Despite the various processing steps involved in V(D)J recombination, which could potentially introduce many biases in the length distribution of complementarity determining region 3 (CDR3) segments, the observed CDR3 length distributions for complete repertoires are very close to a normal-like distribution. This raises the question of whether this distribution is simply a result of the random steps included in the process of gene rearrangement, or has been optimized during evolution. We have addressed this issue by constructing a simulation of gene rearrangement, which takes into account the DNA modification steps included in the process, namely hairpin opening, nucleotide additions, and nucleotide deletions. We found that the near-Gaussian-shape of CDR3 length distribution can only be obtained under a relatively narrow set of parameter values, and thus our model suggests that specific biases govern the rearrangement process. In both B-cell receptor (BCR) heavy chain and T-cell receptor b chain, we obtained a Gaussian distribution using identical parameters, despite the difference in the number and the lengths of the D segments. Hence our results suggest that these parameters most likely reflect the optimal conditions under which the rearrangement process occurs. We have subsequently used the insights gained in this study to estimate the probability of occurrence of two exactly identical BCRs over the course of a human lifetime. Whereas identical rearrangements of the heavy chain are highly unlikely to occur within one human lifetime, for the light chain we found that this probability is not negligible, and hence the light chain CDR3 alone cannot serve as an indicator of B-cell clonality. To cope with the wide range of pathogens encountered during a lifetime, B and T lymphocytes express a diverse repertoire of binding specificities, in the form of B-cell antigen receptors or secreted antibodies, also called immunoglobulins (Ig), in B-cells (BCR), or T-cell receptors (TCR) in T cells. Each B-or T cell expresses a singleantigen specificity, determined early in its differentiation, when the genes encoding the variable regions of Ig heavy and light chains (or TCRb-and -a chains, respectively) are rearranged from gene segments. [1][2][3][4][5][6][7][8][9][10][11] In the BCR light chain or TCRa chain, each V domain is encoded by two separate DNA segments, the V gene segment and the joining or J gene segment. In the BCR heavy chain or TCRb chain V regions, there is a third gene segment called the diversity or D gene segment, which lies between V and J segments. There are multiple copies of V, D, and J gene segments within each receptor gene locus. Sequence variability is not evenly distributed throughout the V region; three areas of particular variability can be identified in both heavy/band light/a chain, designated hypervariable regions. As the three hypervariable loops constitute the binding site for antigen and determine specificity by forming a surface complementary to the antigen, they are more commonly termed t...

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Follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL) and primary central nervous system lymphoma are B cell malignancies. FL and DLBCL have a germinal center origin. We have applied mutational analyses and a novel algorithm for quantifying shape properties of mutational lineage trees to investigate the nature of the diversification, somatic hypermutation and selection processes that affect B cell clones in these malignancies and reveal whether they differ from normal responses. Lineage tree analysis demonstrated higher diversification and mutations per cell in the lymphoma clones. This was caused solely by the longer diversification times of the malignant clones, as their recent diversification processes were similar to those of normal responses, implying similar mutation frequencies. Since previous analyses of antigen-driven selection were shown to yield false positives, we performed a corrected analysis of replacement and silent mutation patterns, which revealed selection against replacement mutations in the framework regions, responsible for the structural integrity of the B cell receptor, but not for positive selection for replacements in the complementary determining regions. Most replacements, however, were neutral or conservative, suggesting that if at all selection operates in these malignancies it is for structural B cell receptor integrity but not for antigen binding.

The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61-89) versus young (24 ± 5 years old, range 18-45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms. Keywords:Aging r B cells r Bone marrow r Immunoglobulin repertoire r Secondary lymphoid tissues Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe age-related changes in the structure and function of the immune system are usually manifested as increased susceptibility Correspondence: Prof. Ramit Mehr e-mail: ramit.mehr@biu.ac.il to infections (both primary and secondary responses) and cancers, poor responsiveness to new or evolving pathogens, reduced efficacy of vaccination and increased incidence of autoimmune diseases [1][2][3][4][5]. This results in increased disease burdens and health- * These authors contributed equally to this work. * * These authors contributed equally to this work as senior authors.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2016. 46: 480-492 Molecular immunology 481 care costs [3,6,7]. Intrinsic changes in primary and secondary lymphoid tissue function, including hematopoietic stem cells in the bone-marrow (BM), are associated with aging. Literature on immunosenescence has focused mainly on T-lineage impairments, as thymus involution is probably the most well-studied age-related immune dysfunction. In addition, there are age-related defects in the ability of CD4 + and CD8 + T cells to respond to T-cell receptor engagement, a propensity of CD4 + cells to differentiate into Th17 cells at the expense of Th1 and Th2 differentiation and an increase in regulatory T-cell numbers and function [4,8].In the B-cell lineage, changes were observed in the composition of B-cell subtypes in both BM and periphery that may result from increased B-cell longe...

B lymphopoiesis has historically been depicted as a unidirectional process, in which cohorts of developing cells transit through successive differentiative stages in an irreversible, synchronous manner. Here, we examine this view by combining kinetic analysis of developing B cell subsets in the bone marrow with mathematical modeling. Our bromo-deoxyuridine (BrdU) labeling data are incompatible with B cell development being a synchronous process, because labeling curves are non-linear. Moreover, we show that B cell development may not be completely unidirectional, because our results support the possibility of a phenotypic "reflux" among the immature to the pre-B cell subsets.