Conversion of anergic non-regulatory into Foxp3- IL-10+ regulatory T cells by dendritic cells in vivo

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T cell anergy is a long known mechanism of T cell tolerance. However, it remains unclear why such T cells with unwanted T cell reactivity are not eliminated, since their passive persistence bears the potential risk of reactivation. We have shown before in vitro that anergic T cells can be converted by immature dendritic cells (DCs) into Foxp3- IL-10+ regulatory T cells (Tr1). Surprisingly, the expression of both CD28 and CTLA-4 molecules was functionally required for this process. Since the expression of CTLA-4 is only transient the anergy-to-Tr1 conversion can occur only in a small time window. This proposal aims to show that anergy-to-Tr1 conversion can occur in vivo in the spleen and requires CD28 and CTLA-4 expression by the anergic T cells. Inducible deletion of either molecule in T cell receptor (TCR)-transgenic T cells will give clear answers to this questions. The use of gene-deficient mice with deletions of DC subset-specific transcription factors will allow to determine the contribution of CD8a+ or CD11b+ conventional DCs or plasmacytoid DCs in anergy induction and anergy-to-Tr1 conversion. Furthermore, we want to demonstrate that in vivo persistent anergic T cells can serve as a reservoir pool of memory T cells awaiting their reactivation into Tr1 cells. Adapted protocols resulting from this knowledge will then be used to suppress experimental autoimmune-encephalomyelitis (EAE) and asthma in mice. Our preliminary data indicate that high antigen doses as used for anergy induction and anergy-to-Tr1 conversion may override the availability of TGF-b to induce Foxp3+ inducible regulatory T cells (iTreg) as we have shown before by implanting osmotic minipumps in mice to deliver low doses of antigen. This question will be addressed by transfer of TCR-transgenic T cells with defective TGF-b receptor or reversely, by injecting high doses of TGF-b into mice with TGF-b receptor competent T cells during anergy induction or anergy-to-Tr1 conversion.Together, the proposed experiments will allow to attribute an active immunological role in T cell tolerance to anergic T cells in vivo as well as the functional role of CD28, CTLA-4 and TGF-b in the anergy-to-Tr1 conversion process and to identify the participating DC subsets.