (JAK) protein family members and signal transducer and activator of transcription 3 (STAT3) (Fig. 2), which are signalling molecules that are involved in cell survival, proliferation, differentiation and apoptosis44. STAT3 is arguably the main transcription factor that regulates the expansion of MDSCs. MDSCs from tumour-bearing mice have markedly increased levels of phosphorylated STAT3 compared with IMCs from naive mice45. Exposure of haematopoietic progenitor cells to tumour-cell-conditioned medium resulted in the activation of JAK2 and STAT3 and was associated with an expansion of MDSCs in vitro, whereas inhibition of STAT3 expression in haematopoietic progenitor cells abrogated the effect of tumour-derived factors on MDSC expansion46. Ablation of STAT3 expression in conditional knockout mice or selective STAT3 inhibitors markedly reduced the expansion of MDSCs and increased T-cell responses in tumour-bearing mice45, 47. STAT3 activation is associated with increased survival and proliferation of myeloid progenitor cells, probably through upregulated expression of STAT3 target genes including B-cell lymphoma XL, (BCL-XL), cyclin D1, MYC and survivin. So, abnormal and persistent activation of STAT3 in myeloid progenitors prevents their differentiation into mature myeloid cells and thereby promotes MDSC expansion.

Recent findings suggest that STAT3 also regulates MDSC expansion through inducing the expression of S100A8 and S100A9 proteins. In addition, it has been shown that MDSCs also express receptors for these proteins on their cell surface. S100A8 and S100A9 belong to the family of S100 calcium-binding proteins that have been reported to have an important role in inflammation48. STAT3-dependent upregulation of S100A8 and S100A9 expression by myeloid progenitor cells prevented their differentiation and resulted in the expansion of MDSCs in the spleens of tumor-bearing and naive S100A9-transgenic mice. By contrast, MDSCs did not expand in the peripheral blood and spleens of mice deficient for S100A9 following challenge with tumour cells or CFA49. In a different study, S100A8 and S100A9 proteins were shown to promote MDSC migration to the tumour site through binding to carboxylated N-glycan receptors expressed on the surface of these cells 50. Blocking the binding of S100A8 and S100A9 to their receptors on MDSCs in vivo with a carboxylated glycan-specific antibody reduced MDSC levels in the blood and secondary lymphoid organs of tumour-bearing mice50. In human colon tumour tissue, and in a mouse model of colon cancer, myeloid progenitor cells expressing S100A8 and S100A9 have been shown to infiltrate regions of dysplasia and adenoma. Furthermore, administration of a carboxylated glycan-specific monoclonal antibody (mAbGB3.1) was found to markedly reduced chronic inflammation and tumorigenesis51. Although the mechanisms involved require further study, these studies suggest that S100A9 and/or S100A8 proteins have a crucial role in regulating MDSC expansion, and may provide a link between inflammation and immune suppression in cancer.

Mechanisms of MDSC activation—Recently, it has become clear that the suppressive activity of MDSCs requires not only factors that promote their expansion but those that induce their activation. The expression of these factors, which are produced mainly by activated T cells and tumour stromal cells, is induced by different bacterial or viral products or as a result of tumour cell death 26. These factors, which include IFNγ, ligands for Toll-like receptors (TLRs), IL-13, IL-4 and transforming growth factor-β (TGFβ), activate several different signalling pathways in MDSCs that involve STAT6, STAT1, and nuclear factor-κB (NF-κB) (Fig. 2).

Blockade of IFNγ, which is produced by activated T cells, abolishes MDSC-mediated T-cell suppression17, 52. STAT1 is the major transcription factor activated by IFNγ-mediated signalling and, in the tumour microenvironment, the upregulation of ARG1 and iNOS expression in MDSCs involved a STAT1-dependent mechanism. Indeed, MDSCs from Stat1-/- mice failed to up regulate ARG1 and iNOS expression and therefore did not inhibit Tcell responses53. Consistent with other findings, IFNγ produced by activated T cells and by MDSCs triggered iNOS expression and synergized with IL-4Rα and ARG1 pathways that have been implicated in the suppressive function of MDSCs20.
An important role for the signalling pathway that involves IL-4 receptor α-chain (IL-4Rα) and STAT6 (which is activated by the binding of either IL-4 or IL-13 to IL-4Rα) in MDSC activation has been demonstrated in several studies. It has been shown that ARG1 expression is induced by culturing freshly isolated MDSCs or cloned MDSC lines with IL-454. In addition, IL-4 and IL-13 upregulate arginase activity, which increases the suppressive function of MDSCs55. In line with these observations, other experiments have shown that STAT6 deficiency prevents signalling downstream of the IL-4Rα and thereby blocks the production of ARG1 by MDSCs56. In addition, the IL-4Rα–STAT6 pathway was also found to be involved in IL-13-induced TGFβ1 production by MDSCs in mice with sarcoma, which resulted in decreased tumour immunosurveillance57. This could be regulated by neutralizing both TGFβ and IL-1357. However, in breast tumor model IL-4Rα knockout mice retain high levels of MDSC after surgery56. In a different study that evaluated the separate role of TGFβ (not involving study of IL-4Rα) TGFβ-specific blocking antibody failed to reverse T-cell anergy in B-cell lymphoma in vitro58. It is possible that, the IL4Rα–STAT6 pathway might not be involved in promoting tumour immunosuppression in all tumour models.

TLRs have a central role in the activation of innate immune responses. Polymicrobial sepsis induced by the ligation and puncture of the caecum, which releases microbial products into the peritoneum and systemic circulation, was shown to result in an expansion of the MDSC population in the spleen that was dependent on the TLR adaptor molecule myeloid differentiation primary-response gene 88 (MyD88)26. However, wild-type mice and mice lacking a functional TLR4 protein had comparable expansion of the MDSC during polymicrobial sepsis, which suggests that signalling through TLR4 is not required for MDSC expansion and that MyD88-dependent signalling pathways that are triggered by other TLRs probably contribute to the expansion of MDSCs in sepsis26. This indicates that the activation of MDSCs is a fundamental outcome of the host innate immune response to pathogens that express TLR ligands.

It is important to note that an increase in the production and/or recruitment of IMCs in the context of acute infectious diseases or following vaccination does not necessarily represent an expansion of an immunosuppressive MDSC population. It is likely that under pathological conditions, the expansion of a suppressive MDSC population is regulated by two different groups of factors that have partially overlapping activity: those that induce MDSC expansion and those that induce their activation (which leads to increased levels of ROS, arginase, and/ or NO). This two-tiered system may allow for flexibility in the regulation of these cells under physiological and pathological conditions.
Mechanisms of MDSC suppressive activity Most studies have shown that the immunosuppressive functions of MDSCs require direct cell– cell contact, which suggests that they act either through cell-surface receptors and/or through the release of short-lived soluble mediators. The following sections describe the several mechanisms that have been implicated in MDSC-mediated suppression of T-cell function.

Arginase and iNOS—Historically, the suppressive activity of MDSCs has been associated with the metabolism of L-arginine. L-arginine serves as a substrate for two enzymes: iNOS, which generates NO, and arginase, which converts L-arginine into urea and L-ornithine. MDSCs express high levels of both arginase and iNOS, and a direct role for both of these enzymes in the inhibition of T-cell function is well established; this has been reviewed recently59, 60. Recent data suggest that there is a close correlation between the availability of arginine and the regulation of T-cell proliferation11, 61. The increased activity of arginase in MDSCs leads to enhanced L-arginine catabolism, which depletes this non-essential amino acid from the microenvironment. The shortage of L-arginine inhibits T-cell proliferation through several different mechanisms, including decreasing their CD3ζ expression62 and preventing their upregulation of the expression of the cell cycle regulators cyclin D3 and cyclin-dependent kinase 4 (CDK4)63. NO suppresses T-cell function through a variety of different mechanisms that involve the inhibition of JAK3 and STAT5 in T cells64, the inhibition of MHC class II expression 65 and the induction of T-cell apoptosis66.

ROS—Another important factor that contributes to the suppressive activity of MDSCs is ROS. Increased production of ROS has emerged as one of the main characteristics of MDSCs in both tumour-bearing mice and patients with cancer6, 10, 13, 53, 67-70. Inhibition of ROS production by MDSCs isolated from mice and patients with cancer completely abrogated the suppressive effect of these cells in vitro10, 13, 67. Interestingly, ligation of integrins expressed on the surface of MDSCs was shown to contribute to increased ROS production following the interaction of MDSCs with T cells10. In addition, several known tumour-derived factors, such as TGFβ, IL-10, IL-6, IL-3, platelet-derived growth factor (PDGF) and GM-CSF, can induce the production of ROS by MDSCs (for review see Ref 71).

The involvement of ROS and NO in mechanisms of MDSC suppression are not restricted to neoplastic conditions, as inflammation and microbial products are also known to induce the development of a MDSC population that produces ROS and NO following interactions with activated T cells15. Similar findings were observed in models of EAE16 and acute Toxoplasmosis infection 16. In addition, it has been observed that MDSCs mediated their suppressive function through IFNγ-dependent NO production in an experimental model of Trypanosoma cruzi infection23.

Peroxynitrite—More recently, it has emerged that peroxynitrite (ONOO-) is a crucial mediator of MDSC-mediated suppression of T-cell function. Peroxynitrite is a product of a chemical reaction between NO and superoxide anoion (O2-) and is one of the most powerful oxidants produced in the body. It induces the nitration and nitrosylation of the amino acids cystine, methionine, tryptophan and tyrosine72. Increased levels of peroxynitrite are present at sites of MDSC and inflammatory-cell accumulation, including sites of ongoing immune reactions. In addition, high levels of peroxynitrite are associated with tumour progression in many types of cancer72, 73,74-78, which has been linked with T-cell unresponsiveness. Bronte and colleagues reported that human prostate adenocarcinomas were infiltrated by terminallydifferentiated CD8+ T cells that were in an unresponsive state. High levels of nitrotyrosine were present in the T cells, which suggested the production of peroxynitrites in the tumour environment. Inhibiting the activity of arginase and iNOS, which are expressed in malignant but not in normal prostate tissue and are key enzymes of L-arginine metabolism,, led to decreased tyrosine nitration and restoration of T-cell responsiveness to tumour antigens79. In addition, we have demonstrated that peroxynitrite production by MDSCs during direct contact with T cells results in nitration of the T-cell receptor (TCR) and CD8 molecules, which alters the specific peptide binding of the T cells and renders them unresponsive to antigen-specific stimulation. However, the T cells maintained their responsiveness to nonspecific stimuli80. This phenomenon of MDSC induced antigen-specific T-cell unresponsiveness was also observed in vivo in tumour-bearing mice53.

Subset-specific suppressive mechanisms?—Recent findings indicate that different subsets of MDSC might use different mechanisms by which to suppress T-cell proliferation. As described earlier, two main subsets of MDSCs have been identified: a granulocytic subset and a monocytic subset. The granulocytic subset of MDSC was found to express high levels of ROS and low levels of NO, whereas the monocytic subset expressed low levels of ROS and high levels of NO and both subsets expressed ARG16 (Fig.3). Interestingly, both populations suppressed antigen-specific T-cell proliferation to an equal extent, despite their different mechanisms of action. Consistent with these observations, Movahedi et al. also reported two distinct MDSC subsets in tumour-bearing mice, one that consisted of mononuclear cells that resembled inflammatory monocytes and a second that consisted of polymorphonuclear cells that were similar to immature granulocytes. Again, both populations were found to suppress antigen-specific T-cell responses, although by using distinct effector molecules and signalling pathways. The suppressive activity of the granulocytic subset was ARG1-dependent, in contrast to the STAT1- and iNOS-dependent mechanism of the monocyte fraction17. Finally, the same trend was observed in Trypanosoma cruzii infection. In this case, monocytic MDSCs produced NO and strongly inhibited T-cell proliferation, and granulocytic MDSCs produced low levels of NO and did not inhibit T-cell proliferation, although they did produce superoxide15. The biological significance of such functional dichotomy of these two MDSC subsets remains to be elucidated.
Induction of TReg cells—Recently, the ability of MDSCs to promote the de novo development of FOXP3+ regulatory T (TReg) cells in vivo has been described18, 19. The induction of TReg cells by MDSCs was found to require the activation of tumour-specific Tcells and the presence of IFNγ and IL-10 but was independent of NO19. In mice bearing 1D8 ovarian tumours, the induction of TReg cells by MDSCs required the expression of cytotoxic lymphocyte antigen 4 (CTLA-4; also known as CD152) by MDSCs18. In a mouse model of lymphoma, MDSCs were shown to induce TReg-cell expansion through a mechanism that required arginase and the capture, processing and presentation of tumour-associated antigens by MDSCs, but not TGFβ58. By contrast, Movahedi et al. found that the percentage of TReg cells was invariably high throughout tumour growth and did not relate to the kinetics of expansion of the MDSC population, suggesting that MDSCs were not involved in TReg-cell expansion17. Furthermore, in a rat model of kidney allograft tolerance that was induced with a CD28-specific antibody, MDSCs that were co-expressing CD80 and CD86 were found to have a limited effect on the expansion of the TReg-cell population81. Although further work is required to resolve these discrepancies and to determine the physiological relevance of these studies, it seems possible that MDSCs are involved in TReg-cell differentiation through the production of cytokines or direct cell–cell interactions. Furthermore, MDSCs and TReg cells might be linked in a common immunoregulatory network (see later).
Tissue-specific effects on MDSCs A major unresolved question in this field is whether MDSCs mediate antigen-specific or nonspecific suppression of T-cell responses. Provided that MDSCs and T cells are in close proximity, the factors that mediate MDSC suppressive function (ROS, arginase and NO) can inhibit T-cell proliferation regardless of the antigen specificity of the T cells. Indeed, numerous in vitro studies have demonstrated the antigen nonspecific nature of MDSC-mediated suppression of T cells82 83. However, whether the situation is the same in vivo is not clear, and evidence suggests that MDSC-mediated immunosuppression in peripheral lymphoid organs is mainly antigen-specific. The idea that MDSC-mediated T-cell suppression occurs in an antigen-specific manner is based on findings that antigen-specific interactions between antigen-presenting cells and T cells result in much more stable and more prolonged cell–cell contact than nonspecific interactions82, 84, 85. Such stable contacts are necessary for MDSCderived ROS and peroxynitrite to mediate effects on the molecules on the surface of T cells that render the T cells unresponsive to specific antigen. It should be noted that such modification of cell-surface molecules does not lead to T-cell death nor prevent nonspecific T-cell activation. Other evidence that supports the idea that MDSCs mediate antigen-specific suppression is the finding that that MDSCs can take up soluble antigens, including tumourassociated antigens, and process and present them to T cells17 80; blockade of MDSC–T-cell interactions with a MHC-class-I-specific antibody abrogated MDSC-mediated inhibition of T cell responses in vitro86. The MHC-class-I-restricted nature of MDSC-mediated CD8+ T-cell suppression has also been demonstrated in vivo in tumor models53 and in the model of inflammatory bowel disease 35. This is consistent with the recent observation that large numbers of tumour-induced MDSCs did not inhibit CD8+ T-cell responses specific for unrelated antigens in a model of sporadic cancer87. Notably, it is currently unclear whether similar antigen-specific mechanisms of MDSC-mediated suppression operate on CD4+ T cells, as published studies have only assessed the effects of MDSCs on CD8+ T cells. Addressing this question is complicated by the fact that only a small proportion of MDSCs in many tumour models expresses MHC class II molecules.

The theory that MDSCs suppress T-cell responses in an antigen-specific manner helps to explain the finding that T cells in the peripheral lymphoid organs of tumour-bearing mice and in the peripheral blood of cancer patients can still respond to stimuli other than tumourassociated antigens, including viruses, lectins, co-stimulatory molecules, IL-2 and CD3- and CD28-specific antibodies21, 80, 88-90. Furthermore, even patients with advanced stage cancer do not have systemic immunodeficiency except in cases in which the patient has received high doses of chemotherapy or is at a terminal stage of the disease.

Evidence suggets that the nature of MDSC-mediated suppression at the tumour site is quite different to that which occurs in the periphery. MDSCs actively migrate into the tumour site10, where they upregulate the expression of ARG1 and iNOS, downregulate the production of ROS and/or rapidly differentiate into tumour-associated macrophages (TAMs) 52. The levels of NO and arginase produced by tumour-associated MDSCs and TAMs are much higher than those of MDSCs found in peripheral lymphoid organs of the same animals. In addition, TAMs produce several cytokines (reviewed in REFs91, 92) that suppress T-cell responses in a nonspecific manner (Fig. 4). The mechanisms by which MDSC functions are regulated within the tumour microenvironment, and how they differ from those that operate at peripheral sites, remain unclear. It is possible that tumour stroma, hypoxia and/or the acidophilic environment have a role.
Therapeutic targeting of MDSCs The recognition that immune suppression has a crucial role in promoting tumour progression and contributes to the frequent failure of cancer vaccines to elicit an immune response has resulted in a paradigm shift with respect to approaches for cancer immunotherapy. Indeed, it has become increasingly clear that successful cancer immunotherapy will be possible only with a strategy that involves the elimination of suppressive factors from the body. As MDSCs are one of the main immunosuppressive factors in cancer and other pathological conditions, several different therapeutic strategies that target these cells are currently being explored (Table 2). Although the studies described below were carried out in tumor-bearing hosts, it is likely that the same strategies will be useful in other pathological conditions in which inhibition or elimination of MDSCs is a therapeutic aim.

Promoting myeloid-cell differentiation—One of the most promising approaches by which to target MDSCs for therapy is to promote their differentiation into mature myeloid cells that do not have suppressive abilities. Vitamin A has been identified as a compound that can mediate this effect: vitamin A metabolites such as retinoic acid have been found to stimulate the differentiation of myeloid progenitors into DCs and macrophages 86, 93. Mice that are deficient in vitamin A94 or that have been treated with a pan-retinoic-acid-receptor antagonist95, show an expansion of MDSCs in the bone marrow and spleen. Conversely, therapeutic concentrations of all-trans retinoic acid (ATRA) results in substantial decrease in the presence of MDSCs in cancer patients and tumour-bearing mice. ATRA induced MDSCs to differentiate into DCs and macrophages in vitro and in vivo 12, 86, 96. It is probable that ATRA preferentially induces the differentiation of the monocytic subset of MDSCs, whereas it causes apoptosis of the granulocytic subset. The main mechanism of ATRA-mediated differentiation involved an upregulation of glutathione synthesis and a reduction in ROS levels in MDSCs 97. Decreasing the number of MDSCs in tumour-bearing mice resulted in increased tumour-specific T-cell responses, and the combination of ATRA and two different types of cancer vaccine prolonged the anti-tumour effect of the vaccine treatment in two different tumour models 96. Moreover, administration of ATRA to patients with metastatic renal cell carcinoma resulted in a substantial decrease in the number of MDSCs in the peripheral blood and improved antigen-specific response of T cells 21. Further studies will lead to identification of other agents that have a similar effect. So far, evidence suggests that Vitamin D3 may be another agent with the potential to decrease MDSC numbers in patients with cancer, as it is also known to promote myeloid-cell differentiation98.

Inhibition of MDSC expansion—Because MDSC expansion is known to be regulated by tumour-derived factors (Table 1), several studies have focused on neutralizing the effects of these factors. Recently, SCF has been implicated in causing MDSC expansion in tumourbearing mice39. Inhibition of SCF-mediated signalling by blocking its interaction with its receptor, c-kit, decreased MDSC expansion and tumor angiogenesis39. VEGF, another tumourderived factor that is involved in promoting MDSC expansion, might also be a useful target by which to manipulate MDSC. However, in a clinical trial of 15 patients with refractory solid tumours, treatment with VEGF–trap (a fusion protein that binds all forms of VEGF-A and placental growth factor) showed no effect on MDSC numbers and did not result in increased T-cell responses99. By contrast, treatment of patients with metastatic renal cell cancer with a VEGF-specific blocking antibody (known as avastin) resulted in a decrease in the size of a CD11b+VEGFR1+ population of MDSCs in the peripheral blood 100. However, whether avastatin treatment resulted in an improvement in antitumour responses in these patients has not been determined. Finally, inhibition of matrix metalloproteinase 9 function in tumorbearing mice decreased the number of MDSCs in the spleen and tumour tissues and resulted in a significant delay in the growth of spontaneous NeuT tumours in transgenic BALB/c mice101. However, the mechanism responsible for this outcome remains to be elucidated.

Inhibition of MDSC function—Another approach by which to inhibit MDSCs is to block the signalling pathways that regulate the production of suppressive factors by these cells. One potential target by which this might be achieved is COX2. COX2 is required for the production of prostaglandin E2, which in 3LL tumour cells61 and mammary carcinoma40 has been shown to induce the upregulation of ARG1 expression by MDSCs, thereby inducing their suppressive function. Accordingly, COX2 inhibitors were found to downregulate the expression of ARG1 by MDSCs, which improved antitumour T-cell responses and enhanced the therapeutic efficacy of immunotherapy102, 103. Similarly, phosphodiesterase-5 inhibitors such as sildenafil were found to downregulate the expression of arginase and iNOS expression by MDSCs, thereby inhibiting their suppressive function in growing tumours104. This resulted in the induction of a measurable anti-tumour immune response and a marked delay of tumour progression in several mouse models 104.
ROS inhibitors have also been shown to be effective for decreasing MDSC-mediated immune suppression in tumour-bearing mice. The coupling of a NO-releasing moiety to a conventional non-steroidal anti-inflammatory drug has proven to be an efficient means by which to inhibit the production of ROS. One such drug, nitroaspirin, was found to limit the activity of ARG1 and iNOS in spleen MDSCs105. In combination with vaccination with endogenous retroviral gp70 antigen, nitroaspirin inhibited MDSCs function and increased the number and function of tumour-antigen-specific T cells105.

Elimination of MDSCs—MDSCs can be directly eliminated in pathological settings by using some chemotherapeutic drugs. Administration of one such drug, gemcitabine, to mice that were bearing large tumours resulted in a dramatic reduction in the number of MDSCs in the spleen and resulted in a marked improvement in the anti-tumour response induced by immunotherapy106, 107. This effect was specific to MDSCs, as a significant decrease in the number of T or B cells was not observed in these animals. Furthermore, in a study of 17 patients with early-stage breast cancer that were treated with doxorubicin–cyclophosphamide chemotherapy, a decrease in the level of MDSCs in the peripheral blood was observed22.

Evidence suggests that there is a broad range of methods that will be effective for targeting of the number and/or function of MDSCs in vivo. These strategies will undoubtedly help to further investigate the biology of these cells as well as expedite clinical applications to treat cancer and other pathological conditions.

MDSCs as regulatory myeloid cells? The wealth of information that has accumulated in recent years regarding the biology of MDSCs suggests that these cells might have evolved as a regulatory component of the immune system. These cells are absent under physiological conditions, as IMCs in naive mice are an intrinsic part of normal haematopoiesis that are not immunosuppressive in an unactivated state. In conditions of acute stress, infection or immunization, there is a transient expansion of this IMC population, which then quickly differentiates into mature myeloid cells. This transient IMC population can mediate the suppressive functions that are characteristic of MDSCs but, because the acute conditions are short-lived, the suppressive functions of this transient population have a minimal impact on the overall immune response. However, these cells probably function as important ‘gatekeepers’ that prevent pathological immune-mediated damage.

