(#19) Preproinsulin-reactive CD4 T cells in the islets of type 1 diabetes organ donors

PRESENTED BY: Maki Nakayama

Authors

First Name	Last Name	Affiliation/Institution
Laurie	Landry	Barbara Davis Center, University of Colorado Denver
Amanda	Anderson	Barbara Davis Center, University of Colorado Denver
Sally	Kent	University of Massachusetts Medical School
Mark	Atkinson	University of Florida
Clayton	Mathews	University of Florida
Aaron	Michels	Barbara Davis Center, University of Colorado Denver
Maki	Nakayama	Barbara Davis Center, University of Colorado Denver

Purpose

Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for developing antigen-specific immunotherapies for type 1 diabetes (T1D). In this study, we sought to comprehensively evaluate pancreatic islet infiltrating CD4 T cell reactivity to preproinsulin from organ donors having T1D.

Methods

We identified T cell receptor (TCR) sequences expressed by CD4 T cells in the islets of six recent-onset T1D organ donors and expressed frequent TCR clonotypes on T-hybridoma cells to analyze their antigen specificity. TCR transductants were tested for the response to 99 truncated preproinsulin peptide pools in the presence of autologous EBV (Epstein-Barr Virus)-transformed B cells. We further determined HLA class I molecules presenting peptides to TCR clonotypes responding to preproinsulin. We also compared frequencies of preproinsulin-reactive CD4 and CD8 T cells in the islets of individual donors.

Summary of Results

Among 187 TCR clonotypes studied, 14 TCRs 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 donors. Only one of the four hot spots, located in insulin A-chain, was overlapped with that for islet CD8 T cells. The 14 TCR clonotypes recognized 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. In terms of frequency of preproinsulin-reactive CD4 T cells in the islets, four of the six T1D donors had at least one proinsulin-reactive CD4 TCR clonotype, and these TCRs were expressed by 10-25% of islet CD4 T cells in each donor. Donors having high frequency of preproinsulin-reactive CD4 T cells did not necessarily have many preproinsulin-reactive CD8 T cells in the islets.

Conclusions

Pancreata of T1D organ donors contain proinsulin-reactive CD4 T cells, but frequencies of proinsulin-reactive T cells varied by individual donors. 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.