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

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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 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 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 *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. In this report, temperature for 10 min and then stained with the indicated Abs for 30 min we further determined the subset of T cells that require CD137 to at 4˚C. For analyzing IGRP206–214-reactive CD8 T cells, cells were incu- fully exert their diabetogenic function. We analyzed the role of bated with MHC class I tetramers and Fc block for 15 min at room tem- perature. Indicated Abs were then added to stain the cells for an additional CD137 in CD4 versus CD8 T cells and further investigated whether 30 min at 4˚C. Stained cells were washed once with the FACS buffer and Downloaded from expression of this costimulatory molecule differentially affects acquired using the FACSCalibur or LSRII flow cytometer (BD Biosci- leukocyte populations in the islet inflammatory environment. ences). All flow cytometric data were analyzed with the FlowJo software (Tree Star, Ashland, OR). Materials and Methods Assessment of T1D and insulitis development Mouse strains T1D and insulitis development were assessed as previously described (25). NOD/ShiLtDvs (NOD), NOD.Cg-Tg(TcraTcrbNY8.3)1Pesa/DvsJ (NOD. In brief, T1D development was monitored weekly using urine glucose http://www.jimmunol.org/ NY8.3), NOD.129S7(B6)-Rag1tm1Mom/J (NOD.Rag12/2), and NOD.B6- strips (Diastix; Bayer) with onset defined by two consecutive readings of Ptprcb/6908MrkTacJ (NOD.CD45.2) mice were obtained from The Jackson .250 mg/dl. At the end of the diabetes incidence study, pancreata of 2 2 Laboratory and subsequently maintained at the Medical College of Wis- nondiabetic NOD.Rag1 / T cell recipients were fixed in a 10% formalin consin by brother–sister mating. NOD.Tnfrsf92/2 mice were generated solution and sectioned at four nonoverlapping levels. Granulated b cells using zinc-finger nucleases as previously described (24) and are currently were stained with aldehyde fuchsin and leukocytes with an H&E coun- maintained at the N4 generation. The generation of NOD.Tnfrsf92/2. terstain. Islets were individually scored as follows: 0, no lesions; 1, peri- Rag12/2 mice was accomplished by outcrossing NOD.Tnfrsf92/2 mice to insular leukocytic aggregates; 2, ,25% islet destruction; 3, .25% islet the NOD.Rag12/2 strain followed by intercrossing. NOD.Rag12/2.NY8.3 destruction; and 4, complete islet destruction. mice were generated through outcrossing NOD.NY8.3 mice to the NOD. 2/2

Rag1 strain, which was then followed by backcrossing the F1 progeny Islet isolation and analysis of infiltrates by guest on September 26, 2021 to NOD.Rag12/2 mice. NOD.Tnfrsf92/2.NY8.3 mice were similarly gen- 2/2 Islet infiltration cells were prepared as previously described (26). Pancreata erated by outcrossing NOD.NY8.3 mice to the NOD.Tnfrsf9 strain. 2 2 from 9- to 12-wk-old NOD and NOD.Tnfrsf9 / mice were inflated with a Female mice were used for all experiments except where indicated. collagenase P solution (5 U/ml; Roche Diagnostics) in HBSS via the bile T cell assays and adoptive transfer studies duct using a 30-gauge needle. The inflated pancreata were incubated at 37˚C for 30 min. After digestion, the pancreata were washed three times with Splenic CD8 and CD4 T cells were isolated by negative selection using the HBSS containing 2% FBS. Each individual pancreas was then resuspended respective CD4 and CD8 T cell isolation kits (Miltenyi Biotec). CD8 and in 5 ml complete RPMI 1640 and plated on a 60 3 15-mm glass petri dish. 2 2 CD4 T cells were individually isolated from NOD and NOD.Tnfrsf9 / Islets from two to seven mice of the same strain were handpicked and 2 2 mice at 6–8 wk of age and admixed in a 2:1 CD4/CD8 ratio. NOD.Rag1 / pooled into a