T cell vaccination (TCV) is proven to be an effective means of regulating autoreactive T cells through regulatory network of CD8+ and CD4+ T cell subsets. Both regulatory T cell (Treg) subsets are thought to contribute to suppression of circulating myelin basic protein (MBP)-reactive T cells and some clinical improvement seen in clinical trials in multiple sclerosis (MS). CD8+ T cells induced by TCV belong to cytotoxic anti-idiotypic T cell subset interacting with the T cell receptors of MBP-reactive T cells. In contrast, the nature of CD4+ Treg subset is poorly understood. They exhibit unique regulatory properties through the production of IL-10 but not Th1 cytokines, resulting in a potent inhibitory effect on MBP-reactive T cells. Their reactivity and inhibition appear restricted to activated T cells, regardless of antigen specificity, but not resting cells. Further evidence suggests that CD4+ Treg cells induced by TCV react with and are activated by endogenously processed IL-2 receptor alpha chain (IL-2Ra). These findings have begun to unfold the unique properties and functional role of CD4+ Treg induced by TCV in the regulation of autoreactive T cells in humans. In this study, we propose to investigate the central hypothesis that TCV induces the activation and in vivo expansion of CD4+ Treg cells through interaction with IL-2Ra expressed on activated autoreactive T cells and that IL-2Ra has an anti-inflammatory property by inducing a CD4+ Treg response. The study has three specific aims.
In Aim 1, the role of TCV in in vivo expansion of pre-existing CD4+ Treg pool will be determined by examining the precursor frequency of CD4+ Treg cells in peripheral blood mononuclear cells (PBMC) derived from MS patients before and after TCV. The results will be analyzed to correlate with serum cytokines, the frequency of MBP-reactive T cells and other parameters in the same patients. Cloned CD4+ Treg cell lines will be characterized in detail to address their functional role and heterogeneity, reactivity and MHC restriction, mode of induction and interaction with various target T cells.
Aim 2 will focus on the reactivity pattern and interaction of CD4+ Treg cells with IL-2Ra using well-characterized T cell lines in two experimental systems by testing the reactivity of CD4+ Treg T cell lines to endogenously processed IL-2Ra using autologous B cell transfectants and to soluble IL-2Rot using recombinant protein in functional assays. Additional experiments will delineate the region(s) of IL-2Ra preferably recognized by CD4+ Treg using target cells transfected with truncated gene segments and overlapping peptides.
In Aim 3, we will define the potency and specificity of IL-2Rc_ protein as a specific agent for the direct induction of CD4+ Treg cell response in PBMC as well as the role of TCV in this process by analyzing the T cell responses, as the frequency of reactive CD4+ Treg cells, to IL-2R(x protein or preferably recognized peptides in pre- and postvaccination PBMC and those of controls. Parallel experiments are designed to evaluate the inhibitory effect of IL-2Ra on MBP-reactive T cell responses through its intrinsic property of inducing CD4+ Treg cell response in MBP-primed PBMC cultures. The overall results will provide new insights into the role of IL- 2Ro_ in regulation of pro-inflammatory T cell response in autoimmune conditions. ? ?
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