Autoimmune diseases are diverse, necessitating a mechanistic understanding of the protracted disease process to improve diagnostic accuracy and therapeutic efficacy. From human and animal model studies, it has become clear that T cells are critical for autoimmune pathogenesis but the involvement of B cells is unmistakable, as evidenced by the efficacy of B cell depletion therapy in alleviating systemic and organ-specific autoimmune conditions. This paper reviews the roles that kinds of murine models have played and continue to play in developing mechanistic understanding of autoimmune diseases, with particular though not exclusive attention to B cells emphasizing their unique contributions to autoimmunity via adaptive and innate mechanisms.

The pathogenesis of Systemic lupus erythematosus (SLE) is driven by autoreactive B and T cells and autoinflammation. Impaired apoptosis in T and B cells has been shown to result in autoimmune disease, and we recently demonstrated that reducing apoptosis in B cells alone, by B cell-specific deletion of Bim (B.Bim f/f) could lead to SLE with prominent features of Sjogren’s Syndrome in C57BL/6 mice. Consistently, B cell targeting therapies are successful in reducing SLE pathogenesis in mouse models, however, their clinical success requires a better understanding of the contributions of B cells and the mechanisms that drive inflammation to autoimmunity. To address the role of inflammation in the development and progression of autoimmunity we generated a novel mouse model that lacks ZFAND6 (Zfand6 −/−) and crossed with the B.Bim f/fmice. ZFAND6 is an A-20 like ZF domain-containing protein with a potential role in the negative regulation of NF-kB activation, however, its physiological function is unknown. The compound mutant mice displayed accelerated and exacerbated overlapping lupus and Sjogren’s autoimmune symptoms with a higher incidence of splenomegaly and kidney pathology than either single mutant alone including the early appearance of anti-SSA, anti-SSB, and anti-RNP autoantibodies. The compound mutant mice displayed increased spontaneous and TLR-induced TNFa and IL-6 and B cells from these mice produced much greater levels of these cytokines in response to TLR7 and TLR9. These results suggest that inappropriate B cell survival and persistent inflammation cooperate in the initiation and severity of lupus pathogenesis. Sylvester Comprehensive Cancer Center, University of Miami, Miami FL

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren’s Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton’s tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Overlap syndromes are inflammatory rheumatic conditions in which patients have clinical manifestations suggestive of multiple distinct immune diseases. The diseases most commonly involved in overlap syndromes include rheumatoid arthritis, lupus, scleroderma, and myositis. The most well-characterized overlap syndrome, mixed connective tissue disease (MCTD), is defined by antiRNP autoimmunity along with features of at least two of these four conditions, and very often includes sufficient lupus manifestations to fulfill lupus classification criteria. Overlap syndromes are generally less common and often more mild than the conditions they encompass. The prevalence of MCTD, for example, is approximately one-twentieth that of SLE, and manifestations of severe lupus such as nephritis are much less common in MCTD. Some autoantigen systems are particularly linked with overlap syndromes, such as RNP and MCTD. Overlap syndromes provide unique opportunities to understand links between autoimmunity and end organ immune targeting. While insights can be extrapolated from studies of disease processes present in overlap, few treatment trials have focused specifically on overlap syndromes themselves.

OBJECTIVE:Previously thought to involve primarily the microvasculature, systemic sclerosis (SSc) has been increasingly linked to macrovascular disease. Cardiovascular (CV) and cerebrovascular disease are responsible for 20-30% of mortality in SSc, but few studies have shown an independent association between SSc and stroke. We assessed whether SSc was an independent risk factor for ischemic stroke. METHODS:We conducted a retrospective cohort study using the national Veterans Affairs (VA) administrative database containing records from 1999 to 2014. We obtained data for all patients with a diagnosis of SSc as well as 2 controls per SSc patient matched on sex, race, smoking status, and VA site. All patients were followed until development of ischemic stroke, death, or last encounter. We used a Cox proportional hazard regression model to estimate risk of ischemic stroke, with adjustments for CV comorbidities (hypertension, diabetes, atrial fibrillation, non-cerebrovascular atherosclerotic disease, hyperlipidemia), baseline medication use (aspirin, nonsteroidal antiinflammatory drugs), and Medicare enrollment. RESULTS:Among 4545 individuals with SSc (83% male, mean age 60.9 yrs), the incidence rate of ischemic stroke was 15.3 per 1000 person-years (vs 12.2 in the control cohort), with an unadjusted HR 1.28 (95% CI 1.11-1.47). The adjusted HR was 1.21 (95% CI 1.05-1.40) after adjusting for baseline CV risk factors, medications, and Medicare enrollment. CONCLUSION:SSc is independently associated with a higher risk of ischemic stroke among US veterans. Patients with SSc represent a population likely to benefit from targeted stroke screening or prevention therapies.

