For mouse data, unpaired, two-tailed, one-way ANOVA with 95% confidence interval was used to compare more than two groups, with Tukeys multiple-comparison test as a posttest ( 0

For mouse data, unpaired, two-tailed, one-way ANOVA with 95% confidence interval was used to compare more than two groups, with Tukeys multiple-comparison test as a posttest ( 0.05). mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine140. The frequency of CD4+ T cells specific for U1-70(131-150):I-Ek (without phosphorylation) correlates with disease severity and Kobe2602 antiCU1-70 autoantibody production. These T cells also express RORt and produce IL-17A. Furthermore, the U1-70Cspecific CD4+ Rabbit polyclonal to NOD1 T cells that produce IL-17A are Kobe2602 detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4+ T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease. Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop high-titer, highly specific, isotype-switched autoantibodies against DNA- and RNA- containing autoantigens (1). U1-70, U1-A, and U1-C, together with U1-RNA and the seven Smith proteins, compose the U1-small nuclear ribonucleoprotein (U1-snRNP) complex. This U1-snRNP complex is one component of the spliceosome (1, 2). A subset of patients with SLE, and all patients with mixed connective tissue disease (MCTD), develop autoantibodies against U1-snRNP, and U1-70 in particular (1, 3C5). Anti-snRNP autoantibodies are detectable before overt disease Kobe2602 in SLE in what is termed a pathogenic autoimmunity phase (6). The role of CD4+ T helper (Th) cells in SLE is a long-standing area of investigation, with evidence of both T-cellCdependent and Cindependent autoantibody production. In support of T-cellCdependent mechanisms, CD4+ T cells are Kobe2602 required for disease in the MRL/murine model of lupus (7, 8), a model in which mice deficient in develop spontaneous autoimmunity (9). MRL/mice with a limited T-cell receptor (TCR) repertoire have increased survival and develop fewer autoantibodies (10), indicating that antigen-specific T-cell help may be required for disease. Furthermore, adoptive transfer of CD4+ T cells from MRL/mice into nonautoimmune anti-snRNP B-cell receptor (BCR) transgenic mice is sufficient for autoantibody synthesis, indicating that cognate T- and B-cell interactions are important for the development of antiCU1-snRNP autoantibodies specifically (11). Despite evidence that antigen-specific T-cell help is required for autoantibody production and full manifestation of disease, T-cellCindependent autoantibody production has been observed in the pristane model of lupus (12), as well as in MRL/mice expressing a transgenic BCR recognizing self-IgG2a (13). In these cases, Toll-like receptor 7 (TLR7) signaling and interferons were required for autoantibodies against RNA-containing antigens. In addition, autoantibodies were sufficient to induce disease in nonautoimmune mice following adoptive transfer of antibodies from the BXD2 murine model of lupus (14); however, in BXD2 mice, treatment with CTLA4Ig before disease onset resulted in long-term suppression of autoantibodies (15), indicating that CD4+ T cells may be important early on, before autoantibody production. Various therapies that target T cells are being investigated in SLE patients (16), including antigen-specific tolerizing therapy using a peptide derived from U1-70 (17). The role of antigen-specific CD4+ T cells in disease remains unclear, however, in part because the field has lacked a reagent for use in studying these cells directly. Here we report the generation of the first MHC class II tetramers to detect autoreactive CD4+ T cells in Mrl/mice. These tetramers were used to identify a population of CD4+ T cells that recognize the self-protein U1-70 and produce the proinflammatory cytokine IL-17A. Such cells appear to be present not only in the MRL/mice, but also in patients with SLE and MCTD. Results U1-70 Tetramers Specifically Detect MRL/CD4+ T Cells. Our approach to generating stable, relevant tetramers to test in MRL/mice was to identify peptides from known lupus autoantigens that (mice, and ((MCC) peptide (88C103), which binds I-Ek (22) (Fig. 1mice and has entered pivotal Phase 3 clinical trials in human SLE patients, where.

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