Recognition of Human Proinsulin Leader Sequence by Class I--Restricted T-Cells in HLA-A*0201 Transgenic Mice and in Human Type 1 Diabetes.

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[sup +] 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/β2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-γ (IFN-γ) 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 peripheral blood mononuclear cells (PBMCs) from 17 of 21 HLA-A2 type 1 diabetic patients. PBMCs from 25 of 38 HLA-A1, -A2, -A24, or -B8 patients produced IFN-γ in response to six preproinsulin peptides covering residues 2-25 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[sup +] 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[sup +] T-cells in human type 1 diabetes. Diabetes 58:394-402, 2009 [ABSTRACT FROM AUTHOR]