The role of the MDSC population in settings of chronic infections and cancer is very different. In these pathological conditions, the prolonged and marked expansion of IMCs and their subsequent activation leads to the expansion of a large population of MDSCs with immunosuppressive abilities. MDSCs accumulate in peripheral lymphoid organs and migrate to tumour sites, where they contribute to immunosuppression. Furthermore, some evidence suggests that MDSCs can also induce expansion of regulatory T cells. Future studies will reveal whether MDSCs can be considered part of a natural immune regulatory network.

Concluding remarks The field of MDSC research has more outstanding questions than answers. The roles of specific MDSC subsets in mediating T-cell suppression, and the molecular mechanisms responsible for inhibition of myeloid-cell differentiation, need to be elucidated. The issue of whether Tcell suppression occurs in an antigen-specific manner remains to be clarified, as do the mechanisms that cause MDSC migration to peripheral lymphoid organs. Some of the main priorities in this field should include a better characterization of human MDSCs and a clear understanding of whether targeting these cells in patients with various pathological conditions will be of clinical significance. Conversely, adoptive cellular therapy with MDSCs may be an attractive opportunity by which to inhibit immune responses in the setting of autoimmune disease or transplantation. The challenge for these approaches will be to devise methods by which to generate these cells ex vivo in clinical-grade conditions such that they are suitable for administration to patients. If the past 5–6 years are an indication of the potential for progress in this area, it is safe to estimate that there will soon be significantly more discoveries that further our understanding about the biology and clinical utility of MDSCs.

Box 1. Definition of myeloid-derived suppressor cells (MDSCs)

• a heterogeneous population of cells of myeloid origin that consist of myeloid progenitors and immature macrophages, immature granulocytes and immature dendritic cells

• present in activated state that is characterized by the increased production of reactive oxygen and nitrogen species, and of arginase

• potent suppressors of various T-cell functions • in mice, their phenotype is CD11b+Gr1+, although functionally distinct subsets within this population have been identified (see main text)

• in humans, their phenotype is Lin-HLA-DR-CD33+ or CD11b+CD14-CD33+.

Human cells do not express a marker homologous to mouse Gr1. MDSC have also been identified within a CD15+ population in human peripheral blood.

• in the steady state, immature myeloid cells lack suppressive activity and are present in the bone marrow, but not in secondary lymphoid organs

• accumulation of MDSCs in lymphoid organs and in tumours in response to various growth factors and cytokines is associated with various pathological conditions (most notably cancer)

• in tumour tissues, MDSCs can be differentiated from tumour-associated macrophages (TAMs) by their high expression of Gr1 (not expressed by TAMs) by their low expression of F4/80 (expressed by TAMs), by the fact that a large proportion of MDSCs have a granulocytic morphology and based the upregulated expression of both arginase and inducible nitric oxide synthase by MDSCs but not TAMs.

References

1. Young MRI, Newby M, Wepsic TH. Hematopoiesis and suppressor bone marrow cells in mice bearing large metastatic Lewis lung carcinoma tumors. Cancer Res 1987;47:100–106. [PubMed: 2947676]
2. Buessow SC, Paul RD, Lopez DM. Influence of mammary tumor progression on phenotype and function of spleen and in situ lymphocytes in mice. J Natl Cancer Inst 1984;73:249–255. [PubMed: 6610791]
3. Seung L, Rowley D, Dubeym P, Schreiber H. Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection. Proc Natl Acad Sci U S A 1995;92:6254–6258. [PubMed: 7603979]
4. Sinha P, Clements VK, Bunt SK, Albelda SM, Ostrand-Rosenberg S. Crosstalk between myeloidderived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. J Immunol 2007;179:977–983. [PubMed: 17617589]
5. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008;8:618–631. [PubMed: 18633355]
6. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumorbearing mice. J Immunol 2008;181:5791–5802. [PubMed: 18832739] Together with reference # 17 this paper described functional differences between subsets of MDSC.
7. Bronte V, et al. Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells. Blood 2000;96:3838. [PubMed: 11090068]
8. Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in cancer: The role of reactive oxygen species. J Leukoc Biol 2003;74:186–196. [PubMed: 12885935]
9. Li Q, Pan PY, Gu P, Xu D, Chen SH. Role of immature myeloid Gr-1+ cells in the development of antitumor immunity. Cancer Res 2004;64:1130–1139. [PubMed: 14871848] …..

 

 

Aurelian Udristioiu commented on your update
“The proto-oncogenic transcription factor Myc is known to promote transcription of genes for the cell cycle as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role to induce the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor HIF1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions. “

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Targeting Cancer Neoantigens and Metabolic Change in T-cells

Curator: Larry H. Bernstein, MD, FCAP

 

Updated 5/28/2016

Updtaed 6/1/2016

Fighting Cancer with Borrowed Immunity

http://www.genengnews.com/gen-news-highlights/fighting-cancer-with-borrowed-immunity/81252754/

Outsource a part of the T cell’s immune value chain, propose cancer immunotherapy researchers, from patient T cells to donor T cells. The novel allogeneic approach could rely on T-cell receptor gene transfer to generate broad and tumor-specific T-cell immune responses. [NIAID]

A new cancer immunotherapy approach could essentially outsource a crucial T-cell function. This function, T-cell reactivity to specific cancer antigens, is sometimes lacking in cancer patients. Yet, according to a new proof-of-principle study, these patients could benefit from T cells provided by healthy donors. Specifically, the healthy donors’ T cells could be used to broaden the T-cell receptor repertoires of the cancer patients’ T cells.

Ultimately, this approach relies on a cancer immunotherapy technique called T-cell receptor (TCR) transfer, or the genetic transfer of TCR chains. TCR transfer can be used to outsource the T cell’s learning function, the process by which a T cell acquires the ability to recognize foreign antigens—in this case, the sort of proteins that can be expressed on the surface of cancer cells. Because cancer cells harbor faulty proteins, they can also display foreign protein fragments, also known as neoantigens, on their surface, much in the way virus-infected cells express fragments of viral proteins.

The approach was detailed in a paper that appeared May 19 in the journal Science, in an article entitled, “Targeting of Cancer Neoantigens with Donor-Derived T Cell Receptor Repertoires.” This article, by scientists based at the Netherlands Cancer Institute and the University of Oslo, describes a novel strategy to broaden neoantigen-specific T-cell responses. Such a strategy would be useful in overcoming a common limitation seen in the immune response to cancer: Neoantigen-specific T-cell reactivity is generally limited to just a few mutant epitopes, even though the number of predicted epitopes is large.

“We demonstrate that T cell repertoires from healthy donors provide a rich source of T cells that specifically recognize neoantigens present on human tumors,” the study’s authors wrote. “Responses to 11 epitopes were observed, and for the majority of evaluated epitopes, potent and specific recognition of tumor cells endogenously presenting the neoantigens was detected.”

First, the researchers mapped all possible neoantigens on the surface of melanoma cells from three different patients. In all three patients, the cancer cells seemed to display a large number of different neoantigens. But when the researchers tried to match these to the T cells derived from within the patient’s tumors, most of these aberrant protein fragments on the tumor cells went unnoticed.

Next, the researchers tested whether the same neoantigens could be seen by T cells derived from healthy volunteers. Strikingly, these donor-derived T cells could detect a significant number of neoantigens that had not been seen by the patients’ T cells.

“Many of the T cell reactivities [among donor T cells] involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes,” the authors of the Science article continued. “T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such ‘outsourced’ immune responses in cancer immunotherapy.”

“In a way, our findings show that the immune response in cancer patients can be strengthened; there is more on the cancer cells that makes them foreign that we can exploit. One way we consider doing this is finding the right donor T cells to match these neoantigens,” said Ton Schumacher, Ph.D., a principal investigator at the Netherlands Cancer Institute. “The receptor that is used by these donor T cells can then be used to genetically modify the patient’s own T cells so these will be able to detect the cancer cells.”

“Our study shows that the principle of outsourcing cancer immunity to a donor is sound,” added Johanna Olweus, M.D., Ph.D., who heads a research group at the University of Oslo. “However, more work needs to be done before patients can benefit from this discovery. Thus, we need to find ways to enhance the throughput.”

“We are currently exploring high-throughput methods to identify the neoantigens that the T cells can ‘see’ on the cancer and isolate the responding cells. But the results showing that we can obtain cancer-specific immunity from the blood of healthy individuals are already very promising.”

 

Targeting of cancer neoantigens with donor-derived T cell receptor repertoires

Erlend Strønen1,2Mireille Toebes3Sander Kelderman3,…., Fridtjof Lund-Johansen2,5Johanna Olweus1,2,*,Ton N. Schumacher3,*,   + Author Affiliations
Science  19 May 2016:                         http://dx.doi.org:/10.1126/science.aaf2288

Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen specific T cell responses are therefore attractive. Here, we demonstrate that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11/57 predicted HLA-A2-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such “outsourced” immune responses in cancer immunotherapy.

Metabolic maintenance of cell asymmetry following division in activated T lymphocytes.

Verbist KC1, Guy CS1, Milasta S1, Liedmann S1, Kamiński MM1, Wang R2, Green DR1
Nature. 2016 Apr 21; 532(7599):389-93.   http://dx. doi.org:/10.1038/nature17442. Epub 2016 Apr 11

Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.

AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5′ AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL.

Glutamine Modulates Macrophage Lipotoxicity.

He L1,2, Weber KJ3,4, Schilling JD5,6,7
Nutrients. 2016 Apr 12;8(4). pii: E215.   http://dx.doi.org:/10.3390/nu8040215
Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α

Sangbin Lim1Hao Liu1,2,*Luciana Madeira da Silva1Ritu Arora1,…., Gary A. Piazza1Oystein Fodstad1,4,*, and Ming Tan1,5,*
C
ancer Res April 5, 2016    http://dx.doi.org:/10.1158/0008-5472.CAN-15-1538

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo. We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3–expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species–dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 1–12. ©2016 AACR.

 

TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

Salerno F1, Guislain A2, …, Wolkers MC2.
J Immunol. 2016 May 1;196(9):3695-705.   http://dx.doi.org:/10.4049/jimmunol.1501997. Epub 2016 Mar 25.
CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.
 Immunometabolism of regulatory T cells 

Newton RPriyadharshini B & Laurence A Turk
Nature Immunology 2016;17:618–625
  http://dx.doi.
doi.org:/10.1038/ni.3466

The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years. Via effects on adipocytes and hepatocytes, immune cells can modulate whole-body metabolism (in metabolic syndromes such as type 2 diabetes and obesity) and, reciprocally, host nutrition and commensal-microbiota-derived metabolites modulate immunological homeostasis. Studies demonstrating the metabolic similarities of proliferating immune cells and cancer cells have helped give birth to the new field of immunometabolism, which focuses on how the cell-intrinsic metabolic properties of lymphocytes and macrophages can themselves dictate the fate and function of the cells and eventually shape an immune response. We focus on this aspect here, particularly as it relates to regulatory T cells.

Figure 1: Proposed model for the metabolic signatures of various Treg cell subsets.

Proposed model for the metabolic signatures of various Treg cell subsets.

(a) Activated CD4+ T cells that differentiate into the Teff cell lineage (green) (TH1 or TH17 cells) are dependent mainly on carbon substrates such as glucose and glutamine for their anabolic metabolism. In contrast to that, pTreg cells…
T-bet is a key modulator of IL-23-driven pathogenic CD4+ T cell responses in the intestine
Krausgruber TSchiering CAdelmann K & Harrison OJ.
Nature Communications 7; Article number:11627    http://dx.doi.org:/10.1038/ncomms11627

IL-23 is a key driver of pathogenic Th17 cell responses. It has been suggested that the transcription factor T-bet is required to facilitate IL-23-driven pathogenic effector functions; however, the precise role of T-bet in intestinal T cell responses remains elusive. Here, we show that T-bet expression by T cells is not required for the induction of colitis or the differentiation of pathogenic Th17 cells but modifies qualitative features of the IL-23-driven colitogenic response by negatively regulating IL-23R expression. Consequently, absence of T-bet leads to unrestrained Th17 cell differentiation and activation characterized by high amounts of IL-17A and IL-22. The combined increase in IL-17A/IL-22 results in enhanced epithelial cell activation and inhibition of either IL-17A or IL-22 leads to disease amelioration. Our study identifies T-bet as a key modulator of IL-23-driven colitogenic responses in the intestine and has important implications for understanding of heterogeneity among inflammatory bowel disease patients.

Th17 cells are enriched at mucosal sites, produce high amounts of IL-17A, IL-17F and IL-22, and have an essential role in mediating host protective immunity against a variety of extracellular pathogens1. However, on the dark side, Th17 cells have also been implicated in a variety of autoimmune and chronic inflammatory conditions, including inflammatory bowel disease (IBD)2. Despite intense interest, the cellular and molecular cues that drive Th17 cells into a pathogenic state in distinct tissue settings remain poorly defined.

The Th17 cell programme is driven by the transcription factor retinoid-related orphan receptor gamma-t (RORγt) (ref. 3), which is also required for the induction and maintenance of the receptor for IL-23 (refs 4, 5). The pro-inflammatory cytokine IL-23, composed of IL-23p19 and IL-12p40 (ref. 6), has been shown to be a key driver of pathology in various murine models of autoimmune and chronic inflammatory disease such as experimental autoimmune encephalomyelitis (EAE)7, collagen induced arthritis8 and intestinal inflammation9, 10, 11, 12. Several lines of evidence, predominantly derived from EAE, suggest that IL-23 promotes the transition of Th17 cells to pathogenic effector cells9, 10, 11, 12. Elegant fate mapping experiments of IL-17A-producing cells during EAE have shown that the majority of IL-17A+IFN-γ+ and IL-17A−IFN-γ+ effector cells arise from Th17 cell progeny13. This transition of Th17 cells into IFN-γ-producing ‘ex’ Th17 cells required IL-23 and correlated with increased expression of T-bet. The T-box transcription factor T-bet drives the Th1 cell differentiation programme14 and directly transactivates the Ifng gene by binding to its promoter as well as multiple enhancer elements15. Indeed, epigenetic analyses have revealed that the loci for T-bet and IFN-γ are associated with permissive histone modifications in Th17 cells suggesting that Th17 cells are poised to express T-bet which could subsequently drive IFN-γ production16, 17.

A similar picture is emerging in the intestine where IL-23 drives T-cell-mediated intestinal pathology which is thought to be dependent on expression of T-bet18 and RORγt (ref. 19) by T cells. In support of this we have recently shown that IL-23 signalling in T cells drives the emergence of IFN-γ producing Th17 cells in the intestine during chronic inflammation20. Collectively these studies suggest a model whereby RORγt drives differentiation of Th17 cells expressing high amounts of IL-23R, and subsequently, induction of T-bet downstream of IL-23 signalling generates IL-17A+IFN-γ+ T cells that are highly pathogenic. Indeed, acquisition of IFN-γ production by Th17 cells has been linked to their pathogenicity in several models of chronic disease13, 21, 22, 23, 24 and a population of T cells capable of producing both IL-17A and IFN-γ has also been described in intestinal biopsies of IBD patients25, 26.

However, in the context of intestinal inflammation, it remains poorly defined whether the requirement for RORγt and T-bet reflects a contribution of Th17 and Th1 cells to disease progression or whether Th17 cells require T-bet co-expression to exert their pathogenic effector functions. Here, we use two distinct models of chronic intestinal inflammation and make the unexpected finding that T-bet is dispensable for IL-23-driven colitis. Rather the presence of T-bet serves to modify the colitogenic response restraining IL-17 and IL-22 driven pathology. These data identify T-bet as a key modulator of IL–23-driven colitogenic effector responses in the intestine and have important implications for understanding of heterogeneous immune pathogenic mechanisms in IBD patients.

Figure 1: IL-23 signalling is required for bacteria-driven T-cell-dependent colitis and the emergence of IL-17A+IFN-γ+ T cells.
C57BL/6 WT and Il23r−/− mice were infected orally with Hh and received weekly i.p. injections of IL-10R blocking antibody. Mice were killed at 4 weeks post infection and assessed for intestinal inflammation. (a) Colitis scores. (b) Typhlitis sores. (c) Representative photomicrographs of colon and caecum (× 10 magnification; scale bars, 200μM). (d) Representative flow cytometry plots of colonic lamina propria gated on viable CD4+ T cells. (e) Frequencies of IL-17A+ and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from two independent experiments (n=12 for WT, n=10 for Il23r−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, ***P<0.001 as calculated by Mann–Whitney U test.

 

IL-23 signals are dispensable for T-bet and RORγt expression 

RORγt but not T-bet is required for T cell transfer colitis

Figure 2: RORγt but not T-bet expression by CD4+ T cells is required for the development of T cell transfer colitis.

C57BL/6 Rag1−/− mice were injected i.p. with 4 × 105 CD4+CD25CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/− T cells developed clinical signs of disease (4–6 weeks) and assessed for intestinal inflammation. (a) Colitis scores. (b) Representative photomicrographs of proximal colon sections (× 10 magnification; scale bars, 200μM). (c) Concentration of cytokines released from colon explants into the medium after overnight culture. Data represent pooled results from two independent experiments (n=14 for WT, n=11 for Rorc−/−, n=14 forTbx21−/−). Bars are the mean and each symbol represents an individual mouse. Bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.

T-bet is dispensable for IL-17A+IFN-γ+ intestinal T cells

Figure 3: T-bet expression by CD4+ T cells is not required for the emergence of IL-17A+IFN-γ+ T cells.

C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of IL-17A and IFN-γ expression in colonic CD4+ T cells. (b) Frequencies of IL-17A+ and/or IFN-γ+ cells among colonic CD4+ T cells. (c) Total numbers of IL-17A+and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from three independent experiments (n=20 for WT, n=18 for Tbx21−/−, n=12 for Rorc−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.

T-bet deficiency promotes an exacerbated Th17-type response

Our transfer of Tbx21−/− T cells revealed a striking increase in the frequency of IL-17A+IFN-γcells (Fig. 3) and we reasoned that T-bet-deficiency could impact on Th17 cell cytokine production. Therefore, we transferred WT or Tbx21−/− CD4+ T cells into Rag1−/− recipients and measured the expression of RORγt, IL-17A, IL-17F and IL-22 by CD4+ T cells isolated from the colon. In agreement with our earlier findings, Tbx21−/− T cells gave rise to significantly increased frequencies of RORγt-expressing T cells capable of producing IL-17A (Fig. 4a). Furthermore, T-bet deficiency also led to a dramatic expansion of IL-17F and IL-22-expressing cells, which constituted only a minor fraction in WT T cells (Fig. 4a,b). By contrast, the frequency of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ producing cells was significantly reduced in T-bet-deficient T cells as compared with WT T cells. When analysed in more detail we noted that the production of IL-17A, IL-17F and IL-22 increased specifically in T-bet-deficient IL-17A+IFN-γ+ T cells as compared with WT T cells whereas IFN-γ production decreased overall in the absence of T-bet as expected (Supplementary Fig. 4A). Similarly, GM-CSF production was also generally reduced in Tbx21−/− CD4+ T cells further suggesting a shift in the qualitative nature of the T cell response.

 

Figure 4: T-bet-deficient CD4+ T cells promote an exacerbated Th17-type inflammatory response.

C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25CD45RBhi T cells from C57BL/6 WT orTbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of cytokines and transcription factors in WT or Tbx21−/− colonic CD4+ T cells. (b) Frequency of IL-17A+, IL-17F+, IL-22+, GM-CSF+ or IFN-γ+ colonic T cells in WT orTbx21−/−. (c) quantitative reverse transcription PCR (qRT-PCR) analysis of mRNA levels of indicated genes in colon tissue homogenates. (d) Total number of neutrophils (CD11b+ Gr1high) in the colon. (e) Primary epithelial cells were isolated from the colon of steady state C57BL/6 Rag1−/− mice and stimulated with 10ngml−1 cytokines for 4h after which cells were harvested and analysed by qRT-PCR for the indicated genes. Data in bd represent pooled results from two independent experiments (n=14 for WT, n=11 for Tbx21−/−). Bars are the mean and error bars represent s.e.m. Data in e are pooled results from four independent experiments, bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01,***P<0.001 as calculated by Mann–Whitney U test.

………

T-bet-deficient colitis depends on IL-23, IL-17A and IL-22

In the present study we show that bacteria-driven colitis is associated with the IL-23-dependent emergence of IFN-γ-producing Th17 cells co-expressing RORγt and T-bet. Strikingly, while RORγt is required for the differentiation of IFN-γ-producing Th17 cells and induction of colitis, T-bet is dispensable for the emergence of IL-17A+IFN-γ+ T cells and intestinal pathology. Our results show that instead of a mandatory role in the colitogenic response, the presence of T-bet modulates the qualitative nature of the IL-23-driven intestinal inflammatory response. In the presence of T-bet, IL-23-driven colitis is multifunctional in nature and not functionally dependent on either IL-17A or IL-22. By contrast, in the absence of T-bet a highly polarized colitogenic Th17 cell response ensues which is functionally dependent on both IL-17A and IL-22. T-bet-deficient T cells are hyper-responsive to IL-23 resulting in enhanced STAT3 activation and downstream cytokine secretion providing a mechanistic basis for the functional changes. These data newly identify T-bet as a key modulator of IL-23-driven colitogenic CD4+ T cell responses.

Contrary to our expectations T-bet expression by CD4 T cells was not required for their pathogenicity. In keeping with the negative effect of T-bet on Th17 differentiation40, 41, 42, we observed highly polarized Th17 responses in T-bet-deficient intestinal T cells. Early studies demonstrated that IFN-γ could suppress the differentiation of Th17 cells40 and thus the reduced IFN-γ production by Tbx21−/−T cells could facilitate Th17 cell generation. However, our co-transfer studies revealed unrestrained Th17 differentiation of Tbx21−/− T cells even in the presence of WT T cells, suggesting a cell autonomous role for T-bet-mediated suppression of the Th17 programme. Indeed, the role of T-bet as a transcriptional repressor of the Th17 cell fate has been described recently. For example, T-bet physically interacts with and sequesters Runx1, thereby preventing Runx1-mediated induction of RORγt and Th17 cell differentiation43. In addition, T-bet binds directly to and negatively regulates expression of many Th17-related genes15, 34 and we identified IL23r to be repressed in a T-bet-dependent manner. In line with this we show here that T-bet-deficient intestinal T cells express higher amounts of Il23r as well as Rorc. This resulted in enhanced IL-23-mediated STAT3 activation and increased production of IL-17A and IL-22. It has also been suggested that T-bet activation downstream of IL-23R signalling is required for pathogenic IL-23-driven T cell responses43, 44. However, we did not find a role for IL-23 in the induction and/or maintenance of T-bet expression and colitis induced by T-bet-deficient T cells was IL-23 dependent. Collectively, these findings demonstrate that T-bet deficiency leads to unrestrained expansion of colitogenic Th17 cells, which is likely mediated through enhanced activation of the IL-23R-STAT3 pathway.

