Recognition of Human Proinsulin Leader Sequence by Class I–Restricted T-Cells in HLA-A*0201 Transgenic Mice and in Human Type 1 Diabetes
- Andréa Toma1,
- Taghrid Laïka12,
- Samy Haddouk1,
- Sandrine Luce1,
- Jean-Paul Briand3,
- Luc Camoin4,
- Francine Connan4,
- Marion Lambert1,
- Sophie Caillat-Zucman1,
- Jean-Claude Carel1,
- Sylviane Muller3,
- Jeannine Choppin4,
- François Lemonnier2 and
- Christian Boitard15
- 1Institut National de la Santé et de la Recherche Médicale U561 et Université Paris N, Hôpital Cochin-Saint Vincent de Paul, Paris, France
- 2Unité d'Immunité Cellulaire Antivirale, Département d'Immunologie, Institut Pasteur, Paris, France
- 3Centre National de la Recherche Scientifique Unité Propre de Recherche 9021, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France
- 4Institut National de la Santé et de la Recherche Médicale U567, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Institut Cochin, Université Paris N, Paris, France
- 5Service d'Immunologie Clinique, Hôpital Cochin-Saint Vincent de Paul, Paris, France
- Corresponding author: Dr. Christian Boitard, christian.boitard{at}inserm.fr
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/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+ 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.
Footnotes
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Published ahead of print at http://diabetes.diabetesjournals.org on 14 November 2008.
A.T. and T.L. contributed equally to this study.
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- Accepted November 6, 2008.
- Received May 2, 2008.
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