B cells contribute to the pathogenesis of chronic autoimmune disorders like systemic lupus erythematosus (SLE) via multiple effector functions. However, B cells are also implicated in regulating SLE and other autoimmune syndromes via release of IL-10. B cells secreting IL-10 have been termed “Breg” and have been proposed as a separate subset of cells, a concept that remains controversial. The balance between pro- and anti-inflammatory effects could determine the success of B cell targeted therapies for autoimmune disorders and it is therefore pivotal to understand the significance of B cell-secreted IL-10 in spontaneous autoimmunity. By lineage specific deletion of Il10 from B cells we demonstrate that B cell-derived IL-10 is ineffective in suppressing the spontaneous activation of self-reactive B and T cells during lupus. Correspondingly, severity of organ disease and survival rates in mice harboring Il10 deficient B cells are unaltered. Genetic marking of cells that transcribe Il10 illustrates that the pool of IL-10 competent cells is dominated by CD4 T cells and macrophages. IL-10 competent cells of the B lineage are rare in vivo and among them short-lived plasmablasts have the highest frequency, suggesting an activation rather than lineage-driven phenotype. Putative Breg phenotypic subsets such as CD1dhiCD5+ and CD21hiCD23hi B cells are not enriched in Il10 transcription. These genetic studies demonstrate that in a spontaneous model of murine lupus, IL-10 dependent B cell regulation does not restrain disease and thus the pathogenic effects of B cells are not detectably counterbalanced by their IL-10 dependent regulatory functions.

A dominant type of spontaneous autoreactive B cell activation in murine lupus is the extrafollicular generation of plasmablasts. The factors governing such activation have been difficult to identify due to the stochastic onset and chronic nature of the response. Thus, the ability to induce a similar autoreactive B cell response with a known autoantigen in vivo would be a powerful tool in deciphering how autoimmune responses are initiated. We report here the establishment and characterization of a system to initiate autoreactive extrafollicular B cell responses that closely mirror the spontaneous response using IgG anti-chromatin Abs. We demonstrate that exogenously administered anti-chromatin Ab, presumably by forming immune complexes (ICs) with released nuclear material, drives activation of RF B cells in AM14 Tg mice. Anti-chromatin elicits autoreactive B cell activation and development into AFCs at the T-zone/red pulp border. Plasmablast generation occurs equally in BALB/c, MRL/+ and MRL/lpr mice, indicating that an autoimmune-prone genetic background is not required for the induced response. Importantly, infused IgG anti-chromatin induces somatic hypermutation (SHM) in the absence of a GC response, thus proving the extrafollicular SHM pathway. This system provides a window on the initiation of an autoantibody response and reveals authentic initiators of it.

Graft-vs.-host disease (GVHD) caused by donor T cells attacking recipient tissues is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (alloSCT). Studies have shown that effector memory T cells (TEM) do not cause GVHD but are capable of immune functions post-transplant, including graft-vs.-leukemia (GVL) effects, but the reasons for this are unclear. In mice, the TEM pool may have a less-diverse T cell receptor (TCR) repertoire than TN with fewer alloreactive clones. We therefore tested whether enhancing the alloreactivity of TEM would restore their ability to cause GVHD. In an MHC-matched system, alloreactive TEM were created by transferring GVHD effector cells into syngeneic recipients and allowing conversion to TEM. Upon retransfer to freshly transplanted recipients, these cells caused only mild GVHD. Similarly, in an MHC-mismatched system, TEM with a proven increased precursor frequency of alloreactive clones only caused limited GVHD. Nonetheless these same cells mounted strong in vitro alloresponses and caused rapid skin graft rejection. TEM created from CD4 cells that had undergone lymphopenia-induced proliferation also caused only mild GVHD. Our findings establish that conversion to TEM significantly reduces GVHD potency, even in cells with a substantially enhanced alloreactive repertoire.

B cells play important roles in autoimmune diseases ranging from multiple sclerosis to rheumatoid arthritis. B cells have long been considered central players in systemic lupus erythematosus. However, anti-CD20 mediated B cell depletion was not effective in two clinical lupus studies, while anti-BLyS, which inhibits B cell survival, was effective. Others and we previously found that anti-CD20 based depletion was surprisingly ineffective in tissues of lupus-prone mice, but that persistent high doses eventually led to depletion and ameliorated lupus. Lupus patients might also have incomplete depletion, as suggested in several studies, and which could have led to therapeutic failure. Here we investigated the mechanism of resistance to Ab-mediated cellular depletion in murine lupus. B cells from lupus-prone mice were easily depleted when transferred into normal environments or in lupus-prone mice that lacked serum Ig. Serum from lupus-prone mice transferred depletion resistance, with the active component being IgG. Because depletion is FcγR-dependent, we assayed macrophages and neutrophils exposed to lupus mouse serum, showing they are impaired in IgG-mediated phagocytosis. We conclude that depletion resistance is an acquired, reversible phagocytic defect depending on exposure to lupus serum IgG. These results have implications for optimizing and monitoring cellular depletion therapy.