The observation that T-bet-deficient T cells retain their colitogenic potential is in stark contrast to earlier studies. Neurath et al.18 convincingly showed that adoptive transfer of Tbx21−/− CD4+ T cells into severe combined immunodeficiency (SCID) recipients failed to induce colitis and this correlated with reduced IFN-γ and increased IL-4 production. Another study revealed that IL-4 plays a functional role in inhibiting the colitogenic potential of Tbx21−/− T cells, as recipients ofStat6−/−Tbx21−/− T cells developed severe colitis37. Importantly, the intestinal inflammation that developed in recipients of Stat6−/−Tbx21−/− T cells could be blocked by administration of IL-17A neutralizing antibody, suggesting that the potent inhibitory effect of IL-4/STAT6 signals on Th17 differentiation normally prevent colitis induced by Tbx21−/− T cells37. Various explanations could account for the discrepancy between our study and those earlier findings. First, in contrast to the published reports, we used naïve Tbx21−/− CD4+ T cells from C57BL/6 mice instead of BALB/c mice. An important difference between Tbx21−/− CD4+ T cells from these genetic backgrounds appears to be their differential susceptibility to suppression by IL-4/STAT6 signals. We found that transfer of Tbx21−/− T cells induced IL-17A-dependent colitis despite increased frequencies of IL-4-expressing cells in the intestine. This discrepancy may be due to higher amounts of IL-4 produced by activated CD4+ T cells from BALB/c versus C57BL/6 mice45, leading to the well-described Th2-bias of the BALB/c strain45. Second, differences in the composition of the intestinal microbiota between animal facilities can have a substantial effect on skewing CD4+ T cells responses. In particular, the Clostridium-related segmented filamentous bacteria (SFB) have been shown to drive the emergence of IL-17 and IL-22 producing CD4+ T cells in the intestine46. Importantly, the ability of naïve CD4+ T cells to induce colitis is dependent on the presence of intestinal bacteria, as germ-free mice do not develop pathology upon T cell transfer47. In line with this, we previously described that colonization of germ-free mice with intestinal microbiota containing SFB was necessary to restore the development of colitis47. Since our Rag1−/− colony is SFB+ and the presence of SFB was not reported in the previous studies, it is possible that differences in SFB colonization status contributed to the observed differences in pathogenicity ofTbx21−/− T cells.

It is important to note that T-bet-deficient T cells did not induce more severe colitis than WT T cells but rather promoted a distinct mucosal inflammatory response. Colitis induced by WT T cells is characterized by a multifunctional response with high amounts of IFN-γ and GM-CSF and a lower IL-17A and IL-22 response. Consistent with this, we have shown that blockade of GM-CSF abrogates T cell transfer colitis48 as well as bacteria-driven intestinal inflammation49 in T-bet sufficiency whereas blockade of IL-17A or IL-22 fails to do so. By contrast T-bet deficiency leads to production of high amounts of IL-17A and IL-22 in the colon and neutralization of either was sufficient to reduce intestinal pathology. Our in vitro experiments suggest that IL-17A and IL-22 synergise to promote intestinal epithelial cell responses, which may in part explain the efficacy of blocking IL-17A or IL-22 in colitis induced by T-bet-deficient T cells. A similar synergistic interplay has been described in the lung where IL-22 served a tissue protective function in homeostasis but induced airway inflammation in the presence of IL-17A (ref. 50). This highlights the complexity of the system in health and disease, and the need for a controlled production of both cytokines. We describe here only one mechanism of how IL-17A/IL-22 induce a context-specific epithelial cell response that potentially impacts on the order or composition of immune cell infiltration. Overall, these results provide a new perspective on T-bet, revealing its role in shaping the qualitative nature of the IL-23-driven colitogenic T cell response.

We also describe here the unexpected finding that a substantial proportion of T-bet-deficient intestinal T cells retain the ability to express IFN-γ. To investigate the potential mechanisms responsible for T-bet-independent IFN-γ production by intestinal CD4+ T cells we focused on two transcription factors, Runx3 and Eomes. Runx3 has been shown to promote IFN-γ expression directly through binding to the Ifng promoter38 and Eomes is known to compensate for IFN-γproduction in T-bet-deficient Th1 cells37. We found IL-23-mediated induction of Runx3 protein in WT and Tbx21−/− T cells isolated from the intestine, thus identifying Runx3 downstream of IL-23R signalling. By contrast, we could only detect Eomes protein and its induction by IL-23 in T-bet-deficient but not WT T cells. Thus, Runx3 and Eomes are activated in response to IL-23 in T-bet-deficient cells and are likely to be drivers of T-bet-independent IFN-γ production. In support of this we found that the majority of T-bet-deficient IL-17AIFN-γ+ T cells expressed Eomes. However, only a minor population of IL-17A+IFN-γ+ T cells stained positive for Eomes, suggesting the existence of alternative pathways for IFN-γ production by Th17 cells. Intriguingly, a recent study identified Runx3 and Runx1 as the transcriptional regulators critical for the differentiation of IFN-γ-producing Th17 cells51. The author’s demonstrated that ectopic expression of Runx transcription factors was sufficient to induce IFN-γ production by Th17 cells even in the absence of T-bet. These findings, combined with our data on Runx3 activation downstream of IL-23R signalling strongly suggest that Runx3 rather than Eomes is driving IFN-γ expression by intestinal Th17 cells.

We have not formally addressed the role of IFN-γ in colitis driven by T-bet-deficient T cells. A recent report by Zimmermann et al.52 found that antibody-mediated blockade of IFN-γ ameliorates colitis induced by WT or T-bet-deficient T cells suggesting IFN-γ also contributes to the colitogneic response mediated by T-bet-deficient T cells as originally described for WT T cells53, 54. By contrast with our results the Zimmerman study found that IL-17A blockade exacerbated colitis following transfer of Tbx21−/− T cells. The reason for the differential role of IL-17A in the two studies is not clear but it is notable that the Zimmerman study was performed in the presence of co-infection with SFB and Hh, and this strong inflammatory drive may alter the pathophysiological role of particular cytokines. Together the data indicate that T-bet deficiency in T cells does not impede their colitogenic activity but that the downstream effector cytokines of the response are context dependent.

In conclusion, our data further underline the essential role for IL-23 in intestinal inflammation and demonstrate that T-bet is an important modulator of the IL–23-driven effector T cell response. The colitogenic T cell response in a T-bet sufficient environment is multifunctional with a dominant GM-CSF and IFN-γ response. By contrast T-bet-deficient colitogenic responses are dominated by IL-17A and IL-22-mediated immune pathology. These results may have significant bearing on human IBD where it is now recognized that differential responsiveness to treatment may reflect considerable disease heterogeneity. As such, identification of suitable biomarkers such as immunological parameters, that allow stratification of patient groups, is becoming increasingly important55. Genome-wide association studies have identified polymorphisms in loci related to innate and adaptive immune arms that confer increased susceptibility to IBD. Among these are Th1 (STAT4, IFNG and STAT1) as well as Th17-related genes (RORC, IL23R and STAT3) (refs56, 57). Thus, detailed profiling of the T cell response in IBD patients may help identify appropriate patient groups that are most likely to benefit from therapeutic blockade of certain effector cytokines. Finally, our studies highlight the importance of IL-23 in the intestinal inflammatory hierarchy and suggest that IL-23 could be an effective therapeutic target across a variety of patient groups.

 

Yale study: How antibodies access neurons to fight infection

Yale scientists have solved a puzzle of the immune system: how antibodies enter the nervous system to control viral infections. Their finding may have implications for the prevention and treatment of a range of conditions, including herpes and Guillain-Barre syndrome, which has been linked to the Zika virus.

Many viruses — such as West Nile, Zika, and the herpes simplex virus — enter the nervous system, where they were thought to be beyond the reach of antibodies. Yale immunobiologists Akiko Iwasaki and Norifumi Iijima used mice models to investigate how antibodies could gain access to nerve tissue in order to control infection.

In mice infected with herpes, they observed a previously under-recognized role of CD4 T cells, a type of white blood cell that guards against infection by sending signals to activate the immune system. In response to herpes infection, CD4 T cells entered the nerve tissue, secreted signaling proteins, and allowed antibody access to infected sites. Combined, CD4 T cells and antibodies limited viral spread.

“This is a very elegant design of the immune system to allow antibodies to go to the sites of infection,” said Iwasaki. “The CD4 T cells will only go to the site where there is a virus. It’s a targeted delivery system for antibodies.”

 

Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help

Norifumi Iijima & Akiko Iwasaki
Nature 533,552–556 (26 May 2016)
    http://dx.
doi.org:/10.1038/nature17979

 

Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood–brain barrier1 and blood–nerve barrier2, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.

 

T Cells Help Reverse Ovarian Cancer Drug Resistance

http://www.genengnews.com/gen-news-highlights/t-cells-help-reverse-ovarian-cancer-drug-resistance/81252753/

T cells (red) attack ovarian cancer cells (green). [University of Michigan Health System]

Researchers at the University of Michigan have recently published the results from a new study that they believe underscores why so many ovarian tumors develop resistance to chemotherapy. The tumor microenvironment is made up of an array of cell types, yet effector T cells and fibroblasts constitute the bulk of the tissue. The investigators believe that understanding the interplay between these two cell types holds the key to how ovarian cancer cells develop resistance.

The new study suggests that the fibroblasts surrounding the tumor work to block chemotherapy, which is why nearly every woman with ovarian cancer becomes resistant to treatment. Conversely, the scientists published evidence that T cells in the microenvironment can reverse the resistance phenotype—suggesting a whole different way of thinking about chemotherapy resistance and the potential to harness immunotherapy drugs to treat ovarian cancer.

“Ovarian cancer is often diagnosed at late stages, so chemotherapy is a key part of treatment,” explained co-senior study author J. Rebecca Liu, M.D., associate professor of obstetrics and gynecology at the University of Michigan. “Most patients will respond to it at first, but everybody develops chemoresistance. And that’s when ovarian cancer becomes deadly.”

Dr. Liu continued, stating that “in the past, we’ve thought the resistance was caused by genetic changes in tumor cells. But we found that’s not the whole story.”

The University of Michigan team looked at tissue samples from ovarian cancer patients and separated the cells by type to study the tumor microenvironment in vitro and in mice. More importantly, the scientists linked their findings back to actual patient outcomes.

The results of this study were published recently in Cell through an article entitled “Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.”

Ovarian cancer is typically treated with cisplatin, a platinum-based chemotherapy. The researchers found that fibroblasts blocked platinum. These cells prevented platinum from accumulating in the tumor and protected tumor cells from being killed off by cisplatin.

Diagram depicting how T cells can reverse chemotherapeutic resistance. [Cell, Volume 165, Issue 5, May 19, 2016]

“We show that fibroblasts diminish the nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy,” the authors wrote. “We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance.”

T cells, on the other hand, overruled the protection of the fibroblasts. When researchers added the T cells to the fibroblast population, the tumor cells began to die off.

“CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts,” the authors explained. “CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xccystine and glutamate antiporter via the JAK/STAT1 pathway.”

By boosting the effector T cell numbers, the researchers were able to overcome the chemotherapy resistance in mouse models. Moreover, the team used interferon, an immune cell-secreted cytokine, to manipulate the pathways involved in cisplatin.

“T cells are the soldiers of the immune system,” noted co-senior study author Weiping Zou, M.D., Ph.D., professor of surgery, immunology, and biology at the University of Michigan. “We already know that if you have a lot of T cells in a tumor, you have better outcomes. Now we see that the immune system can also impact chemotherapy resistance.”

The researchers suggest that combining chemotherapy with immunotherapy may be effective against ovarian cancer. Programmed death ligand 1 (PD-L1) and PD-1 pathway blockers are currently FDA-approved treatments for some cancers, although not ovarian cancer.

“We can imagine re-educating the fibroblasts and tumor cells with immune T cells after chemoresistance develops,” Dr. Zou remarked.

“Then we could potentially go back to the same chemotherapy drug that we thought the patient was resistant to. Only now we have reversed that, and it’s effective again,” Dr. Liu concluded.

 

Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer

Weimin Wang, Ilona Kryczek, Lubomír Dostál, Heng Lin, Lijun Tan, et al.
Cell May 2016;  165, Issue 5:1092–1105.   http://dx.doi.org/10.1016/j.cell.2016.04.009
Highlights
  • Fibroblasts diminish platinum content in cancer cells, resulting in drug resistance
  • GSH and cysteine released by fibroblasts contribute to platinum resistance
  • T cells alter fibroblast GSH and cystine metabolism and abolish the resistance
  • Fibroblasts and CD8+ T cells associate with patient chemotherapy response

 

Summary

Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8+ T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

 

Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation. Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α..
The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.
CD28 co-stimulation further activates the PI3K/Akt/mTOR pathway in particular, and provides a signal for Glut1 expression and cell surface localization.
Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway. The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism.
Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions

 

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Biology, Physiology and Pathophysiology of Heat Shock Proteins

Curation: Larry H. Bernstein, MD, FCAP

 

 

Heat Shock Proteins (HSP)

  1. Exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes

Hsp70 chaperones: Cellular functions and molecular mechanism

M. P. MayerB. Bukau
Cell and Molec Life Sci  Mar 2005; 62:670  http://dx.doi.org:/10.1007/s00018-004-4464-6

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [17]. Hsp70s have thus housekeeping functions in the cell in which they are built-in components of folding and signal transduction pathways, and quality control functions in which they proofread the structure of proteins and repair misfolded conformers. All of these activities appear to be based on the property of Hsp70 to interact with hydrophobic peptide segments of proteins in an ATP-controlled fashion. The broad spectrum of cellular functions of Hsp70 proteins is achieved through

  • the amplification and diversification of hsp70genes in evolution, which has generated specialized Hsp70 chaperones,
  • co-chaperones which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and
  • cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines.

Protein folding processes assisted by Hsp70

The role of Hsp70s in the folding of non-native proteins can be divided into three related activities: prevention of aggregation, promotion of folding to the native state, and solubilization and refolding of aggregated proteins. In the cellular milieu, Hsp70s exert these activities in the quality control of misfolded proteins and the co- and posttranslational folding of newly synthesized proteins. Mechanistically related but less understood is the role of Hsp70s in the disassembly of protein complexes such as clathrin coats, viral capsids and the nucleoprotein complex, which initiates the replication of bacteriophage λ DNA. A more complex folding situation exists for the Hsp70-dependent control of regulatory proteins since several steps in the folding and activation process of these substrates are assisted by multiple chaperones.

Hsp70 proteins together with their co-chaperones of the J-domain protein (JDP) family prevent the aggregation of non-native proteins through association with hydrophobic patches of substrate molecules, which shields them from intermolecular interactions (‘holder’ activity). Some JDPs such as Escherichia coli DnaJ and Saccharomyces cerevisiae Ydj1 can prevent aggregation by themselves through ATP-independent transient and rapid association with the substrates. Only members of the Hsp70 family with general chaperone functions have such general holder activity.

Hsp70 chaperone systems assist non-native folding intermediates to fold to the native state (‘folder’ activity). The mechanism by which Hsp70-chaperones assist the folding of non-native substrates is still unclear. Hsp70-dependent protein folding in vitro occurs typically on the time scale of minutes or longer. Substrates cycle between chaperone-bound and free states until the ensemble of molecules has reached the native state. There are at least two alternative modes of action. In the first mechanism Hsp70s play a rather passive role. Through repetitive substrate binding and release cycles they keep the free concentration of the substrate sufficiently low to prevent aggregation, while allowing free molecules to fold to the native state (‘kinetic partitioning’). In the second mechanism, the binding and release cycles induce local unfolding in the substrate, e.g. the untangling of a misfolded β-sheet, which helps to overcome kinetic barriers for folding to the native state (‘local unfolding’) [8–11]. The energy of ATP may be used to induce such conformational changes or alternatively to drive the ATPase cycle in the right direction.

Hsp70 in cellular physiology and pathophysiology

Two Hsp70 functions are especially interesting, de novo folding of nascent polypeptides and interaction with signal transduction proteins, and therefore some aspects of these functions shall be discussed below in more detail. Hsp70 chaperones were estimated to assist the de novo folding of 10–20% of all bacterial proteins whereby the dependence on Hsp70 for efficient folding correlated with the size of the protein [12]. Since the average protein size in eukaryotic cells is increased (52 kDa in humans) as compared to bacteria (35 kDa in E. coli) [25], it is to be expected that an even larger percentage of eukaryotic proteins will be in need of Hsp70 during de novo folding. This reliance on Hsp70 chaperones increases even more under stress conditions. Interestingly, mutated proteins [for example mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxid dismutase (SOD) 1] seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein [2629]. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations [30], a function demonstrated earlier for Hsp90 [3132]. Such mutations are only uncovered when the overall need for Hsp70 action exceeds the chaperone capacity of the Hsp70 proteins, for example during stress conditions [30], at certain stages in development or during aging, when the magnitude of stress-induced increase in Hsp70 levels declines [3334]. Alternatively, the mutant protein can be targeted by Hsp70 and its co-chaperones to degradation as shown e.g. for CFTRΔF508 and some of the SOD1 mutant proteins [35,36]. Deleterious mutant proteins may then only accumulate when Hsp70 proteins are overwhelmed by other, stress-denatured proteins. Both mechanisms may contribute to pathological processes such as oncogenesis (mutant p53) and neurodegenerative diseases, including amyotrophic, lateral sclerosis (SOD1 mutations), Parkinsonism (α-synuclein mutations), Huntington’s chorea (huntingtin with polyglutamin expansions) and spinocerebellar ataxias (proteins with polyglutamin expansions).

De novo folding is not necessarily accelerated by Hsp70 chaperones. In some cases folding is delayed for different reasons. First, folding of certain proteins can only proceed productively after synthesis of the polypeptide is completed as shown, e.g. for the reovirus lollipop-shaped protein sigma 1 [37]. Second, proteins destined for posttranslational insertion into organellar membranes are prevented from aggregation and transported to the translocation pore [38]. Third, in the case of the caspase-activated DNase (CAD), the active protein is dangerous for the cell and therefore can only complete folding in the presence of its specific inhibitor (ICAD). Hsp70 binds CAD cotranslationally and mediates folding only to an intermediate state. Folding is completed after addition of ICAD, which is assembled into a complex with CAD in an Hsp70-dependent manner [39]. Similar folding pathways may exist also for other potentially dangerous proteins.

As mentioned above Hsp70 interacts with key regulators of many signal transduction pathways controlling cell homeostasis, proliferation, differentiation and cell death. The interaction of Hsp70 with these regulatory proteins continues in activation cycles that also involve Hsp90 and a number of co-chaperones. The regulatory proteins, called clients, are thereby kept in an inactive state from which they are rapidly activated by the appropriate signals. Hsp70 and Hsp90 thus repress regulators in the absence of the upstream signal and guarantee full activation after the signal transduction pathway is switched on [6]. Hsp70 can be titrated away from these clients by other misfolded proteins that may arise from internal or external stresses. Consequently, through Hsp70 disturbances of the cellular system induced by environmental, developmental or pathological processes act on these signal transduction pathways.

In this way stress response and apoptosis are linked to each other. Hsp70 inhibits apoptosis acting on the caspase-dependent pathway at several steps both upstream and downstream of caspase activation and on the caspase-independent pathway. Overproduction of Hsp70 leads to increased resistance against apoptosis-inducing agents such as tumor necrosis factor-α(TNFα), staurosporin and doxorubicin, while downregulation of Hsp70 levels by antisense technology leads to increased sensitivity towards these agents [1840]. This observation relates to many pathological processes, such as oncogenesis, neurodegeneration and senescence. In many tumor cells increased Hsp70 levels are observed and correlate with increased malignancy and resistance to therapy. Downregulation of the Hsp70 levels in cancer cells induce differentiation and cell death [41]. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s corea and spinocerebellar ataxias are characterized by excessive apoptosis. In several different model systems overexpression of Hsp70 or one of its co-chaperones could overcome the neurodegenerative symptoms induced by expression of a disease-related gene (huntingtin, α-synuclein or ataxin) [20,42]. Senescence in cell culture as well as aging in vivo is correlated with a continuous decline in the ability to mount a stress response [3443]. Age-related symptoms and diseases reflect this decreased ability to cope with cellular stresses. Interestingly, centenarians seem to be an exception to the rule, as they show a significant induction of Hsp70 production after heat shock challenge [44].

ATPase domain and ATPase cycle

Substrate binding

The coupling mechanism: nucleotide-controlled opening and closing of the substrate binding cavity

The targeting activity of co-chaperones

J-domain proteins

Bag proteins

Hip, Hop and CHIP

Perspectives

The Hsp70 protein family and their co-chaperones constitute a complex network of folding machines which is utilized by cells in many ways. Despite considerable progress in the elucidation of the mechanistic basis of these folding machines, important aspects remain to be solved. With respect to the Hsp70 proteins it is still unclear whether their activity to assist protein folding relies on the ability to induce conformational changes in the bound substrates, how the coupling mechanism allows ATP to control substrate binding and to what extent sequence variations within the family translate into variations of the mechanism. With respect to the action of co-chaperones we lack a molecular understanding of the coupling function of JDPs and of how co-chaperones target their Hsp70 partner proteins to substrates. Furthermore, it can be expected that more cellular processes will be discovered that depend on the chaperone activity of Hsp70 chaperones.

 

  1. The biochemistry and ultrastructure of molecular chaperones

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Laurence H. Pearl and Chrisostomos Prodromou
Ann Rev of Biochem July 2006;75:271-294
http://dx.doi.org:/10.1146/annurev.biochem.75.103004.142738

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90’s diverse “clientele.” Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

 

  1. Properties of heat shock proteins (HSPs) and heat shock factor (HSF)

Heat shock factors: integrators of cell stress, development and lifespan

Malin Åkerfelt,*‡ Richard I. Morimoto,§ and Lea Sistonen*‡
Nat Rev Mol Cell Biol. 2010 Aug; 11(8): 545–555.  doi:  10.1038/nrm2938

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

In the early 1960s, Ritossa made the seminal discovery of temperature-induced puffs in polytene chromosomes of Drosophila melanogaster larvae salivary glands1. A decade later, it was shown that the puffing pattern corresponded to a robust activation of genes encoding the heat shock proteins (HSPs), which function as molecular chaperones2. The heat shock response is a highly conserved mechanism in all organisms from yeast to humans that is induced by extreme proteotoxic insults such as heat, oxidative stress, heavy metals, toxins and bacterial infections. The conservation among different eukaryotes suggests that the heat shock response is essential for survival in a stressful environment.

The heat shock response is mediated at the transcriptional level by cis-acting sequences called heat shock elements (HSEs; BOX 1) that are present in multiple copies upstream of the HSP genes3. The first evidence for a specific transcriptional regulator, the heat shock factor (HSF) that can bind to the HSEs and induce HSP gene expression, was obtained through DNA–protein interaction studies on nuclei isolated from D. melanogaster cells4,5. Subsequent studies showed that, in contrast to a single HSF in invertebrates, multiple HSFs are expressed in plants and vertebrates68. The mammalian HSF family consists of four members: HSF1,HSF2, HSF3 and HSF4. Distinct HSFs possess unique and overlapping functions (FIG. 1), exhibit tissue-specific patterns of expression and have multiple post-translational modifications (PTMs) and interacting protein partners7,9,10. Functional crosstalk between HSF family members and PTMs facilitates the fine-tuning of HSF-mediated gene regulation. The identification of many targets has further extended the impact of HSFs beyond the heat shock response. Here, we present the recent discoveries of novel target genes and physiological functions of HSFs, which have changed the view that HSFs act solely in the heat shock response. Based on the current knowledge of small-molecule activators and inhibitors of HSFs, we also highlight the potential for pharmacologic modulation of HSF-mediated gene regulation.

