On days 1 and 2, fresh viral supernatant was added and spun at 2500 rpm for 90 min in the presence of 6 g/ml of polybrene. lymphocyte antigen-4 (CTLA-4) is a structural homologue of CD28 and a negative regulator required for T cell homeostasis and tolerance1,2. CTLA-4/mice develop a fatal lymphoproliferative disorder characterized by expansion of CD4+T cells into multiple non-lymphoid tissues35. While the importance of CTLA-4 is clear, several aspects of its mechanism in maintaining self-tolerance are poorly understood6. Specifically, it is not clear if specific self-antigens are involved in the expansion of CD4+T cells or in which lymphocyte populations CTLA-4 must be expressed to prevent the fatal lymphoproliferative disorder. CTLA-4 might regulate T cell activation by several mechanisms. CTLA-4 could exert cell-intrinsic inhibitory actions by competing with CD28 for their shared ligands, B7-1 and B7-27,8, or by delivering inhibitory signals that induce cell cycle arrest and prevent IL-2 production911, or by limiting T cell dwell time with antigen presenting cells (APCs)12. In addition, experiments with mixed bone-marrow (BM) chimeras showed that CTLA-4/T cells can be controlled by wild-type BM-derived cells13, suggesting a cell-extrinsic, dominant action of CTLA-4 in promoting tolerance similar to that exerted by Tregs. Indeed, a recent study demonstrated that CTLA-4-expressing Tregs can regulate CTLA-4/T cellsin vivo14. Although CTLA-4 is not required for all normal Treg activitiesin vitro15,16, it appears essential for Treg functionin vivo, since Treg-specific deletion of CTLA-4 caused spontaneous development of systemic lymphoproliferation and fatal disease17. Some evidence suggests that CTLA-4 expression on Tregs acts to decrease CD80 and CD86 expression on dendritic cells17,18but it is Mogroside IVe unclear whether thein vivostimulatory activity of APCs is affected by CTLA-4. Thus, the cellular site of action of CTLA-4 in controlling tolerance requires further examination. In addition, the antigen-specificity of expanding CD4+T cells in CTLA-4/mice has not been examined. Specifically, it is unclear whether these T cells are reactive generally to self-MHC, reactive to ubiquitous antigens, or reactive to multiple tissue-specific antigens. Some relationship to antigen specificity has been suggested from the observation that introducing rearranged -TCR transgenes onto the CTLA-4/background eliminates the fatal lymphoproliferation1921. However, no direct evidence has supported the interpretation that CTLA-4/mice generate an antigen-specific autoimmune disease. For example, spectratype analysis of Mogroside IVe the TCR CDR3 from T cells expanding in CTLA-4/mice revealed a diverse and unbiased repertoire, and was interpreted as antigen-independent T cell activation22. MYD88 Further, no reports have yet directly analyzed the antigen specificity by cloning of CD4+T cells from CTLA-4/mice. However, resolving the nature of the repertoire of T cells expanding in CTLA-4/mice is important for two reasons. First, mutations in CTLA-4 are associated with several autoimmune diseases, including hypothyroidism and type 1 diabetes23. Second, anti-CTLA-4 treatment is a potential immunotherapeutic approach in treatment of cancer24, so it is important to determine the potential for activating antigen-specific self-reactive T Mogroside IVe cells. To address these issues, we used a similar approach as used in analyzing the repertoire of Treg cells2527. By crossing the DO11.10 TCR transgene28onto CTLA-4/background to restrict TCR-specificity, the lethal multi-organ lymphoproliferative expansion of CD4+T cells in CTLA-4/mice was maintained but the mice showed Mogroside IVe a slightly reduced rate of lethality. In analyzing the specificity of the lymphoproliferative CD4+T cells, we show for the first time that CTLA-4/T cells infiltrating into peripheral non-lymphoid tissues are composed of separate populations of different tissue-specific T cells. We identified one autoantigen recognized by these tissue-infiltrating CTLA-4/T cells, isolated a specific TCR reactive to this antigen, and examined thein vivobehavior of T cell with this specificity. Our results show that CTLA-4 expressed on the antigen-specific effector T cells greatly Mogroside IVe diminishes their pathogenicityin vivo, but that CTLA-4 expression by Tregs is sufficient to control the accumulation of self-reactive tissue-specific effector T cells in target tissues, indicating both cell-intrinsic and cell non-autonomous actions of CTLA-4. == Results == == A fixed TCR repertoire does not eliminate lethal lymphoproliferation in CTLA-4/mice == We compared CTLA-4/mice with CTLA-4/mice expressing the DO11.10 TCR chain (DO) (Fig. 1a). CTLA-4/mice died uniformly by 4 weeks of age, while DOCTLA-4/mice.
On days 1 and 2, fresh viral supernatant was added and spun at 2500 rpm for 90 min in the presence of 6 g/ml of polybrene
