Autoreactive Germinal Centers Research Articles

Invasion of spontaneous germinal centers by naive B cells is rapid and persistent.

In autoreactive germinal centers (GC) initiated by a single rogue B cell clone, wild-type B cells expand and give rise to clones that target other autoantigens, known as epitope spreading. The chronic, progressive nature of epitope spreading calls for early interventions to limit autoimmune pathologies, but the kinetics and molecular requirements for wild-type B cell invasion and participation in GC remain largely unknown. With parabiosis and adoptive transfer approaches in a murine model of systemic lupus erythematosus, we demonstrate that wild-type B cells join existing GCs rapidly, clonally expand, persist, and contribute to autoantibody production and diversification. The invasion of autoreactive GCs by wild-type B cells required TLR7, B cell receptor specificity, antigen presentation, and type I interferon signaling. The adoptive transfer model provides a tool for identifying early events in the breaking of B cell tolerance in autoimmunity.

T cell help shapes B cell tolerance.

T cell help is a crucial component of the normal humoral immune response, yet whether it promotes or restrains autoreactive B cell responses remains unclear. Here, we observe that autoreactive germinal centers require T cell help for their formation and persistence. Using retrogenic chimeras transduced with candidate TCRs, we demonstrate that a follicular T cell repertoire restricted to a single autoreactive TCR, but not a foreign antigen-specific TCR, is sufficient to initiate autoreactive germinal centers. Follicular T cell specificity influences the breadth of epitope spreading by regulating wild-type B cell entry into autoreactive germinal centers. These results demonstrate that TCR-dependent T cell help can promote loss of B cell tolerance and that epitope spreading is determined by TCR specificity.

Haplosufficiency of MHCII in autoreactive germinal center B cells.

Utilizing an autoimmune bone marrow chimera model we determined that B cells depend critically on MHCII expression for participation in the germinal center, but cells displaying a 50% reduction in surface MHCII compete efficiently with their wild-type counterparts. This providesinsights into the requirements for germinal center participation.

Effects of BAMBI deficiency in the development of Systemic Lupus Erythematosus

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies and immune complexes deposition in various organs. Follicular CD4+ T cells play a crucial role in this autoimmune syndrome by activating autoreactive germinal center B cells. The transforming growth factor β (TGBβ) inhibitor BAMBI (Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor) is known to control the differentiation of CD4+ T lymphocytes into either tolerogenic Tregs or pathogenic Th17 cells. BAMBI absence in mutant mice or its pharmacological inhibition with the B101.37 monoclonal antibody promotes an increase in Treg differentiation and activity and at the same time, a reduction in Th17 cell differentiation, resulting in the inhibition of autoimmune disease development. Based on these findings, in the present study we have analysed the effects of BAMBI deficiency in the development of SLE. We have employed the chronic graft-versus-host disease lupus model induced after the transference of 40–60 × 106 lymphocytes from B6 or B6-BAMBI-KO mice into either Bm12 or Bm12-BAMBI-KO recipients. As reported previously, Bm12 mice receiving lymphocytes from B6 mice produce high levels of circulating IgG anti-DNA and anti-nucleosomal autoantibodies and exhibit linear glomerular IgG deposits in their kidneys. In contrast, both Bm12 mice injected with allogeneic B6-BAMBI-KO lymphocytes and Bm12-BAMBI-KO recipients receiving lymphocytes from B6 donors do not produce significant levels of circulating autoantibodies and do not reveal immune complexes deposits. Taken together, these results indicate that the absence of BAMBI in either donor or recipient lymphocytes protects mice against the development of SLE.

