Chetna Soni

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.

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.

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.

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.

B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells

SUMMARY Type 1 interferon (T1IFN) signaling promotes inflammation and lupus pathology, but its role in autoreactive B cell development in the antibody-forming cell (AFC) and germinal center (GC) pathways is unclear. Using a lupus model that allows for focused study of the AFC and GC responses, we show that T1IFN signaling is crucial for autoreactive B cell development in the AFC and GC pathways. Through bone marrow chimeras, DNA-reactive B cell transfer, and GC-specific Cre mice, we confirm that IFNαR signaling in B cells promotes autoreactive B cell development into both pathways. Transcriptomic analysis reveals gene expression alterations in multiple signaling pathways in non-GC and GC B cells in the absence of IFNαR. Finally, we find that T1IFN signaling promotes autoreactive B cell development in the AFC and GC pathways by regulating BCR signaling. These data suggest value for anti-IFNαR therapy in individuals with elevated T1IFN activity before clinical disease onset.

A systemic macrophage response is required to contain a peripheral poxvirus infection

The goal of the innate immune system is to reduce pathogen spread prior to the initiation of an effective adaptive immune response. Following an infection at a peripheral site, virus typically drains through the lymph to the lymph node prior to entering the blood stream and being systemically disseminated. Therefore, there are three distinct spatial checkpoints at which intervention to prevent systemic spread of virus can occur, namely: 1) the site of infection, 2) the draining lymph node via filtration of lymph or 3) the systemic level via organs that filter the blood. We have previously shown that systemic depletion of phagocytic cells allows viral spread after dermal infection with Vaccinia virus (VACV), which infects naturally through the skin. Here we use multiple depletion methodologies to define both the spatial checkpoint and the identity of the cells that prevent systemic spread of VACV. Subcapsular sinus macrophages of the draining lymph node have been implicated as critical effectors in clearance of lymph borne viruses following peripheral infection. We find that monocyte populations recruited to the site of VACV infection play a critical role in control of local pathogenesis and tissue damage, but do not prevent dissemination of virus. Following infection with virulent VACV, the subcapsular sinus macrophages within the draining lymph node become infected, but are not exclusively required to prevent systemic spread. Rather, small doses of VACV enter the bloodstream and the function of systemic macrophages, but not dendritic cells, is required to prevent further spread. The results illustrate that a systemic innate response to a peripheral virus infection may be required to prevent widespread infection and pathology following infection with virulent viruses, such as poxviruses.

Crucial role of Mer tyrosine kinase in the maintenance of SIGN-R1+ marginal zone macrophages

Mer Tyrosine Kinase receptor (Mer) is involved in anti‐inflammatory efferocytosis. Here we report elevated spontaneous germinal center (Spt‐GC) responses in Mer‐deficient mice (Mer−/−) that are associated with the loss of SIGN‐R1+ marginal zone macrophages (MZMs). The dissipation of MZMs in Mer−/− mice occurs independently of reduced cellularity or delocalization of marginal zone B cells, sinusoidal cells or of CD169+ metallophillic macrophages. We find that MZM dissipation in Mer−/− mice contributes to apoptotic cell (AC) accumulation in Spt‐GCs and dysregulation of the GC checkpoint, allowing an expansion of DNA‐reactive B cells in GCs. We further observe that bone marrow derived macrophages from Mer−/− mice produce more TNFα, and are susceptible to cell death upon exposure to ACs compared to WT macrophages. Anti‐TNFα Ab treatment of Mer−/− mice is, however, unable to reverse MZM loss, but results in reduced Spt‐GC responses, indicating that TNFα promotes Spt‐GC responses in Mer−/− mice. Contrary to an anti‐TNFα Ab treatment, treatment of Mer−/− mice with a synthetic agonist for the transcription factor LXRα rescues a significant number of MZMs in vivo. Our data suggest that Mer‐LXRα signaling plays an important role in the differentiation and maintenance of MZMs, which in turn regulate Spt‐GC responses and tolerance.

Mer Receptor Tyrosine Kinase Signaling Prevents Self-Ligand Sensing and Aberrant Selection in Germinal Centers

Mer tyrosine kinase (Mer) signaling maintains immune tolerance by clearing apoptotic cells (ACs) and inducing immunoregulatory signals. We previously showed that Mer-deficient mice (Mer−/−) have increased germinal center (GC) responses, T cell activation, and AC accumulation within GCs. Accumulated ACs in GCs can undergo necrosis and release self-ligands, which may influence the outcome of a GC response and selection. In this study, we generated Mer−/− mice with a global MyD88, TLR7, or TLR9 deficiency and cell type–specific MyD88 deficiency to study the functional correlation between Mer and TLRs in the development of GC responses and autoimmunity. We found that GC B cell–intrinsic sensing of self-RNA, but not self-DNA, released from dead cells accumulated in GCs drives enhanced GC responses in Mer−/− mice. Although self-ligands directly affect GC B cell responses, the loss of Mer in dendritic cells promotes enhanced T cell activation and proinflammatory cytokine production. To study the impact of Mer deficiency on the development of autoimmunity, we generated autoimmune-prone B6.Sle1b mice deficient in Mer (Sle1b.Mer−/−). We observed accelerated autoimmunity development even under conditions where Sle1b.Mer−/− mice did not exhibit increased AC accumulation in GCs compared with B6.Sle1b mice, indicating that Mer immunoregulatory signaling in APCs regulates B cell selection and autoimmunity. We further found significant expansion, retention, and class-switching of autoreactive B cells in GCs under conditions where ACs accumulated in GCs of Sle1b.Mer−/− mice. Altogether, both the phagocytic and immunomodulatory functions of Mer regulate GC responses to prevent the development of autoimmunity.