Objective. Raynaud's phenomenon (RP) is common in anti-RNP-positive patients with rheumatic diseases but is not itself known to be caused by autoimmunity. The aim of this study was to assess autoantibodies that could mediate this process. Methods. Antibodies derived from patient sera and from murine models of anti-RNP autoimmunity were screened for the ability to induce RP-like tissue ischemia and endothelial cell apoptosis in murine models and in vitro systems. Results. RNP-positive sera from RP patients and murine sera from RNP-positive B cell adoptive transfer recipients induced RP-like tissue ischemia and endothelial cell apoptosis. Proteomic analysis identified cytokeratin 10 (K10) as a candidate autoantigen in RP. Monoclonal anti-K10 antibodies reproduced patterns of ischemic tissue loss and endothelial cell apoptosis; K10 knockout or depletion of anti-K10 activity in serum was protective. Cold exposure enhanced K10 expression and in vivo tissue loss. Conclusion. Anti-K10 antibodies are sufficient to mediate RP-like ischemia in murine models and are implicated in the pathogenesis of RP in patients with anti-RNP autoimmunity.

Background Our laboratory has previously characterized defects in humoral B cell responses of aged mice and humans. These defects include: the reduction in activation-induced cytidine deaminase (AID), required for the generation of optimal antibody responses, and the reduction in the percentage/number of the subset of switched memory B cells (1). AID and switched memory B cells have been proposed to be effective predictive biomarkers of vaccine responses (2). Moreover, we have shown that aging is characterized by increased systemic inflammation which induces intrinsic B cell inflammation, measured by intracellular (ic) TNF-α, and this significantly decreases the capacity of the same B cells to make protective antibodies in response to vaccination (3). Other marker of B cell intrinsic inflammation is micro-RNA (miR) expression, particularly miR-16 and miR-155, which is increased in elderly B cells and negatively correlated with B cell function (4). Objectives Our goal for this study was to evaluate B cell phenotype and function in RA patients treated Methotrexate (MTX), alone or together with anti-TNF-α. We hypothesized that patients treated with anti-TNF-α will show improved B cell function due to reduction in icTNF-α. Methods We recruited 9 RA patients, 5 patients on MTX and 4 on MTX/anti-TNF-α. We measured the relevant B cell subsets in blood (Naïve, switched memory, IgM memory and late memory) by flow cytometry. Staining was performed with antibodies specific for CD19, CD27 and IgD. In addition, we isolated blood B cells using magnetic beads, and measured the expression of miR-16 and miR-155 on blood B cells by qPCR. Results Preliminary data showed that the percentages of switched memory (IgD-CD27+) B cells are significantly higher (p<0.003) in patients undergoing MTX/anti-TNF-α therapy. We also observed a significant decrease in naïve B cell percentages (p<0.028). Preliminary results also showed a decrease in the mRNA expression of both miR-16 and miR-155 in patients on combination therapy compared to MTX alone. Conclusions These results support the hypothesis that therapy with anti-TNF-α is beneficial for improving B cell function in RA patients, as compared to MTX therapy alone. Future experiments will seek to evaluate intrinsic B cell TNF levels in these patients and correlate them with measurements of B cell function. Treatment with anti-TNF-α may be able to block the excessive amounts of systemic TNF-α and in turn B cell intrinsic TNF-α which could improve the antibody responses and the risk of infections in RA patients undergoing therapy. References Frasca, D., and B. B. Blomberg. 2013. Age effects on mouse and human B cells. Immunol Res. 57(1–3): 354–360. Frasca, D., A. Diaz, M. Romero, M. Phillips, N. V. Mendez, A. M. Landin, and B. B. Blomberg. 2012. Unique biomarkers for B-cell function predict the serum response to pandemic H1N1 influenza vaccine. Int Immunol. 24(3): 175–182. Frasca, D., M. Romero, A. Diaz, S. Alter-Wolf, M. Ratliff, A. M. Landin, R. L. Riley, and B. B. Blomberg. 2012. A Molecular Mechanism for TNF-alpha-Mediated Downregulation of B Cell Responses. J Immunol 188: 279–286. Frasca, D., A. Diaz, M. Romero, F. Ferraci, and B. B. Blomberg. 2015. MicroRNAs miR-155 and miR-16 Decrease AID and E47 in B Cells from Elderly Individuals. J. Immunol.195 (5): 2134–2140. Disclosure of Interest None declared

