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Effective humoral immune responses to infection and immunization are defined by high-affinity antibodies generated as a result of B cell differentiation and selection that occurs within germinal centers (GC). Within the GC, B cells undergo affinity maturation, an iterative and competitive process wherein B cells mutate their immunoglobulin genes (somatic hypermutation) and undergo clonal selection by competing for T cell help. Balancing the decision to remain within the GC and continue participating in affinity maturation or to exit the GC as a plasma cell (PC) or memory B cell (MBC) is critical for achieving optimal antibody avidity, antibody quantity, and establishing immunological memory in response to immunization or infection. Humoral immune responses during chronic infections are often dysregulated and characterized by hypergammaglobulinemia, decreased affinity maturation, and delayed development of neutralizing antibodies. Previous studies have suggested that poor antibody quality is in part due to deletion of B cells prior to establishment of the GC response.
In fact the impact of chronic infections on B cell fate decisions in the GC remains poorly understood. To address this question, researchers used single-cell transcriptional profiling of virus-specific GC B cells to test the hypothesis that chronic viral infection disrupted GC B cell fate decisions leading to suboptimal humoral immunity. These studies revealed a critical GC differentiation checkpoint that is disrupted by chronic infection, specifically at the point of dark zone re-entry. During chronic viral infection, virus-specific GC B cells were shunted towards terminal plasma cell (PC) or memory B cell (MBC) fates at the expense of continued participation in the GC. Early GC exit was associated with decreased B cell mutational burden and antibody quality. Persisting antigen and inflammation independently drove facets of dysregulation, with a key role for inflammation in directing premature terminal GC B cell differentiation and GC exit. Thus, the present research defines GC defects during chronic viral infection and identify a critical GC checkpoint that is short-circuited, preventing optimal maturation of humoral immunity.
Together, these studies identify a key GC B cell differentiation checkpoint that is dysregulated during chronic infection. Further, it was found that the chronic inflammatory environment, rather than persistent antigen, is sufficient to drive altered GC B cell differentiation during chronic infection even against unrelated antigens. However, the data also indicate that inflammatory circuits are likely linked to perception of antigen stimulation. Nevertheless, this study reveals a B cell-intrinsic program of transcriptional skewing in chronic viral infection that results in shunting out of the cyclic GC B cell process and early GC exit with consequences for antibody quality and hypergammaglobulinemia. These findings have implications for vaccination in individuals with pre-existing chronic infections where antibody responses are often ineffective and suggest that modulation of inflammatory pathways may be therapeutically useful to overcome impaired humoral immunity and foster affinity maturation during chronic viral infections.
4.1.8 Newly Found Functions of B Cell, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 4: Single Cell Genomics
The importance of B cells to human health is more than what is already known. Vaccines capable of eradicating disease activate B cells, cancer checkpoint blockade therapies are produced using B cells, and B cell deficiencies have devastating impacts. B cells have been a subject of fascination since at least the 1800s. The notion of a humoral branch to immunity emerged from the work of and contemporaries studying B cells in the early 1900s.
Efforts to understand how we could make antibodies from B cells against almost any foreign surface while usually avoiding making them against self, led to Burnet’s clonal selection theory. This was followed by the molecular definition of how a diversity of immunoglobulins can arise by gene rearrangement in developing B cells. Recombination activating gene (RAG)-dependent processes of V-(D)-J rearrangement of immunoglobulin (Ig) gene segments in developing B cells are now known to be able to generate an enormous amount of antibody diversity (theoretically at least 1016 possible variants).
With so much already known, B cell biology might be considered ‘‘done’’ with only incremental advances still to be made, but instead, there is great activity in the field today with numerous major challenges that remain. For example, efforts are underway to develop vaccines that induce broadly neutralizing antibody responses, to understand how autoantigen- and allergen-reactive antibodies arise, and to harness B cell-depletion therapies to correct non-autoantibody-mediated diseases, making it evident that there is still an enormous amount we do not know about B cells and much work to be done.
Multiple self-tolerance checkpoints exist to remove autoreactive specificities from the B cell repertoire or to limit the ability of such cells to secrete autoantigen-binding antibody. These include receptor editing and deletion in immature B cells, competitive elimination of chronically autoantigen binding B cells in the periphery, and a state of anergy that disfavors PC (plasma cell) differentiation. Autoantibody production can occur due to failures in these checkpoints or in T cell self-tolerance mechanisms. Variants in multiple genes are implicated in increasing the likelihood of checkpoint failure and of autoantibody production occurring.