Box 1

The heat shock element

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u1.jpg

Heat shock factors (HSFs) act through a regulatory upstream promoter element, called the heat shock element (HSE). In the DNA-bound form of a HSF, each DNA-binding domain (DBD) recognizes the HSE in the major groove of the double helix6. The HSE was originally identified using S1 mapping of transcripts of the Drosophila melanogaster heat shock protein (HSP) genes3 (see the figure; part a). Residues –47 to –66 are necessary for heat inducibility. HSEs in HSP gene promoters are highly conserved and consist of inverted repeats of the pentameric sequence nGAAn132. The type of HSEs that can be found in the proximal promoter regions of HSP genes is composed of at least three contiguous inverted repeats: nTTCnnGAAnnTTCn132134. The promoters of HSF target genes can also contain more than one HSE, thereby allowing the simultaneous binding of multiple HSFs. The binding of an HSF to an HSE occurs in a cooperative manner, whereby binding of an HSF trimer facilitates binding of the next one135. More recently, Trinklein and colleagues used chromatin immunoprecipitation to enrich sequences bound by HSF1 in heat-shocked human cells to define the HSE consensus sequence. They confirmed the original finding of Xiao and Lis, who identified guanines as the most conserved nucleotides in HSEs87,133 (see the figure; part b). Moreover, in a pair of inverted repeats, a TTC triplet 5′ of a GAA triplet is separated by a pyrimidine–purine dinucleotide, whereas the two nucleotides separating a GAA triplet 5′ from a TTC triplet is unconstrained87. The discovery of novel HSF target genes that are not involved in the heat shock response has rendered it possible that there may be HSEs in many genes other than the HSP genes. Although there are variations in these HSEs, the spacing and position of the guanines are invariable7. Therefore, both the nucleotides and the exact spacing of the repeated units are considered as key determinants for recognition by HSFs and transcriptional activation. Part b of the figure is modified, with permission, from REF. 87 © (2004) The American Society for Cell Biology.

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f1.gif

The mammalian HSF machinery

HSFs as stress integrators

A hallmark of stressed cells and organisms is the increased synthesis of HSPs, which function as molecular chaperones to prevent protein misfolding and aggregation to maintain protein homeostasis, also called proteostasis11. The transcriptional activation of HSP genes is mediated by HSFs (FIG. 2a), of which HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance1215. HSF1 is constitutively expressed in most tissues and cell types16, where it is kept inactive in the absence of stress stimuli. Thus, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple PTMs, protein–protein interactions and subcellular localization. HSF1 also has an intrinsic stress-sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a homotrimer in vitro in response to thermal or oxidative stress1719.

Figure 2    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f2.gif

Members of the mammalian HSF family

Functional domains

HSFs, like other transcription factors, are composed of functional domains. These have been most thoroughly characterized for HSF1 and are schematically presented in FIG. 2b. The DNA-binding domain (DBD) is the best preserved domain in evolution and belongs to the family of winged helix-turn-helix DBDs2022. The DBD forms a compact globular structure, except for a flexible wing or loop that is located between β-strands 3 and 4 (REF. 6). This loop generates a protein– protein interface between adjacent subunits of the HSF trimer that enhances high-affinity binding to DNA by cooperativity between different HSFs23. The DBD can also mediate interactions with other factors to modulate the transactivating capacity of HSFs24. Consequently, the DBD is considered as the signature domain of HSFs for target-gene recognition.

The trimerization of HSFs is mediated by arrays of hydrophobic heptad repeats (HR-A and HR-B) that form a coiled coil, which is characteristic for many Leu zippers6,25 (FIG. 2b). The trimeric assembly is unusual, as Leu zippers typically facilitate the formation of homodimers or heterodimers. Suppression of spontaneous HSF trimerization is mediated by yet another hydrophobic repeat, HR-C2628. Human HSF4 lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity29. Positioned at the extreme carboxyl terminus of HSFs is the transactivation domain, which is shared among all HSFs6except for yeast Hsf, which has transactivation domains in both the amino and C termini, and HSF4A, which completely lacks a transactivation domain2931. In HSF1, the transactivation domain is composed of two modules — AD1 and AD2, which are rich in hydrophobic and acidic residues (FIG. 3a) — that together ensures a rapid and prolonged response to stress32,33. The transactivation domain was originally proposed to provide stress inducibility to HSF1 (REFS 34,35), but it soon became evident that an intact regulatory domain, located between the HR-A and HR-B and the transactivation domain, is essential for the responsiveness to stress stimuli32,33,36,37. Because several amino acids that are known targets for different PTMs reside in the regulatory domain33,3842, the structure and function of this domain are under intensive investigation.

Figure 3    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f3.gif

HSF1 undergoes multiple PTMs on activation

Regulation of the HSF1 activation–attenuation cycle

The conversion of the inactive monomeric HSF1 to high-affinity DNA-binding trimers is the initial step in the multistep activation process and is a common feature of all eukaryotic HSFs43,44 (FIG. 3b). There is compelling evidence for HSF1 interacting with multiple HSPs at different phases of its activation cycle. For example, monomeric HSF1 interacts weakly with HSP90 and, on stress, HSF1 dissociates from the complex, allowing HSF1 trimerization45,46 (FIG. 3b). Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex of HSP90, co-chaperone p23 (also known as PTGES3) and immunophilin FK506-binding protein 5 (FKBP52; also known as FKBP4)4651. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation on heat shock52. Activated HSF1 trimers also interact with HSP70 and the co-chaperone HSP40 (also known as DNAJB1), but instead of suppressing the DNA-binding activity of HSF1, this interaction inhibits its transactivation capacity5254. Although the inhibitory mechanism is still unknown, the negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides.

A ribonucleoprotein complex containing eukaryotic elongation factor 1A (eEF1A) and a non-coding RNA, heat shock RNA-1 (HSR-1), has been reported to possess a thermosensing capacity. According to the proposed model, HSR-1 undergoes a conformational change in response to heat stress and together with eEF1A facilitates trimerization of HSF1 (REF. 55). How this activation mode relates to the other regulatory mechanisms associated with HSFs remains to be elucidated.

Throughout the activation–attenuation cycle, HSF1 undergoes extensive PTMs, including acetylation, phosphorylation and sumoylation (FIG. 3). HSF1 is also a phosphoprotein under non-stress conditions, and the results from mass spectrometry (MS) analyses combined with phosphopeptide mapping experiments indicate that at least 12 Ser residues are phosphorylated41,5659. Among these sites, stress-inducible phosphorylation of Ser230 and Ser326 in the regulatory domain contributes to the transactivation function of HSF1 (REFS 38,41). Phosphorylation-mediated sumoylation on a single Lys residue in the regulatory domain occurs rapidly and transiently on exposure to heat shock; Ser303 needs to be phosphorylated before a small ubiquitin-related modifier (SUMO) can be conjugated to Lys298 (REF. 39). The extended consensus sequence ΨKxExxSP has been named the phosphorylation-dependent sumoylation motif (PDSM; FIG. 3)40. The PDSM was initially discovered in HSF1 and subsequently found in many other proteins, especially transcriptional regulators such as HSF4, GATA1, myocyte-specific enhancer factor 2A (MEF2A) and SP3, which are substrates for both SUMO conjugation and Pro-directed kinases40,6062.

Recently, Mohideen and colleagues showed that a conserved basic patch on the surface of the SUMO-conjugating enzyme ubiquitin carrier protein 9 (UBC9; also known as UBE2I) discriminates between the phosphorylated and non-phosphorylated PDSM of HSF1 (REF. 63). Future studies will be directed at elucidating the molecular mechanisms for dynamic phosphorylation and UBC9-dependent SUMO conjugation in response to stress stimuli and establishing the roles of kinases, phosphatases and desumoylating enzymes in the heat shock response. The kinetics of phosphorylation-dependent sumoylation of HSF1 correlates inversely with the severity of heat stress, and, as the transactivation capacity of HSF1 is impaired by sumoylation and this PTM is removed when maximal HSF1 activity is required40, sumoylation could modulate HSF1 activity under moderate stress conditions. The mechanisms by which SUMO modification represses the transactivating capacity of HSF1, and the functional relationship of this PTM with other modifications that HSF1 is subjected to, will be investigated with endogenous substrate proteins.

Phosphorylation and sumoylation of HSF1 occur rapidly on heat shock, whereas the kinetics of acetylation are delayed and coincide with the attenuation phase of the HSF1 activation cycle. Stress-inducible acetylation of HSF1 is regulated by the balance of acetylation by p300–CBP (CREB-binding protein) and deacetylation by the NAD+-dependent sirtuin, SIRT1. Increased expression and activity of SIRT1 enhances and prolongs the DNA-binding activity of HSF1 at the human HSP70.1promoter, whereas downregulation of SIRT1 enhances the acetylation of HSF1 and the attenuation of DNA-binding without affecting the formation of HSF1 trimers42. This finding led to the discovery of a novel regulatory mechanism of HSF1 activity, whereby SIRT1 maintains HSF1 in a state that is competent for DNA binding by counteracting acetylation (FIG. 3). In the light of current knowledge, the attenuation phase of the HSF1 cycle is regulated by a dual mechanism: a dependency on the levels of HSPs that feed back directly by weak interactions with HSF1, and a parallel step that involves the SIRT1-dependent control of the DNA-binding activity of HSF1. Because SIRT1 has been implicated in caloric restriction and ageing, the age-dependent loss of SIRT1 and impaired HSF1 activity correlate with an impairment of the heat shock response and proteostasis in senescent cells, connecting the heat shock response to nutrition and ageing (see below).

HSF dynamics on the HSP70 promoter

For decades, the binding of HSF to the HSP70.1 gene has served as a model system for inducible transcription in eukaryotes. In D. melanogaster, HSF is constitutively nuclear and low levels of HSF are associated with the HSP70promoter before heat shock6466. The uninduced HSP70 promoter is primed for transcription by a transcriptionally engaged paused RNA polymerase II (RNAP II)67,68. RNAP II pausing is greatly enhanced by nucleosome formation in vitro, implying that chromatin remodelling is crucial for the release of paused RNAP II69. It has been proposed that distinct hydrophobic residues in the transactivation domain of human HSF1 can stimulate RNAP II release and directly interact withBRG1, the ATPase subunit of the chromatin remodelling complex SWI/SNF70,71. Upon heat shock, RNAP II is released from its paused state, leading to the synthesis of a full-length transcript. Rapid disruption of nucleosomes occurs across the entire HSP70 gene, at a rate that is faster than RNAP II-mediated transcription72. The nucleosome displacement occurs simultaneously with HSF recruitment to the promoter in D. melanogaster. Downregulation of HSF abrogates the loss of nucleosomes, indicating that HSF provides a signal for chromatin rearrangement, which is required for HSP70 nucleosome displacement. Within seconds of heat shock, the amount of HSF at the promoter increases drastically and HSF translocates from the nucleoplasm to several native loci, including HSP genes. Interestingly, the levels of HSF occupying the HSP70 promoter reach saturation soon after just one minute65,73.

HSF recruits the co-activating mediator complex to the heat shock loci, which acts as a bridge to transmit activating signals from transcription factors to the basal transcription machinery. The mediator complex is recruited by a direct interaction with HSF: the transactivation domain of D. melanogaster HSF binds to TRAP80(also known as MED17), a subunit of the mediator complex74. HSF probably has other macromolecular contacts with the preinitiation complex as it binds to TATA-binding protein (TBP) and the general transcription factor TFIIB in vitro75,76. In contrast to the rapid recruitment and elongation of RNAP II on heat shock, activated HSF exchanges very slowly at the HSP70 promoter. HSF stays stably bound to DNA in vivo and no turnover or disassembly of transcription activator is required for successive rounds of HSP70 transcription65,68.

Functional interplay between HSFs

Although HSF1 is the principal regulator of the heat shock response, HSF2 also binds to the promoters of HSP genes. In light of our current knowledge, HSF2 strictly depends on HSF1 for its stress-related functions as it is recruited to HSP gene promoters only in the presence of HSF1 and this cooperation requires an intact HSF1 DBD77. Nevertheless, HSF2 modulates, both positively and negatively, the HSF1-mediated inducible expression of HSP genes, indicating that HSF2 can actively participate in the transcriptional regulation of the heat shock response. Coincident with the stress-induced transcription of HSP genes, HSF1 and HSF2 colocalize and accumulate rapidly on stress into nuclear stress bodies (NSBs; BOX 2), where they bind to a subclass of satellite III repeats, predominantly in the human chromosome 9q12 (REFS 7880). Consequently, large and stable non-coding satellite III transcripts are synthesized in an HSF1-dependent manner in NSBs81,82. The function of these transcripts and their relationship with other HSF1 targets, and the heat shock response in general, remain to be elucidated.

 

Box 2

Nuclear stress bodies  

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u2.jpg

The cell nucleus is highly compartmentalized and dynamic. Many nuclear factors are diffusely distributed throughout the nucleoplasm, but they can also accumulate in distinct subnuclear compartments, such as nucleoli, speckles, Cajal bodies and promyelocytic leukaemia (PML) bodies136. Nuclear stress bodies (NSBs) are different from any other known nuclear bodies137,138. Although NSBs were initially thought to contain aggregates of denatured proteins and be markers of heat-shocked cells, their formation can be elicited by various stresses, such as heavy metals and proteasome inhibitors137. NSBs are large structures, 0.3–3 μm in diameter, and are usually located close to the nucleoli or nuclear envelope137,138. NSBs consist of two populations: small, brightly stained bodies and large, clustered and ring-like structures137.

NSBs appear transiently and are the main site of heat shock factor 1 (HSF1) and HSF2 accumulation in stressed human cells80. HSF1 and HSF2 form a physically interacting complex and colocalize into small and barely detectable NSBs after only five minutes of heat shock, but the intensity and size of NSBs increase after hours of continuous heat shock. HSF1 and HSF2 colocalize in HeLa cells that have been exposed to heat shock for one hour at 42°C (see the figure; confocal microscopy image with HSF1–green fluorescent protein in green and endogenous HSF2 in red). NSBs form on specific chromosomal loci, mainly on q12 of human chromosome 9, where HSFs bind to a subclass of satellite III repeats78,79,83. Stress-inducible and HSF1-dependent transcription of satellite III repeats has been shown to produce non-coding RNA molecules, called satellite III transcripts81,82. The 9q12 locus consists of pericentromeric heterochromatin, and the satellite III repeats provide scaffolds for docking components, such as splicing factors and other RNA-processing proteins139143.

HSF2 also modulates the heat shock response through the formation of heterotrimers with HSF1 in the NSBs when bound to the satellite III repeats83 (FIG. 4). Studies on the functional significance of heterotrimerization indicate that HSF1 depletion prevents localization of HSF2 to NSBs and abolishes the stress-induced synthesis of satellite III transcripts. By contrast, increased expression of HSF2 leads to its own activation and the localization of both HSF1 and HSF2 to NSBs, where transcription is spontaneously induced in the absence of stress stimuli. These results suggest that HSF2 can incorporate HSF1 into a transcriptionally competent heterotrimer83. It is possible that the amounts of HSF2 available for heterotrimerization with HSF1 influence stress-inducible transcription, and that HSF1–HSF2 heterotrimers regulate transcription in a temporal manner. During the acute phase of heat shock, HSF1 is activated and HSF1–HSF2 heterotrimers are formed, whereas upon prolonged exposures to heat stress the levels of HSF2 are diminished, thereby limiting heterotrimerization83. Intriguingly, in specific developmental processes such as corticogenesis and spermatogenesis, the expression of HSF2 increases spatiotemporarily, leading to its spontaneous activation. Therefore, it has been proposed that HSF-mediated transactivation can be modulated by the levels of HSF2 to provide a switch that integrates the responses to stress and developmental stimuli83 (FIG. 4). Functional relationships between different HSFs are emerging, and the synergy of DNA-binding activities among HSF family members offers an efficient way to control gene expression in a cell- and stimulus-specific manner to orchestrate the differential upstream signalling and target-gene networks.

Figure 4   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f4.gif

 

Interactions between different HSFs provide distinct functional modes in transcriptional regulation

A new member of the mammalian HSF family, mouse HSF3, was recently identified10. Avian HSF3 was shown to be activated at higher temperatures and with different kinetics than HSF1 (REF. 84), whereas in mice, heat shock induces the nuclear translocation of HSF3 and activation of stress-responsive genes other than HSP genes10. Future experiments will determine whether HSF3 is capable of interacting with other HSFs, potentially through heterocomplex formation. HSF4 has not been implicated in the heat shock response, but it competes with HSF1 for common target genes in mouse lens epithelial cells85, which will be discussed below. It is important to elucidate whether the formation of homotrimers or hetero trimers between different family members is a common theme in HSF-mediated transcriptional regulation.

 

HSFs as developmental regulators

Evidence is accumulating that HSFs are highly versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physioogical functions, especially during development. The initial observations using deletion experiments of the D. melanogaster Hsf gene revealed defective oogenesis and larvae development86. These effects were not caused by obvious changes in HSP gene expression patterns, which is consistent with the subsequent studies showing that basal expression of HSP genes during mouse embryogenesis is not affected by the lack of HSF1 (REF. 13). These results are further supported by genome-wide gene expression studies revealing that numerous genes, not classified as HSP genes or molecular chaperones, are under HSF1-dependent control87,88.

Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility13. Fertilized oocytes do not develop past the zygotic stage when HSF1-deficient female mice are mated with wild-type male mice, indicating that HSF1 is a maternal factor that is essential for early post-fertilization development89. Recently, it was shown that HSF1 is abundantly expressed in maturing oocytes, where it regulates specifically Hsp90α transcription90. The HSF1-deficient oocytes are devoid of HSP90α and exhibit a blockage of meiotic maturation, including delayed G2–M transition or germinal vesicle breakdown and defective asymmetrical division90. Moreover, intra-ovarian HSF1-depleted oocytes contain dysfunctional mitochondria and are sensitive to oxidative stress, leading to reduced survival91. The complex phenotype of Hsf1-knockout mice also demonstrates the involvement of HSF1 in placenta formation, placode development and the immune system15,85,92,93, further strengthening the evidence for a protective function of HSF1 in development and survival.

Both HSF1 and HSF2 are key regulators in the developing brain and in maintaining proteostasis in the central nervous system. Disruption of Hsf1 results in enlarged ventricles, accompanied by astrogliosis, neurodegeneration, progressive myelin loss and accumulation of ubiquitylated proteins in specific regions of the postnatal brain under non-stressed conditions94,95. The expression of HSP25 (also known as HSPB1) and α-crystallin B chain (CRYAB), which are known to protect cells against stress-induced protein damage and cell death, is dramatically decreased in brains lacking HSF1 (REF. 13). In contrast to HSF1, HSF2 is already at peak levels during early brain development in mice and is predominantly expressed in the proliferative neuronal progenitors of the ventricular zone and post-mitotic neurons of the cortical plate9699. HSF2-deficient mice have enlarged ventricles and defects in cortical lamination owing to abnormal neuronal migration9799. Incorrect positioning of superficial neurons during cortex formation in HSF2-deficient embryos is caused by decreased expression of the cyclin-dependent kinase 5 (CDK5) activator p35, which is a crucial regulator of the cortical migration signalling pathway100,101. The p35 gene was identified as the first direct target of HSF2 in cortex development99. As correct cortical migration requires the coordination of multiple signalling molecules, it is likely that HSF2, either directly or indirectly, also regulates other components of the same pathway.

 

Cooperativity of HSFs in development

In adult mice, HSF2 is most abundantly expressed in certain cell types of testes, specifically pachytene spermatocytes and round spermatids102. The cell-specific expression of HSF2 in testes is regulated by a microRNA, miR-18, that directly binds to the 3′ untranslated region (UTR) of HSF2 (J.K. Björk, A. Sandqvist, A.N. Elsing, N. Kotaja and L.S., unpublished observations). Targeting of HSF2 in spermatogenesis reveals the first physiological role for miR-18, which belongs to the oncomir-1 cluster associated mainly with tumour progression103. In accordance with the expression pattern during the maturation of male germ cells, HSF2-null male mice display several abnormal features in spermatogenesis, ranging from smaller testis size and increased apoptosis at the pachytene stage to a reduced amount of sperm and abnormal sperm head shape97,98,104. A genome-wide search for HSF2 target promoters in mouse testis revealed the occupancy of HSF2 on the sex chromosomal multi-copy genes spermiogenesis specific transcript on the Y 2 (Ssty2), Sycp3-like Y-linked (Sly) and Sycp3-like X-linked (Slx), which are important for sperm quality104. Compared with the Hsf2-knockout phenotype, disruption of both Hsf1 and Hsf2 results in a more pronounced phenotype, including larger vacuolar structures, more widely spread apoptosis and a complete lack of mature spermatozoa and male sterility105. The hypo thesis that the activities of HSF1 and HSF2 are intertwined and essential for spermatogenesis is further supported by our results that HSF1 and HSF2 synergistically regulate the sex chromosomal multi-copy genes in post-meiotic round spermatids (M.Å., A. Vihervaara, E.S. Christians, E. Henriksson and L.S., unpublished observations). Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are currently the only known transcriptional regulators during post-meiotic repression. These results, together with the earlier findings that HSF2 can also form heterotrimers with HSF1 in testes83, strongly suggest that HSF1 and HSF2 act in a heterocomplex and fine-tune transcription of their common target genes during the maturation of male germ cells.

HSF1 and HSF4 are required for the maintenance of sensory organs, especially when the organs are exposed to environmental stimuli for the first time after birth85,88. During the early postnatal period, Hsf1-knockout mice display severe atrophy of the olfactory epithelium, increased accumulation of mucus and death of olfactory sensory neurons88. Although lens development in HSF4-deficient mouse embryos is normal, severe abnormalities, including inclusion-like structures in lens fibre cells, appear soon after birth and the mice develop cataracts85,106,107. Intriguingly, inherited severe cataracts occurring in Chinese and Danish families have been associated with a mutation in the DBD of HSF4 (REF. 108). In addition to the established target genes, Hsp25Hsp70 and Hsp90, several new targets for HSF1 and HSF4, such as crystallin γF (Crygf), fibroblast growth factor 7 (Fgf7) and leukaemia inhibitory factor (Lif) have been found to be crucial for sensory organs85,88. Furthermore, binding of either HSF1 or HSF4 to the Fgf7 promoter shows opposite effects on gene expression, suggesting competitive functions between the two family members85. In addition to the proximal promoters, HSF1, HSF2 and HSF4 bind to other genomic regions (that is, introns and distal parts of protein-coding genes in mouse lens), and there is also evidence for either synergistic interplay or competition between distinct HSFs occupying the target-gene promoters109. It is possible that the different HSFs are able to compensate for each other to some extent. Thus, the identification of novel functions and target genes for HSFs has been a considerable step forward in understanding their regulatory mechanisms in development.

