CD8 T cell autoreactivity to preproinsulin epitopes with very low human leucocyte antigen class I binding affinity

Abstract

Beta cells presenting islet epitopes are recognized and destroyed by autoreactive CD8 T cells in type 1 diabetes. These islet-specific T cells are believed to react with epitopes binding with high affinity to human leucocyte antigen (HLA) expressed on beta cells. However, this assumption might be flawed in case of islet autoimmunity. We evaluated T cell recognition of the complete array of preproinsulin (PPI) peptides with regard to HLA binding affinity and T cell recognition. In a comprehensive approach, 203 overlapping 9-10mer PPI peptides were tested for HLA-A2 binding and subjected to binding algorithms. Subsequently, a high-throughput assay was employed to detect PPI-specific T cells in patient blood, in which conditional HLA ligands were destabilized by ultraviolet irradiation and HLA molecules refolded with arrays of PPI peptides, followed by quantum-dot labelling and T cell staining. Analysis of patient blood revealed high frequencies of CD8 T cells recognizing very low HLA binding peptides. Of 28 peptides binding to HLA-A2, a majority was predicted not to bind. Unpredicted peptides bound mainly with low affinities. HLA binding affinity and immunogenicity may not correlate in autoimmunity. Algorithms used to predict high-affinity HLA peptide binders discount the majority of low-affinity HLA binding epitopes. Appreciation that peptides binding HLA with very low affinity can act as targets of autoreactive T cells may help to understand loss of tolerance and disease pathogenesis and possibly point to tissue-specific immune intervention targets.

Fig. 1

Predicted and in-vitro preproinsulin (PPI) peptide binding to human leucocyte antigen (HLA)-A2 molecules. Overlapping 9- and 10-mer peptides were analysed by prediction algorithms and compared to results of the in-vitro peptide binding assay. (a) All 9- and (b) 10-mer peptides predicted to bind to HLA-A2 by NetMHC 3·0 (black bars), NetMHC 3·2 (diagonally hatched bars), SYFPEITHI (grey bars), BIMAS (white bars) and MOTIFS (horizontal stripes), and the observed in-vitro binding affinity (1/IC₅₀) were plotted against the amino acid sequence of PPI. Boxes on the PPI amino acid sequence indicate the signal peptide, B chain, cleavage site for conversion from proinsulin to insulin, C-peptide, cleavage site and A chain, respectively.

Fig. 2

High frequency of preproinsulin (PPI)-specific CD8 T cells recognizing peptides with very low human leucocyte antigen (HLA)-A2 binding affinity. PPI peptide HLA (pHLA) multimers were divided into three groups (intermediate, low and very low HLA-A2 binding affinity). (a) Representative dot plots of CD8 T cells recognizing PPI pHLA multimers of intermediate, low and very low affinity (Qdot-605 and -705) and negative control (Qdot-585 and -800). (b) Peripheral blood mononuclear cells (PBMC) of 10 recent-onset type 1 diabetes patients were screened for the presence of CD8 T cells recognizing the PPI peptides or one cytomegalovirus (CMV) epitope. Statistical analysis was performed using the Mann–Whitney U-test.