Autoantibodies are pathogenic in a number of human diseases including SLE (Systemic lupus erythematosus), pemphigus vulgaris, Grave’s disease, and myasthenia gravis. B cell depletion therapy using anti-CD20 antibody has been protective in some of these diseases such as pemphigus vulgaris, but not others such as SLE and this appears to reflect the contribution of SLPC (Short lived plasma cells) versus LLPC (Long lived plasma cells) to autoantibody production and the inability of even prolonged anti-CD20 treatment to eliminate the later. These clinical findings have added to the importance of understanding what factors drive SLPC versus LLPC development and what the requirements are to support LLPCs.
B cell depletion therapy has also been efficacious in several other autoimmune diseases, including multiple sclerosis (MS), type 1 diabetes, and rheumatoid arthritis (RA). While the potential contributions of autoantibodies to the pathology of these diseases are still being explored, autoantigen presentation has been posited as another mechanism for B cell disease-promoting activity.
In addition to autoimmunity, B cells play an important role in allergic diseases. IgE antibodies specific for allergen components sensitize mast cells and basophils for rapid degranulation in response to allergen exposures at various sites, such as in the intestine (food allergy), nose (allergic rhinitis), and lung (allergic asthma). IgE production may thus be favored under conditions that induce weak B cell responses and minimal GC (Germinal center) activity, thereby enabling IgE+ B cells and/or PCs to avoid being outcompeted by IgG+ cells. Aside from IgE antibodies, B cells may also contribute to allergic inflammation through their interactions with T cells.
B cells have also emerged as an important source of the immunosuppressive cytokine IL-10. Mouse studies revealed that B cell-derived IL-10 can promote recovery from EAE (Experimental autoimmune encephalomyelitis) and can be protective in models of RA and type 1 diabetes. Moreover, IL-10 production from B cells restrains T cell responses during some viral and bacterial infections. These findings indicate that the influence of B cells on the cytokine milieu will be context dependent.
The presence of B cells in a variety of solid tumor types, including breast cancer, ovarian cancer, and melanoma, has been associated in some studies with a positive prognosis. The mechanism involved is unclear but could include antigen presentation to CD4 and CD8 T cells, antibody production and subsequent enhancement of presentation, or by promoting tertiary lymphoid tissue formation and local T cell accumulation. It is also noteworthy that B cells frequently make antibody responses to cancer antigens and this has led to efforts to use antibodies from cancer patients as biomarkers of disease and to identify immunotherapy targets.
Malignancies of B cells themselves are a common form of hematopoietic cancer. This predilection arises because the gene modifications that B cells undergo during development and in immune responses are not perfect in their fidelity, and antibody responses require extensive B cell proliferation. The study of B cell lymphomas and their associated genetic derangements continues to be illuminating about requirements for normal B cell differentiation and signaling while also leading to the development of targeted therapies.
Overall this study attempted to capture some of the advances in the understanding of B cell biology that have occurred since the turn of the century. These include important steps forward in understanding how B cells encounter antigens, the co-stimulatory and cytokine requirements for their proliferation and differentiation, and how properties of the B cell receptor, the antigen, and helper T cells influence B cell responses. Many advances continue to transform the field including the impact of deep sequencing technologies on understanding B cell repertoires, the IgA-inducing microbiome, and the genetic defects in humans that compromise or exaggerate B cell responses or give rise to B cell malignancies.
Other advances that are providing insight include:
single-cell approaches to define B cell heterogeneity,
glycomic approaches to study effector sugars on antibodies,
new methods to study human B cell responses including CRISPR-based manipulation, and
the use of systems biology to study changes at the whole organism level.
With the recognition that B cells and antibodies are involved in most types of immune response and the realization that inflammatory processes contribute to a wider range of diseases than previously believed, including, for example, metabolic syndrome and neurodegeneration, it is expected that further
basic research-driven discovery about B cell biology will lead to more and improved approaches to maintain health and fight disease in the future.
Novel clinically-available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies.
Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection.
Abstract
The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded (FFPE), blood and bone marrow samples with high accuracy in a clinically relevant timeframe, which is performed in our CLIA-certified CAP-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs and gene fusions, with similar accuracy to lower-throughput assays which focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically-relevant genomic alterations with therapeutic relevance.
In addition to the concordance analysis, genomic profiling of the 76 test samples using FoundationOne Heme also identified 126 additional somatic alterations including clinically relevant genomic alterations in KRAS, TET2, EZH2, and DNMT3A.
Importantly, the study also showed that the molecular information supplied by the test can help accurately match patients with a particular targeted therapy.
In the study Foundation Medicine shared clinical data from genomic profiling of 3,696 hematologic malignancies submitted to its CLIA-certified, NYS-approved lab.
More than 90 percent of the specimens — 3,433 out of 3696 — were successfully characterized. The test identified at least one driver alteration in 95 percent of the tumor specimens, and results showed that 77 percent of the cases harbored at least one alteration linked to a commercially available targeted therapy or one that is in clinical development, the MSKCC researchers reported.
