Human CD8 responses to a complete epitope set from preproinsulin: implications for approaches to epitope discovery.
OBJECTIVE
In this study, we explored the breadth of CD8 T cell reactivity to preproinsulin (PPI) in type 1 diabetes.


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).


CONCLUSIONS
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.
Baker
Caroline
C
Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Petrich de Marquesini
Liliana G
LG
Bishop
Amanda J
AJ
Hedges
Alan J
AJ
Dayan
Colin M
CM
Wong
F Susan
FS
D000328:Adult; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005260:Female; D005952:Glucose-6-Phosphatase / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000276:immunology; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000276:immunology*; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D019151:Peptide Library; D011498:Protein Precursors / Q000276:immunology*
D001323:Autoantibodies; D001324:Autoantigens; D018984:Epitopes, T-Lymphocyte; C096322:ICA512 autoantibody; D007328:Insulin; D019151:Peptide Library; D011498:Protein Precursors; C013737:preproinsulin; D005952:Glucose-6-Phosphatase; C492194:G6PC2 protein, human; D053804:Granzymes; D005968:Glutamate Decarboxylase