Cytosolic DNA-sensing pathways that signal via Stimulator of interferon genes (STING) mediate immunity to pathogens and also promote autoimmune pathology in DNaseII- and DNaseIII-deficient mice. In contrast, we report here that STING potently suppresses inflammation in a model of systemic lupus erythematosus (SLE). Lymphoid hypertrophy, autoantibody production, serum cytokine levels, and other indicators of immune activation were markedly increased in STING-deficient autoimmune-prone mice compared with STING-sufficient littermates. As a result, STING-deficient autoimmune-prone mice had significantly shorter lifespans than controls. Importantly, Toll-like receptor (TLR)-dependent systemic inflammation during 2,6,10,14-tetramethylpentadecane (TMPD)-mediated peritonitis was similarly aggravated in STING-deficient mice. Mechanistically, STING-deficient macrophages failed to express negative regulators of immune activation and thus were hyperresponsive to TLR ligands, producing abnormally high levels of proinflammatory cytokines. This hyperreactivity corresponds to dramatically elevated numbers of inflammatory macrophages and granulocytes in vivo. Collectively these findings reveal an unexpected negative regulatory role for STING, having important implications for STING-directed therapies.

Age-associated B cells (ABCs) are formed under inflammatory conditions and are considered a type of memory B cell (MBC) expressing the transcription factor T-bet. In SLE, ABC frequency is correlated with disease, and they are thought to be the source of autoantibody-secreting cells. However, in inflammatory conditions, whether autoreactive B cells can become resting MBCs is uncertain. Further, the phenotypic identity of ABCs and their relationship to other B cell subsets, such as plasmablasts, is unclear. Whether ABCs directly promote disease is untested. Here we report, in the MRL/lpr SLE model, unexpected heterogeneity among ABC-like cells for expression of the integrins CD11b and CD11c, T-bet, and memory or plasmablast markers. Transfer and labeling studies demonstrated that ABCs are dynamic, rapidly turning over. scRNA-seq identified B cell clones present in multiple subsets, revealing that ABCs can be plasmablast precursors or undergo cycles of reactivation. Deletion of CD11c-expressing B cells revealed a direct role for ABC-like B cells in lupus pathogenesis.

Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B-MHC II(-/-)), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)-specific B cell receptor (BCR; IgH(MOG-mem)) but cannot secrete antibodies. B-MHC II(-/-) mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell- and B cell-dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B-MHC II(-/-) mice. In the absence of antibodies, IgH(MOG-mem) mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle-like structures were observed in IgH(MOG-mem) mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell-targeted therapies.

NADPH oxidase deficiency exacerbates lupus in murine models and patients, but the mechanisms remain unknown. It is hypothesized that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, nonautoimmune mice. Prior work implicated cytochrome b-245, β polypeptide (CYBB), a component of the NADPH oxidase complex, and the RUN and cysteine-rich domain-containing Beclin 1-interacting protein (RUBICON) as requisite for LAP. To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Faslpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite - RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradict the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found that LAP is not dependent on either of these 2 pathways. To our knowledge, our data reveal RUBICON as a novel regulator of SLE, possibly by a B cell-intrinsic mechanism, but do not support a role for LAP in lupus.

The inducible T cell costimulator (ICOS) is a potent promoter of organ inflammation in murine lupus. ICOS stimulates T follicular helper cell differentiation in lymphoid tissue, suggesting that it might drive autoimmunity by enhancing autoantibody production. Yet the pathogenic relevance of this mechanism remains unclear. It is also unknown whether other ICOS-induced processes might contribute to lupus pathology. Here we show that selective ablation of ICOS ligand (ICOSL) in CD11c(+) cells, but not in B cells, dramatically ameliorates kidney and lung inflammation in lupus-prone MRL.Fas(lpr) mice. Autoantibody formation was largely unaffected by ICOSL deficiency in CD11c(+) cells. However, ICOSL display by CD11c(+) cells in inflamed organs had a nonredundant role in protecting invading T cells from apoptosis by elevating activity of the PI3K-Akt signaling pathway, thereby facilitating T cell accrual. These findings reveal a mechanism that locally sustains organ inflammation in lupus.

