Colocalization of expansion of the splenic marginal zone population with abnormal B cell activation and autoantibody production in B6 mice with an introgressed New Zealand Black chromosome 13 interval

Polyclonal B cell activation is a prominent feature of the lupus-prone New Zealand Black (NZB) mouse strain. We have previously demonstrated linkage between a region on NZB chromosome 13 and increased costimulatory molecule expression on B cells. In this study we have produced C57BL/6 congenic mice with an introgressed homozygous NZB interval extending from approximately 24 to 73 cM on chromosome 13 (denoted B6.NZBc13). We show that B6.NZBc13 female mice not only have enhanced B cell activation but also share many other B cell phenotypic characteristics with NZB mice, including expansion of marginal zone and CD5+ B cell populations, increased numbers of IgM ELISPOTs, and increased serum levels of total IgM and IgM autoantibodies. In addition these mice have increased T cell activation, increased numbers of germinal centers, mild glomerulonephritis, and produce high-titer IgM and IgG anti-chromatin Abs. Male B6.NZBc13 mice have a less pronounced cellular phenotype, lacking expansion of the marginal zone B cell population and IgG anti-chromatin Ab production, indicating the presence of gender dimorphism for this locus. Thus, we have identified a genetic locus that recapitulates with fidelity the B cell phenotypic abnormalities in NZB mice, and we demonstrate that this locus is sufficient to induce an autoimmune phenotype. The data provide further support to the contention that immune abnormalities leading to altered B cell activation and selection contribute to the development of autoimmunity in NZB mice.     

Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naive and Ag experienced B cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. In this study, using PCR to analyze the Ab response to phenyl-oxazolone in the mouse, we show that the Ab repertoire of CD21(hi)/CD23(-) marginal zone B cells shows persistent increase in levels of particular IgM after immunization with foreign Ag. Further, we show that these IgMs have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analyzed. We also detect an effect of Ag on the follicular B cell repertoire that is less persisting. These results provide evidence consistent with the signal-strength model of mature B cell development being extended to include stimulation by foreign Ag, and also further the known zone of influence of foreign Ag on the B cell compartment.     

The Yaa mutation promoting murine lupus causes defective development of marginal zone B cells

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.     

Split tolerance in peripheral B cell subsets in mice expressing a low level of Igkappa-reactive ligand

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.     

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.     

Impaired antibody response to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient mice

Group B Streptococcus (GBS) is the foremost bacterial cause of serious neonatal infections. Protective immunity to GBS is mediated by specific Abs to the organism's capsular polysaccharide Ags. To examine the role of complement in the humoral immune response to type III GBS capsular polysaccharide (III-PS), mice deficient in C3 or in CD21/CD35 (i.e., complement receptors 1 and 2; CR1/CR2) were immunized with III-PS. Mice deficient in C3 or Cr2 had an impaired primary immune response to III-PS. The defective response was characterized by low IgM levels and the lack of an isotype switch from IgM to IgG Ab production. Compared with wild-type mice, C3- and Cr2-deficient mice exhibited decreased uptake of III-PS by follicular dendritic cells within the germinal centers and impaired localization of III-PS to the marginal zone B cells. Complement-dependent uptake of capsular polysaccharide by marginal zone B cells appears necessary for an effective immune response to III-PS. The normal immune response in wild-type mice may require localization of polysaccharide to marginal zone B cells with subsequent transfer of the Ag to follicular dendritic cells.     

Natural IgM is required for suppression of inflammatory arthritis by apoptotic cells

The clearance of dying cells is vital for re-establishing tolerance during inflammation and has potent immunoregulatory consequences. Because natural IgM plays a key role in the removal of apoptotic cells, we investigated whether the immune modulatory properties of apoptotic cells depended on its presence. Using an Ab-independent, Ag-induced model of inflammatory arthritis, we tested whether natural IgM is essential for the arthritis-suppressing properties of apoptotic cells. Whereas administration of apoptotic cells reduced joint inflammation and damage in normal mice accompanied by suppression of the Th17 response, no protection was afforded in secreted IgM-deficient (Sμ(-)) mice. The enhanced production of IL-10 by T cells from draining lymph nodes and splenic marginal zone B cells, driven by the infusion of apoptotic cells, was abrogated in the absence of natural IgM. Apoptotic cells were present shortly after administration in the splenic marginal zone, but their removal was substantially delayed in the absence of natural IgM. Incubation of apoptotic cells with natural IgM in vitro restored their arthritis-suppressing properties in Sμ(-) mice. Moreover, these IgM-coated apoptotic cells were cleared rapidly after injection from the spleens of Sμ(-) mice. Our results demonstrate that natural IgM is a critical factor in a chain of events triggered by the administration of apoptotic cells that promote IL-10-secreting B and T cells and restrain the development of inflammation.     

