Ziaur S. M. Rahman

Normal Induction but Attenuated Progression of Germinal Center Responses in BAFF and BAFF-R Signaling–Deficient Mice

The factors regulating germinal center (GC) B cell fate are poorly understood. Recent studies have defined a crucial role for the B cell–activating factor belonging to TNF family (BAFF; also called BLyS) in promoting primary B cell survival and development. A role for this cytokine in antigen-driven B cell responses has been suggested but current data in this regard are limited. A BAFF receptor expressed by B cells (BAFF-R/BR3) is defective in A/WySnJ mice which exhibit a phenotype similar to BAFF-deficient (BAFF−/−) animals. Here, we show that although GC responses can be efficiently induced in both A/WySnJ and BAFF−/− mice, these responses are not sustained. In BAFF−/− mice, this response is rapidly attenuated and accompanied by perturbed follicular dendritic cell development and immune complex trapping. In contrast, analysis of the A/WySnJ GC response revealed a B cell autonomous proliferative defect associated with reduced or undetectable Ki67 nuclear proliferation antigen expression by GC B cells at all stages of the response. These data demonstrate a multifaceted role for the BAFF pathway in regulating GC progression.

The Fas-Associated Death Domain Protein Is Required in Apoptosis and TLR-Induced Proliferative Responses in B Cells

The Fas-associated death domain protein (FADD)/Mort1 is a signaling adaptor protein which mediates the activation of caspase 8 during death receptor-induced apoptosis. Disruption of FADD in germ cells results in death receptor-independent embryonic lethality in mice. Previous studies indicated that in addition to its function in apoptosis, FADD is also required in peripheral T cell homeostasis and TCR-induced proliferative responses. In this report, we generated B cell-specific FADD-deficient mice and showed that deletion of FADD at the pro-B cell stage had minor effects on B cell development in the bone marrow, and resulted in increased splenic and lymph node B cell numbers and decreased peritoneal B1 cell numbers. As in T cells, a FADD deficiency inhibited Fas-induced apoptosis in B cells. However, B cell-proliferative responses induced by stimulation of the BCR and CD40 using anti-IgM or anti-CD40 Abs were unaffected by the absence of FADD. Further analyses revealed that FADD-deficient B cells were defective in proliferative responses induced by treatments with dsRNA and LPS which stimulate TLR3 and TLR4, respectively. Therefore, in addition to its apoptotic function, FADD also plays a role in TLR3- and TLR4-induced proliferative responses in B cells.

B Cell–Intrinsic TLR7 Signaling Is Essential for the Development of Spontaneous Germinal Centers

Spontaneous germinal center (Spt-GC) B cells and follicular helper T cells generate high-affinity autoantibodies that are involved in the development of systemic lupus erythematosus. TLRs play a pivotal role in systemic lupus erythematosus pathogenesis. Although previous studies focused on the B cell–intrinsic role of TLR-MyD88 signaling on immune activation, autoantibody repertoire, and systemic inflammation, the mechanisms by which TLRs control the formation of Spt-GCs remain unclear. Using nonautoimmune C57BL/6 (B6) mice deficient in MyD88, TLR2, TLR3, TLR4, TLR7, or TLR9, we identified B cell–intrinsic TLR7 signaling as a prerequisite to Spt-GC formation without the confounding effects of autoimmune susceptibility genes and the overexpression of TLRs. TLR7 deficiency also rendered autoimmune B6.Sle1b mice unable to form Spt-GCs, leading to markedly decreased autoantibodies. Conversely, B6.yaa and B6.Sle1b.yaa mice expressing an extra copy of TLR7 and B6.Sle1b mice treated with a TLR7 agonist had increased Spt-GCs and follicular helper T cells. Further, TLR7/MyD88 deficiency led to compromised B cell proliferation and survival after B cell stimulation both in vitro and in vivo. In contrast, TLR9 inhibited Spt-GC development. Our findings demonstrate an absolute requirement for TLR7 and a negative regulatory function for TLR9 in Spt-GC formation under nonautoimmune and autoimmune conditions. Our data suggest that, under nonautoimmune conditions, Spt-GCs initiated by TLR7 produce protective Abs. However, in the presence of autoimmune susceptibility genes, TLR7-dependent Spt-GCs produce pathogenic autoantibodies. Thus, a single copy of TLR7 in B cells is the minimal requirement for breaking the GC-tolerance checkpoint.