Synthetic oligonucleotides containing CpG motifs (CpG ODNs) have been shown to induce proliferation, differentiation and cytokine production in B cells, macrophages and DCs through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) ODNs display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce pDCs to produce high levels of IFNα, while CpG-B is a potent B cell mitogen. Here we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate to distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared to CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation.

The paucity of murine memory B cell markers has been a significant impediment to the study of memory. The most commonly used marker is IgG, which is neither sensitive nor specific, because activated nonmemory cells can be IgG+, and memory cells can be IgM+. In this article, we show that, together, PD-L2 (CD273), CD80, and CD73 define at least five phenotypic subsets of murine memory B cells. These subsets are generated from naive cells bearing a single BCR in response to a single T-dependent Ag. This diversity is independent of class switch, because IgG1- and IgM-bearing memory cells are found within each compartment. Memory subsets defined by PD-L2, CD80, and CD73 are biologically distinct from one another, because they differ in ontogeny and selection. Together, these distinctions suggest that there is a spectrum of memory B cells and progressive acquisition from more naive-like to more memory-like properties.

Summary

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.Faslpr 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.

Recipient antigen presenting cells (APCs) are required for CD8-mediated GVHD and have an important and nonredundant role in CD4-mediated GVHD in mouse MHC-matched allogeneic bone marrow transplantation (alloBMT). However, the precise roles of specific recipient APCs — dendritic cells, macrophages, and B cells — are not well defined. If recipient B cells are important APCs they could be depleted with Rituximab, an anti-CD20 monoclonal antibody. On the other hand, B cells can downregulate T cell responses and consequently B cell depletion could exacerbate GVHD. Patients with B cell lymphomas undergo allogeneic hematopoietic stem cell transplantation (alloSCT) and many are B-cell-deficient due to prior Rituximab. We therefore studied the role of recipient B cells in MHC-matched murine models of CD8- and CD4-mediated GVHD by using recipients genetically deficient in B cells and with antibody-mediated depletion of host B cells. In both CD4-and CD8-dependent models, B cell deficient recipients developed clinical and pathologic GVHD. However, although CD8-mediated GVHD was clinically less severe in hosts genetically deficient in B cells, it was unaffected in anti-CD20-treated recipients. These data indicate that recipient B cells are not important initiators of GVHD and that efforts to prevent GVHD by APC depletion should focus on other APC subsets.

SUMMARY

Mice lacking epidermal Langerhans cells (LC) develop exaggerated contact-hypersensitivity (CHS) responses due to the absence of LC during sensitization/initiation. Examination of T cell responses reveals that the absence of LC leads to increased numbers of hapten-specific CD4 and CD8 T cells but does not alter cytokine expression or development of Treg. CHS responses and antigen-specific T cells are increased in mice in which MHC-II is ablated specifically in LC suggesting that direct cognate interaction between LC and CD4 cells is required for LC suppression. LC-derived IL-10 is also required for optimal inhibition of CHS. Both LC-derived IL-10 mediated suppression and full LC activation require LC expression of MHC-II. These data support a model in which cognate interaction of LC with CD4 T cells enables LC to inhibit expansion of antigen-specific responses via elaboration of IL-10.

The ability to detect selection by analyzing mutation patterns in experimentally derived immunoglobulin (Ig) sequences is a critical part of many studies. Such techniques are useful not only for understanding the response to pathogens, but also to determine the role of antigen-driven selection in autoimmunity, B cell cancers and the diversification of pre-immune repertoires in certain species. Despite its importance, quantifying selection in experimentally derived sequences is fraught with difficulties. The necessary parameters for statistical tests (such as the expected frequency of replacement mutations in the absence of selection) are non-trivial to calculate, and results are not easily interpretable when analyzing more than a handful of sequences. We have developed a web server that implements our previously proposed Focused binomial test for detecting selection. Several features are integrated into the web site in order to facilitate analysis, including V(D)J germline segment identification with IMGT alignment, batch submission of sequences and integration of additional test statistics proposed by other groups. We also implement a Z-score-based statistic that increases the power of detecting selection while maintaining specificity, and further allows for the combined analysis of sequences from different germlines. The tool is freely available at http://clip.med.yale.edu/selection.

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive BCR transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1/RAGE interaction was important for this response [1]. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1/DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE− AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.

