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.     

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.     

Safety of rituximab in the treatment of B cell malignancies: implications for rheumatoid arthritis

The chimeric anti-CD20 monoclonal antibody rituximab has been used extensively in the treatment of B cell malignancies, and more recently it has emerged as a potential treatment for rheumatoid arthritis (RA), via selective B lymphocyte depletion. Experience in oncology shows that rituximab is well tolerated in a variety of settings, with mild-to-moderate infusion related reactions following the first infusion being the most common adverse event. Current data suggest that the safety profile of rituximab in patients with RA is similar to that in oncology, but that the adverse events are less frequent and less severe in patients with RA.     

B cell non-Hodgkin's lymphoma: rituximab safety experience

A substantial body of data supports use of rituximab as first-line and maintenance therapy for the treatment of indolent non-Hodgkin's lymphoma. With 7 years of postmarketing surveillance experience and more than 370,000 patient exposures, the safety profile of rituximab is well defined. Several multicenter trials suggest that infusion reactions associated with rituximab administration are well characterized and generally associated with the first infusion; toxicity is reduced with subsequent doses. Since some adverse events are related to circulating tumor loads of non-Hodgkin's lymphoma, fewer events are anticipated in rheumatoid arthritis. Low infection rates in oncology would indicate similar safety in patients with rheumatoid arthritis.     

The diversity of the secondary Salmonella typhimurium-specific B cell repertoire

This report describes the first analysis of the expressed B cell repertoire specific for a bacterium. In this study, responses to an acetone-killed and dried preparation of Salmonella typhimurium strain TML (AKD-TML) are described. The results show that AKD-TML can stimulate splenic B cells from primed CBA/Ca mice over a wide dose range. The average frequency of secondary TML-specific B cells is 16.4 per 10(5) splenic B cells. This frequency is similar to that observed for another complex, natural antigen, the hemagglutinin of influenza virus. The majority of all secondary TML-specific B cells (greater than 70%) secrete immunoglobulin M, but most of these clones also secrete other isotypes of which immunoglobulins G2 and A are the most prevalent. Analysis of the specificity of secondary TML-specific B cells showed that the vast majority of these B cells were specific for the lipopolysaccharide (LPS) molecule. Moreover, fine specificity analysis demonstrated that approximately two-thirds of these anti-LPS-specific B cell clones are directed against the core polysaccharides or lipid A regions of the LPS molecule, while only about one-third are directed toward the O antigen region. Since anti-S. typhimurium serum antibodies are directed primarily against the O antigens, these studies suggest that the serum levels of antibodies to a given epitope on a bacterial antigen may not be a true reflection of the expressed B cell repertoire when analyzed at the single B cell level. These studies also suggest that the role of antibodies to lipid A molecules in the development of protective immunity to S. typhimurium be reevaluated.     

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.     

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.     

Intrathymic T cell repertoire after prenatal trinitrobenzene-sulfonic acid-treatment

It is still a matter of debate, whether tolerance toward self-non-MHC antigens is due to intrathymic deletion or to regulatory processes in the periphery. To further pursue this question, responsiveness toward TNP and an anti-TNP monoclonal antibody (Sp6) carrying a recurrent idiotype was evaluated in prenatally trinitrobenzenesulfonic acid (TNBS)-treated mice. In prenatally untreated as well as in TNBS-treated mice, thymocytes proliferating in the absence of nominal antigen were double negative (L3T4-/Lyt2-), but antigen-specific thymocytes were single positive (L3T4+/Lyt2- or L3T4-/Lyt2+). TNBS-treated mice differed from controls inasmuch as in their first week of life T cells proliferating in response to TNP were found in the thymus and detected at increased frequencies in the spleen. The frequency of TNP-specific thymocytes and spleen cells declined rapidly, finally reaching in the spleen a level of 20-30% of controls. Furthermore, after antigenic stimulation, the frequency of thymocytes and spleen cells proliferating in response to TNP was found to be increased in control mice, but TNP-specific T cell were no more recovered in the thymus or the spleen of tolerized mice. The same accounted for thymic and splenic T cells proliferating in response to Sp6. They were expanded in control mice after antigenic stimulation, but were undetectable in TNBS-treated mice. Thus, T cells with specificity for an internal (Sp6) and an external (TNP) antigen, provided the latter was present during ontogeny, were detected in the thymus of control and, transiently, in the thymus of tolerized mice. But, the fate of antigen-specific thymocytes was different in prenatally untreated and TNBS-treated mice. The data are interpreted in the sense that tolerance toward non-MHC antigens may be acquired subsequently to tolerance toward self-MHC antigens and possibly after imprinting of antigen specificity.     