Objective The objective of this paper is to develop novel classification criteria to distinguish between unclear systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) cases. Methods A total of 205 variables from 111 SLE and 55 MCTD patients were evaluated to uncover unique molecular and clinical markers for each disease. Binomial logistic regressions (BLRs) were performed on currently used SLE and MCTD classification criteria sets to obtain six reduced models with power to discriminate between unclear SLE and MCTD patients that were confirmed by receiving operating characteristic (ROC) curve. Decision trees were employed to delineate novel classification rules to discriminate between unclear SLE and MCTD patients. Results SLE and MCTD patients exhibited contrasting molecular markers and clinical manifestations. Furthermore, reduced models highlighted SLE patients exhibiting prevalence of skin rashes and renal disease while MCTD cases show dominance of myositis and muscle weakness. Additionally decision tree analyses revealed a novel classification rule tailored to differentiate unclear SLE and MCTD patients (Lu-vs-M) with an overall accuracy of 88%. Conclusions Validation of our novel proposed classification rule (Lu-vs-M) includes novel contrasting characteristics (calcinosis, CPK elevated and anti-IgM reactivity for U1-70K, U1A and U1C) between SLE and MCTD patients and showed a 33% improvement in distinguishing these disorders when compared to currently used classification criteria sets. Pending additional validation, our novel classification rule is a promising method to distinguish between patients with unclear SLE and MCTD diagnosis.

Introduction Anti-RNP autoantibodies occur either in mixed connective tissue disease (MCTD) (with a frequently favorable prognosis), or in systemic lupus erythematosus (SLE) cases with aggressive major organ disease. It is uncertain how to assess for the risk of severe disease in anti-RNP + patients. Methods Following institutional review board-approved protocols, clinical data and blood were collected from patients with known or suspected anti-RNP autoimmunity and normal controls in a cohort study. Samples were screened for parameters of immune activation. Groups were compared based on clinical diagnoses, disease classification criteria, disease activity and specific end-organ clinical manifestations. Results Ninety-seven per cent of patients satisfying Alarcon-Segovia MCTD criteria also met Systemic Lupus International Collaborating Clinic (SLICC) SLE criteria, while 47% of the anti-RNP + SLE patients also met MCTD criteria. Among SLICC SLE patients, MCTD criteria were associated with reduced rates of renal disease (odds ratio (OR) 4.3, 95% confidence interval (CI) 1.3–14.0), increased rates of Raynaud's phenomenon (OR 3.5, 95% CI 1.3–9.5) and increased serum B-cell maturation antigen, transmembrane activator and CAML interactor and TNFα levels. Circulating immune markers and markers of type I interferon activation were not effective at distinguishing clinical subgroups. Conclusions Among anti-RNP patients, the question of MCTD versus SLE is not either/or: most MCTD patients also have lupus. MCTD classification criteria (but not a broad set of immune markers) distinguish a subset of SLE patients at reduced risk for renal disease.

T cells have been shown to mediate aspects of anti-ribonucleoprotein (RNP) autoimmunity, and are a potential target of therapy in lupus and related diseases. In this study, we assessed the relevance of a conserved class of anti-RNP T cells to autoimmune disease expression and therapy. Our data show that anti-RNP T cell selection induced a limited set of homologous CDR3 motifs at high frequency. Homologous CDR3 motifs have been reported in other autoimmune diseases. Vaccination with irradiated anti-RNP (but not anti–tetanus toxoid) CD4 + cells induced remission of anti-RNP–associated nephritis in ‡80% of treated mice, even with donor/ recipient MHC class II mismatch, and in both induced and spontaneous autoimmunity. Vaccine responder sera inhibited anti-70k T cell proliferation and bound hybridomas expressing the conserved CDR3 motifs. Our data indicate that a limited set of TCR CDR3 motifs may be important for the pathogenesis of anti-RNP lupus and other autoimmune diseases. The ability to target a consistent set of pathogenic T cells between individuals and across class II restrictions may allow for the more practical development of a standardized anti-RNP T cell vaccine preparation useful for multiple patients. A utoimmunity to small nuclear ribonucleoprotein (snRNP) autoantigens occurs in systemic lupus erythematosus (SLE) and is particularly prevalent in early-onset and severe cases (1, 2). We have developed an induced murine model of anti-snRNP autoimmunity by immunizing mice with the 70kD subunit of the U1 snRNP (70k) along with the RNA component of this ribonucleoprotein (RNP), U1-RNA, a known TLR3 and TLR7 agonist (3, 4). This model shares clinical and immune features of human anti-snRNP autoimmunity, including spreading of the immune response from the 70kD subunit of the U1 snRNP (70k) to multiple other autoantigens, as well as the potential to induce nephritis (5, 6). Anti-RNP responses and nephritis (in the absence of anti-dsDNA or anti-chromatin Abs) have also been reported to develop spontaneously in the Trex1 deficiency model of SLE (7). These models provide opportunities to develop new therapeutic approaches to anti-RNP autoimmunity. CD4 + T cells play an important role in anti-RNP autoimmunity and have previously been shown to be sufficient to induce RNP-associated antigenic spreading (8). Anti-RNP CD4 + T cells appear at high frequency (compared with healthy controls) in PBMCs in SLE patients and in the spleen and lesional tissues of anti-RNP autoimmune mice (9, 10). Adoptive transfer of anti-RNP–specific CD4 + T cells is sufficient to transfer nephritis …