Evidence That a Peptide Spanning the B-C Junction of Proinsulin Is an Early Autoantigen Epitope in the Pathogenesis of Type 1 Diabetes This information is current as of September 24, 2021. Wei Chen, Isabelle Bergerot, John F. Elliott, Leonard C. Harrison, Norio Abiru, George S. Eisenbarth and Terry L. Delovitch J Immunol 2001; 167:4926-4935; ; doi: 10.4049/jimmunol.167.9.4926 Downloaded from http://www.jimmunol.org/content/167/9/4926 References This article cites 50 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/167/9/4926.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Evidence That a Peptide Spanning the B-C Junction of Proinsulin Is an Early Autoantigen Epitope in the Pathogenesis of Type 1 Diabetes1 Wei Chen,2* Isabelle Bergerot,2* John F. Elliott,‡ Leonard C. Harrison,§ Norio Abiru,¶ George S. Eisenbarth,¶ and Terry L. Delovitch3*† The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the Downloaded from periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24–33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in http://www.jimmunol.org/ female NOD mice. Our findings support the notion that proinsulin p24–33 may be a primary autoantigen epitope in the patho- genesis of T1D in NOD mice. The Journal of Immunology, 2001, 167: 4926–4935. utoimmunity to insulin and/or isoforms of glutamic acid GAD65 delays/prevents insulitis and T1D (4, 5). However, NOD decarboxylase (GAD65 and GAD67)4 is detectable in T cell clones exist that can induce insulitis and T1D and yet do not A humans and nonobese diabetic (NOD) mice with type 1 react with GAD65 or insulin. Thus, it remains unclear whether diabetes (T1D) (1). Insulin-specific T cells are found preferentially insulin, GAD65, or another protein is an early autoantigen that in islet infiltrates in NOD mice between 4 and 12 wk of age, and elicits T cell-mediated islet ␤ cell destruction. by guest on September 24, 2021 insulin B chain (B9–23)-specific T cell clones both accelerate di- Accumulating evidence suggests that proinsulin may be an early abetes in young NOD mice and adoptively transfer T1D to NOD. autoantigen in the pathogenesis of T1D. Proinsulin gene transcripts scid mice (2, 3). GAD65-reactive splenic T cells are detectable in have been found in the neonatal mouse and human thymus (6, 7). NOD mice as early as 3 wk, and tolerization of NOD mice with Furthermore, proinsulin is the only islet ␤ cell-specific autoanti- gen, and aberrant transcription or processing of a cell-specific product would be a logical explanation for targeted autoimmunity. *Autoimmunity/Diabetes Group, The John P. Robarts Research Institute and †De- Indeed, we have shown that altered processing of human insulin partments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario, Canada; ‡Department of Medical Microbiology and Im- occurs in B cell APC from a patient with T1D (8). This notion is munology, University of Alberta, Edmonton, Canada; §Autoimmunity and Transplan- further supported by evidence that T cell epitopes of insulin de- tation Division, The Walter and Eliza Hall Institute of Medical Research, Royal Mel- bourne Hospital PO, Parkville, Victoria, Australia; and ¶Barbara Davis Center for fined in HLA-DR4 transgenic NOD mice are actually derived from Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO preproinsulin and proinsulin (9). A 13-aa sequence homology be- 80262 tween human proinsulin residues 24–36 and human GAD65 Received for publication March 8, 2001. Accepted for publication August 24, 2001. (hGAD65) residues 506–518 was identified (10). This 13-mer se- The costs of publication of this article were defrayed in part by the payment of page quence of proinsulin contains a binding motif for the I-Ag7 MHC charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. class II molecule of NOD mice (11). T cell reactivity to this pro- 1 This work was supported by grants from the Canadian Institutes of Health Research insulin peptide may therefore represent an early autoimmune event (MT-5729), Juvenile Diabetes Research Foundation International (JDRFI), Canadian in T1D, and as a result of molecular mimicry and cross-reactivity Institutes of Health Research /JDRFI Diabetes Interdisciplinary Research Program, may give rise to T cell reactivity to the similar GAD peptide (5). and the National Health and Medical Research Council of Australia. W.C. was the ϩ recipient of a postdoctoral fellowship from the JDRFI. I.B. was the recipient of post- Interestingly, rat CD4 T cell lines specific for proinsulin peptides doctoral fellowships from the Foundation pour la Recherche´Me´dicale and Canadian (located between B chain and C peptide of proinsulin), but not the Diabetes Association. similar GAD65 peptide, adoptively transfer insulitis to syngeneic 2 W.C. and I.B. contributed equally to this work. naive rats (12). Moreover, GAD65 and insulin B chain peptide 3 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Autoim- (9–23) are not primary autoantigens for the development of T1D munity/Diabetes Group, The John P. Robarts Research Institute, 1400 Western Road, London, Ontario, Canada N6G 2V4. E-mail address: [email protected] in Bio-Breeding rats (13). T cell reactivity to proinsulin occurs in 4 Abbreviations used in this paper: GAD, glutamic acid decarboxylase; BGL, blood individuals at risk for T1D (10, 14), and transgenic expression of glucose level; EAE, experimental allergic encephalomyelitis; hGAD, human GAD; mouse proinsulin by MHC class II-bearing cells prevents T1D in IAA, insulin autoantibody; ICA69, islet cell Ag p69; mGAD, mouse GAD; NOD, nonobese diabetic; PLN, pancreatic lymph node; PLP, proteolipid protein; PmB, NOD mice (15). These findings support the possibility that proinsulin Polymyxin B Sulfate; SI, stimulation index; T1D, type 1 diabetes. may be a pathogenic autoantigen early in the development of T1D. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 4927 A disproportionately elevated release of proinsulin, but not in- SDS-PAGE (4–20% gradient gel; Invitrogen Canada, Buglington, Can- sulin, is found in human islets in response to cytokines (16). It may ada) analysis of the purified recombinant mouse proinsulin II under non- be clinically relevant that circulating proinsulin levels can be more reducing conditions demonstrated that it consists mainly of monomers, a lesser amount of dimers, and a rather small amount of multimers (includes than 2-fold higher in recently diagnosed diabetic patients than in hexamers) (Fig. 1A). In contrast, human proinsulin (Sigma, St. Louis, MO; normal healthy control individuals (17, 18). As proinsulin is ex- expressed in E. coli and purified by HPLC) consists mainly of hexamers. pressed in the thymus and in highest concentration in islet ␤ cells, The mouse proinsulin II preparation did not contain another detectable proinsulin rather than its processed products (insulin and C pep- recombinant protein(s). Considering that a low percentage of proinsulin multimers may alter the immunogenicity of the protein preparation, mouse tide) might be a better candidate for an early autoantigen in T1D. proinsulin II was further purified by electroelution from a SDS-PAGE gel. To test this possibility, we analyzed whether peripheral T cells in The electroeluted protein was dialyzed against Ͼ200 vol of 20 mM sodium perinatal vs adult female NOD mice respond to proinsulin and its carbonate, 4 mM DTT (pH 10.6) at 23°C (two steps of 4 h each) and then immunodominant peptides, and whether perinatal immunization at 4°C (two steps of 4 h each). Purified proinsulin II was present in mo- with proinsulin accelerates or delays progression to the onset nomeric form (Fig.