Impaired Apoptotic Cell Clearance in the Germinal Center by Mer-Deficient Tingible Body Macrophages Leads to Enhanced Antibody-Forming Cell and Germinal Center Responses

Germinal centers (GCs) are specialized microenvironments that generate high-affinity Ab-forming cells (AFCs) and memory B cells. Many B cells undergo apoptosis during B cell clonal selection in GCs. Although the factors that regulate the AFC and GC responses are not precisely understood, it is widely believed that dysregulated AFCs and GCs contribute to autoimmunity. The Mer receptor tyrosine kinase (Mer) facilitates macrophage clearance of apoptotic cells. The Tyro-3, Axl, and Mer receptors, including Mer, suppress TLRs and cytokine-mediated inflammatory responses. We report in this study that tingible body macrophages (TBMϕs) in GCs express Mer. Compared to C57BL/6 (B6) controls, Mer-deficient (Mer−/−) mice had significantly higher AFC, GC, and Th1-skewed IgG2 Ab (especially IgG2c) responses against the T cell-dependent Ag (4-hydroxy-3-nitrophenyl) acetyl-chicken γ globulin. Mer−/− mice had a significantly higher percentage of GC B cells on days 9, 14, and 21 postimmunization compared with B6 controls. Significantly increased numbers of apoptotic cells accumulated in Mer−/− GCs than in B6 GCs, whereas the number of TBMϕs remained similar in both strains. Our data are the first, to our knowledge, to demonstrate a critical role for Mer in GC apoptotic cell clearance by TBMϕs and have interesting implications for Mer in the regulation of B cell tolerance operative in the AFC and GC pathways.

B Cells Expressing Bcl-2 and a Signaling-Impaired BAFF-Specific Receptor Fail to Mature and Are Deficient in the Formation of Lymphoid Follicles and Germinal Centers

The TNF family cytokine B cell-activating factor belonging to the TNF family (BAFF) (BLyS) plays a fundamental role in regulating peripheral B cell survival and homeostasis. A BAFF-specific receptor (BAFF-R; BR3) appears to mediate these functions via activation of the NF-κB2 pathway. Signaling by the BAFF-R is also required to sustain the germinal center (GC) reaction. Engagement of this receptor results in the induction of Bcl-2, suggesting that this antiapoptotic factor acts downstream of the BAFF-R and NF-κB2 pathway to promote peripheral B cell survival during primary and Ag-driven development. To test this idea, we created lines of mice coexpressing a Bcl-2 transgene and a signaling-deficient form of the BAFF-R derived from the B lymphopenic A/WySnJ strain. Surprisingly, although dramatically elevated numbers of B cells accumulate in the periphery of these mice, these B cells exhibit extremely perturbed primary development, formation of lymphoid microenvironments, and GC and IgG responses. Moreover, mice expressing the bcl-2 transgene alone display a loss of marginal zone B cells, an expansion of follicular B cells that appear immature, and alterations of the GC reaction. These results suggest that the BAFF-R and Bcl-2 regulate key and nonoverlapping aspects of peripheral B cell survival and development.

IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity

B cell–intrinsic IFN-γ receptor signaling through STAT1 is required for the generation of spontaneous germinal centers, which can lead to pathogenic autoantibody production.

The Lupus Susceptibility Locus Sle1 Breaches Peripheral B Cell Tolerance at the Antibody-Forming Cell and Germinal Center Checkpoints

We have described a line of VH knock-in mice termed HKIR in which the transgenic Igh locus partially encodes “dual-reactive” antichromatin and anti-p-azophenylarsonate (Ars) BCRs. HKIR B cells termed canonical, expressing a particular Vκ L chain, evade central tolerance by down-regulating BCR levels. Canonical HKIR B cells can be recruited into the primary germinal center (GC) and Ab-forming cell (AFC) compartments via Ars immunization. However, their participation in the GC response rapidly wanes and they do not efficiently contribute to the memory compartment, indicating that they are regulated by a GC tolerance checkpoint. We analyzed the influence of the Sle1 genetic interval, shown to break tolerance of chromatin-reactive B cells, on the behavior of HKIR B cells during the anti-Ars response. Canonical B cells from congenic HKIR.Sle1 mice gave rise to elevated short and long-lived AFC responses, and the attenuated GC and memory responses characteristic of these B cells were relieved in adoptive, wild-type recipients. HKIR GC B cells containing Sle1 expressed increased levels of Bcl-2 and c-FLIP and decreased levels of Fas RNA compared with HKIR controls, suggesting direct alteration of the regulation of the GC response by Sle1. High titers of canonical and anti-dsDNA Abs spontaneously developed in many aged HKIR.Sle1 mice. Together, these data indicate that Sle1 perturbs the action of peripheral tolerance checkpoints operative on antinuclear Ag B cells in both the AFC and GC pathways in a cell autonomous fashion.