In addition, 61 percent of the cases harbored at least one alteration with known prognostic relevance in that tumor type.
In discussion of the results, the study authors argued that clinical merit of the test was underscored by the demonstrated ability to identify genetic lesions with prognostic and therapeutic relevance in specific diseases.
For example, the authors wrote, “In the case of B-cell ALL … the challenge has been that the critical genes … can be altered by whole gene/intragenic deletions, DNA base-pair substitutions, and larger indels, as well as chromosomal, intergenic, and cryptic rearrangements, which lead to expression of fusion transcripts.”
“Currently, most centers use an amalgam of DNA, FISH, and gene-specific RNA approaches to identify a subset of the most critical genetic lesions in B-ALL. Our assay provides a single profiling platform that can reliably identify all known actionable disease alleles relevant to B-ALL to improve diagnosis and risk-adapted therapy for B-ALL patients,” they wrote.
Germinal centres support antibody affinity maturation and memory formation1. Follicular T-helper cells promote proliferation and differentiation of antigen-specific B cells inside the follicle2, 3. A genetic deficiency in the inducible co-stimulator (ICOS), a classic CD28 family co-stimulatory molecule highly expressed by follicular T-helper cells, causes profound germinal centre defects4, 5, leading to the view that ICOS specifically co-stimulates the follicular T-helper cell differentiation program2, 6, 7. Here we show that ICOS directly controls follicular recruitment of activated T-helper cells in mice. This effect is independent from ICOS ligand (ICOSL)-mediated co-stimulation provided by antigen-presenting dendritic cells or cognate B cells, and does not rely on Bcl6-mediated programming as an intermediate step. Instead, it requires ICOSL expression by follicular bystander B cells, which do not present cognate antigen to T-helper cells but collectively form an ICOS-engaging field. Dynamic imaging reveals ICOS engagement drives coordinated pseudopod formation and promotes persistent T-cell migration at the border between the T-cell zone and the B-cell follicle in vivo. When follicular bystander B cells cannot express ICOSL, otherwise competent T-helper cells fail to develop into follicular T-helper cells normally, and fail to promote optimal germinal centre responses. These results demonstrate a co-stimulation-independent function of ICOS, uncover a key role for bystander B cells in promoting the development of follicular T-helper cells, and reveal unsuspected sophistication in dynamic T-cell positioning in vivo.
Acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells, affects both children and adults,
with peak prevalence between the ages of 2 and 5 years (2). Acute lymphocytic leukemia (ALL) is a heterogeneous disease, both in terms of its pathology and the populations that it affects. Disease pathogenesis involves a number of deregulated pathways controlling cell proliferation, differentiation, and survival that are important determinants of treatment response (3). Approximately 5200 new cases of ALL are estimated to have occurred in the United States in 2007 and survival varies with age and disease biology (3). Although five-year survival rates for ALL approach 90 percent with available chemotherapy treatments, the harmful side effects of the drugs, including secondary cancers and fertility, cognitive, hearing, and developmental problems, present significant concern for survivors and their families.
Biological and Clinical Prognostic Factors in ALL: Setting the Stage for Risk-Adapted Therapy
Of the many variables that influence prognosis the genetic subsets, initial white blood cell count (WBC), age at diagnosis, and early treatment response are the most important.
Childhood Acute Lymphoblastic Leukemia
Pathobiology
Acute lymphoblastic leukaemia is thought to originate from various important genetic lesions in blood-progenitor cells that are committed to differentiate in the T-cell or B-cell pathway, including mutations that impart the capacity for unlimited self-renewal and those that lead to precise stage-specific developmental arrest. In some cases, the first mutation along the multistep pathway to overt acute lymphoblastic leukaemia might arise in a haemopoietic stem cell possessing multilineage developmental capacity.
The dominant theme of contemporary research in pathobiology of acute lymphoblastic leukaemia is to understand the outcomes of frequently arising genetic lesions, in terms of their effects on cell proliferation, differentiation, and survival, and then to devise selectively targeted treatments against the altered gene products to which the leukaemic clones have become addicted (2).
Prognostic factors used in pediatric and adult clinical trials
The Table illustrates the different prognostic factors in children and adults that may be used for risk stratification in current clinical trials (3).
Genetics
Chromosomal translocations that activate specifi c genes
are a defi ning characteristic of human leukaemias and
of acute lymphoblastic leukaemia in particular.
About 25% of cases of B-cell precursor acute lymphoblastic leukaemia, the most frequent form of acute leukaemia in children, harbour the TEL-AML1 fusion gene—generated by the t(12;21)(p13;q22) chromosomal translocation.
