CD137 Plays Both Pathogenic and Protective Roles in Type 1 Diabetes Development in NOD Mice This information is current as Matthew H. Forsberg, Ashley E. Ciecko, Kyle J. Bednar, of September 26, 2021. Arata Itoh, Kritika Kachapati, William M. Ridgway and Yi-Guang Chen J Immunol published online 31 March 2017 http://www.jimmunol.org/content/early/2017/03/31/jimmun ol.1601851 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/03/31/jimmunol.160185 Material 1.DCSupplemental 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 • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 31, 2017, doi:10.4049/jimmunol.1601851 The Journal of Immunology CD137 Plays Both Pathogenic and Protective Roles in Type 1 Diabetes Development in NOD Mice Matthew H. Forsberg,* Ashley E. Ciecko,* Kyle J. Bednar,† Arata Itoh,† Kritika Kachapati,† William M. Ridgway,† and Yi-Guang Chen*,‡,x We previously reported that CD137 (encoded by Tnfrsf9) deficiency suppressed type 1 diabetes (T1D) progression in NOD mice. We also demonstrated that soluble CD137 produced by regulatory T cells contributed to their autoimmune-suppressive function in this model. These results suggest that CD137 can either promote or suppress T1D development in NOD mice depending on where it is expressed. In this study, we show that NOD.Tnfrsf92/2 CD8 T cells had significantly reduced diabetogenic capacity, whereas absence of CD137 in non-T and non-B cells had a limited impact on T1D progression. In contrast, NOD.Tnfrsf92/2 CD4 T cells highly promoted T1D development. We further demonstrated that CD137 was important for the accumulation of b cell– autoreactive CD8 T cells but was dispensable for their activation in pancreatic lymph nodes. The frequency of islet-infiltrating Downloaded from CD8 T cells was reduced in NOD.Tnfrsf92/2 mice in part because of their decreased proliferation. Furthermore, CD137 deficiency did not suppress T1D development in NOD mice expressing the transgenic NY8.3 CD8 TCR. This suggests that increased precursor frequency of b cell–autoreactive CD8 T cells in NY8.3 mice obviated a role for CD137 in diabetogenesis. Finally, blocking CD137–CD137 ligand interaction significantly delayed T1D onset in NOD mice. Collectively, our results indicate that one important diabetogenic function of CD137 is to promote the expansion and accumulation of b cell–autoreactive CD8 T cells, and in the absence of CD137 or its interaction with CD137 ligand, T1D progression is suppressed. The Journal of Immunology, 2017, http://www.jimmunol.org/ 198: 000–000. he costimulatory molecule CD137 belongs to the TNFR is incomplete (12). The Idd9.3 locus was mapped to a 1.2-Mb superfamily (1). Expression of CD137 is induced on ac- region at the distal end of chromosome 4 (13). The C57BL/10 T tivated T cells, and it interacts with CD137 ligand (B10)-derived Idd9.3 region confers T1D resistance in NOD mice (CD137L) expressed on APCs. Engagement of CD137 recruits (14, 15). Compared with the B10 allele, two nonsynonymous TRAF1 and TRAF2; activates ERK, JNK, p38 MAPK, and NF-kB single nucleotide polymorphisms and one 3-bp insertion have pathways; and leads to enhanced activation and survival of T cells been reported in the coding region of NOD Tnfrsf9, a major by guest on September 26, 2021 (2–8). In addition to T cells, CD137 is also expressed on myeloid candidate gene for the Idd9.3 region (14). CD137 (the protein cells and activated NK cells to elicit various immune-modulating product of Tnfrsf9) in NOD mice is hypofunctional. NOD T cells functions (9, 10). Previous studies have also indicated a role of stimulated via CD137 proliferated less, and produced significantly CD137 in the regulation of several autoimmune diseases including lower levels of IL-2, than those isolated from the B10-derived type 1 diabetes (T1D) (11). Idd9.3 congenic strain (NOD.Idd9.3) (13). Genetic susceptibility contributes to T1D development. In the Many costimulatory molecules have been found on T cells (11, NOD mouse model, .30 insulin-dependent diabetes susceptibility 16). However, it is not completely known whether individual co- (Idd) loci have been identified, although our understanding of the stimulatory molecules play distinct roles in promoting the diabe- underlying genes and the mechanisms by which they regulate T1D togenic activity of T cells. In the NOD model of T1D, CD137 has been mostly studied for its role in FOXP3+ CD4+ regulatory T cell *Department of Microbiology and Immunology, Medical College of Wisconsin, (Treg)–mediated diabetes suppression. Previous studies have Milwaukee, WI 