The B cell repertoire

The hallmark of the immune system is its ability to produce a seemingly infinite variety of antigen-binding receptors. This is made possible by molecular and cellular mechanisms uniquely suited to continuously generate a large number of individual receptor molecules and to select some for further expansion. The well-studied genetic rearrangement that results in the juxtaposition of germ line-encoded variable, diversity, and joining elements remains the foundation for diversification on which the repertoire is built. Many of the rules that regulate this phenomenon have been described, although the underlying enzymatic machinery responsible for these events remains to be elucidated. Recent progress in categorizing the immunoglobulin heavy-chain variable region genes into families as well as studies establishing their utilization in both fetal and adult life is helping to further refine these rules. Subsequent cellular interactions 1) permit the discriminant expansion of clones expressing relevant antibody molecules, 2) allow the active affinity alterations needed for effective ongoing immune responses, and 3) limit the potential deleterious effect of autoreactive cells.     

B cell repertoire diversity to PR8 influenza virus does not decrease with age

The affect of aging on the B cell repertoire was investigated at the clonal level by studying the response of 24- to 26-mo-old BALB/c mice to the PR8 influenza virus (H1N1). By using the splenic fragment culture technique, it was found that the frequency of B cells specific for either the intact virion or the HA does not change with age. The anti-HA monoclonal antibodies obtained from culture were additionally analyzed for fine specificity by binding in RIA to a panel of six H1 variant viruses. This analysis showed a considerable similarity in the distribution and diversity of reactivity patterns between monoclonal antibodies derived from splenic B cells of young vs aged mice. These findings indicate that repertoire expression per se may not be truncated in aged mice, and imply that reductions in the diversity of serum antibodies in aged mice may be due to environmental regulatory mechanisms.     

Heterogeneity of the human phosphocholine-specific B cell repertoire

We have utilized a highly efficient method of culturing small numbers of Epstein Barr virus (EBV)-infected cells to analyze the heterogeneity of human antibodies specific for phosphocholine (PC). Lymphocytes from peripheral blood or tonsils of individuals who had no evidence of recent pneumococcal infection were infected with EBV and cultured at limiting dilution. After correction for the cloning efficiency, between 1/1500 and 1/10,000 B cells produced specific anti-PC antibodies by our criteria. Examination of the heterogeneity of these antibodies revealed that most individuals had an overwhelming predominance of anti-PC antibodies with kappa-light chain. Fine specificity analysis of 39 monoclonal anti-PC antibodies demonstrated that the IgM antibodies examined displayed significant binding site diversity, whereas the IgA PC-specific clones were much less heterogeneous. In general, the human anti-PC antibodies had a much higher relative affinity (Krel) for choline and glycerophosphocholine than the murine antibody families. Through examination of the human PC-specific B cell repertoire we have drawn some interesting parallels with the well-defined murine clonotype families and have begun to dissect the human response to this naturally occurring antigenic determinant.     

Tolerance and B cell repertoire establishment

We have probed the mechanism by which immature B cells are uniquely susceptible to antigen-induced inactivation. Our studies have demonstrated that this tolerance trigger is an active process that requires both energy metabolism and the biosynthesis of various macromolecules, including protein, RNA, and DNA. However, the tolerance trigger is resistant to inhibitors of patching and capping, as well as an inhibitor of mitosis. The tolerance trigger requires a high-affinity interaction between a multivalent antigen and the cells' Ig receptor, but apparently does not require interactions with other cell surface molecules, or interactions with T cells or macrophages. Our efforts to demonstrate the physiological applicability of this tolerance trigger have concentrated on an attempt to demonstrate potentially self-reactive cells within the immature bone marrow population that do not appear in the mature splenic B cell population. To date we have identified prereceptor B cells of several specificities whose frequency is much lower in the spleen and whose elimination appears to involve tolerance rather than antiidiotypic regulation. However, the demonstration that such cells are eliminated by contact with self-antigens has not as yet been accomplished.     

