Type 1 diabetes (T1D) is caused by aberrant activation of autoreactive T cells specific for the islet beta cells. How islet-specific T cells evade tolerance to become effector T cells is unknown, but it is believed that an altered gut microbiota plays a role. Possible mechanisms include bystander activation of autoreactive T cells in the gut or "molecular mimicry" from cross-reactivity between gut microbiota-derived peptides and islet-derived epitopes. To investigate these mechanisms, we use two islet-specific CD8 + T cell clones and the non-obese diabetic mouse model of type 1 diabetes. Both insulin-specific G9C8 cells and IGRP-specific 8.3 cells underwent early activation and proliferation in the pancreatic draining lymph nodes but not in the Peyer's patches or mesenteric lymph nodes. Mutation of the endogenous epitope for G9C8 cells abolished their CD69 upregulation and proliferation, ruling out G9C8 cell activation by a gut microbiota derived peptide and molecular mimicry. However, previously activated islet-specific effector memory cells but not na€ ıve cells migrated into the Peyer's patches where they increased their cytotoxic function. Oral delivery of butyrate, a microbiota derived antiinflammatory metabolite, reduced IGRP-specific cytotoxic function. Thus, while initial activation of islet-specific CD8 + T cells occurred in the pancreatic lymph nodes, activated cells trafficked through the gut lymphoid tissues where they gained additional effector function via non-specific bystander activation influenced by the gut microbiota.

The autoimmune disease type 1 diabetes is predominantly mediated by CD8+ cytotoxic T‐cell destruction of islet beta cells, of which islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP)206–214 is a dominant target antigen specificity. Previously, we found that a liposome‐based antigen‐specific immunotherapy encapsulating the CD4+ T‐cell islet epitope 2.5mim together with the nuclear factor‐κB inhibitor calcitriol induced regulatory T cells and protected from diabetes in NOD mice. Here we investigated whether the same system delivering IGRP206–214 could induce antigen‐specific CD8+ T‐cell‐targeted immune regulation and delay diabetes. Subcutaneous administration of IGRP206–214/calcitriol liposomes transiently activated and expanded IGRP‐specific T‐cell receptor transgenic 8.3 CD8+ T cells. Liposomal co‐delivery of calcitriol was required to optimally suppress endogenous IGRP‐specific CD8+ T‐cell interferon‐γ production and cytotoxicity. Concordantly, a short course of IGRP206–214/calcitriol liposomes delayed diabetes progression and reduced insulitis. However, when IGRP206–214/calcitriol liposomes were delivered together with 2.5mim/calcitriol liposomes, disease protection was not observed and the regulatory effect of 2.5mim/calcitriol liposomes was abrogated. Thus, tolerogenic liposomes that target either a dominant CD8+ or a CD4+ T‐cell islet epitope can delay diabetes progression but combining multiple epitopes does not enhance protection.