single petri dish under a dissecting microscope. Islets were mice (6–8 wk) were injected i.v. with a total of 6 3 106 CD4 and CD8 then transferred to a 1.5-ml microcentrifuge tube and spun down. After the T cells to test their diabetogenic activity. In separate experiments, splenic removal of the supernatant, islets were resuspended in 500 ml of enzyme- CD4 T cells (3 3 106) isolated from 6- to 8-wk-old NOD or NOD. free Life Technologies cell dissociation buffer to obtain single-cell sus- 2 2 2 2 Tnfrsf9 / mice were infused into NOD.Rag1 / .NY8.3 recipients. To pension. Cells were then washed once with 1 ml of the HBSS, resuspended evaluate the function of CD137 in non-T and non-B cells, we isolated total in FACS buffer, and stained with the indicated fluorochrome-conjugated splenic T cells (5 3 106) from 6- to 8-wk-old NOD mice by negative Abs. Samples were then analyzed on the LSRII flow cytometer. For in- selection using a Pan T cell isolation II (Miltenyi Biotec) and trans- tracellular staining with anti–Ki-67, samples were first stained for surface 2 2 2 2 2 2 ferred them into 6-wk-old NOD.Rag1 / and NOD.Tnfrsf9 / .Rag1 / markers and then fixed/permeabilized for 2–4 h using the FOXP3 staining mice. The purity of the isolated T cell populations was routinely .92% as buffer set from eBioscience. After fixation, the cells were washed twice determined by flow cytometry. For in vivo cell proliferation experiments, with permeabilization buffer and then stained with anti–Ki-67 for 30 min. CD8 T cells were isolated from 7- to 10-wk-old NOD.NY8.3 and NOD. After two additional washes, samples were analyzed by flow cytometry. Tnfrsf92/2.NY8.3 mice and labeled with 5 mM eFluor670 (eBioscience, San Diego, CA) in HBSS at 37˚C for 10 min. After washing, 5 3 106 Immunofluorescence staining labeled CD8 T cells were then transferred into 7- to 9-wk-old NOD re- 2/2 cipients. At 6 d posttransfer, spleens and pancreatic lymph nodes (PLNs) Pancreata from 10-wk-old NOD and NOD.Tnfrsf9 mice were removed were harvested and analyzed via flow cytometry to determine cell prolif- and placed in Tissue-Tek O.C.T. compound from Sakura Finetek (Tor- eration. For CFSE CD8 T cell proliferation studies, miniMACS purified rance, CA) and flash-frozen in isopentane cooled with liquid nitrogen for m CD8 T cells were labeled with CFSE, and proliferation was calculated as 60 s. Sections were cut at 5 m and allowed to air-dry for at least 30 min. described previously (22). Some cells were treated with recombinant Samples were fixed in 75% acetone/25% EtoH for 5 min at room tem- soluble CD137, which was produced as described previously (22). perature. Slides were then immediately moved into TBST, at which point sections were placed on slides and fixed. Serum-free protein block from Generation of mixed bone marrow chimeras DAKO (Carpinteria, CA) was applied for 5 min, after which samples were incubated with primary Ab mixture diluted with Ab diluent (DAKO) for Bone marrow (BM) cells were collected from femurs and tibias of 7- to 11- 90 min at room temperature. Primary Abs used were anti-CD8a (sc-18913; wk-old NOD, NOD.Tnfrsf92/2, and NOD.CD45.2 mice. T cells were Santa Cruz, Dallas, TX), anti-CD4 (sc-13573; Santa Cruz), insulin depleted by using anti-CD3e microbeads (Miltenyi Biotec). (NOD 3 (A0564; DAKO), and B220 (14-0542-85; eBioscience). After a 5-min NOD.CD45.2) F1 mice (6–8 wk) were lethally irradiated (1100 rad) and wash, slides were incubated with secondary Ab for 45 min at room tem- infused with T cell–depleted BM cells (5 3 106) isolated from the indi- perature. Secondary Abs used were donkey anti-rat (A21208; Invitrogen, The Journal of Immunology 3