Stochastic niche structure and diversity maintenance in the T cell repertoire

The reliability of the immune response to pathogenic challenge depends critically on the size and diversity of the T cell repertoire. We study naïve T cell repertoire diversity maintenance by a stochastic model that incorporates the concept of competition between T cells for survival stimuli emanating from self-antigen presenting cells (APCs). In the mean field approximation we show that clonotype extinction is certain and compute mean extinction times. We introduce the concept of mean niche overlap and show that clones with a mean niche overlap greater than one have a short repertoire lifespan. This selection differential induces minimal recognition commonality between T cell receptors (TCRs) resulting in a diverse T cell repertoire.     

Clonal analysis of the BALB/c secondary B cell repertoire specific for a self-antigen, cytochrome c

mAb to rat cytochrome c (cyt c), totaling 556, were produced by individual clones of secondary B lymphocytes from nine groups of five BALB/c mice each in vitro using the splenic focus culture system. Inasmuch as rat and mouse cyt c are identical, these B cells can be considered specific for a self-antigen. The mAb were categorized into specificity groups based on their reactivities with a panel of seven cyts c that differ at two to six amino acid residues. The number of distinct specificities for the native protein was restricted to fewer than 20. Different groups of mice expressed the same specificities at comparable frequencies, including a single dominant one, and the total number of secondary cyt c-specific B cells was constant among groups of mice. This suggests that the acquisition of the secondary B cell specificity repertoire for this self-antigen is regulated. However, it is indeed possible that each specificity group may comprise a number of distinct mAb molecules that have arisen stochastically. Specificities expressed by as few as 1% of the total mAb were observed. Thus, it is likely that the identified specificities reflect the secondary B cell specificity repertoire for rat cyt c. The dominant specificity expressed by 50% of the mAb was characterized by elimination of antigen recognition as a result of replacement of aspartic acid by glutamic acid at position 62. Minor specificities expressed by 19% of the mAb were characterized by more subtle affects of an amino acid change at position 62 and/or an amino acid substitution from rat cyt c at position 60. Antibodies in other specificity groups reacted with epitopes in the region of residues 44 and 47. Whereas substitutions at positions 44, 47, 60, and 62 eliminated recognition by most of the mAb, changes at position 92 and at 103 also appeared to affect the binding of some mAb in the region around residues 60 and 62. The amino acid residues implicated in the recognition by murine mAb of murine cyt c have been shown previously to be involved in the epitopes of foreign mammalian cyt c. Therefore, self-tolerance cannot fully explain the restriction of the epitopes to these regions on foreign mammalian cyt c.     

The many important facets of T-cell repertoire diversity

In the thymus, a diverse and polymorphic T-cell repertoire is generated by random recombination of discrete T-cell receptor (TCR)-alphabeta gene segments. This repertoire is then shaped by intrathymic selection events to generate a peripheral T-cell pool of self-MHC restricted, non-autoaggressive T cells. It has long been postulated that some optimal level of TCR diversity allows efficient protection against pathogens. This article focuses on several recent advances that address the required diversity for the generation of an optimal immune response.     

The naive B cell repertoire predisposes to antigen-induced systemic lupus erythematosus

It is clear that the development of an autoimmune disease usually depends on both a genetic predisposition and an environmental trigger. In this study, we demonstrate that BALB/c mice develop a lupus-like serology following immunization with a peptide mimetope of DNA, while DBA/2 mice do not. We further demonstrate that the critical difference resides within the B cell compartment and that the naive B cell repertoire of DBA/2 mice has fewer B cells specific for the DNA mimetope. Differences in the strength of B cell receptor signaling exist between these two strains and may be responsible for the difference in disease susceptibility. BALB/c mice possess more autoreactive cells in the native repertoire; they display a weaker response to Ag and exhibit less Ag-induced apoptosis of B cells. DBA/2 mice, in contrast, display a stronger B cell receptor signal and more stringent central tolerance. This correlates with resistance to lupus induction. Thus, the degree to which autoreactive B cells have been eliminated from the naive B cell repertoire is genetically regulated and may determine whether a nonspontaneously autoimmune host will develop autoimmunity following exposure to Ag.     

