Project 1: In previous studies we have defined a potentially important set of human regulatory T cells that recognize self MHC and are therefore called self-MHC reactive (autoreactive) T cells. In essence, we showed that such cells exert their regulation (suppression) via interactions with activated B cells and the production of TGF-beta and IL-10. Furthermore, we showed that TGF-beta secretion required interaction between Beta7 costimulatory molecules on the B cell and CTLA-4 on the self-MHC reactive T cell. In the current studies we explored the interactions between self-MHC reactive regulatory T cells and stimulatory B cells necessary for the secretion of IL-10. We found that regulatory T cell lines stimulated by activated B cells produced increased amounts of IL-10 when cultured with IL-12. In addition, we found that secretion of IL-10 by such cells were inhibited if the latter are cultured in the presence of ICOS-Fc, a reagent that blocks interaction between ICOS on the T cell surface and ICOS ligand on the stimulatory B cell surface. In further studies we showed that patients with active SLE who have been previously shown to be prone to disturbances in regulatory cell function, have cells expressing increased expression of ICOS and suppression of autologous MLR's which was reversed by addition of ICOS-Fc. These studies thus link IL-10 production in regulatory T cells to a particular co-stimulatory molecule interaction and may imply that autoimmunity is, at least in part, controlled by regulatory T cells employing this mechanism. Project 2: In previous studies, we observed that treatment of mice with TNBS-colitis, a Th1 T cell-mediated colitis resembling human Crohn's disease, with anti-IL-12 induce apoptosis of Th1 T cells in the inflamed colon. This effect is reduced in mice carrying the lpr gene and in SJL/J mice treated with Fas-Fc suggesting that the induction of apoptosis by anti-IL-12 is mediated by the Fas signaling pathway, i.e., IL-12 normally prevents Fas-mediated apoptosis of Th1 T cells bearing the IL-12R. In the present studies, we investigated the biochemical basis of the anti-apoptotic effect of IL-12 using a human cell line, Kit225 cells, bearing an IL-12R and having susceptibility to Fas-mediated apoptosis. In initial studies we showed that IL-12 substantially reduced apoptosis induced by anti-Fas antibody; importantly, this effect was not due to IL-12 down-regulation of surface Fas expression. In addition, we showed that IL-12 inhibited apoptosis mediated by Trail, a TNF-receptor superfamily molecule that induces apoptosis via DR4 and DR5 (but not Fas). In further studies we analyzed discrete steps of the Fas apoptotic pathway to determine the mechanism of the anti-apoptotic effects of IL-12. First we showed that IL-12 does not interfere with the formation of the DISC (death-inducing signaling complex) which forms as a result of Fas-signaling; this was done by showing via immunoprecipitation (IP) studiesthat cells cultured with IL-12 contains IP's containing FLICE, FADD, and Fas. In further studies, we showed that the anti-apoptotic effect of IL-12 does not involve mitochondrial apoptotic mechanisms since IL-12 does not suppress UV irradiation-induced apoptosis and IL-12 did not affect Bcl-2 or Bcl-Lx levels as determined by Western blot. In a final series of studies designed to localize the effect of IL-12, we showed that IL-12 treatment of Kit225 cells results in the major down-regulation of caspase 8 activity as well as some down-regulation of caspase 3 activity, suggesting that the anti-apoptotic effect is specific to a caspase that functions early in the caspase cascade. One possible mechanism of this effect on caspase 8 activity is the activation of an IAP protein specific for its caspase. Indeed, we showed that IL-12 treatment resulted in upregulation of IAP-2 protein and we are provisionally considering this effect to be the mechanism of IL-12 anti-apoptosis.