Human CD8 responses to a complete epitope set from preproinsulin: Implications for approaches to epitope discovery

Purpose: In this study, we explored the breadth of CD8 T cell reactivity to preproinsulin (PPI) in type 1 diabetes. Materials and Methods: We tested a complete peptide set in pools covering all 406 potential 8-11mer epitopes of PPI and 61 algorithm-predicted human leukocyte antigen (HLA)-A2-specific epitopes (15 pools) from islet-specific glucose-6-phophatase catalytic subunit-related protein (IGRP), using a CD8-specific granzyme B enzyme-linked immunosorbent spot assay. Results: Responses were seen to 64 of the 102 PPI pools in two or more newly diagnosed patients (63%) compared to 11 pools in the control subjects (11%, p<0.0001, Fisher's exact test). We identified five pools containing 20 peptides, which distinguished patients from control subjects, most of which had predicted low-affinity binding to HLA class I molecules. In contrast, fewer (5 of 15=33%) IGRP peptide pools, selected by higher binding affinity for HLA-A2 (present in seven of eight patients and five of seven control subjects), stimulated responses in two or more patients, and none stimulated responses in more than two control subjects (p=0.042, Fisher's exact test). Conclusion: Thus, we conclude that CD8 T cell reactivity to PPI in patients with type 1 diabetes can be much broader than shown previously and more diverse than seen in control subjects. Furthermore, responses were often stimulated by peptides with low predicted HLA-binding affinities.