53226; †Division of Immunology, Allergy and Rheumatology, Uni- versity of Cincinnati College of Medicine, Cincinnati, OH 45221; ‡Max McGee demonstrated that a subset of Tregs constitutively expresses National Research Center for Juvenile Diabetes, Medical College of Wisconsin, CD137 (17–21). The hypofunctional NOD Tnfrsf9 allele was as- x Milwaukee, WI 53226; and Department of Pediatrics, Medical College of Wiscon- sociated with significantly decreased numbers of CD137+ Tregs in sin, Milwaukee, WI 53226 NOD mice compared with NOD.Idd9.3 (21). CD137+ Tregs were ORCID: 0000-0001-5804-3415 (K.J.B.). more suppressive than the CD1372 subset in vitro and were the Received for publication October 31, 2016. Accepted for publication March 6, 2017. primary cellular source of soluble CD137 (21). Compared with the This work was supported by National Institutes of Health Grants DK107541 (to Y.-G. Idd9.3 C. and W.M.R.), DK097605 (to Y.-G.C.), AI110963 (to Y.-G.C.), and AI125879 (to NOD. congenic strain, NOD mice also had significantly Y.-G.C.), and an American Diabetes Association grant (to Y.-G.C. and W.M.R.). lower levels of serum soluble CD137 (21). Importantly, soluble Address correspondence and reprint requests to Dr. Yi-Guang Chen, Department of CD137 actively suppressed CD4 T cell activation in vitro in an Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, APC-independent but CD137L-dependent fashion, and it pro- WI 53226. E-mail address: [email protected] tected NOD mice from T1D (21, 22). Collectively, these results The online version of this article contains supplemental material. indicate that CD137 has a diabetes-protective role most likely Abbreviations used in this article: B10, C57BL/10; BM, bone marrow; CD137L, through a Treg-dependent mechanism, and its functional impair- CD137 ligand; DP, double-positive; Idd, insulin-dependent diabetes susceptibility; IGRP, islet-specific glucose-6-phosphatase catalytic subunit–related protein; PLN, ment contributes to T1D development in NOD mice. In contrast, pancreatic lymph node; SP, single-positive; T1D, type 1 diabetes; Treg, regulatory NOD mice transgenically expressing an agonistic anti-CD137 T cell. single-chain variable domain in islets developed accelerated Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 T1D, suggesting a diabetes-promoting role of CD137 (23). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601851 2 DUAL ROLES OF CD137 IN TYPE 1 DIABETES To further test the role of CD137 in T1D development, we used cated strains. After 10–12 wk, spleens from recipients were removed and zinc-finger nucleases to target Tnfrsf9 and generate two different analyzed by flow cytometry. knockout alleles directly in NOD mice (24). Both mutant alleles Flow cytometry analysis abolished CD137 protein expression (24). Compared with wild-type NOD mice, T1D development was significantly suppressed in the Fluorochrome-labeled Abs specific for CD8 (53–6.72), CD4 (RM4-5), CD3 (145-2C11), CD44 (IM7.8.1), CD45.1 (A20), CD45.2 (104), and Ki-67 absence of CD137, indicating a diabetes-promoting role of this (So1A15) were purchased from BD Biosciences (San Jose, CA), Bio- costimulatory molecule (24). T1D suppression in CD137-deficient Legend (San Diego, CA), or eBioscience. MHC class I (Kd) tetramers NOD mice was found to be in part caused by reduced diabetogenic loaded with an islet-specific glucose-6-phosphatase catalytic subunit– activity of their T cells (24). These results indicate that CD137 related protein (IGRP) peptide (VYLKTNVFL, aa residues 206–214) were obtained from the National Institutes of Health Tetramer Core Facility. plays a nonredundant role among other costimulatory molecules in Single-cell suspension was prepared from the spleen, PLN, thymus, and supporting the pathogenic function of b cell–autoreactive T cells. BM of mice at the indicated age. BM was isolated from both femurs of Because CD137 contributes to Treg-mediated immunosuppression, individual mice by injecting HBSS in a prepared opening at the proximal our observation that unfractionated CD137-deficient T cells were end of the bone, resulting in the efflux of marrow through a prepared hole less diabetogenic than the wild-type counterparts also suggested a made in the distal end. RBCs were lysed with the ACK lysis buffer. Cells were then resuspended in FACS buffer for MHC class I tetramer or Ab critical disease-promoting role of this costimulatory molecule in at staining. Cells were first blocked with Fc block (BD Biosciences) at room least a subset of b cell–autoreactive T effector cells.