B cells in the balance: Offsetting self-reactivity avoidance with protection against foreign

Antibodies are theoretically limitless in their diversity and specificity to foreign antigens; however they are constrained by the need to avoid binding to self. Germinal centers (GC) allow diversification and maturation of the antibody response towards the foreign antigen. While self-tolerance mechanisms controlling self-reactivity during B cell maturation are well recognized, the mechanisms by which GCs balance self-tolerance and foreign binding especially in the face of cross-reactivity between self and foreign, remain much less well defined. In this review we explore the extent to which GC self-tolerance restricts affinity maturation. We present studies suggesting that the outcome is situationally dependent, affected by affinity and avidity to self-antigen, and the extent to which self-binding and foreign-binding are interdependent. While auto-reactive GC B cells can mutate away from self while maturing towards the foreign antigen, if no mutational trajectories allow for self-reactive redemption, self-tolerance prevails and GC responses to the foreign pathogen are restricted, except when self-tolerance checkpoints are relaxed. Finally, we consider whether polyreactivity is subject to the same level of restriction in GC responses, especially if polyreactivity is linked to an increase in foreign protection, as occurs in certain broadly neutralizing antibodies. Overall, the outcomes for GC B cells that bind self-antigen can range from redemption, transient relaxation in self-tolerance or restriction of the antibody response to the foreign pathogen.

T cell help in the autoreactive germinal center.

The germinal center serves as a site of B cell selection and affinity maturation, critical processes for productive adaptive immunity. In autoimmune disease tolerance is broken in the germinal center reaction, leading to production of autoreactive B cells that may propagate disease. Follicular T cells are crucial regulators of this process, providing signals necessary for B cell survival in the germinal center. Here, we review the emerging roles of follicular T cells in the autoreactive germinal center. Recent advances in immunological techniques have allowed study of the gene expression profiles and repertoire of follicular T cells at unprecedented resolution. These studies provide insight into the potential role follicular T cells play in preventing or facilitating germinal center loss of tolerance. Improved understanding of the mechanisms of T cell help in autoreactive germinal centers provides novel therapeutic targets for diseases of germinal center dysfunction.

B Cell Intrinsic STING Signaling Is Not Required for Autoreactive Germinal Center Participation.

Germinal centers (GCs) are induced microanatomical structures wherein B cells undergo affinity maturation to improve the quality of the antibody response. Although GCs are crucial to appropriate humoral responses to infectious challenges and vaccines, many questions remain about the molecular signals driving B cell participation in GC responses. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an important mediator of type I interferon and proinflammatory cytokine responses during infection and cellular stress. Recent studies have reported important roles for STING in B cell responses, including an impact on GC B cells and downstream antibody responses, which could have great consequences for vaccine design and understanding STING-associated interferonopathies. GCs are also involved in untoward reactions to autoantigens in a plethora of autoimmune disorders, and it is generally thought that these responses coopt the mechanisms used in foreign antigen-directed GCs. Here, we set out to investigate the importance of the cGAS-STING pathway in autoreactive B cell responses. In a direct competition scenario in a murine mixed bone marrow chimera model of autoreactive GCs, we find that B cell intrinsic deficiency of cGAS, STING, or the type I interferon receptor IFNAR, does not impair GC participation, whereas Toll-like receptor (TLR)-7 deficiency mediates a near-complete block. Our findings suggest that physiological B cell responses are strictly sustained by signals linked to BCR-mediated endocytosis. This wiring of B cell signals may enable appropriate antibody responses, while at the same time restricting aberrant antibody responses during infections and in autoimmune or autoinflammatory settings.

TLR7 Signaling in Lupus B Cells: New Insights into Synergizing Factors and Downstream Signals.

Systemic lupus erythematosus (SLE) is driven by nucleic acid-containing antigens that stimulate endosomal TLRs. We review new advances in our understanding of how TLR7 signaling in B cells drives autoimmunity. Pathogenic B cell responses to TLR7 engagement are shaped by the disease-associated cytokine environment. TLR7, IFNγ, and IL-21 together promote the formation of autoreactive germinal centers and the ABC/DN2 B cell subset. BAFF and type 1 IFNs enhance autoantibody production from transitional B cells in concert with TLR7. TLR7 signaling components STAT1, BANK1, IRF5, SLC15A4, and CXorf21/TASL are associated genetically with SLE and important for lupus development in mice, while role of T-bet is controversial. Proper control of TLR7 trafficking by UNC93B1, syntenin-1, and αvβ3 integrin is critical for preventing autoimmunity. A better understanding of TLR7 signaling has revealed potential new therapeutic approaches for SLE, several of which are being tested in animal models or clinical trials.