 

HSFs and lifespan

The lifespan of an organism is directly linked to the health of its tissues, which is a consequence of the stability of the proteome and functionality of its molecular machineries. During its lifetime, an organism constantly encounters environmental and physiological stress and requires an efficient surveillance of protein quality to prevent the accumulation of protein damage and the disruption of proteostasis. Proteotoxic insults contribute to cellular ageing, and numerous pathophysiological conditions, associated with impaired protein quality control, increase prominently with age11. From studies on the molecular basis of ageing, in which a wide range of different model systems and experimental strategies have been used, the insulin and insulin-like growth factor 1 receptor (IGF1R) signalling pathway, which involves the phosphoinositide 3-kinase (PI3K) and AKT kinases and the Forkhead box protein O (FOXO) transcription factors (such as DAF-16 in Caenorhabditis elegans), has emerged as a key process. The downregulation of HSF reduces the lifespan and accelerates the formation of protein aggregates in C. elegans carrying mutations in different components of the IGF1R-mediated pathway. Conversely, inhibition of IGF1R signalling results in HSF activation and promotes longevity by maintaining proteostasis110,111. These results have prompted many laboratories that use other model organisms to investigate the functional relationship between HSFs and the IGF1R signalling pathway.

The impact of HSFs on the lifespan of whole organisms is further emphasized by a recent study, in which proteome stability was examined during C. elegansageing112. The age-dependent misfolding and downregulation of distinct metastable proteins, which display temperature-sensitive missense mutations, was examined in different tissues. Widespread failure in proteostasis occurred rapidly at an early stage of adulthood, coinciding with the severely impaired heat shock response and unfolded protein response112. The age-dependent collapse of proteostasis could be restored by overexpression of HSF and DAF-16, strengthening the evidence for the unique roles of these stress-responsive transcription factors to prevent global instability of the proteome.

Limited food intake or caloric restriction is another process that is associated with an enhancement of lifespan. In addition to promoting longevity, caloric restriction slows down the progression of age-related diseases such as cancer, cardiovascular diseases and metabolic disorders, stimulates metabolic and motor activities, and increases resistance to environmental stress stimuli113. To this end, the dynamic regulation of HSF1 by the NAD+-dependent protein deacetylase SIRT1, a mammalian orthologue of the yeast transcriptional regulator Sir2, which is activated by caloric restriction and stress, is of particular interest. Indeed, SIRT1 directly deacetylates HSF1 and keeps it in a state that is competent for DNA binding. During ageing, the DNA-binding activity of HSF1 and the amount of SIRT1 are reduced. Consequently, a decrease in SIRT1 levels was shown to inhibit HSF1 DNA-binding activity in a cell-based model of ageing and senescence42. Furthermore, an age-related decrease in the HSF1 DNA-binding activity is reversed in cells exposed to caloric restriction114. These results indicate that HSF1 and SIRT1 function together to protect cells from stress insults, thereby promoting survival and extending lifespan. Impaired proteostasis during ageing may at least partly reflect the compromised HSF1 activity due to lowered SIRT1 expression.

 

Impact of HSFs in disease

The heat shock response is thought to be initiated by the presence of misfolded and damaged proteins, and is thus a cell-autonomous response. When exposed to heat, cells in culture, unicellular organisms, and cells in a multicellular organism can all trigger a heat shock response autonomously115117. However, it has been proposed that multicellular organisms sense stress differently to isolated cells. For example, the stress response is not properly induced even if damaged proteins are accumulated in neurodegenerative diseases like Huntington’s disease and Parkinson’s disease, suggesting that there is an additional control of the heat shock response at the organismal level118. Uncoordinated activation of the heat shock response in cells in a multicellular organism could cause severe disturbances of interactions between cells and tissues. In C. elegans, a pair of thermosensory neurons called AFDs, which sense and respond to temperature, regulate the heat shock response in somatic tissues by controlling HSF activity119,120. Moreover, the heat shock response in C. elegans is influenced by the metabolic state of the organism and is reduced under conditions that are unfavourable for growth and reproduction121. Neuronal control may therefore allow organisms to coordinate the stress response of individual cells with the varying metabolic requirements in different tissues and developmental stages. These observations are probably relevant to diseases of protein misfolding that are highly tissue-specific despite the often ubiquitous expression of damaged proteins and the heat shock response.

Elevated levels of HSF1 have been detected in several types of human cancer, such as breast cancer and prostate cancer122,123. Mice deficient in HSF1 exhibit a lower incidence of tumours and increased survival than their wild-type counterparts in a classical chemical skin carcinogenesis model and in a genetic model expressing an oncogenic mutation of p53. Similar results have been obtained in human cancer cells lines, in which HSF1 was depleted using an RNA interference strategy124. HSF1 expression is likely to be crucial for non-oncogene addiction and the stress phenotype of cancer cells, which are attributes given to many cancer cells owing to their high intrinsic level of proteotoxic and oxidative stress, frequent spontaneous DNA damage and aneuploidy125. Each of these features may disrupt proteostasis, raising the need for efficient chaperone and proteasome activities. Accordingly, HSF1 would be essential for the survival of cancer cells that experience constant stress and develop non-oncogene addiction.

 

HSFs as therapeutic targets

Given the unique role of HSF1 in stress biology and proteostasis, enhanced activity of this principal regulator during development and early adulthood is important for the stability of the proteome and the health of the cell. However, HSF1 is a potent modifier of tumorigenesis and, therefore, a potential target for cancer therapeutics125. In addition to modulating the expression of HSF1, the various PTMs of HSF1 that regulate its activity should be considered from a clinical perspective. As many human, age-related pathologies are associated with stress and misfolded proteins, several HSF-based therapeutic strategies have been proposed126. In many academic and industrial laboratories, small molecule regulators of HSF1 are actively being searched for (see Supplementary information S1 (table)). For example, celastrol, which has antioxidant properties and is a natural compound derived from the Celastreace family of plants, activates HSF1 and induces HSP expression with similar kinetics to heat shock, and could therefore be a potential candidate molecule for treating neurodegenerative diseases127,128. In a yeast-based screen, a small-molecule activator of human HSF1 was found and named HSF1A129. HSF1A, which is structurally distinct from the other known activators, activates HSF1 and enhances chaperone expression, thereby counteracting protein misfolding and cell death in polyQ-expressing neuronal precursor cells129. Triptolide, also from the Celastreace family of plants, is a potent inhibitor of the transactivating capacity of HSF1 and has been shown to have beneficial effects in treatments of pancreatic cancer xenografts130,131. These examples of small-molecule regulators of HSF1 are promising candidates for drug discovery and development. However, the existence of multiple mammalian HSFs and their functional interplay should also be taken into consideration when planning future HSF-targeted therapies.

 

Concluding remarks and future perspectives

HSFs were originally identified as specific heat shock-inducible transcriptional regulators of HSP genes, but now there is unambiguous evidence for a wide variety of HSF target genes that extends beyond the molecular chaperones. The known functions governed by HSFs span from the heat shock response to development, metabolism, lifespan and disease, thereby integrating pathways that were earlier strictly divided into either cellular stress responses or normal physiology.

Although the extensive efforts from many laboratories focusing on HSF biology have provided a richness of understanding of the complex regulatory mechanisms of the HSF family of transcription factors, several key questions remain. For example, what are the initial molecular events (that is, what is the ‘thermometer’) leading to the multistep activation of HSFs? The chromatin-based interaction between HSFs and the basic transcription machinery needs further investigation before the exact interaction partners at the chromatin level can be established. The activation and attenuation mechanisms of HSFs require additional mechanistic insights, and the roles of the multiple signal transduction pathways involved in post-translational regulation of HSFs are only now being discovered and are clearly more complex than anticipated. Although still lacking sufficient evidence, the PTMs probably serve as rheostats to allow distinct forms of HSF-mediated regulation in different tissues during development. Further emphasis should therefore be placed on understanding the PTMs of HSFs during development, ageing and different protein folding diseases. Likewise, the subcellular distribution of HSF molecules, including the mechanism by which HSFs shuttle between the cytoplasm and the nucleus, remains enigmatic, as do the movements of HSF molecules in different nuclear compartments such as NSBs.

Most studies on the impact of HSFs in lifespan and disease have been conducted with model organisms such as D. melanogaster and C. elegans, which express a single HSF. The existence of multiple members of the HSF family in mammals warrants further investigation of their specific and overlapping functions, including their extended repertoire of target genes. The existence of multiple HSFs in higher eukaryotes with different expression patterns suggests that they may have functions that are triggered by distinct stimuli, leading to activation of specific target genes. The impact of the HSF family in the adaptation to diverse biological environments is still poorly understood, and future studies are likely to broaden the prevailing view of HSFs being solely stress-inducible factors. To this end, the crosstalk between distinct HSFs that has only recently been uncovered raises obvious questions about the stoichiometry between the components in different complexes residing in different cellular compartments, and the mechanisms by which the factors interact with each other. Interaction between distinct HSF family members could generate new opportunities in designing therapeutics for protein-folding diseases, metabolic disorders and cancer.

 

  1. Role in the etiology of cancer

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Dan Tang,1 Md Abdul Khaleque,2 Ellen L. Jones,1 Jimmy R. Theriault,2 Cheng Li,3 Wing Hung Wong,3 Mary Ann Stevenson,2 and Stuart K. Calderwood1,2,4
Cell Stress Chaperones. 2005 Mar; 10(1): 46–58. doi:  10.1379/CSC-44R.1

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.

Heat shock proteins (HSPs) were first discovered as a cohort of proteins that is induced en masse by heat shock and other chemical and physical stresses in a wide range of species (Lindquist and Craig 1988Georgopolis and Welch 1993). The HSPs (Table 1) have been subsequently characterized as molecular chaperones, proteins that have in common the property of modifying the structures and interactions of other proteins (Lindquist and Craig 1988Beckmann et al 1990;Gething and Sambrook 1992Georgopolis and Welch 1993Netzer and Hartl 1998). Molecular chaperone function dictates that the HSP often interact in a stoichiometric, one-on-one manner with their substrates, necessitating high intracellular concentrations of the proteins (Lindquist and Craig 1988Georgopolis and Welch 1993). As molecules that shift the balance from denatured, aggregated protein conformation toward ordered, functional conformation, HSPs are particularly in demand when the protein structure is disrupted by heat shock, oxidative stress, or other protein-damaging events (Lindquist and Craig 1988;Gething and Sambrook 1992Georgopolis and Welch 1993). The HSP27, HSP40,HSP70, and HSP110 genes have therefore evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation (Voellmy 1994). HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress (Hickey and Weber 1982Landry et al 1982Li and Werb 1982Subjeck et al 1982Henics et al 1999) (Zhao et al 2002). Heat shock factor (HSF) proteins have been shown to interact with the promoters of many HSP genes and ensure prompt transcriptional activation in stress and equally precipitous switch off after recovery (Sorger and Pelham 1988Wu 1995). The hsf gene family includes HSF1 (hsf1), the molecular coordinator of the heat shock response, as well as 2 less well-characterized genes, hsf2 and hsf4(Rabindran et al 1991Schuetz et al 1991) (Nakai et al 1997). In addition to the class of HSPs induced by heat, cells also contain a large number of constitutively expressed HSP homologs, which are also listed in Table 1. The constitutive HSPs are found in a variety of multiprotein complexes containing both HSPs and cofactors (Buchner 1999). These include HSP10-HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with regulatory proteins within the cell (Netzer and Hartl 1998). HSP90 plays a particularly versatile role in cell regulation, forming complexes with a large number of cellular kinases, transcription factors, and other molecules (Buchner 1999Grammatikakis et al 2002).

 

Table 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1074571/bin/i1466-1268-10-1-46-t01.jpg

 

Heat shock protein family genes studied by microchip array analysis

Many tumor types contain high concentrations of HSP of the HSP28, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca et al 1993Yano et al 1999Cornford et al 2000Strik et al 2000Ricaniadis et al 2001Ciocca and Vargas-Roig 2002). We have concentrated here on HSP gene expression in prostate carcinoma. The progression of prostatic epithelial cells to the fully malignant, metastatic phenotype is a complex process and involves the expression of oncogenes as well as escape from androgen-dependent growth and survival (Cornford et al 2000). There is a molecular link between HSP expression and tumor progression in prostate cancer in that HSP56, HSP70, and HSP90 regulate the function of the androgen receptor (AR) (Froesch et al 1998Grossmann et al 2001). Escape from AR dependence during tumorigenesis may involve altered HSP-AR interactions (Grossmann et al 2001). The role of HSPs in tumor development may also be related to their function in the development of tolerance to stress (Li and Hahn 1981). Thermotolerance is induced in cells preconditioned by mild stress coordinately with the expression of high HSP levels (Landry et al 1982Li and Werb 1982Subjeck et al 1982). Elevated HSP expression appears to be a factor in tumor pathogenesis, and, among other mechanisms, this may involve the ability of individual HSPs to block the pathways of apoptosis and permit malignant cells to arise despite the triggering of apoptotic signals during transformation (Volloch and Sherman 1999). De novo HSP expression may also afford protection of cancer cells from treatments such as chemotherapy and hyperthermia by thwarting the proapoptotic influence of these modalities (Gabai et al 1998Hansen et al 1999Blagosklonny 2001Asea et al 2001Van Molle et al 2002). The mechanisms underlying HSP induction in tumor cells are not known but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment, which would be expected to lead to cellular stress.

Here, we have examined expression of hsf and HSP genes in immortalized normal human prostate epithelial cells and a range of prostate carcinoma cells obtained from human tumors at the mRNA and protein levels. Our aim was to determine whether hsf-HSP expression profiles are conserved in cells that express varying degrees of malignancy, under resting conditions and after heat and ionizing radiation. In addition, we have compared HSP expression profiles of a metastatic human prostate carcinoma cell line growing either in monolayer culture or as a tumor xenograft in nude mice. These studies were prompted by findings in our laboratory that prostate carcinoma cells are considerably more sensitive to heat-induced apoptosis in vivo growing as tumors compared with similar cells growing in tissue culture in vitro. Our studies show that, although the hsf-HSP expression profiles are similar in normal and malignant prostate-derived cells at the mRNA level, expression at the protein level was very different. HSF1 and HSP protein expression was highest in the 3 aggressively metastatic prostate cancer cell lines (PC-3, DU-145, and CA-HPV-10). Although the gene expression patterns of constitutive HSP differ enormously in PC-3 cells in vitro and in xenografts in vivo, stress induction of HSP genes is not markedly altered by exposure to the tumor microenvironment, indicating the hierarchical rank of the stress response that permits it to override other forms of regulation. ……

The experiments described here are largely supportive of the notion that HSP gene expression and HSF activity and expression are increased in more advanced stages of cancer (Fig 4). The most striking finding in the study was the elevation of HSF1 and HSP levels in aggressively malignant prostate carcinoma cell lines (Fig 4). It is significant that these changes in HSF and HSP levels would not have been predicted from microarray studies of HSF (Fig 3) and HSP (Fig 1) mRNA levels. The increased HSF levels observed in the metastatic prostate carcinoma cell lines in particular appear to be due to altered regulation of either mRNA translation or protein turnover (or both) (Figs 3 and ​and4).4). Although we do not at this stage know the mechanisms involved, 1 candidate could be differential activity of the proteosome in the metastatic cell lines: both HSF1 and HSF2 are targets for proteosomal degradation (Mathew et al 1998). Despite these differences in HSP expression between cells of varying degrees of malignancy under growth conditions, stress caused a major shift in HSP gene expression and activation of HSP40-1, HSP70-1A, HSP70-1B, HSP70-6 (HSP70B), DNA-J2–like, and HSP105 in all cells (Fig 2). Even in LnCap cells with minimal HSF1 and HSF2 expression, heat-inducible HSP70 protein expression was observed (Fig 4). Interestingly, we observed minimal induction of the HSP70B gene in LnCap cells: because the HSP70B promoter is known to be almost exclusively induced by stress through the HSE in its promoter, the findings may suggest that a mechanism for HSP70 induction alternative to HSF1 activation may be operative in LnCap cells (Schiller et al 1988). Increased HSP expression in cancer patients has been shown to signal a poor response to treatment by a number of modalities, suggesting that HSP expression is involved with development of resistance to treatment in addition to being involved in the mechanisms of malignant progression (Ciocca et al 1993Cornford et al 2000Yamamoto et al 2001Ciocca and Vargas-Roig 2002;Mese et al 2002). In addition, subpopulations of LnCap-derived cells, selected for enhanced capacity to metastasize, have been shown to express elevated levels of HSF1, HSP70, and HSP27 compared with nonselected controls (Hoang et al 2000). This may be highly significant because our studies indicate minimal levels of HSF1 and HSP in the poorly metastatic parent LnCap cells (Figs 1 and ​and4).4). Previous studies have also indicated that elevated HSP70 expression occurs at an early stage in cellular immortalization from embryonic stem cells (Ravagnan et al 2001). We had to use immortalized prostatic epithelial cells for our normal controls and may have missed a very early change in HSP expression during the immortalization process.

As indicated by the kinetic studies (Figs 5–7), HSPs are activated at a number of regulatory levels by stress in addition to transcriptional activation, and these may include stress-induced mRNA stabilization, differential translation, and protein stabilization (Hickey and Weber 1982Zhao et al 2002). HSF1 activity and HSP expression appear to be subject to differential regulation by a number of pathways at normal temperatures but are largely independent of such regulation when exposed to heat shock, which overrides constitutive regulation and permits prompt induction of this emergency response.

Growth of PC-3 cells in vivo as tumor xenografts was accompanied by a marked decrease in constitutive HSP expression (Figs 8 and ​and11).11). Decreased HSP expression was part of a global switch in gene expression that accompanies the switch of PC-3 cells from growth as monolayers in tissue culture to growth as tumors in vivo (D. Tang and S.K. Calderwood, in preparation). Many reports indicate changes in a wide range of cellular properties as cells grow as tumors, and these properties may reflect the remodeling of gene expression patterns. These changes may reflect adaptation to the chemical nature of the tumor microenvironment and the alterations in cell-cell interaction in growth as a tumor in vivo. Our studies also indicate the remarkable sturdiness of the heat shock response that remains intact in the PC-3 cells growing in vivo despite the global rearrangements in other gene expressions mentioned above (Figs 10 and ​and1111).

The elevation in HSF1 and HSP levels in cancer shown in our studies and in those of others and its association with a poor prognosis and inferior response to therapy suggests the strategy of targeting HSP in cancer therapy. Treatment with HSP70 antisense oligonucleotides, for instance, can cause tumor cell apoptosis on its own and can synergize with heat shock in cell killing (Jones et al 2004). Indeed, it has been shown that antagonizing heat-inducible HSP expression with quercitin, a bioflavonoid drug that inhibits HSF1 activation, or by using antisense oligonucleotides directed against HSP70 mRNA further sensitizes PC-3 cells to heat-induced apoptosis in vitro and leads to tumor regression in vivo (Asea et al 2001Lepchammer et al 2002Jones et al 2004) (A. Asea et al, personal communication). The strategy of targeting HSP expression or function in cancer cells may thus be indicated. Such a strategy might prove particularly effective because constitutive HSP expression is reduced in tumors, and this might be related to increased killing of PC-3 tumor cells by heat (Fig 12).

 

  1. Molecular chaperones in aging

Aging and molecular chaperones

Csaba So˝ti*, Pe´ter Csermely
Exper Geront 2003; 38:1037–1040  http://195.111.72.71/docs/pcs/03exger.pdf

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Molecular chaperones Chaperones are ubiquitous, highly conserved proteins (Hartl, 1996), either assisting in the folding of newly synthesized or damaged proteins in an ATP-dependent active process or working in an ATP-independent passive mode sequestering damaged proteins for future refolding or digestion. Environmental stress leads to proteotoxic damage. Damaged, misfolded proteins bind to chaperones, and liberate the heat shock factor (HSF) from its chaperone complexes. HSF is activated and transcription of chaperone genes takes place (Morimoto, 2002). Most chaperones, therefore, are also called stress or (after the archetype of experimental stress) heat shock proteins (Hsp-s).

Aging proteins—proteins of aging organisms During the life-span of a stable protein, various posttranslational modifications occur including backbone and side chain oxidation, glycation, etc. In aging organisms, the disturbed cellular homeostasis leads to an increased rate of protein modification: in an 80-year old human, half of all proteins may become oxidized (Stadtman and Berlett, 1998). Susceptibility to various proteotoxic damages is mainly increased due to dysfunction of mitochondrial oxidation of starving yeast cells (Aguilaniu et al., 2001). In prokaryotes, translational errors result in folding defects and subsequent protein oxidation (Dukan et al., 2000), which predominantly takes place in growth arrested cells (Ballesteros et al., 2001). Additionally, damaged signalling networks loose their original stringency, and irregular protein phosphorylation occurs (e.g.: the Parkinson disease-related a-synuclein also becomes phosphorylated, leading to misfolding and aggregation; Neumann et al., 2002).

Aging protein degradation Irreversibly damaged proteins are recognized by chaperones, and targeted for degradation. Proteasome level and function decreases with aging, and some oxidized, aggregated proteins exert a direct inhibition on proteasome activity. Chaperones also aid in lysosomal degradation. The proteolytic changes are comprehensively reviewed by Szweda et al. (2002). Due to the degradation defects, damaged proteins accumulate in the cells of aged organisms, and by aggregation may cause a variety of protein folding diseases (reviewed by So˝ti and Csermely, 2002a).

Aging chaperones I: defects in chaperone induction Damaged proteins compete with the HSF in binding to the Hsp90-based cytosolic chaperone complex, which may contribute to the generally observed constitutively elevated chaperone levels in aged organisms (Zou et al., 1998; So˝ti and Csermely, 2002b). On the contrary, the majority of the reports showed that stress-induced synthesis of chaperones is impaired in aged animals. While HSF activation does not change, DNA binding activity may be reduced during aging (Heydari et al., 2000). A number of signaling events use an overlapping network of chaperones not only to establish the activation-competent state of different transcription factors (e.g. steroid receptors), but also as important elements in the attenuation of respective responses. HSF transcriptional activity is also negatively influenced by higher levels of chaperones (Morimoto, 2002). Differential changes of these proteins in various organisms and tissues may lead to different extents of (dys)regulation. More importantly, the cross-talk between different signalling pathways through a shared pool of chaperones may have severe consequences during aging when the cellular conformational homeostasis is deranged (see below).

Aging chaperones II: defects in chaperone function   Direct studies on chaperone function in aged organisms are largely restricted to a-crystallin having a decreased activity in aged human lenses (Cherian and Abraham, 1995; Cherian-Shaw et al., 1999). In a recent study, an initial test of passive chaperone function of whole cytosols was assessed showing a decreased chaperone capacity in aged rats compared to those of young counterparts (Nardai et al., 2002). What can be the mechanism behind these deleterious changes in chaperone function? Chaperones may also be prone to oxidative damage, as GroEL is preferentially oxidized in growth-arrested E. coli (Dukan and Nystro¨m, 1999). Macario and Conway de Macario (2002) raised the idea of ‘sick chaperones’ in aged organisms in a recent review. Indeed, chaperones are interacting with a plethora of other proteins (Csermely, 2001a), which requires rather extensive binding surfaces. These exposed areas may make chaperones a preferential target for proteotoxic damage: chaperones may behave as ‘suicide proteins’ during aging, sacrificing themselves instead of ‘normal’ proteins. The high abundance of chaperones (which may constitute more than 5% of cellular proteins), and their increased constitutive expression in aged organisms makes them a good candidate for this ‘altruistic courtesy.’ It may be especially true for mitochondrial Hsp60, the role of which would deserve extensive studies.