Quantitative clonal analysis of the B cell repertoire in human lupus

To gain further insight into the origin of autoantibody hyperproduction in human lupus, we quantitated the B cell repertoire toward exogenous and self-antigens. Using the Spot-ELISA method and two panels of nine exogenous and 10 self-antigens, we found that the normal human immune repertoire comprises a high frequency of B cell precursors secreting IgM antibodies to self- and exogenous determinants. This repertoire was markedly deficient in precursors producing IgG able to bind self-antigens. In lupus patients, the absolute numbers of clone precursors of the immune repertoire expressing IgM receptors whose paratopes impart affinity to self- and exogenous determinants were higher than in control individuals. Additionally, IgG antibody-forming cell precursors with binding specificity for lupus-associated antigens were detectable in the repertoire of these patients. Based on these results, we propose that hyperproduction of human lupus-associated autoantibodies arises in a two-stage mechanism whereby a general activation of the multireactive immune B cell repertoire precedes an oligospecific expansion of selected B cell clonotypes.     

Reconstitution of the adult B cell repertoire after treatment with rituximab

B cells play diverse and fundamental roles in the pathogenesis of autoimmune diseases. Consequently, therapeutic targeting of B cells is gaining prominence in our clinical armamentarium for an ever expanding array of autoimmune and neoplastic disorders. Therefore, it is of great importance to understand the mechanism of action of B cell depletion. Given that the ideal consequence of B cell depletion would be the subsequent re-establishment of immunologic tolerance, a detailed analysis of the properties of the emerging repertoire will be required. The results presented by Rouzière and coworkers in their study of rheumatoid arthritis patients shed some light on this question and are discussed in this commentary.     

Aging-dependent exclusion of antigen-inexperienced cells from the peripheral B cell repertoire

Aging is accompanied by greatly reduced B cell production in the bone marrow, yet peripheral B cell numbers do not decline. We hypothesize that this may reflect filling of the peripheral pool with B cells that are long-lived as a consequence of specificity for, and chronic stimulation by, environmental Ags. To begin to explore this possibility, we analyzed the effects of aging on B cell population dynamics in the anti-H2(k/b) 3-83 mu-delta Ig-transgenic mouse. We predicted that, because they presumably do not bind environmental Ags, B cells bearing the transgenic receptor may be lost in aged animals. As seen in nontransgenic animals, total splenic B cell numbers remained constant with age in the Ig-transgenic animals despite reduced B cell production. Importantly, although the few newly produced B cells in the bone marrow of aged mice are 3-83 positive, the peripheral compartment of these mice is dominated by B cells that express endogenous Ig genes rather than the transgenes. This population includes large numbers of marginal zone-like and CD21(low/-)CD23(low/-)IgM(low) B cells, as well as elevated numbers of CD5+ B cells. Many of these cells express only non-B220 CD45 isoforms, suggesting that they may be memory cells. A significant proportion of aged transgenic animals produce autoantibodies that are reactive with ssDNA, dsDNA, or histones. Results support the hypothesis that, in the face of severely reduced production with age, B cells are selected based on reactivity to environmental Ags, accumulate, and display activated phenotypes. Cells bearing 3-83-transgenic receptors are excluded from this population due to their specificity. Beyond their importance in aging, these findings define a novel form of receptor revision in which B cells are selected rather than deleted based on Ag reactivity.     

Dissecting the anti-desmoglein autoreactive B cell repertoire in pemphigus vulgaris patients

Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. V(H) gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two V(H) genes. V(L) gene segment use was diverse even among IgG hybridomas suggesting that the V(L) is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in V(H) use, is somatically mutated, and is Ag selected.     

The molecular repertoire of the 'almighty' stem cell

Stem cells share the defining characteristics of self-renewal, which maintains or expands the stem-cell pool, and multi-lineage differentiation, which generates and regenerates tissues. Stem-cell self-renewal and differentiation are influenced by the convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the surrounding stem-cell niche, but the specific signals involved are poorly understood. Recently, several studies have sought to identify the genetic mechanisms that underlie the stem-cell phenotype. Such a molecular road map of stem-cell function should lead to an understanding of the true potential of stem cells.     