A recent advance in the treatment and understanding of autoimmune disease has been the efficacy of B cell targeted therapy. Such therapies are effective for several such diseases, with systemic autoimmunity being a prototypical example. The mechanism of action is not fully defined, but blocking B cell Ag presentation to T cells is likely to be important. T-B interactions probably engender a positive feedback loop that amplifies and sustains autoimmunity. But how is self-tolerance first broken to initiate this loop? I propose, based on recent data, a model in which autoreactive B cells are activated first, independent of T cells, but dependent upon BCR and TLR signals. These activated B cells then break T celltolerance, initiating full-blown autoimmunity.

Memory B and plasma cells (PCs) are generated in the germinal center (GC). As PD-1 is highly expressed in T follicular helper cells (TFH), we investigated the role of PD-1 signaling in the humoral response. We found that PD-L1 and PD-L2 are upregulated on GC B cells. Pdcd1lg2−/−, CD274−/−Pdcd1lg2−/− and Pdcd1−/− mice had reduced numbers of long-lived PCs. The mechanism involved increased GC cell death and decreased TFH cytokine production in the absence of PD-1; the effect was selective, as remaining PCs had higher affinity. PD-1 expression on T cells and PD-L2 expression on B cells controlled TFH and PC numbers. Thus, PD-1 regulates selection and survival in the GC, impacting the quantity and quality of long-lived PCs.

Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR−/− B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1−/− B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-β feedback loop and constitutively low expression of TLR7 in the IFNAR1−/− B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.

In humans and non-obese diabetic (NOD) mice, defects in immune tolerance result in the spontaneous development of type-1-diabetes. Recent studies have ascribed a breakdown in tolerance to dysfunction in regulatory T-cells (Tregs) that is secondary to reduced IL-2 production by T-cells having the NOD diabetes susceptibility region insulin-dependent diabetes 3 (Idd3). Here we demonstrate a peripheral tolerance defect in the dendritic cells (DCs) of NOD mice that is independent of Tregs. NOD CD8 T-cells specific for islet antigens fail to undergo deletion in the pancreatic lymph nodes. Deletion was promoted by expression of the protective alleles of both Idd3 (Il2) and Idd5 in DCs. We further identify a second tolerance defect that involves endogenous CD4 T-cell expression of the disease promoting NOD alleles of these genetic regions. Pervasive insulitis can be reduced by expression of the Idd3 and Idd5 protective alleles by either the antigen-presenting cell or lymphocytes.

B cells can influence T cell responses by directly presenting antigen or by secreting antibody that binds to antigen to form immunogenic complexes. Conflicting evidence suggests that persisting antigen/antibody complexes propagate long-term T cell memory; yet other data indicate that memory cells can survive without specific antigen or MHC. Here, the roles of B cells and antigen/antibody complexes in T cell responses to lymphocytic choriomeningitis virus (LCMV) infection were investigated using B cell-deficient or B cell-competent mice. Despite normal lymphocyte expansion after acute infection, B cell-deficient mice rapidly lost CD4+ T cell memory – but not CD8+ T cell memory – during the contraction phase. To determine whether antigen/antibody complexes sustain CD4+ T cell memory, T cell responses were followed in B cell-transgenic (mIg-Tg) mice that have B cells but neither LCMV-specific antibody nor LCMV-immune complex deposition. In contrast to B cell-deficient mice, mIg-Tg mice retained functional T-helper cell memory, indicating that B cells selectively preserve CD4+ T cell memory independently of immune-complex formation. An in vivo consequence of losing CD4+ T cell memory was that B cell-deficient mice were unable to resolve chronic virus infection. These data implicate a B cell function other than antibody production that induces long-term protective immunity.

Ixodes scapularis ticks transmit a number of human pathogens, including the Lyme disease spirochete Borrelia burgdorferi. I. scapularis suppresses host immunity in the skin to promote feeding and systemically skew T-helper (Th)-cell differentiation toward Th2 cells in secondary lymphoid organs. Although components of tick saliva are known to influence Th-cell polarization, the mechanism whereby tick feeding in the skin modulates regional and systemic Th-cell responses is unknown. In this study, the role of the epidermal Langerhans cell (LC) subset of skin dendritic cells in tick-mediated Th1/Th2-cell immunomodulation was assessed. Mice deficient in LCs (Langerin-DTA mice) exhibited enhanced lymph node (LN) concanavalin A (ConA)-induced Th1 responses after tick infestation in comparison to results for uninfested Langerin-DTA or wild-type (WT) mice, whereas effects on Th2-cell production of interleukin 4 were more variable. Nonetheless, the altered T-cell response did not impact tick feeding or refeeding. Gamma interferon production by ConA-stimulated LN cells of both WT and LC-deficient mice was enhanced by as much as fourfold after B. burgdorferi-infected-tick feeding, indicating that immunomodulatory effects of tick saliva were not able to attenuate the Th1 immune responses induced by this pathogen. Taken together, these findings show a requirement for LCs in the tick-mediated attenuation of Th1 responses in regional lymph nodes but not in the spleens of mice and show that the presence of a pathogen can overcome the Th1-inhibitory effects of tick feeding on the host.