Aging chaperones III: defects in capacity, the chaperone overload Another possible reason of decreased chaperone function is chaperone overload (Csermely, 2001b). In aging organisms, the balance between misfolded proteins and available free chaperones is grossly disturbed: increased protein damage, protein degradation defects increase the amount of misfolded proteins, while chaperone damage, inadequate synthesis of molecular chaperones and irreparable folding defects (due to posttranslational changes) decrease the amount of available free chaperones. Chaperone overload occurs, where the need for chaperones may greatly exceed the available chaperone capacity (Fig. 1). Under these conditions, the competition for available chaperones becomes fierce and the abundance of damaged proteins may disrupt the folding assistance to other chaperone targets, such as: (1) newly synthesized proteins; (2) ‘constantly damaged’ (mutant) proteins; and (3) constituents of the cytoarchitecture (Csermely, 2001a). This may cause defects in signal transduction, protein transport, immune recognition, cellular organization as well as the appearance of previously buffered, hidden mutations in the phenotype of the cell (Csermely, 2001b). Chaperone overload may significantly decrease the robustness of cellular networks, as well as shift their function towards a more stochastic behavior. As a result of this, aging cells become more disorganized, their adaptation is impaired.

Fig. 1. Chaperone overload: a shift in the balance between misfolded proteins and available free chaperones in aging organisms. The accumulation of chaperone substrates along with an impaired chaperone function may exhaust the folding assistance to specific chaperone targets and leads to deterioration in vital processes. Chaperone overload may significantly decrease the robustness of cellular networks, and compromise the adaptative responses. See text for details.

Senescent cells and chaperones The involvement of chaperones in aging at the cellular level is recently reviewed (So˝ti et al., 2003). Non-dividingsenescent-peripheral cells tend to have increased chaperone levels (Verbeke et al., 2001), and cannot preserve the induction of several chaperones (Liu et al., 1989), similarly to cells from aged animals. Activation and binding of HSF to the heat shock element is decreased in aged cells (Choi et al., 1990). Interestingly, cellular senescence seems to unmask a proteasomal activity leading to the degradation of HSF (Bonelli et al., 2001). Chaperone induction per se seems to counteract senescence. Repeated mild heat shock (a kind of hormesis) has been reported to delay fibroblast aging (Verbeke et al., 2001), though it does not seem to extend replicative lifespan. A major chaperone, Hsp90 is required for the correct function of telomerase, an important enzyme to extend the life-span of cells (Holt et al., 1999). Mortalin (mtHsp70/Grp75), a member of the Hsp70 family, produces opposing phenotypic effects related to its localization. In normal cells, it is pancytoplasmically distributed, and its expression causes senescence. Its upregulation and perinuclear distribution, however, is connected to transformation, probably via p53 inactivation. Mortalin also induces life-span extension in human fibroblasts or in C. elegans harboring extra copies of the orthologous gene (Kaul et al., 2002).

Aging organisms and chaperones: age-related diseases Unbalanced chaperone requirement and chaperone capacity in aged organisms helps the accumulation of aggregated proteins, which often cause folding diseases, mostly of the nervous system, due to the very limited proliferation potential of neurons. Over expression of chaperones often delays the onset or diminishes the symptoms of the disease (So˝ti and Csermely, 2002b). Other aging diseases, such as atherosclerosis and cancer are also related to chaperone action. Here space limitation precludes a detailed description of these rapidly developing fields, however, numerous recent reviews were published on these subjects, where the interested readers may find a good summary and several hints for further readings (Ferreira and Carlos, 2002; Neckers, 2002; Sarto et al., 2000; Wick and Xu, 1999).

 

Chaperones and Longevity

Increased chaperone induction leads to increased longevity (Tatar et al., 1997). Moreover, a close correlation exists between stress resistance and longevity in several long-lived C. elegans and Drosophila mutants (Lithgow and Kirkwood, 1996). As the other side of the same coin, damaged HSF has been found as an important gene to cause accelerated aging in C. elegans (Garigan et al., 2002). Caloric restriction, the only effective experimental manipulation known to retard aging in rodents and primates (Ramsey et al., 2000), restores age-impaired chaperone induction, while reversing the age-induced changes in constitutive Hsp levels (see So˝ti and Csermely, 2002a,b). These examples confirm the hypothesis that a better adaptation capacity to various stresses greatly increases the chances to reach longevity. 10. Conclusions and perspectives Aging can be defined as a multicausal process leading to a gradual decay of self-defensive mechanisms, and an exponential accumulation of damage at the molecular, cellular and organismal level. The protein oxidation, damage, misfolding and aggregation together with the simultaneously impaired function and induction of chaperones in aged organisms disturb the balance between chaperone requirement and availability. There are several important aspects for future investigation of this field: † the measurement of active chaperone function (i.e. chaperone-assisted refolding of damaged proteins) in cellular extracts does not have a well-established method yet; † we have no methods to measure free chaperone levels; † among the consequences of chaperone overload, changes in signal transduction, protein transport, immune recognition and cellular organization have not been systematically measured and/or related to the protein folding homeostasis of aging organisms and cells.

 

  1. Extracellular HSPs in inflammation and immunity

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1

Amir Kol,* Andrew H. Lichtman,† Robert W. Finberg,‡ Peter Libby,*† and Evelyn A. Kurt-Jones2‡
J  Immunol 2000; 164: 13–17.  https://www.researchgate.net/profile/Robert_Finberg/publication/12696457_Cutting_Edge_Heat_Shock_Protein_(HSP)_60_Activates_the_Innate_Immune_Response_CD14_Is_an_Essential_Receptor_for_HSP60_Activation_of_Mononuclear_Cells/links/53ee00460cf23733e80b21c0.pdf

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

There is increasing interest in the role of nontraditional mediators of inflammation in atherosclerosis (1). Recent studies from our laboratory have shown that chlamydial and human heat shock protein 60 (HSP60)3 colocalize in human atheroma (2), and either HSP60 induces adhesion molecule and cytokine production by human vascular cells and macrophages, in a pattern similar to that induced by Escherichia coli LPS (3, 4). These results suggested that HSP60 and LPS might share similar signaling mechanisms. CD14 is the major high-affinity receptor for bacterial LPS on the cell membrane of mononuclear cells and macrophages (5, 6). In addition to LPS, CD14 functions as a signaling receptor for other microbial products, including peptidoglycan from Gram-positive bacteria and mycobacterial lipoarabinomann (7, 8). CD14 is considered a pattern recognition receptor for microbial Ags and, with Toll-like receptor (TLR) proteins, an important mediator of innate immune responses to infection (9–14). We have examined the role of CD14 in the response of human monocytes and macrophages to HSP60.  …..

HSP may play a central role in the innate immune response to microbial infections. Because both microbes and stressed or injured host cells produce abundant HSP (36), and dying cells likely release these proteins, it is conceivable that HSP furnish signals that inform the innate immune system of the presence of infection and cell damage. The findings reported here, that human HSP60 induces IL-6 production by mononuclear cells and macrophages via the CD14, supports this hypothesis, suggesting that human HSP60 may act together with LPS or other microbial products to provoke innate immune responses.

Inflammation and immunity can contribute to the pathogenesis and complications of atherosclerosis (37). Moreover, the search for novel risk factors for atherosclerosis has revived the concept that microbial products might substantially contribute to the inflammatory reaction in the atheromatous vessel wall (38, 39). We have shown that chlamydial HSP60 colocalizes with human HSP60 in the macrophages of human atheroma (2). Therefore, bacterial and human HSP60, released from dying or injured cells during atherogenesis (40) or myocardial injury (41), may further promote local inflammation and possibly activate the innate immune system. Previous reports that immunization with mycobacterial HSP65 enhances atheroma formation in rabbits (42), have suggested an important role for HSPs in atherogenesis, particularly because the high degree of homology between HSPs of the same m.w. among different species might stimulate autoimmunity (43).

In conclusion, our findings, that CD14 mediates cellular activation induced by human HSP60 provide further insight into the molecular mechanisms by which HSP may activate the innate immune system and participate in atherogenesis and other inflammatory disorders.

DAMPs, PAMPs and alarmins: all we need to know about danger

Marco E. Bianchi1
J. Leukoc. Biol. 81: 1–5; 2007.   http://aerozon.ru/documents/publications/37_Bianche.pdf

Multicellular animals detect pathogens via a set of receptors that recognize pathogen associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term “alarmin” is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage associated molecular patterns (DAMPs).

Multicellular animals must distinguish whether their cells are alive or dead and detect when microorganisms intrude, and have evolved surveillance/defense/repair mechanisms to this end. How these mechanisms are activated and orchestrated is still incompletely understood, and I will argue that that these themes define a unitary field of investigation, of both basic and medical interest.

A complete system for the detection, containment, and repair of damage caused to cells in the organism requires warning signals, cells to respond to them via receptors and signaling pathways, and outputs in the form of physiological responses. Classically, a subset of this system has been recognized and studied in a coherent form: pathogen-associated molecular patterns (PAMPs) are a diverse set of microbial molecules which share a number of different recognizable biochemical features (entire molecules or, more often, part of molecules or polymeric assemblages) that alert the organism to intruding pathogens [1]. Such exogenous PAMPs are recognized by cells of the innate and acquired immunity system, primarily through toll-like receptors (TLRs), which activate several signaling pathways, among which NF-kB is the most distinctive. As a result, some cells are activated to destroy the pathogen and/or pathogen-infected cells, and an immunological response is triggered in order to produce and select specific T cell receptors and antibodies that are best suited to recognize the pathogen on a future occasion. Most of the responses triggered by PAMPs fall into the general categories of inflammation and immunity.

However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Tissues can be ripped, squashed, or wounded by mechanical forces, like falling rocks or simply the impact of one’s own body hitting the ground. Animals can be wounded by predators. In addition, tissues can be damaged by excessive heat (burns), cold, chemical insults (strong acids or bases, or a number of different cytotoxic poisons), radiation, or the withdrawal of oxygen and/or nutrients. Finally, humans can also be damaged by specially designed drugs, such as chemotherapeutics, that are meant to kill their tumor cells with preference over their healthy cells. Very likely, we would not be here to discuss these issues if evolution had not incorporated in our genetic program ways to deal with these damages, which are not caused by pathogens but are nonetheless real and common enough. Tellingly, inflammation is also activated by these types of insults. A frequently quoted reason for the similarity of the responses evoked by pathogens and trauma is that pathogens can easily breach wounds, and infection often follows trauma; thus, it is generally effective to respond to trauma as if pathogens were present. In my opinion, an additional reason is that pathogens and trauma both cause tissue and cell damage and thus trigger similar responses.

None of these considerations is new; however, a new awareness of the close relationship between trauma- and pathogenevoked responses emerged from the EMBO Workshop on Innate Danger Signals and HMGB1, which was held in February 2006 in Milano (Italy); many of the findings presented at the meeting are published in this issue of the Journal of Leukocyte Biology. At the end of the meeting, Joost Oppenheim proposed the term “alarmin” to differentiate the endogenous molecules that signal tissue and cell damage. Together, alarmins and PAMPs therefore constitute the larger family of damage-associated molecular patterns, or DAMPs.

Extranuclear expression of HMGB1 has been involved in a number of pathogenic conditions: sepsis [44], arthritis [45, 46], atherosclerosis [10], systemic lupus erythematosus (SLE) [47], cancer [48] and hepatitis [49, this issue]. Uric acid has been known to be the aethiologic agent for gout since the 19th century. S100s may be involved in arthritis [31, this issue] and psoriasis [50]. However, although it is clear that excessive alarmin expression might lead to acute and chronic diseases, the molecular mechanisms underlying these effects are still largely unexplored.

The short list of alarmins presented above is certainly both provisional and incomplete and serves only as an introduction to the alarmin concept and to the papers published in this issue of JLB. Other molecules may be added to the list, including cathelicidins, defensins and eosinophil-derived neurotoxin (EDN) [51], galectins [52], thymosins [53], nucleolin [54], and annexins [55; and 56, this issue]; more will emerge with time. Eventually, the concept will have to be revised and adjusted to the growing information. Indeed, I have previously argued that any misplaced protein in the cell can signal damage [57], and Polly Matzinger has proposed that any hydrophobic surface (“Hyppo”, or Hydrophobic protein part) might act as a DAMP [58]. As most concepts in biology, the alarmin category serves for our understanding and does not correspond to a blueprint or a plan in the construction of organisms. Biology proceeds via evolution, and evolution is a tinkerer or bricoleur, finding new functions for old molecules. In this, the reuse of cellular components as signals for alerting cells to respond to damage and danger, is a prime example.

 

  1. Role of heat shock and the heat shock response in immunity and cancer

 

Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

Stuart K. Calderwood,1,* Ayesha Murshid,1 and Jianlin Gong1
Front Immunol. 2012; 3: 75.  doi:  10.3389/fimmu.2012.00075

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock protein, vaccine, inflammation, antigen presentation

Heat shock proteins (HSP) were first discovered as a group of polypeptides whose level of expression increases to dominate the cellular proteome after stress (Lindquist and Craig, 1988). These increases in HSPs synthesis correlate with a marked resistance to potentially toxic stresses such as heat shock (Li and Werb,1982). The finding that such proteins have extracellular immune functions suggested that, as highly abundant intracellular proteins they could be prime candidates as danger signals to the immune response (Srivastava and Amato,2001). There are several human HSP gene families with known immune significance and their classification is reviewed in Kampinga et al. (2009). These include the HSPA (Hsp70) family, which includes the HPA1A and HSPA1B genes encoding the two major stress-inducible Hsp70s, that together are often referred to as Hsp72. When referring to Hsp70 in this chapter, we generally refer to the products of these two genes. The Hsp70 family also includes two other members with immune function – HSPA8 and HSPA5 genes, whose protein products are known as Hsc70 the major constitutive Hsp70 family member and Grp78, a key ER-resident protein. In addition two more Hsp70 related genes have immune significance and these include HSPH2 (Hsp110) and HSPH4 the ER-resident class H protein Grp170. The Hsp90 family also has major functions in tumor immunity and these include HSPC2 and HSPC3, which encode the major cytoplasmic proteins Hsp90a and Hsp90b, and HSPC4 that encodes ER chaperone Grp94. In addition, the product of the HSPD1 gene, the mitochondrial chaperone Hsp60 has some immunological functions. Mice have been shown to encode orthologs of each of these genes (Kampinga et al., 2009).

It has been suggested that many of the HSPs have the property of damage associated molecular patterns (DAMPs), inducers of sterile inflammation and innate immunity (Kono and Rock, 2008). The additional discovery that intracellular HSPs function as molecular chaperones and can bind to a wide spectrum of intracellular polypeptides further indicated that they could play a broad role in the immune response and might mediate both innate immunity due to their status as DAMPs and adaptive immunity by chaperoning antigens.

Heat shock proteins are currently employed as vaccines in cancer immunotherapy (Tamura et al., 1997; Murshid et al., 2011a). The rationale behind the approach is that if HSPs can be extracted from tumor tissue bound to the polypeptides which they chaperone during normal metabolism, they may retain antigenic peptides specific to the tumor (Noessner et al., 2002; Srivastava, 2002; Wang et al., 2003; Enomoto et al., 2006; Gong et al., 2010). Indeed, vaccines based on Hsp70, Hsp90, Grp94, Hsp110, and Grp170 polypeptide complexes have been used successfully to immunize mice to a range of tumor types and Hsp70 and Grp94 vaccines have undergone recent clinical trials (rev: Murshid et al., 2011a). These effects of the HSP vaccines on tumor immunity appear to be mediated largely to the associated, co-isolated tumor polypeptides, although in the case of Grp94 this question is still controversial and tumor regression was observed in mice treated with the chaperone devoid of its peptide binding domain (Udono and Srivastava, 1993; Srivastava, 2002; Nicchitta, 2003; Chandawarkar et al., 2004; Nicchitta et al.,2004). Use of such HSP vaccines is potentially a powerful approach to tumor immunotherapy as the majority of the antigenic repertoire of most individual tumor cells is unknown (Srivastava and Old, 1988; Srivastava, 1996). Individual cancer cells are likely to take a lone path in accumulating a spectrum of random mutations. Although some mutations are functional, permitting cells to become transformed and to progress into a highly malignant state, many such changes are likely to be passenger mutations not required to drive tumor growth (Srivastava and Old, 1988; Srivastava, 1996). Some of these individual mutant sequences will be novel antigenic epitopes and together with the few known shared tumor antigens comprise an “antigenic fingerprint” for each individual tumor (Srivastava,1996). Accumulation of mutations in cancer appears to be related to, and may drive the increases in HSPs observed in many tumors (Kamal et al., 2003; Whitesell and Lindquist, 2005; Trepel et al., 2010). As the mutant conformations of tumor proteins are “locked in” due to the covalent nature of the alterations, cancer cells appear to be under permanent proteotoxic stress and rich in HSP expression (Ciocca and Calderwood, 2005). For tumor immunology these conditions may offer a therapeutic opportunity as individual HSPs, whose expression is expanded in cancer will chaperone a cross-section of the “antigenic fingerprint” of the individual tumors (Murshid et al., 2011a). This approach was first utilized by Srivastava (20002006) and led to the development of immunotherapy using HSP–peptide complexes.

In addition to using HSP–peptide complexes extracted from tumors, in cases where tumor antigens are known, these can be directly loaded onto purified or recombinant HSPs and the complex used as a vaccine. This procedure has been carried out successfully in the case of the “large HSPs,” Hsp110 and Grp170 (Manjili et al., 20022003). A variant of this approach employs the molecular engineering of tumor antigens in order to produce molecular chaperone-fusion genes which encode products in which the HSP is fused covalently to the antigen. The fusion proteins are then employed as vaccines. This approach was pioneered by Young et al. who showed that a fusion between mycobacterial Hsp70 and ovalbumin could induced cytotoxic lymphocytes (CTL) in mice with the capacity to kill Ova-expressing cancer cells (Suzue et al., 1997). The vaccines could be used effectively without adjuvant and adjuvant properties were ascribed to the molecular chaperone component of the fusion protein. Subsequent studies have confirmed the utility of the approach in targeting common tumor antigens such as the melanoma antigen Mage3 (Wang et al., 2009).

HSPs and Immunosurveillance in Cancer

The question next arises as to the role of endogenous HSPs, with or without bound antigens in immunosurveillance of cancer cells. Although the immune system can recognize tumor antigens and generate a CTL response, most cancers evade immune cell killing by a range of strategies (van der Bruggen et al., 1991; Pardoll,2003). These include the down-regulation of surface MHC class I molecules by individual tumor cells and release of immunosuppressive IL-10 by tumors (Moller and Hammerling, 1992; Chouaib et al., 2002). Tumors in vivo also appear to attract a range of hematopoietic cells with immunosuppressive action including regulatory CD4+CD25+FoxP3+ T cells (Treg), M2 macrophages, myeloid-derived suppressor cells (MDSC) and some classes of natural killer cells (Pekarek et al.,1995; Terabe et al., 2005; Mantovani et al., 2008; Marigo et al., 2008). The tumor milieu also contain a small fraction of cells of mesenchymal origin identified by surface fibroblast activation protein-a (FAP cells) that suppress antitumor immune responses (Kraman et al., 2010). Endogenous tumor HSPs may also participate in immune suppression. Although the majority of the HSPs function as intracellular molecular chaperones, a fraction of these proteins can be released from cells even under unstressed conditions and may participate in immune functions (rev: Murshid and Calderwood, 2012). Intracellular Hsp70 can be actively secreted from tumor cells in either free form or packaged into lipid-bounded structures called exosomes (Mambula and Calderwood, 2006b; Chalmin et al., 2010). In addition Hsp70 and Hsp90 can also be found associated with the surfaces of tumor cells where they can function as molecular chaperones or as recognition structures for immune cells (Sidera et al., 2008; Qin et al., 2010; Multhoff and Hightower, 2011). As Hsp70 was shown in a number of earlier studies to be pro-inflammatory due to its interaction with pattern recognition receptors such as Toll-like receptors 2 and 4 (TLR2 and TLR4), these findings might suggest, as mentioned above, that Hsp70 released by tumors could be pro-inflammatory and possess the properties of DAMPs (Asea et al., 20002002; Vabulas et al., 2002). However, subsequent studies indicated that a portion of the TLR4 activation detected in the earlier reports, involving exposure of monocytes, macrophages, or dendritic cells (DC) to HSPs in vitro may be due to trace contamination with bacterial pathogen associated molecular patterns (PAMPs), potent TLR activators (Tsan and Gao,2004). In spite of these drawbacks, an overwhelming amount of evidence now seems to indicate the interaction of Hsp70 and other HSPs with TLRs (particularly TLR4) in vivo – in a wide range of physiological and pathological conditions, leading to acute inflammation in many conditions (Chase et al., 2007; Wheeler et al., 2009; see Appendix for a full list of references). Thus both TLR2 and TLR4 appear to be important components of inflammatory responses to Hsp70 under many pathophysiological conditions. In cancer therapy it has been shown that autoimmunity can be triggered in mice through necrotic killing of melanocytes engineered to overexpress Hsp70; such treatment led to the concomitant immune destruction of B16 melanoma tumors that share patterns of antigen expression with the killed melanocytes (Sanchez-Perez et al., 2006). Hsp70 appears to play an adjuvant role in this form of therapy through its interaction with TLR4 and induction of the cytokine TNF-a (Sanchez-Perez et al., 2006). However, despite these findings it has also been shown that depletion of Hsp70 in cancer cells can, in the absence of other treatments lead to tumor regression by inducing antitumor immunity (Rerole et al., 2011). This effect appears to be due to the secretion by cancer cells of immunosuppressive exosomes containing Hsp70 that activate MDSC and lead to local immunosuppression (Chalmin et al., 2010). Under normal circumstances therefore, release of endogenous Hsp70 into the extracellular microenvironment may be a component of the tumor defenses against immunosurveillance. Extracellular Hsp60 has also been shown be immunomodulatory and can increase levels of FoxP3 Treg in vitro and suppress T cell-mediated immunity (de Kleer et al., 2010; Aalberse et al., 2011).

The pro-inflammatory properties of extracellular HSPs may be more evident underin vivo situations particularly in the context of tissue damage (Sanchez-Perez et al.,2006). For instance when elevated temperatures were used to boost Hsp70 release from Lewis Lung carcinoma cells in vivo, antitumor immunity was activated along with release of chemokines CCL2, CCL5, and CCL10, in a TLR4-dependent manner, leading to attraction of DC and T cells into the tumor (Chen et al., 2009). Thus under resting conditions, the tumor milieu appears to be a specialized immunosuppressive environment, rich in inhibitory cells such as Treg, MDSC, and M2 macrophages and inaccessible to “exhausted” CD8+ T cells that often fail to penetrate the tumor microcirculation. However, under inflammatory conditions involving necrotic cell killing of tumor cells, extracellular HSPs may be able to amplify the anticancer immune response, intracellular HSPs may be released to further increase such a response and CTL may triggered to penetrate the tumor milieu, inducing antigen-specific cancer cell killing (Evans et al., 2001; Mambula and Calderwood, 2006a; Sanchez-Perez et al., 2006; Chen et al., 2009).

