Combinatorial ex vivo analysis of beta cell specific CD4+ T-cell responses reveals a predominance of proinsulin specific cells

Abstract

Abstract CD4+ T cell responses are thought to play a key role in the progression of type 1 diabetes (T1D). However, effective monitoring and characterization of T cells that respond to beta cell epitopes in subjects with T1D has been limited by technical obstacles, including the inherently low frequencies of autoreactive CD4+ T cells and the variable responsiveness of individual subjects to single epitopes. We have recently implemented a combinatorial staining approach that allows direct ex vivo characterization of multiple CD4+ T-cell specificities in a single sample. We applied this combinatorial HLA class II multimer assay to directly measure the frequency and phenotype of beta cell specific CD4+ T cells. For this work we utilized five peptides that were previously identified as naturally processed DRB1*04:01 restricted epitopes from proinsulin, GAD65, IA-2, and IGRP. Although responses to each of these peptides can be readily detected after in vitro expansion culture, our results indicated that only proinsulin specific T cells were consistently present at high frequencies in subjects with T1D. Phenotypic analysis of beta cell specific CD4+ T cells revealed that subjects with T1D had a higher frequency of proinsulin specific T cells with a memory phenotype than HLA matched controls. The majority of these memory cells were CXCR3 positive and an increased percentage were CCR7 negative, suggesting that Th1-like effector memory responses are present in subjects with T1D. Finally, this approach is compatible with combinatorial class I multimer analysis, facilitating the characterization of self-reactive CD4+ and CD8+ T cells using a single sample.