IL-17 receptor A signaling on B cells promotes B-cell responses to TLR7 and enhances autoreactive germinal center B-cell development

Abstract IL-17 receptor A (IL-17RA) expression has been identified in B cells and macrophages. There was low expression of IL-17RA on T cells. Surprisingly, we previously showed that IL-17RA signaling through NF-κB p65/p50 has a complex effect on both B and T-cell localization and germinal center (GC) architecture, making it difficult to dissect the specific effects of IL-17RA on B cells. To isolate the effects of the absence of IL-17RA specifically on B cells, we have generated a B-cell specific IL-17RA knockout (Il17rafloxed/floxed x Cd19.Cre) BXD2 mice. Flow cytometry confirmed the loss of IL-17RA expression in only CD19+ B cells but not CD3+ T cells or CD11b+ macrophages. There was a 2-fold decreased spontaneous GC B cell population in Il17rafloxed/floxed x Cd19.Cre BXD2, compared to Il17rawt/floxed x Cd19.Cre BXD2 mice. Interestingly, the abnormal activation and expansion of T-follicular helper cell (Tfh) phenotype in BXD2-Il17ra−/− mice was abrogated in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. In the BXD2-Il17ra−/− mice, B cells were anergic to TLR7 stimulation and exhibited a strong stimulus-specific transcription repressor p50/p50 homodimer and a lower NF-κB phospho-p65. To demonstrate that this anergy is due specifically to IL-17RA on B cells but not other cells, we have identified that there was a lower TLR7-induced CD69 in vitro in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. Taken together, our results suggest that IL-17RA signaling directly regulates B cell autoimmune phenotype in the BXD2 mice through an enhancement of B-cell responses stimulated by the NF-κB signaling pathway. A deficiency of such signal enforces B cell anergy and prevents autoreactive GC formation.

Recent Advances in Lupus B Cell Biology: PI3K, IFNγ, and Chromatin.

In the autoimmune disease Systemic Lupus Erythematosus (SLE), autoantibodies are formed that promote inflammation and tissue damage. There has been significant interest in understanding the B cell derangements involved in SLE pathogenesis. The past few years have been particularly fruitful in three domains: the role of PI3K signaling in loss of B cell tolerance, the role of IFNγ signaling in the development of autoimmunity, and the characterization of changes in chromatin accessibility in SLE B cells. The PI3K pathway coordinates various downstream signaling molecules involved in B cell development and activation. It is governed by the phosphatases PTEN and SHIP-1. Murine models lacking either of these phosphatases in B cells develop autoimmune disease and exhibit defects in B cell tolerance. Limited studies of human SLE B cells demonstrate reduced expression of PTEN or increased signaling events downstream of PI3K in some patients. IFNγ has long been known to be elevated in both SLE patients and mouse models of lupus. New data suggests that IFNγR expression on B cells is required to develop autoreactive germinal centers (GC) and autoantibodies in murine lupus. Furthermore, IFNγ promotes increased transcription of BCL6, IL-6 and T-bet in B cells, which also promote GC and autoantibody formation. IFNγ also induces epigenetic changes in human B cells. SLE B cells demonstrate significant epigenetic reprogramming, including enhanced chromatin accessibility at transcription factor motifs involved in B cell activation and plasma cell (PC) differentiation as well as alterations in DNA methylation and histone modifications. Histone deacetylase inhibitors limit disease development in murine lupus models, at least in part via their ability to prevent B cell class switching and differentiation into plasma cells. This review will discuss relevant discoveries of the past several years pertaining to these areas of SLE B cell biology.

