Abstract
Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for development of antigen-specific immunotherapies for type 1 diabetes (T1D). In this study, we sought to comprehensively evaluate reactivity to preproinsulin by CD4 T cells originally isolated from pancreatic islets of organ donors having T1D. We analyzed 187 T cell receptor (TCR) clonotypes expressed by CD4 T cells obtained from six T1D donors and determined their response to 99 truncated preproinsulin peptide pools, in the presence of autologous B cells. We identified 14 TCR clonotypes from four out of the six donors that responded to preproinsulin peptides. Epitopes were found across all of proinsulin (insulin B-chain, C-peptide, and A-chain) including four hot spot regions containing peptides commonly targeted by TCR clonotypes derived from multiple T1D donors. Of importance, these hot spots overlap with peptide regions to which CD4 T cell responses have previously been detected in the peripheral blood of T1D patients. The 14 TCR clonotypes recognized proinsulin peptides presented by various HLA class II molecules, but there was a trend for dominant restriction with HLA-DQ, especially T1D risk alleles DQ8, DQ2, and DQ8-trans. The characteristics of the tri-molecular complex including proinsulin peptide, HLA-DQ molecule, and TCR derived from CD4 T cells in islets, provides an essential basis for developing antigen-specific biomarkers as well as immunotherapies.
Highlights
Type 1 diabetes (T1D) is a disease resulting from dysregulation of adaptive immune responses targeting pancreatic beta cells (1, 2)
T cell receptor (TCR) (7.5%), derived from four donors, that responded to preproinsulin peptide pools
As the peptide concentration used for the screening was supraphysiologic to maximize the detection of preproinsulin-reactive TCRs, some
Summary
Type 1 diabetes (T1D) is a disease resulting from dysregulation of adaptive immune responses targeting pancreatic beta cells (1, 2). A high proportion of T1D patients have HLA-DR4-DQ8 and/or DR3-DQ2 haplotypes (3–7). This strong genetic association within the HLA class II gene locus suggests a crucial role of CD4 T cells in the development of this disease. Multiplex efforts spare samples and reagents necessary for the assay, thereby allowing analysis of reactivity to hundreds of peptides within a relatively short period of time. In this current study, we used this multiplex assay system to test responses against preproinsulin peptides in the presence of autologous B cells transformed with
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