Dendritic cells (DCs) initiate and control the adaptive immune response against infections. However, their contributions to the anti-self adaptive immune response in autoimmune disorders like systemic lupus erythematosus are uncertain. By constitutively deleting DCs in MRL.Fas(lpr) mice, we show that they have complex roles in murine lupus. The net effect of DC deletion was to ameliorate disease. DCs were crucial for the expansion and differentiation of T cells but, surprisingly, not required for their initial activation. Correspondingly, kidney interstitial infiltrates developed in the absence of DCs, but failed to progress. DC deletion concomitantly decreased inflammatory and regulatory T cell numbers. Unexpectedly, plasmablast numbers and autoantibody concentrations depended on DCs, in contrast to total serum immunoglobulin concentrations, suggesting an effect of DCs on extrafollicular humoral responses. These findings reveal that DCs operate in unanticipated ways in murine lupus and validate them as a potential therapeutic target in autoimmunity.

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies, we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system, which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor, yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures, revealing that the reaction engenders both MBC subsets with different immune effector function and, ultimately, LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity, for vaccine development, and for understanding long-term pathogen resistance.

We previously found that kidney-infiltrating T cells (KITs) in murine lupus nephritis (LN) resembled dysfunctional T cells that infiltrate tumors. This unexpected finding raised the question of how to reconcile the "exhausted" phenotype of KITs with ongoing tissue destruction in LN. To address this, we performed single-cell RNA-Seq and TCR-Seq of KITs in murine lupus models. We found that CD8+ KITs existed first in a transitional state, before clonally expanding and evolving toward exhaustion. On the other hand, CD4+ KITs did not fit into current differentiation paradigms but included both hypoxic and cytotoxic subsets with a pervasive exhaustion signature. Thus, autoimmune nephritis is unlike acute pathogen immunity; rather, the kidney microenvironment suppresses T cells by progressively inducing exhausted states. Our findings suggest that LN, a chronic condition, results from slow evolution of damage caused by dysfunctional T cells and their precursors on the way to exhaustion. These findings have implications for both autoimmunity and tumor immunology.

Germinal centers (GC) are crucial for the formation of long-lived humoral immunity. Many pathogens suppress GC, including Salmonella enterica serovar Typhimurium (STm), but the mechanisms driving suppression remain unknown. We report that neither plasmablasts nor STm-specific B cells are required for GC suppression in mice. Rather, we identify that interleukin-12 (IL-12), but not interferon-γ (IFN-γ), directly suppresses T follicular helper (Tfh) cell differentiation of T cells intrinsically. Administering recombinant IL-12 during nitrophenyl-Chicken Gamma Globulin (NP-CGG) immunization also suppresses Tfh cell differentiation and GC B cells, indicating that IL-12 is sufficient to suppress Tfh cell differentiation independent of STm infection. Recombinant IL-12 induces high levels of T-bet, and T-bet is necessary for Tfh cell suppression. Therefore, IL-12 induced during STm infection in mice contributes to GC suppression via suppression of Tfh cell differentiation. More broadly, these data suggest that IL-12 can tailor the proportions of humoral (Tfh cell) and cellular (T helper type 1 [Th1] cell) immunity to the infection, with implications for IL-12 targeting therapies in autoimmunity and vaccination.

Systemic autoimmune disease in humans and mice is characterized by loss of immunologic tolerance to a restricted set of self-nuclear antigens. Autoantigens, such as double-stranded (ds) DNA and the RNA-containing Smith antigen (Sm), may be selectively targeted in systemic lupus erythematosus because of their ability to activate a putative common receptor. Toll-like receptor 9 (TLR9), a receptor for CpG DNA, has been implicated in the activation of autoreactive B cells in vitro, but its role in promoting autoantibody production and disease in vivo has not been determined. We show that in TLR9-deficient lupus-prone mice, the generation of anti-dsDNA and antichromatin autoantibodies is specifically inhibited. Other autoantibodies, such as anti-Sm, are maintained and even increased in TLR9-deficient mice. In contrast, ablation of TLR3, a receptor for dsRNA, did not inhibit the formation of autoantibodies to either RNA- or DNA-containing antigens. Surprisingly, we found that despite the lack of anti-dsDNA autoantibodies in TLR9-deficient mice, there was no effect on the development of clinical autoimmune disease or nephritis. These results demonstrate a specific requirement for TLR9 in autoantibody formation in vivo and indicate a critical role for innate immune activation in autoimmunity.