Expression of the autoimmune Fcgr2b NZW allele fails to be upregulated in germinal center B cells and is associated with increased IgG production

The inhibitory receptor FcγRIIb regulates B-cell functions. Genetic studies have associated Fcgr2b polymorphisms and lupus susceptibility in both humans and murine models, in which B cells express reduced FcγRIIb levels. Furthermore, FcγRIIb absence results in lupus on the appropriate genetic background, and lentiviral-mediated FcγRIIb overexpression prevents disease in the NZM2410 lupus mouse. The NZM2410/NZW allele Fcgr2b is, however, located in-between Sle1a and Sle1b, two potent susceptibility loci, making it difficult to evaluate Fcr2bNZW independent contribution. By using two congenic strains that each carries only Sle1a (B6.Sle1a(15–353)), or Fcr2bNZW in the absence of Sle1a or Sle1b (B6.Sle1(111–148)), we show that the Fcr2bNZW allele does not upregulate its expression on germinal center B cells and plasma cells, as does the C57BL/6 allele on B6.Sle1a(15–353) B cells. Furthermore, in the absence of the flanking Sle1a and Sle1b, Fcr2bNZW does not produce an autoimmune phenotype, but is associated with an increased number of class-switched plasma cells. These results show that while a lower level of FcγRIIb does not by itself induce the development of autoreactive B cells, it has the potential to amplify the contribution of autoreactive B cells induced by other lupus-susceptibility loci by enhancing the production of class-switched plasma cells.

Failed Up-Regulation of the Inhibitory IgG Fc Receptor FcγRIIB on Germinal Center B Cells in Autoimmune-Prone Mice Is Not Associated with Deletion Polymorphisms in the Promoter Region of the FcγRIIB Gene

FcγRIIB, a low-affinity FcR for IgG, inhibits BCR-mediated activation when these two receptors are cocross-linked by Ags and IgG-containing immune complexes. Although a role for FcγRIIB in the germinal center (GC) reaction has been proposed, conflicting results have been published regarding the levels of FcγRIIB expressed on GC B cells in normal and autoimmune-prone mice and humans. In the present study, we investigate this issue in detail in mice by using multiple GC B cell markers, two different antigenic systems, primary and secondary GC responses, and by excluding the influence of splenic influx of immature B cells and passive acquisition of FcγRIIB from follicular dendritic cells. Our results are in concordance with previous data indicating that FcγRIIB expression is up-regulated on GC B cells in normal mice. In contrast, we observe comparable levels of FcγRIIB on GC and non-GC B cells in New Zealand White, New Zealand Black, and B6.Sle1 autoimmune-prone strains. Therefore, we suggest that these strains exhibit failed up-regulation of FcγRIIB on GC B cells, rather than down-regulation, as previously suggested. Also, in contrast to previous indications, this perturbed regulation is not uniquely associated with deletion polymorphisms in the promoter region of the FcγRIIB gene but does appear to be independent of genetic background. Finally, we present evidence indicating that FcγRIII, a low-affinity activating IgG FcR, is expressed on the GC B cells of normal but not autoimmune-prone mice.

FcγRIIB Regulates Autoreactive Primary Antibody-Forming Cell, but Not Germinal Center B Cell, Activity

The low-affinity FcR for IgG FcγRIIB suppresses the development of IgG autoantibodies and autoimmune disease in normal individuals, but how this effect is mediated is incompletely understood. To investigate this issue, we created FcγRIIB-deficient versions of two previously described targeted BCR-transgenic lines of mice that contain follicular B cells with specificity for the hapten arsonate, but with different levels of antinuclear autoantigen reactivity. The primary development and tolerance of both types of B cells were unaltered by the absence of FcγRIIB. Moreover, the reduced p-azophenylarsonate-driven germinal center and memory responses characteristic of the highly autoreactive clonotype were not reversed by an intrinsic FcγRIIB deficiency. In contrast, the p-azophenylarsonate-driven primary Ab-forming cell responses of both clonotypes were equivalently increased by such a deficiency. In total, our data do not support the idea that FcγRIIB directly participates in the action of primary or germinal center tolerance checkpoints. In contrast, this receptor apparently contributes to the prevention of autoimmunity by suppressing the production of autoreactive IgGs from B cells that have breached tolerance checkpoints and entered the Ab-forming cell pathway due to spontaneous, or cross-reactive, Ag-mediated activation.