40. Transgene Product-Specific Treg Are Initially Induced in the Celiac Lymph Node During Tolerance Induction by Hepatic AAV Gene Transfer

Previous work by us and several other laboratories has shown that liver-directed gene transfer can induce transgene product-specific immune tolerance. Induction of CD4+CD25+FoxP3+ regulatory T cells (Treg) is a critical component of the tolerance mechanism. Gaps in knowledge exist about the identity of the antigen-presenting cells (APCs) and the origin of peripherally induced Treg. To characterize the interaction of adeno-associated virus (AAV) antigen expression in the liver with immune cells, we used AAV8-EF1a vectors, which have a high tropism for murine liver, expressing either a secreted ovalbumin (AAV8-ova) or cytoplasmic ovalbumin (AAV8-Cytoova). In Treg-deficient mice transgenic for an ova-specific CD4+ T cell receptor (DO11.10-tg Rag-2-/- BALB/c), Treg were induced with either vector. For secreted ova, multiple organs (liver, spleen, lymph nodes [LN]) showed a similar level and time course of Treg induction, with greater than 1% of the CD4+ T cells being Treg starting at 4 weeks. Substantial T cell activation using the very early activation marker CD69 was also observed in these tissues starting at 3 weeks after AAV8-ova. However, Treg induction was observed in the celiac LN (one of the liver-draining LN) as early as 3 weeks after AAV8-Cyto-ova gene transfer, followed by induction of Treg in other tissues by 4 weeks. Interestingly, T cell activation was notably higher in liver and celiac LN after AAV8-Cyto-ova with over 70% activated CD4+ T cells after 4 weeks. In addition, T cell activation was observed earlier in all tissues after AAV8-Cyto-ova when compared to AAV8-ova. Literature data document a role for tolerogenic antigen presentation in the liver itself via sinusoidal endothelial cells and possibly hepatocytes. Our data suggest an important role of conventional mechanisms of adaptive immunity involving draining LNs that survey tissue antigens leading to early T cell activation and Treg induction of hepatic expressed non-systemic antigens. To start defining the critical APCs, BALBc mice were given AAV8-ova vector two weeks prior to adoptive transfer of CellTrace Violet-labeled CD4+ T cells from DO11.10 donor mice and their proliferation was measured using flow cytometry after 5 days. Half of these mice had their Kupffer cells and M1 macrophages inactivated with Gadolinium chloride for two days prior to DO11.10 adoptive transfer. DO11.10 cells proliferated in an antigen-specific manner, and proliferation was blocked by Gadolinium chloride, supporting a role for professional APCs in presentation of hepatic transgene product. Our data suggest and reinforce the importance of Kupffer cells as key APCs for hepatic antigens and further characterize the impact of the route of antigen expression on T cell activation and Treg induction.