MHCII density only weakly impacts autoreactive germinal center B cell fitness

Abstract B cells generate high-affinity antibodies within induced microanatomical structures called germinal centers (GCs). Each GC is, in essence, a Darwinian microcosm, in which B cells undergo consecutive rounds of division, hypermutation, and selection based on antigen affinity. The consensus has been that this selection is governed by T follicular helper (Tfh) cell signals, determined by the amount of cognate peptide presented by B cells on major histocompatibility complex (MHC) class II. This view was recently challenged by observations in a foreign antigen model system suggesting that peptide-MHCII density dictates the degree of initial GC entry, but does not impact B cell selection once in the GC. GCs are central to the development of pathogenic autoantibodies in a breadth of autoimmune diseases, but it is unclear to what extent autoreactive GC behavior mimics that of foreign-antigen-reactive GCs. The question of peptide-MHCII in autoreactive GC fitness is particularly relevant because the linked recognition requirement provides a safeguard against inadvertent autoreactivity. Accordingly, we investigated the role of peptide-MHCII density in self-reactive GC B cell selection in a chimeric mouse model of epitope spreading. Bone marrow chimeras were generated, in which either MHCII null/b or MHCII null/null cells were competing with MHCII b/b cells for participation in chronic autoreactive GCs. Flow cytometry and confocal analysis of GCs revealed that whereas complete MHCII deficiency blocked B cell participation in the GC, partial MHCII deficiency had only a minor impact on GC fitness. Thus, our results confirm, in an autoreactive setting, the recently reported observation of MHCII haplosufficiency in foreign antigen responses.

VSV-primed CD8+ T cells suppress autoreactive germinal centers

Abstract Pathogenic infections are important environmental modifiers of autoimmune disease as they can alter the immune system in a way that either promotes or reduces autoimmune responses. We tested the effect of infection with vesicular stomatitis virus (VSV) on the FcγR2B−/− (R2) mouse strain that spontaneously develops lupus disease. Infecting FcγR2B−/− mice with live VSV reduces autoantibody levels and inflammatory pathology and prolongs survival. We showed that VSV-mediated protection can be transferred in a CD8 cell adoptive transfer experiment. After further testing the transfer of various CD8 T cell subsets, we have identified CD8+CD122+NKG2D+ cells as mediator in VSV-induced Lupus amelioration. RNA Sequencing data for gene expression profiles among various CD8 subsets, indicate that the protective cells express higher level of genes linked to memory phenotype. Our working hypothesis is that the viral infection may induce the expansion of long lived CD8 populations that regulate the antibody response. In support of the idea we initiated to identify the target of the VSV-primed CD8 T cells in vivo. We examined the effect of VSV-primed CD8 T cells on the immune phenotypes of recipient R2 mice at the initial stages, e.g. after 1, 2 or 3 weeks of transfer. Interestingly, we found that Tfh and germinal center B cells are significantly reduced after three weeks of VSV-CD8 transfer. Thus, the protective CD8 T cells from VSV-infected mice might target autoreactive germinal center to regulate the antibody response. How VSV-CD8 T cells might control germinal center & why it takes about 3 weeks for controlling is under investigation.

IL-23 is essential for the switching between Tfh-IFN-γ and Tfh-IL-17 in lupus

Abstract The role of IL-23 in promoting pathogenic autoantibody development in autoimmune disease is unclear. The purposes of this study are to use the IL-23p19 knockout in the BXD2, a lupus mouse strain, to determine: (i) if IL-23 is required for germinal center (GC) and pathogenic autoantibody development; and (ii) if IL-23 acts through follicular T-helper cells (Tfh) lineage switching to induce autoantibody formation. Sera autoantibodies were measured by ELISA. GC development and the expression of AID were determined by confocal imaging. IL-17 and IFN-γ producing Tfh cells were detected by intracellular staining. The expression of GC program genes and T cell program genes was measured by qRT-PCR. Exogenous IL-23 was administered to B6 mice using an IL-23 expressing adenovirus (AdIL-23). There was significantly increased IgM but decreased IgG autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. The expression of AID was decreased in BXD2-p19−/− mice. Surprisingly, the size and number of GC were increased in BXD2-p19−/− mice. There was also similar percentage of PD1+CXCR5+Tfh cells in BXD2 and BXD2-p19−/− mice. However, the percentage of IL-17A expressing Tfh cells was suppressed but the IFN-γ producing Tfh was increased in BXD2-p19−/− mice. Consistent with this, AdIL-23 administration suppressed the expression of Tbet, Ifng, Bcl6, Il21, Gata3, and Il4 but enhanced the expression Il17a, Il17f in Tfh cells. Our data suggest Tfh-IFN-γ and Tfh-IL-17 each plays an important role to determine the development of autoantibody producing B cells. While IL-23-mediated Tfh-IL-17 does not promote the size of autoreactive GCs, it regulates the induction of AID and thereby plays an important role to determine autoantibody affinity maturation.