 

HSP-Based Anticancer Vaccines

It is apparent that a number of HSP types, conjugated to peptide complexes (HSP.PC) from cancer cells form effective bases for immunotherapy approaches with unique properties, as mentioned above (Calderwood et al., 2008; Murshid et al., 2011a). The immunogenicity of most HSP.PC appears to involve the ability of the HSPs to sample the tumor “antigenic fingerprint,” deliver the antigens to antigen presenting cells (APC) such as DC and stimulate activation of CTL (Tamura et al., 1997; Singh-Jasuja et al., 2000b; Wang et al., 2003; Murshid et al.,2010). A number of studies show that HSPs can chaperone tumor antigens and deliver them to the appropriate destination – MHC class I molecules on the DC surface (Singh-Jasuja et al., 2000a,b; Srivastava and Amato, 2001; Delneste et al.,2002; Enomoto et al., 2006; Gong et al., 2009). In addition, Hsp70 has been shown to chaperone viral antigenic peptides and increase cross priming of human CTL under ex vivo conditions (Tischer et al., 2011). However, it is still far from clear how the process of HSP-mediated cross priming unfolds. For instance, the CD8+ expressing DC subpopulation in lymph nodes is regarded as the primary cross-presenting APC (Heath and Carbone, 2009). It is not however currently known whether the CD8+ DC subset or other peripheral or lymph-node resident, DC interact with HSP vaccines to induce cross presentation. HSPs appear to be able to enter APC, such as mouse bone marrow derived DC (BMDC) and human DC in a receptor-mediated manner (Basu et al., 2001; Delneste et al., 2002; Gong et al.,2009; Murshid et al., 2010). However, no unique endocytosing HSP receptor has emerged and HSP–antigen complexes appear instead to be taken up by proteins with “scavenger” function such as LOX-1, SRECI, and CD91 that can each take up a wide range of extracellular ligands (Basu et al., 2001; Delneste et al., 2002; Theriault et al., 2006; Murshid et al., 2010). A pathway for Hsp90–peptide (Hsp90.PC) uptake has been characterized in mouse BMDC by scavenger receptor SRECI (Murshid et al., 2010). SRECI is able to mediate the whole process of Hsp90.PC endocytosis, trafficking through the cytoplasm to the sites of antigen processing and presentation of antigens to CD8+ T lymphocytes on MHC class I molecules (Murshid et al., 2010). This process is known as antigen cross presentation (Kurts et al., 2010). It is not currently clear what the relative contribution to antigen cross presentation of the various HSP receptors might be under in vivo conditions. It may be that each receptor class contributes to an individual aspect of CTL activation by HSP peptide complexes although a definitive understanding may await studies in mice deficient in each receptor class.

 

HSPs and CTL Programming

It is evident that that HSPs can mediate antigen cross presentation and activate CD8+ T lymphocytes. However, presentation of tumor antigens by DC is not sufficient for CTL programming and, in the absence of co-stimulatory molecules and innate immunity, the “helpless” CD8+ cells will cease to proliferate abundantly and will most likely undergo apoptosis (Schurich et al., 2009; Kurts et al., 2010). One mechanism for enhancing CTL programming involves activation of the TLR pathways that lead to synthesis of co-stimulatory molecules (Rudd et al.,2009; Yamamoto and Takeda, 2010). The co-stimulatory molecules, including CD80 and CD86 then become expressed on the DC cell surface and amplify the signals induced by binding of the T cell receptor on CD8+ T cells to MHC class I peptide complexes on the presenting DC (Parra et al., 1995; Rudd et al., 2009). This process is important in pathogen infection in which microbially derived antigens are encountered in the presence of inflammatory PAMPs that can activate innate immune transcriptional networks. Originally it had been thought that HSPs could provide analogous stimulation through their suspected activity as DAMPs and their inbuilt ability to trigger innate immunity through TLR2 and TLR4 on DC (Asea et al., 20002002; Vabulas et al., 2002). (The potential role of HSPs as DAMPs has been the subject of a recent review: van Eden et al., 2012). Subsequent studies on the capacity of HSPs to bind TLRs do not indicate avid binding of Hsp70 to either TLR2 or TLR4 when expressed in cells deficient in HSP receptors in vitro (Theriault et al., 2006). In vivo however, TLR signaling is essential for Hsp70 vaccine-induced tumor cell killing. Studies of tumor-bearing mice treated with an Hsp70 vaccine in vivo indicated that vaccine function is depleted by knockout of the TLR signaling intermediate Myd88 and completely abrogated by double knockout of TLR2 and TLR4 (Gong et al., 2009). These findings were somewhat complicated by the fact that TLR4 is involved in upstream regulation of the expression of Hsp70 receptor SRECI, but do strongly implicate a role for these receptors in amplifying immune signaling by Hsp70 vaccines and Hsp70-based immunotherapy (Sanchez-Perez et al., 2006; Gong et al., 2009). It is still not clear to what degree HSPs are capable of providing a sturdy DC maturing signal through TLR2/TLR4. The potency of HSP anticancer vaccines could potentially be improved by addition of PAMPs such as CpG DNA shown to activate TLR9, or double stranded RNA that can activate TLR3 (Murshid et al., 2011a). As mentioned, one contradictory factor in the earlier studies was that, although TLR2 and TLR4 are required for a sturdy Hsp70 vaccine-mediated immune response, direct binding of Hsp70 to these receptors was not observed (Theriault et al., 2006; Gong et al., 2009; Murshid et al., 2012). A rationale for these findings might be that HSPs can activate TLR signaling indirectly through primary binding to established HSP receptors such as LOX-1 and SRECI which secondarily recruit and activate the TLRs (Murshid et al., 2011b). Both of these scavenger receptors bind to TLR2 upon stimulation and activate TLR2-based signaling (Jeannin et al., 2005; A. Murshid and SK Calderwood, in preparation). In addition, we have found that Hsp90–SRECI complexes move to the lipid raft compartment of the cell, an environment highly enriched in TLR2 and TLR4 (Triantafilou et al., 2002; Murshid et al., 2010).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342006/bin/fimmu-03-00075-g001.jpg

Heat shock protein–peptide complexes extracted from tumor cells interact with endocytosing receptors (HSP-R) such as SRECI or signaling receptors (TLR) such as TLR4 on DC. SREC1 mediates uptake and intracellular processing of antigens and the presentation of resulting peptides on surface MHC class I and MHC class II proteins. MHC class II receptor–peptide complexes then bind to T cell receptors on CD4+ cells. One consequence of binding is interaction of CD40 ligand on the MHC class II cell with CD40 on the DC leading to the licensing interaction that results in enhanced expression of co-stimulatory proteins on the DC cell surface. The licensed DC may then interact with CD8+ T cells through T cell interaction with MHC class I peptide complexes. This effect will be enhanced by simultaneous interaction of CD80 or Cd86 co-stimulatory complexes on the DC with CD28 on the CD8+ cells, leading to effective CD8+ CTL that can lyse tumor cells. T cell programming can also be amplified by signals emanating from activated TLR that can boost levels of CD80 and CD86 as well as inflammatory cytokines (not shown).

 

Hsp70, Cell Damage, and Inflammation

The question of whether Hsp70 acts as DAMP and could by itself induce an inflammatory response in cancer patients in vivo is still open. However, some recent studies by Vile et al. using a gene therapy approach may shed some light on the inflammatory role of Hsp70 in tumor therapy. In this approach, as mentioned above, normal murine tissues were engineered to express high Hsp70 levels then subjected to treatments that lead to necrotic killing. The aim was to stimulate an autoimmune response that could lead to bystander immune killing of tumor cells that share the antigenic repertoire as the killed normal cells (Sanchez-Perez et al.,2006). In the initial studies, normal melanocytes were preloaded with Hsp70 plasmids and then necrotic cell death was triggered (Daniels et al., 2004). This treatment led to T cell-mediated immune killing of syngeneic B16 melanoma cells transplanted at a distant site in the mouse, presumably in response to antigens shared by the killed normal melanocytes and melanoma cell (Daniels et al., 2004). This effect only occurred when melanocytes were induced to undergo necrosis and Hsp70 levels were elevated, indicating a role for high levels of Hsp70 in the tumor specific immune response. Interestingly, these conditions did not lead to a prolonged autoimmune response, an effect mediated by the induction of a delayed Treg response (Srivastava, 2003; Daniels et al., 2004). It is notable that some early studies of chaperone-based tumor vaccines in animal models demonstrated a primary CTL response to tumors in response to treatment followed by delayed activation of a Treg reaction, and that chaperone levels must be carefully titrated for effective induction of tumor immunity (Udono and Srivastava, 1993; Liu et al.,2009). The role of Hsp70 in autoimmune rejection of tumors was also investigated in prostate cancer (Kottke et al., 2007). Ablation of normal prostate cells by necrotic killing with fusogenic viruses in the absence of Hsp70 elevation led to the induction of the cytokines IL-10 and TGF-b in the mouse prostate and a Treg response. However, when Hsp70 levels were elevated in these cells, IL-10, TGF-b, and IL-6 were induced simultaneously, the IL-6 component leading to further induction of IL-17, a profound Th17 response and tumor rejection (Kottke et al.,2007). Thus elevated levels of Hsp70, presumably released from cells undergoing necrosis can influence the local cytokine patterns and lead to an inflammatory statein vivo. Interestingly, these results seem to be tissue specific as inflammatory killing of pancreatic cells even in the presence of elevated Hsp70 did not provoke IL-6 release, a Th17 response or tumor rejection and the Treg response dominated under these conditions (Kottke et al., 2009). Thus the role of Hsp70 in tissue inflammation and tumor rejection seems to require elevated concentrations of extracellular chaperones, significant levels of necrotic cell killing, and tissue specific cytokine release.

Conclusion

  • Earlier studies investigating HSP vaccines considered such structures to be the “Swiss penknives” of immunology able to deliver antigens directly to APC and confer a maturing signal that could render DC able to effectively program CTL (Srivastava and Amato, 2001; Noessner et al., 2002). It is well established now that Hsp70, Hsp90, Hsp110, and GRP170 can chaperone tumor antigens and activate antigen cross presentation (Murshid et al., 2011a). In addition, HSPs were thought to be DAMPs with ability to strongly activate TLR signaling and innate immunity (Asea et al., 2000). However, although there is compelling evidence to indicate that Hsp70, for instance can interact with TLR4 under a number of pathological situations (see Appendix, Sanchez-Perez et al., 2006), it remains unclear whether free Hsp70 binds directly to the Toll-like receptor and induces innate immunity in the absence of other treatments in vitro(Tsan and Gao, 2004).
  • Elevated levels of extracellular HSPs appear to have the capacity to amplify the effects of inflammatory signals emanating from necrotic cells in vivoin a TLR4-dependent manner (Daniels et al., 2004; Sanchez-Perez et al., 2006; Kottke et al., 2007). In the presence of cell injury and death, elevated levels of Hsp70 appear to increase the production of inflammatory signals that involve cytokines such as IL-6 and IL-17 and lead to a specific T cell-mediated immune response to tumor cells sharing antigens with the dying cells (Kottke et al., 2007). The mechanisms involved in these processes are not clear although one possibility is that HSPs can induce the engulfment of necrotic cells. Hsp70 has been shown to increase bystander engulfment of a variety of structures (Wang et al., 2006a,b). In addition, tumor cells treated with elevated temperatures release inflammatory chemokines in an Hsp70 and TLR4-dependent mechanisms and this effect may be significant in CTL programming and tumor cell killing (Chen et al., 2009). Our studies indicate that CTL induction by Hsp70 vaccines in vivo has an absolute requirement for TLR2 and TLR4 suggesting that at least in vivo HSPs can trigger innate immunity through TLR signaling (Gong et al., 2009).
  • HSPs appear also to be able to direct antigen presentation through the class II pathway in DC and may stimulate T helper cells (Gong et al., 2009). It may thus be possible that HSPs participate in DC licensing and reinforce CTL programming during exposure to HSP vaccines. Future studies will address these questions.
  • A further interesting consideration is whether HSPs released from untreated tumor cells enhance or depress tumor immunity. One initial study shows that Hsp70 released from tumor cells in exosomes can strongly decrease tumor immunity through effects on MDSC (Chalmin et al., 2010). Further studies will be required to make a definitive statement on these questions.

 

  1. Protein aggregation disorders and HSP expression

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Christopher J. Cummings1,5, Michael A. Mancini3, Barbara Antalffy4, Donald B. DeFranco7, Harry T. Orr8 & Huda Y. Zoghbi1,2,6
Nature Genetics 19, 148 – 154 (1998) http://dx.doi.org:/10.1038/502

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease

Andreas Wyttenbach, Jenny Carmichael, Jina Swartz, Robert A. Furlong, Yolanda Narain, Julia Rankin, and David C. Rubinsztein*
https://www.researchgate.net/profile/David_Rubinsztein/publication/24447892_Effects_of_heat_shock_heat_shock_protein_40_(HDJ2)_and_proteasome_inhibition_on_protein_aggregation_in_cellular_models_of_Huntington’s_disease/links/00b7d528b80aab69bb000000.pdf

Huntington’s disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGypolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2yHSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2yHSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2yHSDJ in protein folding.

 

  1. Hsp70 in blood cell differentiation.

 

Apoptosis Versus Cell Differentiation -Role of Heat Shock Proteins HSP90, HSP70 and HSP27

David Lanneau, Aurelie de Thonel, Sebastien Maurel, Celine Didelot, and Carmen Garrido
Prion. 2007 Jan-Mar; 1(1): 53–60.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs

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Introduction

Stress or heat shock proteins (HSPs) were first discovered in 19621 as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.2 Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.3 Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α’s functions.4 HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.7 HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.8 This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.9

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.10 Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.11 One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.12Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases15 (Fig. 1).

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/figure/F1/

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and 

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.16 It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.

 

HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.2,1921 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.2224 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).

 

HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.25

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.26 A direct interaction between Akt and HSP90 was reported later.27 Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.26 Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.30 When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.27 Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.26 Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.31 HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.32 Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.33 The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p5334 and the transcription factors Her2 and Hif1α.35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.37

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,23 suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.38

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.

 

HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.39Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.40 In cancer cells, depletion of HSP70 results in spontaneous apoptosis.41

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation44 in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.45

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.46

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.47However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.48

HSP70 also prevents cell death in conditions in which caspase activation does not occur.49 Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.52 AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.53 HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation54 but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.55 During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.56

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).57 Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-150 and AIF binding,51 it is dispensable for JNK60 or GATA-161binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.61

 

HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.6466 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.65 In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.67

HSP27 can also interfere with caspase activation upstream of the mitochondria.66This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin68preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release66 (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,69 to decrease reactive oxygen species cell content,19 and to neutralize the toxic effects of oxidized proteins.70 These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.71

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.72 HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.73 In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).74 Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival75 while that of p27kip1facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.76

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.77 Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.78 In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary73,79 and it is its phosphorylated form that protects against neurotoxicity.80

 

HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.81 In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.82 In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.83

In leukemic cells HSP27 has been described as a pre-differentiation marker84because its induction occurs early during differentiation.8588 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes89 and for C2C12 muscle cells.90 This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G0/G1).19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells’ formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.91 HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.61 HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.18

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate92 and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.93 This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,94 RIP32 or Rb.95 Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.96 In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.97 In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.98 In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.4

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.99 In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G1 block. These G1 arrested cells do not differentiate but instead die by apoptosis.100

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.101Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.102 In breast cancer cells, 17-AAG-induced G1 block is accompanied by differentiation followed by apoptosis.103 The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.104

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.105 Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.16,17Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.61 Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.106It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.18 Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.

 

Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells’ aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells’ tumorigenicty and their depletion can induce a spontaneous regression of the tumors.24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.108 However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.

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HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death inC. elegans

A new pathway for non-apoptotic cell death

The results presented here allow us to construct a model for the initiation and execution of LCD in C. elegans (Figure 7). The logic of the LCD pathway may be similar to that of developmental apoptotic pathways. In C. elegans and Drosophila, where the control of specific cell deaths has been primarily examined, cell lineage or fate determinants control the expression of specific transcription factors that then impinge on proteins regulating caspase activation (Fuchs and Steller, 2011). Likewise, LCD is initiated by redundant determinants that require a transcription factor to activate protein degradation genes.

Figure 7.

https://elife-publishing-cdn.s3.amazonaws.com/12821/elife-12821-fig7-v3-480w.jpg

Figure 7. Model for linker cell death.

Green, upstream regulators. Orange, HSF-1. Purple, proteolytic components.    DOI: http://dx.doi.org/10.7554/eLife.12821.016

 

Our data suggest that three partially redundant signals control LCD initiation. The antagonistic Wnt pathways we describe may provide positional information to the linker cell, as the relevant ligands are expressed only near the region where the linker cell dies. The LIN-29 pathway, which controls timing decisions during the L4-adult molt, may ensure that LCD takes place only at the right time. Finally, while the TIR-1/SEK-1 pathway could act constitutively in the linker cell, it may also respond to specific cues from neighboring cells. Indeed, MAPK pathways are often induced by extracellular ligands. We propose that these three pathways, together, trigger activation of HSF-1. Our data support a model in which HSF-1 is present in two forms, HSF-1LC, promoting LCD, and HSF-1HS, protecting cells from stresses, including heat shock. We postulate that the redundant LCD initiation pathways tip the balance in favor of HSF-1LC, allowing this activity to bind to promoters and induce transcription of key LCD effectors, including LET-70/UBE2D2 and other components of the ubiquitin proteasome system (UPS), functioning through E3 ligase complexes consisting of CUL-3, RBX-1, BTBD-2, and SIAH-1.

Importantly, the molecular identification of LCD components and their interactions opens the door to testing the impact of this cell death pathway on vertebrate development. For example, monitoring UBE2D2 expression during development could reveal upregulation in dying cells. Likewise, genetic lesions in pathway components we identified may lead to a block in cell death. Double mutants in apoptotic and LCD genes would allow testing of the combined contributions of these processes.

The proteasome and LCD

As is the case with caspase proteases that mediate apoptosis (Pop and Salvesen, 2009), how the UPS induces LCD is not clear, and remains an exciting area of future work. That loss of BTBD-2, a specific E3 ligase component, causes extensive linker cell survival suggests that a limited set of targets may be required for LCD. Previous work demonstrated that BTBD2, the vertebrate homolog of BTBD-2, interacts with topoisomerase I (Khurana et al., 2010; Xu et al., 2002), raising the possibility that this enzyme may be a relevant target, although other targets may exist.

The UPS has been implicated in a number of cell death processes in which it appears to play a general role in cell dismantling, most notably, perhaps, in intersegmental muscle remodeling during metamorphosis in moths (Haas et al., 1995). However, other studies suggest that the UPS can have specific regulatory functions, as with caspase inhibition by IAP E3 ligases (Ditzel et al., 2008).

During Drosophila sperm development, caspase activity is induced by the UPS to promote sperm individualization, a process that resembles cytoplasm-specific activation of apoptosis (Arama et al., 2007). While C. elegans caspases are dispensible for LCD, it remains possible that they participate in linker cell dismantling or serve as a backup in case the LCD program fails.

Finally, the proteasome contains catalytic domains with target cleavage specificity reminiscent of caspases; however, inactivation of the caspase-like sites does not, alone, result in overt cellular defects (Britton et al., 2009), suggesting that this activity may be needed to degrade only specific substrates. Although the proteasome generally promotes proteolysis to short peptides, site-specific cleavage of proteins by the proteasome has been described (Chen et al., 1999). It is intriguing to speculate, therefore, that caspases and the proteasome may have common, and specific, targets in apoptosis and LCD.

A pro-death developmental function for HSF-1

Our discovery that C. elegans heat-shock factor, HSF-1, promotes cell death is surprising. Heat-shock factors are thought to be protective proteins, orchestrating the response to protein misfolding induced by a variety of stressors, including elevated temperature. Although a role for HSF1 has been proposed in promoting apoptosis of mouse spermatocytes following elevated temperatures (Nakai et al., 2000), it is not clear whether this function is physiological. In this context, HSF1 induces expression of the gene Tdag51 (Hayashida et al., 2006). Both pro- and anti-apoptotic activities have been attributed to Tdag51 (Toyoshima et al., 2004), and which is activated in sperm is not clear. Recently, pathological roles for HSF1 in cancer have been detailed (e.g. Mendillo et al., 2012), but in these capacities HSF1 still supports cell survival.

Developmental functions for HSF1 have been suggested in which HSF1 appears to act through transcriptional targets different from those of the heat-shock response (Jedlicka et al., 1997), although target identity remains obscure. Here, we have shown that HSF-1 has at least partially non-overlapping sets of stress-induced and developmental targets. Indeed, typical stress targets of HSF-1, such as the small heat-shock gene hsp-16.49 as well as genes encoding larger chaperones, likehsp-1, are not expressed during LCD, whereas let-70, a direct transcriptional target for LCD, is not induced by heat shock. Interestingly, the yeast let-70 homologs ubc4 and ubc5 are induced by heat shock (Seufert and Jentsch, 1990), supporting a conserved connection between HSF and UBE2D2-family proteins. However, the distinction between developmental and stress functions is clearly absent in this single-celled organism, raising the possibility that this separation of function may be a metazoan innovation.

What distinguishes the stress-related and developmental forms of HSF-1? One possibility is that whereas the stress response appears to be mediated by HSF-1 trimerization, HSF-1 monomers or dimers might promote LCD roles. Although this model would nicely account for the differential activities in stress responses and LCD of the HSF-1(R145A) transgenic protein, which would be predicted to favor inactivation of a larger proportion of higher order HSF-1 complexes, the identification of conserved tripartite HSEs in the let-70 and rpn-3 regulatory regions argues against this possibility. Alternatively, selective post-translational modification of HSF-1 could account for these differences. In mammals, HSF1 undergoes a variety of modifications including phosphorylation, acetylation, ubiquitination, and sumoylation (Xu et al., 2012), which, depending on the site and modification, stimulate or repress HSF1 activity. In this context, it is of note that p38/MAPK-mediated phosphorylation of HSF1 represses its stress-related activity (Chu et al., 1996), and the LCD regulator SEK-1 encodes a MAPKK. However, no single MAPK has been identified that promotes LCD (E.S.B., M.J.K. unpublished results), suggesting that other mechanisms may be at play.

Our finding that POP-1/TCF does not play a significant role in LCD raises the possibility that Wnt signaling exerts direct control over HSF-1 through interactions with β-catenin. However, we have not been able to demonstrate physical interactions between these proteins to date (M.J.K, unpublished results).

Finally, a recent paper (Labbadia and Morimoto, 2015) demonstrated that in young adult C. elegans, around the time of LCD, global binding of HSF-1 to its stress-induced targets is reduced through changes in chromatin modification. Remarkably, we showed that chromatin regulators play a key role in let-70 induction and LCD (J.A.M., M.J.K and S.S., manuscript in preparation), suggesting, perhaps, that differences in HSF-1 access to different loci may play a role in distinguishing its two functions.

