RECOGNITION OF HUMAN PROINSULIN LEADER SEQUENCE BY CLASS I-RESTRICTED T CELLS IN HLA-A*0201 TRANSGENIC MICE AND IN HUMAN TYPE 1 DIABETES

Abstract

Objective A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8⁺ T cells. Our objective is to characterize HLA class I-restricted epitopes located within the preproinsulin leader sequence.

Research Design and Methods Seven 8- to 11-mer preproinsulin peptides carrying anchoring residues for HLA-A1, -A2, -A24 and -B8 were selected from databases. HLA-A2-restricted peptides were tested for immunogenicity in transgenic mice expressing a chimeric HLA-A*0201/beta2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying C-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon gamma ELISpot assay.

Results Five HLA-A2-restricted peptides were immunogenic in transgenic mice. Murine T cell clones specific for these peptides were cytotoxic against cells transfected with the preproinsulin gene. They were recognized by PBMCs from 17/21 HLA-A2 type 1 diabetic patients. PBMCs from 25/38 HLA-A1, -A2, -A24 or -B8 patients produced interferon gamma in response to six preproinsulin peptides covering residues 2 to 25 within the preproinsulin region. In most patients, the response was against several class I-restricted peptides. T cells recognizing preproinsulin peptide were characterized as CD8⁺ T cells by staining with peptide/HLA-A2 tetramers.

Conclusions We defined class I-restricted epitopes located within the leader sequence of human preproinsulin, through in vivo (transgenic mice) and ex vivo (diabetic patients) assays, illustrating the possible role of preproinsulin-specific CD8⁺ T cells in human type 1 diabetes.