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs)6 through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a reactive B cells. By contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR-crosslinking alone is insufficient to activate low affinity autoreactive B cells. Importantly, priming B cells with IFN-α lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-α can contribute to the pathogenesis of systemic lupus erythematosus (SLE).

Autoantigens that contain DNA, RNA, or self-IgG are preferred targets for autoantibodies in Systemic Lupus Erythematosus (SLE). B cells promote SLE pathogenesis by: producing autoantibodies, activating autoreactive T cells, and secreting cytokines. We discuss how certain autoreactive B cells are selectively activated, with emphasis on the roles of key Toll-like receptors (TLRs). Although TLR7, which recognizes ssRNA, promotes autoimmune disease, TLR9, which recognizes DNA, unexpectedly regulates disease, despite being required for the secretion of anti-chromatin autoantibodies. We describe positive feedback loops involving B cells, T cells, DCs and soluble mediators and how these networks are regulated by TLR signals.

Langerhans cells (LCs) are bone marrow (BM)–derived epidermal dendritic cells (DCs) that develop from precursors found in the dermis. Epidermal LCs are absent in transforming growth factor (TGF) β1-deficient mice. It is not clear whether TGFβ1 acts directly on LC precursors to promote maturation or whether it acts on accessory cells, which in turn affect LC precursors. In addition, the physiologic source of TGFβ1 is uncertain because BM chimera experiments showed that neither hematopoietic nor nonhematopoietic-derived TGFβ1 is required for LC development. To address these issues, we created mice transgenic for a bacterial artificial chromosome (BAC) containing the gene for human Langerin into which Cre recombinase had been inserted by homologous recombination (Langerin-Cre). These mice express Cre selectively in LCs, and they were bred to floxed TGFβRII and TGFβ1 mice, thereby generating mice with LCs that either cannot respond to or generate TGFβ1, respectively. Langerin-Cre TGFβRII mice had substantially reduced numbers of epidermal LCs, demonstrating that TGFβ1 acts directly on LCs in vivo. Interestingly, Langerin-Cre TGFβ1 mice also had very few LCs both in the steady state and after BM transplantation. Thus, TGFβ1 derived from LCs acts directly on LCs through an autocrine/paracrine loop, and it is required for LC development and/or survival.

The study of murine memory B cells has been limited by small cell numbers and the lack of a definitive marker. We have addressed some of these difficulties with hapten-specific transgenic (Tg) mouse models that yield relatively large numbers of antigen-specific memory B cells upon immunization. Using these models, along with a 5-bromo-2′-deoxyuridine (BrdU) pulse-label strategy, we compared memory cells to their naive precursors in a comprehensive flow cytometric survey, thus revealing several new murine memory B cell markers. Most interestingly, memory cells were phenotypically heterogeneous. Particularly surprising was the finding of an unmutated memory B cell subset identified by the expression of CD80 and CD35. We confirmed these findings in an analogous V region knock-in mouse and/or in non-Tg mice. There also was anatomic heterogeneity, with BrdU+ memory cells residing not just in the marginal zone, as had been thought, but also in splenic follicles. These studies impact the current understanding of murine memory B cells by identifying new phenotypes and by challenging assumptions about the location and V region mutation status of memory cells. The apparent heterogeneity in the memory compartment implies either different origins and/or different functions, which we discuss.

The precise roles of B cells in promoting the pathogenesis of type 1 diabetes remain undefined. Here, we demonstrate that B cell depletion in mice can prevent or delay diabetes, reverse diabetes after frank hyperglycemia, and lead to the development of cells that suppress disease. To determine the efficacy and potential mechanism of therapeutic B cell depletion, we generated a transgenic NOD mouse expressing human CD20 (hCD20) on B cells. A single cycle of treatment with an antibody specific for hCD20 temporarily depleted B cells and significantly delayed and/or reduced the onset of diabetes. Furthermore, disease established to the point of clinical hyperglycemia could be reversed in over one-third of diabetic mice. Why B cell depletion is therapeutic for a variety of autoimmune diseases is unclear, although effects on antibodies, cytokines, and antigen presentation to T cells are thought to be important. In B cell–depleted NOD mice, we identified what we believe is a novel mechanism by which B cell depletion may lead to long-term remission through expansion of Tregs and regulatory B cells. Our results demonstrate clinical efficacy even in established disease and identify mechanisms for therapeutic action that will guide design and evaluation of parallel studies in patients.

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603–607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837–847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-α, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

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.