The presence of the TEL-AML1 fusion
protein in B-cell progenitors seems to lead to disordered
early B-lineage lymphocyte development, a hallmark of
leukaemic lymphoblasts.
Analysis of TEL-AML1-induced cord blood cells suggests that the fusion gene serves as a first-hit mutation by endowing the preleukemic cell with altered self-renewal and survival properties.
In adults, the most frequent chromosomal translocation is t(9;22), or the Philadelphia chromosome, which causes fusion of the BCR signalling protein to the ABL non-receptor tyrosine kinase, resulting in constitutive tyrosine kinase activity and complex interactions of this fusion protein with many other transforming elements. BCR-ABL off ers an attractive therapeutic target, and imatinib mesilate, a small-molecule inhibitor of the ABL kinase, has proven effective against leukaemias that express BCR-ABL
More than 50% of cases of T-cell acute lymphoblastic leukaemia have activating mutations that involve NOTCH1. NOTCH1, which translocates to the nucleus and regulates by transcription a diverse set of responder genes, including the MYC oncogene. The precise mechanisms by which aberrant NOTCH signalling (due to mutational activation) causes T-cell acute lymphoblastic leukaemia are still unclear but probably entail constitutive expression of oncogenic responder genes, such as MYC, and cooperation with other signalling pathways (pre-TCR [T-cell receptor for antigen] and RAS, for example). Interference with NOTCH signalling by small-molecule inhibition of γ-secretase activity has the potential to induce remission of T-cell acute lymphoblastic leukemia.
Additionally A recent discussion has aimed to reveal the genetic origin of the disease (1). Several of these genes, including ARID5B, IKZF1, and CEBPE, have been implicated in processes such as hematopoietic differentiation and development of ALL. These gene obviously adds up to a number of other gene mutations and translocation already discovered and are associated with disease progression (2) “The fact that alterations in these genes lead to ALL raises the question of what would happen if we restore these pathways in ALL and also make them possible exciting therapeutic targets as well.”
Nanotechnology and therapeutic
Dr. Rajasekaran, director and head of the Membrane Biology Laboratory University of Delaware, says that there are currently seven or eight drugs that are used for chemotherapy to treat leukemia in children. They are all toxic and do their job by killing rapidly dividing cells. these drugs don’t differentiate cancer cells from other healthy cells. “The good news is that these drugs are 80 to 90 percent effective in curing leukemia. The bad news is that many chemotherapeutic treatments cause severe side effects, especially in children. In preclinical models of leukemia, Dr. Rajasekaran research team have created NP with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The researches have been encapsulated with dexamethasone as one third of the typical dose, with good treatment results and no discernible side effects.In addition, the mice that received the drugs delivered via nanoparticles survived longer than those that received the drug administered in the traditional way (4).
In another preclinical study Uckun F et al developed nanoparticle (NP) constructs of WHI-P131. WHI-P131 (CAS 202475-60-3) is a dual-function inhibitor of JAK3 tyrosine kinase that demonstrated potent in vivo anti-inflammatory and anti-leukemic activity in several preclinical animal models (5). Notably, WHI-P131-NP was capable of causing apoptotic death in primary leukemia cells from chemotherapy-resistant acute lymphoblastic leukemia (ALL) as well as chronic lymphocytic leukemia (CLL) patients. WHI-P131-NP was also active in the RS4;11 SCID mouse xenograft model of chemotherapy-resistant B-lineage ALL. The life table analysis showed that WHI-P131-NP was more effective than WHI-P131 (P = 0.01), vincristine (P<0.0001), or vehicle (P<0.0001). These experimental results demonstrate that the nanotechnology-enabled delivery of WHI-P131 shows therapeutic potential against leukemias with constitutive activation of the JAK3-STAT3/STAT5 molecular target (5).
Summary:
Acute Lymphoblastic Leukemia (ALL) is a pediatric type of cancer that affects adults to lesser degree. The current rate of cure if 80% in children whereas in adults only 30-40% will survive. Much of the success is due to understanding the mechanisms that lead to the development and progression of cancer. Several gene mutations and gene-translocation have already been identified, and targeting them enabled some of the major success in curing these kids.
Thus far, nanotechnology has been mainly focusing on solid tumors affecting adults. Not much attention is been made on childhood cancer in general and hematopoietic types per se. Two examples of preclinical studies have been discussed above and although they show promise in treatment and reduction of side effects, yet additional research is needed to evaluated their effect in human clinical trials.
5. Uckun FM, Dibirdik I, Qazi S, Yiv S. Therapeutic nanoparticle constructs of a JAK3 tyrosine kinase inhibitor against human B-lineage ALL cells. Arzneimittelforschung 2010; 60(4): 210-217.
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