Cutting Edge: Hormonal milieu, not antigenic specificity, determines the mature phenotype of autoreactive B cells

Although both marginal zone and follicular B cells produce anti-DNA Abs in murine models of systemic lupus erythematosus, it has been unclear whether these distinct B cell subsets make identical or different Abs. Single-cell analysis demonstrates that the same DNA-reactive B cells can mature to either subset, depending on the hormonal environment. Anti-DNA B cells in estradiol-treated mice become marginal zone cells while identical cells from prolactin-treated mice become follicular cells. The B cell receptor signaling pathway is influenced by hormonal milieu. Thus, hormonal milieu and perhaps B cell receptor signaling, but not antigenic specificity, correlates with the differentiation pathway. These observations have implications for the pathogenesis and treatment of autoimmune disease.     

Recirculating and marginal zone B cell populations can be established and maintained independently of primary and secondary follicles

In normal spleen, most recirculating na?ve IgM+IgDhi B cells are located within primary follicles and mantle zones of secondary follicles. By contrast, the marginal zone contains a heterogeneous population of IgMhiIgDlo/- B cells that are mostly non-recirculating. Although these are dynamic populations they are maintained at a constant size, the fundamental homeostatic mechanisms remain uncertain. One possibility is that the presence and turnover of each of the B cell populations is dependent on their location within discrete splenic compartments. To investigate this, we have characterized immature, non-recirculating, mature recirculating, marginal zone and B-1 cell populations in TNF-/- and TNF/lymphotoxin(LT)-alpha-/- mice that have disorganized splenic architecture. Labelling with 5-bromo-2'-deoxyuridine revealed that turnover of B cells in TNF-/- mice is normal, but is diminished in TNF/LT-alpha-/- mice. The recirculating B cell populations in both mutant strains are normal in proportion and phenotype. Marginal zone B cells are not seen in TNF/LT-alpha-/- mice, but this population appears normal in TNF-/- mice, even though they lack germinal centres. These findings indicate that peripheral B cell subsets can be established and maintained independently of normal follicular architecture.     

Cutting edge: expansion and activation of a population of autoreactive marginal zone B cells in a model of estrogen-induced lupus

We have demonstrated previously that 17 beta-estradiol (E2) treatment of BALB/c mice transgenic for the heavy chain of a pathogenic anti-DNA Ab induces a lupus-like phenotype with expansion of anti-DNA B cells, elevation of anti-DNA Ab titers, and glomerular immunoglobulin deposition. To understand this loss of B cell tolerance, the effects of E2 on B cell development and activation were examined. A sustained increase in E2 resulted in an altered distribution of B cell subsets, with a diminished transitional population and an increase in marginal zone B cells. Depletion of CD4+ T cells did not abrogate these effects. Furthermore, the B cells that spontaneously secreted anti-DNA Abs displayed a marginal zone phenotype. Thus, a sustained increase in E2 alters B cell development, leading to the survival, expansion, and activation of a population of autoreactive marginal zone B cells implicating this B cell subset in autoimmunity.     

Crucial Role for BAFF-BAFF-R Signaling in the Survival and Maintenance of Mature B Cells

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.     

IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells.

Ag encounter will recruit Ag-specific cells from the pool of mature B lymphocytes in the spleen and activate them to perform effector functions: generation of Ab-forming cells (plasma cells) and presentation of Ag to T cells. We have compared the ability of mature follicular and marginal zone cells to develop into effector B cells. The generation of marginal zone B cells and their localization in the marginal sinus area are T cell and CD40 ligand independent, suggesting that they do not represent a postgerminal center population. Compared with mature recirculating follicular B cells, they express several characteristics of previous antigenic experience, including higher levels of B7.1 (CD80) and B7.2 (CD86) when freshly isolated and following in vitro stimulation. After a brief 6- to 8-h in vitro stimulation with LPS or anti-CD40 Abs, marginal zone B cells become potent APCs. In addition, their ability to proliferate and differentiate into plasma cells in response to low doses of T-independent polyclonal stimuli (LPS) is far greater than that of follicular B cells. These findings indicate a functional heterogeneity within splenic mature B lymphocytes, with marginal zone B cells having the capacity to generate effector cells in early stages of the immune response against particulate Ags scavenged efficiently in this special anatomical site.     

TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses

Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-alphabeta receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the sphingosine-1-phosphate (S1P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.     

Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the V(H)1-69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)CD95(-)). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated V(H)1-69(+) B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal V(H)1-69(+) B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.     

Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease

Invasive pneumococcal infection is an important cause of morbidity and mortality in HIV-1-infected individuals. B cells play an important role in maintaining serologic memory after infection. IgM memory B cells are significantly reduced in HIV-1-infected patients and their frequency is similar to that observed in other patient groups (splenectomized individuals and patients with primary Ab deficiency) who are also known to have an increased risk of invasive pneumococcal infection. Antiretroviral therapy does not restore marginal zone B cell percentages. Immunization with the 23-valent polysaccharide pneumococcal vaccine shows that HIV-1-infected patients have impaired total IgM and IgG pneumococcal vaccines compared with healthy controls. Loss of switched memory B cells was associated with impaired tetanus toxoid IgG vaccine responses. Results of this study demonstrate that defects in B cell memory subsets are associated with impaired humoral immune responses in HIV-1 patients who are receiving antiretroviral therapy and may be a contributory factor to the increased risk of invasive pneumococcal infection observed in HIV-1 infection.     

The resolution of relapsing fever borreliosis requires IgM and is concurrent with expansion of B1b lymphocytes

The rate of pathogen clearance is a critical determinant of morbidity and mortality. We sought to characterize the immune response responsible for the remarkably rapid clearance of individual episodes of bacteremia caused by the relapsing fever bacterium, Borrelia hermsii. SCID or Rag(-/-) mice were incapable of resolving B. hermsii infection, indicating a critical role for T and/or B cells. TCR(-/-) mice, which lack T cells, and IL-7(-/-) mice, which are deficient in both T cells and follicular B cells, but not in B1 cells and splenic marginal zone (MZ) B cells, efficiently cleared B. hermsii. These findings suggested that B1 cells and/or MZ B cells, two B cell subsets that are known to participate in rapid, T-independent responses, might be involved. The efficient resolution of the episodes of moderate level bacteremia by splenectomized mice suggested that MZ B cells do not play the primary role in clearance of this bacterium. In contrast, xid mice, which are deficient in B1 cells, suffered more severe episodes of bacteremia than wild-type mice. The hypothesis that B1 cells are critical for clearance of B. hermsii was further supported by a selective expansion of the B1b (i.e., IgM(high), IgD(-/low), Mac1(+) CD23(-), and CD5(-)) cell subset in infected xid mice, which coincided with the eventual resolution of infection. Finally, mice selectively incapable of secreting IgM, the dominant isotype produced by B1 cells, were completely unable to clear B. hermsii. Together these results support the model that B1b cells generate the T-independent IgM required for the control and resolution of relapsing fever borreliosis.     

Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.     

Complement Receptors 1 and 2 in Murine Antibody Responses to IgM-Complexed and Uncomplexed Sheep Erythrocytes

Early complement components are important for normal antibody responses. In this process, complement receptors 1 and 2 (CR1/2), expressed on B cells and follicular dendritic cells (FDCs) in mice, play a central role. Complement-activating IgM administered with the antigen it is specific for, enhances the antibody response to this antigen. Here, bone marrow chimeras between Cr2(-/-) and wildtype mice were used to analyze whether FDCs or B cells must express CR1/2 for antibody responses to sheep erythrocytes (SRBC), either administered alone or together with specific IgM. For robust IgG anti-SRBC responses, CR1/2 must be expressed on FDCs. Occasionally, weak antibody responses were seen when only B cells expressed CR1/2, probably reflecting extrafollicular antibody production enabled by co-crosslinking of CR2/CD19/CD81 and the BCR. When SRBC alone was administered to mice with CR1/2(+) FDCs, B cells from wildtype and Cr2(-/-) mice produced equal amounts of antibodies. Most likely antigen is then deposited on FDCs in a way that optimizes engagement of the B cell receptor, making CR2-facilitated signaling to the B cell superfluous. SRBC bound to IgM will have more C3 fragments, the ligands for CR1/2, on their surface than SRBC administered alone. Specific IgM, forming a complex with SRBC, enhances antibody responses in two ways when FDCs express CR1/2. One is dependent on CR1/2(+) B cells and probably acts via increased transport of IgM-SRBC-complement complexes bound to CR1/2 on marginal zone B cells. The other is independent on CR1/2(+) B cells and the likely mechanism is that IgM-SRBC-complement complexes bind better to FDCs than SRBC administered alone. These observations suggest that the immune system uses three different CR1/2-mediated effector functions to generate optimal antibody responses: capture by FDCs (playing a dominant role), transport by marginal zone B cells and enhanced B cell signaling.     

CD36 is differentially expressed on B cell subsets during development and in responses to antigen

Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.     

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4(+) T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-x(L) or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc(-/-) mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.