Antigen-specific peptide immunotherapy for treatment and prevention of type 1 diabetes

Abstract

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic b cells, involving CD4+and CD8+T cells.nCurrently, there is no treatment for T1D, and disease management relies on insulin replacement therapy associated with major complications. Therefore, there is an urgent need for effective T1D therapy without the need for systemic immunosuppression. The ideal immunotherapy would be safe, cheap and specificallyninactivate or eliminate pathogenic islet-specific T cells and/ornincrease islet-specific regulatory T cells (Tregs). To this end, I have investigated the utility of liposome delivered antigen-specific therapy for T1D.Using the non-obese diabetic (NOD) mouse model of T1D, the Hamilton-Williams lab has shown previously that liposomes co-encapsulating a CD4-targeted islet antigen and NF-kB inhibitor induced antigen-specific induction of regulatory CD4+T cells in mice and delay disease progression. As islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is a major CD8-T cells diabetogenic antigen in NOD mice, I now aimed to test the efficacy of an IGRP-specific immunotherapy targeting CD8+ T cells for inducing tolerance and protecting from disease.For this current model, I have used liposomes co-delivering the CD8 epitope IGRP206-214and NF-kB inhibitor calcitriol to treat NOD mice. Liposome delivered peptide was presented by antigen-presenting cells to T cells as IGRP-specific CD8+transgenic 8.3 T cells proliferated in draining lymph nodes after treatment. IGRP-specific 8.3 T cells expanded at day 4 and then contracted by day 10 following delivery of IGRP206-214only and IGRP206-214/D3 liposomes. Furthermore, the upregulation of activation and anergy markers such as CD44, LAG-3, and PD-1 on the 8.3 T cells following the liposomal treatment was observed. In contrast, endogenous IGRP-specific CD8+population did not increase in frequency or upregulate of activation markers after one liposomal treatment. However, after two treatments the endogenous IGRP-specific cells significantly upregulated CD44 and PD-1. Consistent with the cells gaining a tolerogenic phenotype, IFN-g production was suppressed in endogenous IGRP-specific CD8+T cells following IGRP206-214/D3 but not IGRP206-214only liposome treatment. These data suggest that endogenous IGRP-specific CD8+T cells get activated in response to the liposome delivered antigen, but they gain an unresponsive phenotype.I then tested the efficacy of the therapy firstly using an accelerated transfer model of diabetes. IGRP206-214/D3 subcutaneous liposomal treatment significantly delayed the transfer of diabetes compared to control mice and IGRP206-214only liposome treatment. We then tested the efficacy of this treatment in spontaneous disease using both pre-diabetic NOD mice and mice treated at the onset of hyperglycaemia.nT1D progression was delayed in all animals treated with IGRP206-214/D3 compared to control groups both in the prediabetic and disease onset regimens. This demonstrates that immunotherapy targeting a single dominant CD8 epitope is effective but codelivery of immunomodulator calcitriol was required to enforce tolerance.I next sought to improve this therapy by combining the IGRP-targeted liposomes with the CD4 T cell chromogranin-A (BDC2.5mim/D3) targeting liposomes our lab has previously shown protect from disease by induction of a population of IL-10 producing antigen-specific Tregs. I then co-delivered IGRP206-214/D3 and BDC2.5mim/D3, hypothesising that this multi-epitope delivery will offer more robust tolerance by inducing production of Tregs and simultaneously inactivating pathogenic CD8+nT cells. Treatment at hyperglycaemia delayed the disease progression but did not improve the outcome compared to a single treatment with IGRP206-214/D3 liposome, whilst no disease protection was achieved in pre-diabetic mice. Furthermore, co-delivery of IGRP and BDC2.5mim liposomes actually increased the expansion of CD8+IGRP-specific cells and prevented induction of a regulatory response suggesting that this combination may, in fact, impede effective tolerance induction.In summary, our IGRP-specific liposomal treatment results in inactivation and anergy of pathogenic CD8+nT cells evidenced by increased expression of PD-1 and LAG3 as well as significant reduction of IFN-g production. While we did not find good evidence for complete deletion of the IGRP-specific T cells, the expansion of these cells was blunted, and this may have been mediated by depletion of the cross-presenting CD8+T cell population. While multi-epitope liposomal delivery did not induce antigen-specific Tregs or synergise to increase disease protection, disease development was still delayed when treated at the onset of hyperglycaemia. Further investigation is required to improve the efficacy of this treatment.nThese data establish a model in NOD mice for analysis of diabetogenic antigen presentation and response to antigen-specific liposome immunotherapy to complement studies of immunotherapy to prevent or treat diabetes.