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Clonal Evolution of Autoreactive Germinal Centers

Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive Bcells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers.

Neutrophils are well characterized as mediators of peripheral tissue damage in lupus, but it remains unclear whether they influence loss of self-tolerance in the adaptive immune compartment. Lupus neutrophils produce elevated levels of factors known to fuel autoantibody production, including IL-6 and B cell survival factors, but also reactive oxygen intermediates, which can suppress lymphocyte proliferation. To assess whether neutrophils directly influence the progression of autoreactivity in secondary lymphoid organs (SLOs), we characterized the localization and cell-cell contacts of splenic neutrophils at several stages in the progression of disease in the NZB/W murine model of lupus. Neutrophils accumulate in SLO over the course of lupus progression, preferentially localizing near T lymphocytes early in disease and B cells with advanced disease. RNA sequencing reveals that the splenic neutrophil transcriptional program changes significantly over the course of disease, with neutrophil expression of anti-inflammatory mediators peaking during early-stage and midstage disease, and evidence of neutrophil activation with advanced disease. To assess whether neutrophils exert predominantly protective or deleterious effects on loss of B cell self-tolerance in vivo, we depleted neutrophils at different stages of disease. Neutrophil depletion early in lupus resulted in a striking acceleration in the onset of renal disease, SLO germinal center formation, and autoreactive plasma cell production. In contrast, neutrophil depletion with more advanced disease did not alter systemic lupus erythematosus progression. These results demonstrate a surprising temporal and context-dependent role for neutrophils in restraining autoreactive B cell activation in lupus.

Osteopontin in Spontaneous Germinal Centers Inhibits Apoptotic Cell Engulfment and Promotes Anti-Nuclear Antibody Production in Lupus-Prone Mice.

Disposal of apoptotic cells is important for tissue homeostasis. Defects in this process in immune tissues may lead to breakdown of self-tolerance against intracellular molecules, including nuclear components. Development of diverse anti-nuclear Abs (ANAs) is a hallmark of lupus, which may arise, in part, due to impaired apoptotic cell clearance. In this work, we demonstrate that spontaneous germinal centers (GCs) in lupus-prone mice contain significantly elevated levels of unengulfed apoptotic cells, which are otherwise swiftly engulfed by tingible body macrophages. We indicate that osteopontin (OPN) secreted by CD153(+) senescence-associated T cells, which selectively accumulate in the GCs of lupus-prone mice, interferes with phagocytosis of apoptotic cells specifically captured via MFG-E8. OPN induced diffuse and prolonged Rac1 activation in phagocytes via integrin αvβ3 and inhibited the dissolution of phagocytic actin cup, causing defective apoptotic cell engulfment. In wild-type B6 mice, administration of TLR7 ligand also caused spontaneous GC reactions with increasing unengulfed apoptotic cells and ANA production, whereas B6 mice deficient for Spp1 encoding OPN showed less apoptotic cells and developed significantly reduced ANAs in response to TLR7 ligand. Our results suggest that OPN secreted by follicular CD153(+) senescence-associated T cells in GCs promotes a continuous supply of intracellular autoantigens via apoptotic cells, thus playing a key role in the progression of the autoreactive GC reaction and leading to pathogenic autoantibody production in lupus-prone mice.

Interleukin-21 promotes germinal center reaction by skewing the follicular regulatory T cell to follicular helper T cell balance in autoimmune BXD2 mice.

Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21(-/-) mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21(-/-) mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21(-/-) mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21(-/-) mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell-mediated inhibition of antibody secretion in the Tfh cell-B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice.

SP0011 Follicular Dendritic Cells in Immunity and Autoimmunity

Follicular dendritic cells (FDCs) are unique immune cells that contribute to the regulation of humoral immune responses. These cells are located in the B cell follicles of secondary and tertiary...