Tahsin N. Khan

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.

Prolonged Apoptotic Cell Accumulation in Germinal Centers of Mer-Deficient Mice Causes Elevated B Cell and CD4+ Th Cell Responses Leading to Autoantibody Production

Mer receptor tyrosine kinase is a member of the Tyro-3/Axl/Mer (TAM) subfamily of receptor tyrosine kinases, and its expression on phagocytes facilitates their clearance of apoptotic cells (ACs). Mer expression in germinal centers (GCs) occurs predominantly on tingible body macrophages. B and T cells do not express Mer. In this study, we show that Mer deficiency ((Mer−/−) resulted in the long-term accumulation of ACs primarily in GCs and not in the T cell zone, marginal zone, or red pulp areas of the spleen. AC accumulation in GCs led to augmented Ab-forming cell, GC, and IgG2 Ab responses in Mer−/− mice, which were sustained for at least 80 d. Enhanced responses in Mer−/− mice were due to increased activation and proliferation of B cells and CD4+ Th cells, including follicular helper T cells, which resulted in high titers of anti-nuclear Abs in Mer−/− mice compared with wild-type controls. Secondary IgG-producing Ab-forming cell, total IgG, and IgG2 Ab responses were also increased in Mer−/− mice. Finally, compared with wild-type controls, Mer−/− mice had increased percentage of IFN-γ–producing CD4+ Th cells and elevated levels of Th1 (i.e., IL-2 and IFN-γ) and proinflammatory (i.e., TNF and IL-6) cytokines, consistent with elevated levels of Th1-biased IgG2 Abs in Mer−/− mice. Together, our results demonstrate that Mer deficiency induces prolonged accumulation of ACs in GCs, resulting in dysregulation of GC B cell and CD4+ Th cell responses and Th1 cytokine production, leading to alteration of B cell tolerance and the development of autoantibodies.

The Lupus-Prone NZM2410/NZW Strain–Derived Sle1b Sublocus Alters the Germinal Center Checkpoint in Female Mice in a B Cell–Intrinsic Manner

C57BL/6 (B6) mice carrying the Sle1b sublocus (named B6.Sle1b), which harbors the lupus-associated NZM2410/NZW SLAM family genes, produce antinuclear Abs (ANAs). However, the role and mechanism(s) involved in the alteration of the germinal center (GC) tolerance checkpoint in the development of ANAs in these mice is not defined. In this study, we show significantly higher spontaneously formed GCs (Spt-GCs) in B6.Sle1b female mice compared with B6 controls. We also found a significant increase in CD4+CXCR5hiPD-1hi spontaneously activated follicular Th cells in B6.Sle1b female mice. Compared with B6 controls, B6.Sle1b female mice had increased numbers of proliferating B cells predominantly located in Spt-GCs. The elevated Spt-GCs in B6.Sle1b female mice were strongly associated with increased ANA-specific Ab-forming cells and ANA titers. The increased numbers of Spt-GCs and spontaneously activated follicular Th cells in B6.Sle1b mice were not the result of a generalized defect in B cells expressing Sle1b. Consistent with the elevated spontaneous response in B6.Sle1b mice, the attenuated GC response characteristic of DNA and p-azophenylarsonate reactive B cells from Ig VH knock-in mice (termed HKIR) were relieved in adoptively transferred recipients in the presence of Sle1b. Finally, by generating mixed bone marrow chimeras, we showed that the effect of Sle1b on Spt-GC, follicular Th cell, and autoantibody responses in B6.Sle1b mice was B cell autonomous. These data indicate that the NZM2410/NZW-derived Sle1b sublocus in conjunction with the female sex primarily affects B cells, leading to the alteration of the GC tolerance checkpoint and the generation of ANA-specific Ab-forming cells.

B Cell–Intrinsic CD84 and Ly108 Maintain Germinal Center B Cell Tolerance

Signaling lymphocyte activation molecules (SLAMs) play an integral role in immune regulation. Polymorphisms in the SLAM family receptors are implicated in human and mouse model of lupus disease. The lupus-associated, somatically mutated, and class-switched pathogenic autoantibodies are generated in spontaneously developed germinal centers (GCs) in secondary lymphoid organs. The role and mechanism of B cell–intrinsic expression of polymorphic SLAM receptors that affect B cell tolerance at the GC checkpoint are not clear. In this study, we generated several bacterial artificial chromosome–transgenic mice that overexpress C57BL/6 (B6) alleles of different SLAM family genes on an autoimmune-prone B6.Sle1b background. B6.Sle1b mice overexpressing B6-derived Ly108 and CD84 exhibit a significant reduction in the spontaneously developed GC response and autoantibody production compared with B6.Sle1b mice. These data suggest a prominent role for Sle1b-derived Ly108 and CD84 in altering the GC checkpoint. We further confirm that expression of lupus-associated CD84 and Ly108 specifically on GC B cells in B6.Sle1b mice is sufficient to break B cell tolerance, leading to an increase in autoantibody production. In addition, we observe that B6.Sle1b B cells have reduced BCR signaling and a lower frequency of B cell–T cell conjugates; the reverse is seen in B6.Sle1b mice overexpressing B6 alleles of CD84 and Ly108. Finally, we find a significant decrease in apoptotic GC B cells in B6.Sle1b mice compared with B6 controls. Our study establishes a central role for GC B cell–specific CD84 and Ly108 expression in maintaining B cell tolerance in GCs and in preventing autoimmunity.

Distinct and synergistic roles of FcγRIIB deficiency and 129 strain-derived SLAM family proteins in the development of spontaneous germinal centers and autoimmunity

The inhibitory IgG Fc receptor (FcγRIIB) deficiency and 129 strain-derived signaling lymphocyte activation molecules (129-SLAMs) are proposed to contribute to the lupus phenotype in FcγRIIB-deficient mice generated using 129 ES cells and backcrossed to C57BL/6 mice (B6.129.RIIBKO). In this study, we examine the individual contributions and the cellular mechanisms by which FcγRIIB deficiency and 129-derived SLAM family genes promote dysregulated spontaneous germinal center (Spt-GC) B cell and follicular helper T cell (Tfh) responses in B6.129.RIIBKO mice. We find that B6 mice congenic for the 129-derived SLAM locus (B6.129-SLAM) and B6 mice deficient in FcγRIIB (B6.RIIBKO) have increased Spt-GC B cell responses compared to B6 controls but significantly lower than B6.129.RIIBKO mice. These data indicate that both FcγRIIB deficiency and 129-SLAMs contribute to elevated Spt-GC B cell responses in B6.129.RIIBKO mice. However, only 129-SLAMs contribute significantly to augmented Tfh responses in B6.129.RIIBKO mice, and do so by a combination of T cell-dependent effects and enhanced B cell and DC-dependent antigen presentation to T cells. Elevated Spt-GC B cell responses in mice with FcγRIIB deficiency and polymorphic 129-SLAMs were associated with elevated metabolic activity, improved GC B cell survival and increased differentiation of naïve B cells into GC B cell phenotype. Our data suggest that the interplay between 129-SLAM expression on B cells, T cells and DCs is central to the alteration of the GC tolerance checkpoint, and that deficiency of FcγRIIB on B cells is necessary to augment Spt-GC responses, pathogenic autoantibodies, and lupus disease.