Leveraging Human iPSC-derived beta-cells to Probe Antigen Specificity of Anti-islet Memory T Cells in T1D - PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease, yet the relevant autoantigens for β-cell destruction are poorly defined, especially in humans. There is evidence that insulin is the driving antigen in early anti-islet autoimmunity, however, with disease progression antibodies against other antigens arise, indicating that islet damage is accompanied by antigen spreading. Therefore, the antigens relevant for β-cell destruction during disease initiation might differ from those leading to advanced disease. Knowledge of which antigens mediate β-cell autoimmunity in established T1D has significant clinical importance for emerging cell replacement strategies using autologous induced pluripotent stem cells (iPSCs). IPSC-β-cells will likely trigger expansion of memory anti-β-cell T cells leading to graft destruction. The identification of dominant antigens and epitopes for autograft rejection could find immediate therapeutic applications as iPSC-β-cells invisible to the immune system of people with T1D could be produced by gene editing. To probe antigen specificity of anti-β-cell memory CD4+ T cells and assess whether functionally relevant antigen spreading occurs, we have developed relevant tools. These include MHC-II tetramers, single-cell assays to characterize activation states of CD4+ T cells and paired TCR sequencing, NOD mice carrying edited autoantigens, as well as an autologous system of iPSC-β-cells and peripheral blood monocytes (PBMCs) derived from people carrying the T1D-associated human leukocyte antigen class II DQ8 variant. The proposed project will build on these tools to study MHC-II-mediated antigen presentation and the memory CD4+ T cell response in people with long-standing T1D. To accomplish this, in Aim 1 we will compare the CD4+ T cell repertoire in young non-diabetic and diabetic NOD mice during graft rejection and evaluate the extent of antigen spreading. To guide the effort of discovering new antigens in the memory repertoire, we will conduct immunopeptidome studies in β-cells and functionally test candidate antigens in presentation assays. These experiments will evaluate the memory CD4+ T cell repertoire in mice and provide insight into functionally relevant antigen spreading in a preclinical model. In Aim 2, we will use a similar approach to study the human memory CD4+ T cell repertoire by establishing co-cultures and a humanized mouse model of iPSC-β-cells and PBMCs. We will then edit candidate autoantigens and epitopes in iPSCs and test their function in memory CD4+ T cell stimulation and graft rejection. This high-risk, high-reward project, co-led by a T1D immunologist and an expert in β-cell biology and iPSCs, has substantial translational impact and builds upon technological advances that our team has developed.