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.     

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.     

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.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

Ig repertoire of human polyspecific antibodies and B cell ontogeny.

A total of 463 EBV Ig-secreting clones were derived from embryonic tissues, cord blood, and adult peripheral blood. Subcloning and analysis of the H and K loci (germline vs rearranged DNA status) of 44 primary clones insured clonality in at least 92% of cases. Whatever the cell origin, a somewhat constant proportion of clones (i.e., 11 to 16%) expressed polyspecific antibodies when tested on a panel of nine Ag, including self-Ag. The VH and VK repertoires have been studied using VH1-VH6 and VK1-VK4 family-specific probes. For all EBV clones the VH and VK utilization was similar to that of the normal untransformed population. A correlation was observed between the level of expression and the gene number for VH, whereas a clear distortion appeared for VK. Moreover, the usage pattern of VH and VK families of the polyspecific clones did not significantly differ from that of clones of unknown specificity, suggesting that polyspecificity was not linked to a restricted repertoire.     

Cross-reactive idiotype family observed in the phthalate-specific B cell repertoire of adult BALB/c mice: diversity of IgM compared with IgG monoclonal anti-phthalate antibodies

A highly conserved clonotype has been identified within the repertoire of B cells specific for the negatively charged hapten phthalate. The prototype of this phthalate-specific clonotype is a primary-response hybridoma (2E9) that produces a mu,kappa anti-phthalate antibody. The 2E9 monoclonal antibody was found to share idiotypic determinants with several other independently-derived mu,kappa and gamma 1,kappa anti-phthalate monoclonal antibodies and with a significant proportion of conventional anti-phthalate antibodies derived from all of the BALB/c mice immunized with phthalate-keyhole limpet hemocyanin. Competitive RIA analysis of the 2E9 idiotypic relatedness between primary and secondary response antibodies was consistent with the hypothesis that the primary response mu,kappa antibodies represent a conserved germ-line product, whereas the secondary response to gamma 1,kappa antibodies reflect somatic variants of the 2E9 clonotype. Further analysis with a site-specific anti-idiotype reagent suggests that the idiotypic differences between mu,kappa and gamma 1,kappa monoclonal antibodies occur at positions outside of the combining site. Fine specificity analysis of the monoclonal antibodies expressing the 2E9 cross-reactive idiotype (CRI) also supports this hypothesis. Seven to 35% of the anti-phthalate antibodies after a single immunization with phthalate-KLH and 1 to 10% of the antibodies after a second immunization express the 2E9 CRI. The 2E9 CRI was also found in several other strains of mice, and its expression was associated exclusively with anti-phthalate antibodies.     

T cell repertoire development in humans with SCID after nonablative allogeneic marrow transplantation

Transplantation of HLA-identical or haploidentical T cell-depleted allogeneic bone marrow (BM) into SCID infants results in thymus-dependent T cell development in the recipients. Immunoscope analysis of the TCR V beta repertoire was performed on 15 SCID patients given BM transplants. Before and within the first 100 days after bone marrow transplantation (BMT), patients' PBMC displayed an oligoclonal or skewed T cell repertoire, low TCR excision circles (TREC) values, and a predominance of CD45RO(+) T cells. In contrast, the presence of high numbers of CD45RA(+) cells in the circulation of SCID patients >100 days post-BMT correlated with active T cell output by the thymus as revealed by high TREC values and a polyclonal T cell repertoire demonstrated by a Gaussian distribution of V beta-specific peaks. Ten years after BMT, we observed a decrease of the normal polyclonal T cell repertoire and an increase of a more skewed T cell repertoire. A decline of TREC levels and a decrease in the number of CD45RA(+) cells beyond 10 years after BMT was concomitant with the detection of oligoclonal CD3(+)CD8(+)CD45RO(+) cells. The switch from a polyclonal to a more skewed repertoire, observed in the CD3(+)CD8(+)CD45RO(+) T cell subset, is a phenomenon that occurs normally with decreased thymic output during aging, but not as rapidly as in this patient population. We conclude that a normal T cell repertoire develops in SCID patients as a result of thymic output and the repertoire remains highly diverse for the first 10 years after BMT. The TCR diversity positively correlates in these patients with TREC levels.