LCD and neurodegeneration

Previous studies from our lab raised the possibility that LCD may be related to degenerative processes that promote vertebrate neuronal death. Nuclear crenellation is evident in dying linker cells and in degenerating cells in polyQ disease (Abraham et al., 2007) and the TIR-1/Sarm adapter protein promotes LCD in C. elegans as well as degeneration of distal axonal segments following axotomy in Drosophila and vertebrates (Osterloh et al., 2012). The studies we present here, implicating the UPS and heat-shock factor in LCD, also support a connection with neurodegeneration. Indeed, protein aggregates found in cells of patients with polyQ diseases are heavily ubiquitylated (Kalchman et al., 1996). Chaperones also colocalize with protein aggregates in brain slices from SCA patients, and HSF1 has been shown to alleviate polyQ aggregation and cellular demise in both polyQ-overexpressing flies and in neuronal precursor cells (Neef et al., 2010). While the failure of proteostatic mechanisms in neurodegenerative diseases is generally thought to be a secondary event in their pathogenesis, it is possible that this failure reflects the involvement of a LCD-like process, in which attempts to engage protective measures instead result in activation of a specific cell death program.

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Natural Killer Cell Response: Treatment of Cancer

Curator: Larry H. Bernstein, MD, FCAP

 

Molecular mechanisms of natural killer cell activation in response to cellular stress

C J Chan1,2,3, M J Smyth1,2,3,4,5 and L Martinet1,2,4,5        Edited by M Piacentini

Cell Death and Differentiation (2014) 21, 5–14;    http://www.nature.com/cdd/journal/v21/n1/full/cdd201326a.htm

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.

 

  • Stress induces specific intrinsic and extrinsic physiological mechanisms within cells that lead to their identification as functionally abnormal
  • Sources of cellular stress can be nutritional, physical, pathogenic, or oncogenic
  • Intrinsic responses to cellular stress include activation of the DNA-damage response, tumor-suppressor genes, and senescence
  • The extrinsic response to cellular stress is activation of the immune system, such as natural killer cells
  • Intrinsic responses to cellular stress can directly upregulate factors that can activate the immune system, and the immune system been shown to be indispensable for the efficacy of some chemotherapy

Further critical determinants of intrinsic responses to stress and cell death that can activate the immune system must be identified

  • Identification of the different cellular pathways and molecular determinants controlling the immunogenicity of different cancer therapies is required
  • How can we harness the ability of therapeutic agents to activate both the intrinsic and extrinsic responses to cellular stress to achieve more specific and safer approaches to cancer treatment?

Any insult to a cell that leads to its abnormal behavior or premature death can be defined as a source of stress. As the turnover and maintenance of cells in all multi-cellular organisms is tightly regulated, it is essential that stressed cells be rapidly identified to avoid widespread tissue damage and to maintain tissue homeostasis. Various intrinsic cellular mechanisms exist within cells that become activated when they are exposed to stress. These include activation of DNA-damage response proteins, senescence programs, and tumor-suppressor genes.1 Extrinsic mechanisms also exist that combat cellular stress, through the upregulation of mediators that can activate different components of the immune system.2 Although frequently discussed separately, much recent evidence has indicated that intrinsic and extrinsic responses to cellular stress are intimately linked.3

As the link between cell intrinsic and extrinsic responses to stress have been uncovered, these observations are now being harnessed therapeutically, particularly in the context of cancer.4 Indeed, various chemotherapeutic agents and radiotherapy are critically dependent on the immune system to elicit their full therapeutic benefit.5, 6 The mechanisms by which this occurs may be twofold: (i) the induction of intrinsic cellular stress mechanisms activates innate immunity and (ii) the release and presentation of tumor-specific antigens engages an inflammatory adaptive immune response.

NK cells are the major effector lymphocyte of innate immunity found in all the primary and secondary immune compartments as well as various mucosal tissues.7 Through their ability to induce direct cytotoxicity of target cells and produce pro-inflammatory cytokines such as interferon-gamma, NK cells are critically involved in the immune surveillance of tumors8, 9, 10 and microbial infections.11, 12 The major mechanism that regulates NK cell contact-dependent functions (such as cytotoxicity and recognition of targets) is the relative contribution of inhibitory and activating receptors that bind to cognate ligands.

Under normal physiological conditions, NK cell activity is inhibited through the interaction of their inhibitory receptors with major histocompatibility complex (MHC) class I.13, 14 However, upon instances of cellular stress that are frequently associated with viral infection and malignant transformation, ligands for activating receptors are often upregulated and MHC class I expression may be downregulated. The upregulation of these activating ligands and downregulation of MHC class I thus provides a signal for NK cells to become activated and display effector functions. Activating receptors are able to provide NK cells with a strong stimulus in the absence of co-stimulation due to the presence of adaptor molecules such as DAP10, DAP12, FcRγ, and CD3ζ that contain immunoreceptor tyrosine-based activating motifs (ITAMs).15, 16,17 By contrast, inhibitory receptors contain inhibitory motifs (ITIMs) within their cytoplasmic tails that can activate downstream targets such as SHP-1 and SHP-2 and directly antagonize those signaling pathways activated through ITAMs.18, 19, 20 The specific details of individual classes of inhibitory and activating receptors and their ligands are summarized in Figure 1 and have been extensively reviewed elsewhere.14, 21 Instead, this review will more focus on the relevant activating receptors that are primarily involved in the direct regulation of NK cell-mediated recognition of cellular stress: natural killer group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1).

Figure 1.

Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorNK cell receptors and their cognate ligands. Major inhibitory and activating receptors on NK cells and their cognate ligands on targets are depicted. BAT3, human leukocyte antigen (HLA)-B-associated transcript 3; CRTAM, class I-restricted T-cell-associated molecule; HA, hemagglutinin; HLA-E, HLA class I histocompatibility antigen, alpha chain E; IgG, immunoglobulin G; LFA-1, leukocyte function-associated antigen-1; LLT1, lectin-like transcript 1; TIGIT, T cell immunoglobulin and ITIM domain

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NK Cell-Mediated Recognition of Cellular Stress by NKG2D and DNAM-1

NKG2D is a lectin-like type 2 transmembrane receptor expressed as a homodimer in both mice and humans by virtually all NK cells.22, 23 Upon interaction with its ligands, NKG2D can trigger NK cell-mediated cytotoxicity against their targets. The ligands for NKG2D are self proteins related to MHC class I molecules.24 In humans, these ligands consist of the MHC class I chain-related protein (MIC) family (e.g., MICA and MICB) and the UL16-binding protein (ULBP1-6) family.25, 26 In mice, ligands for NKG2D include the retinoic acid early inducible (Rae) gene family, the H60 family, and mouse ULBP-like transcript-1 (MULT-1).27, 28, 29 NKG2D ligands are generally absent on the cell surface of healthy cells but are frequently upregulated upon cellular stress associated with viral infection and malignant transformation.3, 30 Indeed, NKG2D ligand expression has been found on many transformed cell lines, and NKG2D-dependent elimination of tumor cells expressing NKG2D ligands has been well documented in vitro and in tumor transplant experiments.25, 30, 31, 32, 33 In humans, NKG2D ligands have been described on different primary tumors34, 35 and specific NKG2D gene polymorphisms are associated with susceptibility to cancer.36 Finally, blocking NKG2D through gene inactivation or monoclonal antibodies leads to an increased susceptibility to tumor development in mouse models,37, 38demonstrating the key role played by NKG2D in immune surveillance of tumors. NKG2D can also contribute to shape tumor immunogenicity, a process called immunoediting, as demonstrated by the frequent ability of tumor cells to avoid NKG2D-mediated recognition through NKG2D ligand shedding, as discussed later in this review.38, 39, 40

DNAM-1 is a transmembrane adhesion molecule constitutively expressed on T cells, NK cells, macrophages, and a small subset of B cells in mice and humans.41, 42, 43 DNAM-1 contains an extracellular region with two IgV-like domains, a transmembrane region and a cytoplasmic region containing tyrosine- and serine-phosphorylated sites that is able to initiate downstream activation cascades.41, 44 There is accumulating evidence showing that DNAM-1 not only promotes adhesion of NK cells and CTLs but also greatly enhances their cytotoxicity toward ligand-expressing targets.41, 45, 46, 47, 48, 49, 50 The ligands for DNAM-1 are the nectin/nectin-like family members CD155 (PVR, necl-5) and CD112 (PVRL2, nectin-2).45, 46 Like NKG2D ligands, DNAM-1 ligands are frequently expressed on virus-infected and transformed cells.51, 52DNAM-1 ligands, especially CD155, are overexpressed by many types of solid and hematological malignancies and blocking DNAM-1 interactions with its ligands reduces the ability of NK cells to kill tumor cells in vitro.41, 49, 53, 54, 55, 56, 57 Further evidence of the role of DNAM-1 in tumor immune surveillance is provided by studies using experimental and spontaneous models of cancer in vivo showing enhanced tumor spread in the absence of DNAM-1.47, 48, 49, 50, 58

As NKG2D and DNAM-1 ligands are frequently expressed on stressed cells, many studies have sought to determine the mechanisms that underpin these observations. The guiding hypothesis for these studies is that cell-intrinsic responses to stress are directly linked to cell-extrinsic responses that can trigger rapid NK cell surveillance and elimination of stressed cells. Indeed, major cell-intrinsic responses to cellular stress can directly lead to NK cell-activating ligand upregulation and are outlined in the following sections.

The DNA-Damage Response

Cellular stress caused by the activation of the DNA-damage response leads to downstream apoptosis or cell-cycle arrest. The activation of DNA-damage checkpoints occurs when there are excessive DNA strand breaks and replication errors, thereby representing an important tumorigenesis barrier that can slow or inhibit the progression of malignant transformation.59, 60 Two major transducers of the DNA-damage response are the PI3-kinase-related protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM and ATR can modulate numerous signaling pathways such as checkpoint kinases (Chk1 and Chk2, which inhibit cell-cycle progression and promote DNA repair) and p53 (which mediates cell-cycle arrest and apoptosis).61

In addition to the induction of cell-cycle arrest and apoptosis, activation of the DNA-damage response has been shown to promote the expression of several activating ligands that are specific for NK cell receptors, primarily those of the NKG2D receptor. These findings have shown a critical direct link between cellular transformation, apoptosis, and surveillance by the immune system.62 The first evidence of this link between DNA damage and immune cell activation was provided by Raulet and colleagues who showed that NKG2D ligands were upregulated by genotoxic stress and stalled DNA replication conditions known to activate either ATM or ATR.63 These observations have now been extended by several other studies that have defined further DNA-damaging conditions (e.g., genotoxic drugs/chemotherapy, deregulated proliferation, or oxidative stress) that can promote NKG2D ligand upregulation.64, 65, 66, 67

The role of the DNA-damage response in controlling NKG2D ligand expression and subsequent NK cell activation has also been demonstrated in the context of anti-viral immunity, specifically in Abelson murine leukemia virus infection.68 This pathogen was shown to induce activation-induced cytidine deaminase (AID) expression outside the germinal center, resulting in generalized hypermutation, DNA-damage checkpoint activation, and Chk1 phosphorylation. The genotoxic activity of virally induced AID not only restricted the proliferation of infected cells but also induced the expression of NKG2D ligands. More recently, another member of APOBEC-AID family of cytidine deaminases, A3G, has been shown to promote the recognition of HIV-infected cells by NK cells after DNA-damage response activation.69 In this study, viral protein Vpr-mediated repair processes, which generate nicks, gaps, and breaks of DNA, activate an ATM/ATR DNA-damage response that leads to NKG2D ligand expression.

The DNA-damage sensors ATM and ATR have also been shown to regulate other key NK cell-activating ligands such as the DNAM-1 ligand, CD155.58, 65, 70 For example, in the Eμ-myc spontaneous B-cell lymphoma model, activation of the DNA-damage response leads to the upregulation of CD155 in the early-stage transformed B cells, subsequently activating spontaneous tumor regression in an NK cell- and T-cell-dependent manner.58 The DNA-damage response can also regulate the expression of the death receptor DR5.71 The engagement of DR5 by the effector molecule TRAIL, which is expressed by NK cells and T cells, can induce apoptosis of target cells and has been shown to have a key role in immune surveillance against tumors.72 Collectively, these results suggest that the detection of DNA damage, primarily through ATM and ATR, may represent a conserved protection mechanism governing the immunogenicity of infected or transformed cells, leading to direct recognition by NK cells (Figure 2).

Figure 2.

Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorOverview of the molecular pathways leading to NK cell recognition of intrinsic cellular stress. Oncogenic transformation and viral infection can activate intrinsic cellular responses to stress. These responses include activation of the DNA-damage response, senescence, tumor suppressors, and the presentation and/or release of HSPs that, in turn, can activate NK cells through various receptor–ligand interactions. Senescent cells can also release pro-inflammatory cytokines that can recruit NK cells and other innate immunity, such as macrophages. CCL2, C-C motif chemokine ligand 2; CXCL11, C-X-C motif chemokine ligand 11; DR, death receptor 5; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

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As a result of these studies, many therapeutic agents known to induce DNA damage have been evaluated for their ability to increase the immunogenicity of cancer cells for a more targeted therapeutic approach using NK cells.64, 65 For example, treatment of multiple myeloma cells with doxorubicin, melphalan, or bortezomib can lead to DNAM-1 and NKG2D ligand upregulation.65Indeed, many chemotherapeutic agents commonly used, especially in hematological malignancies, can trigger the DNA-damage pathway. Therefore, it is reasonable to speculate that there is a general role of ATM and ATR in the induction of NK cell activation as a therapeutic effect of these agents.

Senescence

Cellular senescence is generally defined as a growth-arrest program in mammalian cells that limits their lifespan.73 The major type of cellular senescence is replicative senescence that occurs due to telomere shortening. However, it is now generally accepted that premature senescence can also occur due to oncogene activation (oncogene-induced senescence) and/or the loss/gain of tumor-suppressor gene function, in the absence of telomere shortening.74 Thus, premature senescence is an important barrier against malignant transformation.59 Upon engagement of the senescence program, although cells are in growth arrest, they remain metabolically active and can produce many pro-inflammatory cytokines, as well as upregulate adhesion molecules and activating ligands to alert the immune system.75, 76, 77Activation of the immune system, in particular innate immunity, has a critical role in the clearance of senescent cells.78, 79, 80, 81More specifically, in a model of hepatocellular carcinoma, it has been shown that reactivation of p53 can induce a senescence program, resulting in tumor regression through the activation of NK cells, macrophages, and neutrophils. Of note, intercellular adhesion molecule (ICAM)-1, which can trigger both adhesion and cytotoxicity of NK cells,82 and interleukin-15, a cytokine that can promote NK cell effector function,83 were both upregulated in senescent tumors. More recently, the potential contribution of NK cells was also shown in the clearance of senescent hepatic stellate cells, a mechanism important in limiting liver fibrosis in response to a fibrogenic agent.80 ICAM-1, NKG2D ligands (MICA and ULPB2), and DNAM-1 ligands (CD155) were all upregulated on senescent hepatic stellate cells.

The specific mechanisms linking the senescence program to immune activation are not yet fully understood. However, the intracellular molecular mechanisms that govern induction of senescence may provide possible indications. Both replicative senescence and premature senescence (e.g., oncogene-induced senescence) have been shown to have common molecular determinants, such as the activation of the DNA-damage response pathway (e.g., ATM and ATR) and downstream activation of p53 and p16INK4A.1, 59, 84, 85, 86 Activation of the DNA-damage response would presumably initiate the upregulation of NK cell-activating ligands as previously discussed. However, how senescence may be linked to the induction of pro-inflammatory cytokine release is a more compelling question and requires further investigation (Figure 2). Nevertheless, induction of pro-inflammatory cytokines is an important protective mechanism in order to recruit immune cells that can rapidly recognize and remove senescent cells. Interestingly, activation of NK cells by senescent cells has been observed in a clinical context when multiple myeloma cells were treated with chemotherapy and genotoxic agents.65 In this setting, NKG2D and DNAM-1 ligands were both upregulated through a mechanism that required activation of the DNA-damage pathway initiated by ATM and ATR.65

Tumor Suppressors: p53

p53 is a potent tumor suppressor and central regulator of apoptosis, DNA repair, and cell proliferation, that is activated in response to DNA damage, oncogene activation, and other cellular stress.87 The number of identified cellular functions that p53 regulates has greatly increased over the past few years, and there is now a vast array of evidence that shows that p53 can be induced by viral infection88 to limit pathogen spread by inducing apoptosis.89, 90 Furthermore, p53 not only acts as an intrinsic barrier against tumorigenesis or pathogenic spread but can also lead to increased cellular immunogenicity. For example, p53 reactivation in a hepatocellular carcinoma can promote tumor regression mediated by innate immunity.78 A direct link between p53 expression and immune cell recognition was recently provided by Textor et al.91 where expression of p53 in lung cancer cell lines strongly upregulated the NKG2D ligands ULBP1 and 2, resulting in NK cell activation. Subsequently, p53-responsive elements were found to directly regulate ULBP1 and 2 expression, the deletion of which abolished the capacity of p53 to mediate ULBP1 and 2 upregulation. Another recent report that used a pharmacological activator of p53 confirmed the ability of p53 to directly induce ULBP2 expression that was independent of ATM/ATR.92 However, it has also been shown that miR34a and miR34C microRNAs (miRNAs) induced by p53 can target ULBP2 mRNA and reduce its cell-surface expression, suggesting that p53 may have a dual role in regulating ULBP2 expression.93 Finally, early work showed that NKG2D ligands can be upregulated by ATR/ATM in the total absence of p53 in tumor cell lines,62, 63 suggesting the existence of ATM/ATR-dependent and p53-independent pathways that regulate NKG2D ligand expression in response to cellular stress.

In addition to regulating NK cell ligand expression, genetic reactivation of p53 in tumors can also induce a wide array of pro-inflammatory mediators ranging from adhesion receptor (ICAM-1) expression to the production of various chemokines (CXCL11 and monocyte chemoattractant protein-1) and cytokines (interleukin-15).78 Furthermore, recent studies in anti-viral immunity indicate that several interferon-inducible genes and Toll-like receptor-3 expression are direct transcriptional targets of p53 and that p53 contributes to production of type I interferon by virally infected cells.94, 95, 96 All together, these studies suggest that p53 accumulation could represent a key determinant of the immunogenicity of stressed cells that are infected or undergoing malignant transformation through its ability to regulate innate immune activation.

Oncogenes

Malignant transformation is a complex process that frequently involves the activation of one or more oncogenes in addition to the inactivation or mutation of tumor-suppressor genes (e.g., p53). Oncogene activation is a powerful inducer of cellular stress that is able to activate intrinsic cellular programs that lead to cell apoptosis or senescence (e.g., activation of the DNA-damage response and p53).1 In addition, many recent reports have also shown that major oncogenes can activate extrinsic responses to cellular stress through inducing the upregulation of NK cell-activating ligands.63, 97, 98 This suggests that oncogene activation can represent a key cellular event in alerting the immune system to ongoing cellular transformation (Figure 3).

Figure 3.

Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorMolecular mechanisms that regulate the cell surface expression of NKG2D ligands. The major group of NK cell-activating ligands that are upregulated by intrinsic cellular responses to stress are those that bind the NKG2D receptor. Activation of the DNA-damage response, senescence, oncogenes, tumor suppressors, or sensing of deregulated proliferation can induce NKG2D ligand gene transcription and increase mRNA translation, leading to extracellular protein expression. MMP, matrix metalloproteases

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The enhanced expression of the proto-oncogene Myc has been described as a critical event leading to cellular transformation and is a frequently found genetic alteration in cancer.99 In a recent study, again using the Eμ-myc model, Medzhitov and colleagues demonstrated the ability of c-Myc to alert NK cells to early oncogenic transformation through the upregulation of Rae-1.97 In this study, the induction of Rae-1 was dependent on the direct regulation of Rae-1 transcription by Myc through its interaction with the Raet1 epsilon gene. Collectively, these results provide a possible direct molecular mechanism to explain the increased susceptibility of NKG2D gene-targeted mice to lymphoma development in the Eμ-myc model.38

Recent evidence suggests that several oncogenic mutations of Ras (H-Ras, N-Ras, and K-Ras) can also regulate NKG2D ligand expression in both mice and humans.98 Interestingly, in this case, NKG2D ligands were regulated through MAPK/MEK and PI3K pathways downstream of oncogenic H-RasV12. The activation of PI3K pathways, and more particularly the p110α subunits by virus-encoded proteins, has also been shown to induce the Rae-1 family of ligands.100 As many viruses can manipulate the PI3K pathway101 and tumors often bear Ras and p110α oncogene mutations,102 collectively, this data suggests that there is the existence of a common molecular mechanism by which NK cells sense cellular stress mediated by PI3K-dependent regulation of NKG2D ligands.

Interestingly, whereas Myc was involved in the transcriptional regulation of NKG2D ligands, PI3K can increase NKG2D ligand expression by increasing the translation of Rae-1 mRNA.98 This involved the induction of eIF4E, a protein that enhances the translation of mRNA.103 As number of tumors and viruses can upregulate host translation initiation machinery through the overexpression of eIF4E,104, 105 this may represent an important means by which NK cells can discriminate tumor- and virus-infected cells from normal cells.

Heat-Shock Proteins (HSPs)

HSPs are highly conserved intracellular chaperone molecules that are present in most prokaryotic and eukaryotic cells that mediate protection against cellular damage under conditions of stress. HSPs are distributed in most intracellular compartments of cells where they support the correct folding of nascent polypeptides, prevent protein aggregation, and assist in protein transport across membranes.106 Many tumors display overexpression of HSPs as a response to cellular stress induced by oncogenic transformation.107, 108 HSPs can also be mobilized to the plasma membrane, or even released from cells, under conditions of stress.109

Although intracellular HSPs can promote cell survival by interfering with different apoptosis components, many studies have reported that membrane-bound or soluble HSPs can directly stimulate innate immunity.110 A major immunostimulatory function of HSPs is to promote the presentation of tumor-specific antigens by MHC class I to CD8 T cells.111, 112, 113 Soluble and membrane-bound HSPs can also induce antigen-presenting cell maturation and the resultant secretion of pro-inflammatory cytokines.114, 115, 116Finally, HSPs may directly activate NK cells as HSP70, when overexpressed on tumor cells, can induce a selective dose-dependent increase in NK cell-mediated cytotoxicity in vitro.117 NK cells may directly recognize HSP70 through a 14-amino-acid oligomer (TKD) that is localized in the C-terminal domain of the protein through CD94.118, 119 Tumor-specific HSP70 that is either presented at the cell surface or secreted on exosomes can also enhance NK cell activity against diverse types of cancer in vivo.120, 121 Most importantly, hepatocellular carcinoma cells that are treated with various chemotherapeutic agents can become more susceptible to NK cell-mediated cytotoxicity through their release of HSP-containing exosomes, giving the aforementioned findings a therapeutic context.122 Collectively, these results suggest that HSP translocation to the plasma membrane or secretion during cellular stress may represent a potent danger signal that can stimulate NK cell activity, particularly in the context of cancer.

 

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