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(+) T-cells. Our objective is to characterize HLA class I-restricted epitopes located within the preproinsulin leader sequence.


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 COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-gamma (IFN-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 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-gamma 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(+) 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.
Toma
Andréa
A
Institut National de Santé et de Recherche Médicale U561 et Université Paris N, Hôpital Cochin-Saint Vincent de Paul, Paris, France.
Laïka
Taghrid
T
Haddouk
Samy
S
Luce
Sandrine
S
Briand
Jean-Paul
JP
Camoin
Luc
L
Connan
Francine
F
Lambert
Marion
M
Caillat-Zucman
Sophie
S
Carel
Jean-Claude
JC
Muller
Sylviane
S
Choppin
Jeannine
J
Lemonnier
François
F
Boitard
Christian
C
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010446:Peptide Fragments / Q000276:immunology; D011384:Proinsulin / Q000276:immunology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism
D000939:Epitopes; D006680:HLA Antigens; D010446:Peptide Fragments; D011384:Proinsulin; D046988:Proteasome Endopeptidase Complex