Unraveling the tolerogenic potential of lymph node fibroblastic reticular networks in autoimmune diabetes

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Project Summary: Impaired central and peripheral tolerance cause type-1 diabetes (T1D); b-cell antigen-reactive T cells escape negative selection in the thymus and regulation/deletion in lymph nodes. The role of professional antigen- presenting cells and regulatory T cells in maintaining peripheral tolerance has been demonstrated and is currently being evaluated for therapy of autoimmune diseases, including T1D. Conversely, non-professional antigen-presenting cells like lymph node stromal cells are more stable tolerogenic cells but their potential for therapeutic tolerance induction in T1D has not been tested yet. Fibroblastic reticular cells (FRCs) are a subtype of lymph node stromal cells that support and remodel the lymph node and the integrity of FRC reticula is critical for adaptive immunity. In transgenic mice, expression and presentation of model antigens by FRCs to specific T cells induced their proliferation followed by deletion and absence of MHC-II on lymph node stromal cells was associated with progressive development of autoimmunity. Thus, FRC engineering for peripheral overexpression of tissue-specific antigens, including those not expressed in the thymus, may be exploited for tolerance induction in the therapeutic treatment of autoimmunity. We showed that expression of insulin, a major b-cell antigen, the relative FRC frequency and the FRC reticular remodeling properties are decreased in T1D. Thus, to exploit the capability of FRCs to promote antigen-specific tolerance for therapeutic treatment of T1D, FRCs need to be engineered to (i) increase their frequency by therapeutic transplantation and (ii) overexpress disease-relevant antigens. We recently developed genetic engineering approaches to overexpress T1D-relevant antigens, including hybrid peptides, in FRCs and tissue engineering approaches to fabricate three-dimensional FRC reticula that recapitulate FRC organization in the lymph node paracortex. Engineered FRC reticula overexpressing b-cell antigens and lacking expression of costimulatory molecules engraft in mice to recruit antigen-specific T cells and in vitro, engineered FRCs promote engagement of specific T cells, anergy, and regulatory T cell expansion, which could promote antigen-specific tolerance in vivo. Thus, in aim 1.1, we will test the therapeutic application of our engineered FRC reticula for tolerance induction and mechanisms in vitro and in vivo in a murine T1D model with selected disease-relevant antigens. For clinical translation, in aim 1.2, we will evaluate whether reticula engineered with b-cell antigen-expressing FRCs from human lymph nodes can engage human T cells engineered for antigen specificity, which is important for clinical translation of our approach. Peripheral expression and presentation of tissue-specific antigens by lymph node stromal cells to autoreactive T cells escaping thymic deletion in lymph nodes could contribute to peripheral tolerance. In aim 2, we will test the contribution of b-cell antigen expression and presentation by lymph node FRCs on tolerization of b-cell antigen autoreactive CD8 T cells in a mouse model of T1D.