Expression of Preproinsulin-2 Gene Shapes the Immune Response to Preproinsulin in Normal Mice 1 Be ´atrice Faideau,* Jean-Paul Briand, ‡ Chantal Lotton,* Isabelle Tardivel,* Philippe Halbout,* Jacques Jami, § John F. Elliott, ¶ Patricia Krief,* Sylviane Muller, ‡ Christian Boitard,* and Jean-Claude Carel 2 * † Deciphering mechanisms involved in failure of self tolerance to preproinsulin-2 is a key issue in type 1 diabetes. We used non- autoimmune 129SV/Pas mice lacking preproinsulin-2 to study the immune response to preproinsulin-2. In these mice, a T cell response was detected after immunization with several preproinsulin-2 peptides and confirmed by generating hybridomas. Ac- tivation of some of these hybridomas by wild-type (wt) islet cells or recombinant murine proinsulin-2 demonstrated that two epitopes can be generated from the naturally expressed protein. Although T cells from wt mice responded to preproinsulin-2 peptides, we could not detect a response to the naturally processed epitopes in these mice. Moreover, after immunization with recombinant whole proinsulin-2, a T cell response was detected in preproinsulin-2-deficient but not in wt mice. This suggests that islet preproinsulin-2-autoreactive T cells are functionally eliminated in wt mice. We used a transplantation model to evaluate the relevance of reactivity to preproinsulin-2 in vivo. Wild-type preproinsulin-2-expressing islets transplanted in preproinsulin-2- deficient mice elicited a mononuclear cell infiltration and insulin Abs. Graft infiltration was further increased by immunization with preproinsulin-2 peptides. Preproinsulin-2 expression thus shapes the immune response and prevents self reactivity to the islet. Moreover, islet preproinsulin-2 primes an immune response to preproinsulin-2 in deficient mice. The Journal of Immunology, 2004, 172: 25–33. A utoimmunity results from the failure of the different mechanisms maintaining immune tolerance to autoanti- gens (1–3). Understanding these mechanisms is essential to decipher how autoimmunity occurs and design strategies to pre- vent its development. Type 1 diabetes, which results from loss of tolerance to  cell autoantigens, has been extensively studied as a model disease for development of autoimmunity (4 –7). Studies in the nonobese diabetic (NOD) 3 mouse (4), a spontaneous model of type 1 diabetes, have identified proinsulin, glutamate decarboxyl- ase, tyrosine phosphatase, and several other  cell autoantigens as targets of autoimmunity in this model. Autoimmunity in human diabetes and in the NOD mouse results from a multigenic process in which numerous immune and  cell defects associate to drive the diabetogenic process (5). Several models have been established to address the issue of self tolerance to specific Ags expressed by  cells in the absence of spontaneous autoimmunity. In a majority of models, transgenes expressed on  cells have been used to study how these new self- Ags shape tolerance (8, 9). These studies have greatly enhanced our understanding of immune response regulation, but generalizing their results is limited by artifacts due to the transgenic process. For instance, variable thymic expression of the same transgene in different transgenic mouse lines was observed, leading to differ- ences in the level of autoreactivity after infection with a virus expressing the same Ag (10). Transgenic expression of B7-1 in  cells was able to induce an autoimmune reaction against a  cell transgene but not against  cell native Ags (11). Proinsulin is thought to be a major autoantigen in type 1 dia- betes for several reasons. Insulin (and proinsulin)-specific Abs and autoreactive T cells have been detected in NOD mice and in pa- tients with diabetes or prediabetes. In young children at risk for developing the disease, insulin autoantibodies are detected first (12). Allelic variations in the variable number of tandem repeats region flanking the insulin gene are important determinants of the genetic susceptibility to diabetes (IDDM2) and influence  cell insulin secretion and proinsulin expression in the thymus (13–16). Protection from diabetes is obtained in the NOD mouse by inject- ing insulin, the insulin B-chain or B-chain peptides (17–19). Un- derstanding how loss of tolerance to preproinsulin occurs and how IDDM2 regulates diabetes autoimmunity is therefore essential. In the NOD mouse, a progressive loss of tolerance to multiple au- toantigens occurs, and deciphering the respective role of each of them is complex. Therefore, evaluating self tolerance to autoanti- gens such as preproinsulin in nonautoimmune animals allows a better understanding of the mechanisms involved. *Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 561, and † Depart- ment of Pediatric Endocrinology, Groupe Hospitalier Cochin-Saint Vincent de Paul, Paris, France; ‡ Centre National de la Recherche Scientifique, Unite ´ Propre de Re- cherche 9021, Institut de Biologie Mole ´culaire et Cellulaire, Strasbourg, France; § In- stitut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 567, Institut Cochin, Paris, France; and ¶ Department of Medical Microbiology and Immunology, Univer- sity of Alberta, Edmonton, Canada Received for publication November 20, 2002. Accepted for publication October 7, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The study was supported by an Institut National de la Sante ´ et de la Recherche Me ´dicale Grant, Juvenile Diabetes Foundation Grant 1-2001-751, Ministe `re de la Recherche et de la Technologie Action Concerte ´e et Incitative Grant 1A011G and a grant from Association Franc ¸aise des Diabe ´tiques (2002). We thank Association Dia’parole and Comite ´ d’Entreprise des Ae ´roports de Paris for their generous dona- tions. B.F. was supported by Institut National de la Sante ´ et de la Recherche Me ´dicale and Assistance Publique-Ho ˆpitaux de Paris (poste d’accueil Institut National de la Sante ´ et de la Recherche Me ´dicale 2001). 2 Address correspondence and reprint requests to Dr. Jean-Claude Carel, Institut Na- tional de la Sante ´ et de la Recherche Me ´dicale Unite ´ 561, Groupe Hospitalier Cochin- Saint Vincent de Paul, 82 avenue Denfert Rochereau, 75014 Paris, France. E-mail address: carel@paris5.inserm.fr 3 Abbreviations used in this paper: NOD, nonobese diabetic; LNC, lymph node cell; wt, wild type. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00