PROJECT SUMMARY/ABSTRACT
Type 1 diabetes (T1D) is an autoimmune disease, yet the relevant autoantigens for ß-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 ß-cell destruction during disease
initiation might differ from those leading to advanced disease. Knowledge of which antigens mediate ß-cell
autoimmunity in established T1D has significant clinical importance for emerging cell replacement strategies
using autologous induced pluripotent stem cells (iPSCs). IPSC-ß-cells will likely trigger expansion of memory
anti-ß-cell T cells leading to graft destruction. The identification of dominant antigens and epitopes for autograft
rejection could find immediate therapeutic applications as iPSC-ß-cells invisible to the immune system of people
with T1D could be produced by gene editing. To probe antigen specificity of anti-ß-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-ß-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 ß-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-ß-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 ß-cell biology and iPSCs, has substantial translational impact and builds
upon technological advances that our team has developed.