Preferential autoantibody reactivity of the preimmune B cell repertoire in normal mice

Naturally occurring autoantibodies are frequently found in the sera of healthy individuals. They usually exhibit low binding affinities for autoantigens and often react with multiple antigenic determinants. To determine whether their frequencies have been overestimated by sensitive testing procedures, the germ-line B cell repertoire of strain A mice was examined for reactivity with a panel of auto- and foreign Ag. If the high frequencies of autoantibodies result from testing procedures, equally high frequencies would be expected for foreign Ag specificities detected in the same manner. The presence of specific autoantibodies was confirmed in this study by the disparate frequencies observed for antibodies reactive with individual Ag. The frequencies were highest for autoantigens associated with SLE, indicating a bias toward autoreactivity in the preimmune repertoire. Analysis of VH gene usage did not indicate any selection in V gene expression with autoreactivity.     

Impaired receptor editing in the primary B cell repertoire of BASH-deficient mice

The editing of B cell Ag receptor (BCR) through successive rearrangements of Ig genes has been considered to be a major mechanism for the central B cell tolerance, which precludes appearance of self-reactive B cells, through studies using anti-self-Ig transgenic/knock-in mouse systems. However, contribution of the receptor editing in the development of the normal B cell repertoire remains unclear. In addition, the signaling pathway directing this event is unknown. In this study, we demonstrate that receptor editing in anti-DNA Ig knock-in mice is impaired in the absence of an adaptor protein BASH (BLNK/SLP-65) that is involved in BCR signaling. Remarkably, the supposed hallmarks of receptor editing such as Iglambda chain expression, recombination sequence rearrangements at Igkappa loci, and presence of in-frame VkappaJkappa joins in the Igkappa loci inactivated by the recombination sequence rearrangements, were all diminished in BASH-deficient mice with unmanipulated Ig loci. BCR ligation-induced Iglambda gene recombination in vitro was also impaired in BASH-deficient B cells. Furthermore, the BASH-deficient mice showed an excessive Ab response to a DNA carrier immunization, suggesting the presence of unedited DNA-reactive B cells in the periphery. These results not only define a signaling pathway required for receptor editing but indicate that the BCR-signaled receptor editing indeed operates in the development of normal B cell repertoire and contributes to establishing the B cell tolerance.     

Development of the autoimmune B cell repertoire in MRL-lpr/lpr mice

The processes responsible for the production of autoantibodies have been shown to include both Ag-specific and generalized (polyclonal) forms of B cell activation. The relative contribution and temporal association of these processes to the genesis of systemic autoimmunity are incompletely understood. To study this relationship, the B cell repertoires of MRL-lpr/lpr mice were analyzed by ELISA spot assay over an 8-mo period. Between 6 and 12 wk of age, the number of splenic lymphocytes producing antibodies reactive with both autoantigens and conventional Ag increased proportionately. The repertoires of MRL-lpr/lpr mice under 12 wk were dominated by IgM-secreting B cells that showed no bias toward the production of specific autoantibodies. From 12 to 38 wk of age, an increasing proportion of animals developed repertoires dominated by IgG-secreting B cells that were skewed toward reactivity against one or very few (auto)antigens. Although there was no single Ag against which all mice developed skewed reactivity, 55% of MRL-lpr/lpr adults had increased numbers of B cells producing antibodies to the Sm Ag and 13 to 16% developed increased reactivity toward DNA, myosin, histone, thyroglobulin, or T cells. These data indicate that generalized (polyclonal) B cell activation dominates early repertoire development whereas (auto)-antigen-specific responses become increasingly important during the latter stages of disease in these autoimmune-prone mice.     

The murine B cell repertoire is severely selected against endogenous cellular prion protein

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnp o/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnp o/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnp o/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnp o/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnp o/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.     

Inferring processes underlying B-cell repertoire diversity

We quantify the VDJ recombination and somatic hypermutation processes in human B cells using probabilistic inference methods on high-throughput DNA sequence repertoires of human B-cell receptor heavy chains. Our analysis captures the statistical properties of the naive repertoire, first after its initial generation via VDJ recombination and then after selection for functionality. We also infer statistical properties of the somatic hypermutation machinery (exclusive of subsequent effects of selection). Our main results are the following: the B-cell repertoire is substantially more diverse than T-cell repertoires, owing to longer junctional insertions; sequences that pass initial selection are distinguished by having a higher probability of being generated in a VDJ recombination event; somatic hypermutations have a non-uniform distribution along the V gene that is well explained by an independent site model for the sequence context around the hypermutation site.