Using a co-culture system in which pathogenic and regulatory T cells could be distinguished by allotypic markers, we have demonstrated that BV8S2- but not BV10-specific T cells could recognize cellular determinants and inhibit proliferation, migration into the CNS, and encephalitogenic activity of MBP-specific T cells. The regulatory functions of the TCR-specific T cells could be activated by direct cell-cell contact with the MBP-specific T cells, and more extensive activation with soluble BV8S2-39-59 peptide did not produce additional effects. In a previous study utilizing a similar co-culture system, we observed a lesser degree of activation induced by cell-cell contact, with additional effects of secreted factors released after activation with soluble TCR peptide. These differences probably reflect variations in expression of BV8S2 by the encephalitogenic MBP-specific T cells, and the properties and degree of activation of the TCR-reactive T cells. It is important to note that the MBP-specific T cell lines used in the current experiments typically contained about 30% BV8S2+ T cells, and the profound inhibitory effects induced by the BV8S2-specific T cells after specific cell-cell interaction would require production of soluble factors that would affect bystander MBP-specific T cells expressing BV genes other than BV8S2. Because no regulatory effects were produced by BV10-specific T cells, we conclude that activation of the regulatory TCR-specific T cells was highly specific, and involved production of inhibitory cytokines that affected local activation of MBP-specific T cells. The TCR-reactive T cells strongly inhibited proliferation of the MBP-specific T cells, resulting in a reduction in cell number and percentage of cells in the mixed culture. However, the remaining MBP-specific T cells did not have significant changes in expression of activation and adhesion molecules, with the exception of L-selectin, which was enhanced after co-culture with either BV8S2- or BV10-specific T cells. Given the reduced number and function of the MBP-specific T cells after co-culture with BV8S2-specific T cells, the remaining MBP-reactive T cells had a strongly reduced capacity to transfer EAE on a per/cell basis. This event was reflected by the almost total lack of transferred MBP-specific T cells in the CNS of protected rats, and a substantial reduction in recruitment of host inflammatory cells. Activated TCR-specific T cells that were co-transferred into recipient rats also migrated into the CNS, but there was essentially no difference in the rate of migration of BV8S2- and BV10-specific cells. However, in the presence of the BV8S2-reactive T cells, few if any transferred MBP-specific or recruited CD4+ T cells appeared in the CNS of protected rats, suggesting that the BV8S2-specific T cells provided ongoing regulation of inflammation within the CNS. It is noteworthy that the rate of migration into CNS of MBP-specific T cells was about sevenfold higher than the rate of migration of BV8S2-specific T cells, suggesting a greater degree of retention due possibly to encountering higher concentrations of specific antigen. What are the implications of the current study with regard to our ongoing clinical studies using TCR peptides to produce immune regulation in MS patients? Foremost is the demonstration that TCR-reactive T cells specific for dominant BV peptides can specifically recognize cellular determinants on activated MBP-reactive T cells, leading to the production of as-yet-unidentified inhibitory cytokines that can prevent activation and encephalitogenic activity of BV8S2+ as well as bystander T cells. Moreover, we have demonstrated directly that BV8S2-reactive T cells can migrate into the CNS, where they further regulate entry and reactivation of inflammatory CD4+ T cells. This result suggests that the TCR-reactive T cells can exert regulation within the affected target organ, and thus may influence ongoing disease. This interpretation is consistent with our observations that successful vaccination of MS patients with TCR BV5S2 peptides can stabilize or reverse clinical progression, and that BV5S2-specific T cells can become activated by cell-cell contact with BV5+ T cells and produce soluble inhibitory factors for Th1 cells. The major challenges confronting the application of TCR vaccination in MS include the identification of disease-associated target V genes, and the induction of biologically meaningful frequencies of regulatory TCR-specific T cells that can seek out and regulate encephalitogenic specificities. To this end, we are continuing our quest to identify potentially relevant CNS target antigens and associated V gene expression by reactive T cells, as well as to develop multi-component, highly antigenic TCR peptide vaccines.
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