Carlsbad, CA) and goat anti-guinea pig (ab175714; Abcam, Cambridge, the role of CD137 in the different subsets (24). A subset of U.K.). After washing, slides were stained with DAPI for 10 min. Cover- FOXP3+ Tregs constitutively expresses CD137 on the cell surface slips were applied with Prolong Gold (Thermo Fisher, Waltham, MA) and and is the main cellular source of soluble CD137 (21). NOD mice allowed to cure for 30 min. Whole-slide scanning was performed by using + the Olympus VS120 virtual slide microscope (Olympus, Center Valley, have reduced frequencies and numbers of CD137 Tregs, as well PA). Islets with a diameter of $100 mm were analyzed for the presence of as the level of soluble CD137 in the serum, compared with the B cells, as well as CD4 and CD8 T cells. The area of each islet was diabetes-resistant NOD.Idd9.3 congenic strain (21). Furthermore, calculated (in square micrometers) using ImageJ (27). The cell counts per recombinant soluble CD137 suppressed CD4 T cell proliferation 100 mm2 of CD4 T cells, CD8 T cells, and B cells were then determined for each islet. in vitro and T1D progression in NOD mice (22). Contrary to these results, we showed that CD137 promoted the diabetogenic activity Anti-CD137L injection of unfractionated T cells in an adoptive transfer system (24). One Starting at 9 wk of age, female NOD mice were injected i.p. with 250 mgof possibility that can reconcile these seemingly inconsistent findings anti-CD137L (TKS-1) or an isotype control Ab (2A3) every other week for is that CD137 plays distinct roles in different T cell subsets. a total of 10 wk (five injections). Both anti-CD137L and the isotype Absence of CD137 signaling has been shown to exert distinct control Ab were purchased from BioXCell (West Lebanon, NH). Mice effects in CD4 and CD8 T cells (28). Thus, we tested the function were observed every week for the development of T1D as described earlier. of CD137 in CD4 and CD8 T cells independently. CD4 and CD8 T cells were individually isolated from NOD or NOD.Tnfrsf92/2 Statistical analysis mice, mixed in a “crisscross” design (Fig. 1A), and transferred 2 2 Mann–Whitney U test, unpaired t test, or Wilcoxon matched-pairs signed into NOD.Rag1 / recipients. As expected, CD4 and CD8 T cells test was used for comparison between two groups as indicated. Log- from NOD mice induced high incidence of diabetes, but NOD. Downloaded from rank test was used for the analysis of T1D incidence. All statistical anal- Rag12/2 recipients of both cell types from NOD.Tnfrsf92/2 do- yses were performed using the GraphPad Prism 6 software (La Jolla, CA). nors did not (Fig. 1B). Interestingly, T1D development in NOD. Rag12/2 recipients of NOD.Tnfrsf92/2 CD8 and NOD CD4 Results T cells was also strongly suppressed (Fig. 1B). In contrast, dia- CD137 expression in CD4 and CD8 T cells, respectively, betes development was significantly accelerated in NOD.Rag12/2 suppresses and promotes T1D development 2/2 recipients of NOD CD8 and NOD.Tnfrsf9 CD4 T cells com- http://www.jimmunol.org/ We previously demonstrated that T1D development in NOD. pared with those infused with both cell populations from the NOD 2 2 Tnfrsf9 / mice was suppressed in part because of the reduced donors (Fig. 1B). To rule out the possibility that differences in T1D diabetogenic activity of their T cells; however, we did not identify progression in the four recipient groups were due to differential by guest on September 26, 2021

FIGURE 1. CD137 expression in CD4 and CD8 T cells differentially controls their diabetogenic activity. (A) Schematic diagram showing the exper- imental design of the CD8 and CD4 T cell cotransfer into NOD.Rag12/2 recipients. (B) T1D incidence study of NOD.Rag12/2 recipients depicted in (A). CD4 and CD8 T cells isolated from 6- to 8-wk-old female NOD and/or NOD.Tnfrsf92/2 mice were injected into 6- to 8-wk-old NOD.Rag12/2 females. Recipients were analyzed for the development of T1D weekly over the course of 20 wk posttransfer. (C) Histological analysis of NOD.Rag12/2 recipients in (B) that did not progress to T1D at the end of incidence study. Islets were scored 0–4 based on the severity of insulitis: 0, no lesions; 1, peri-insular leukocytic aggregates; 2, ,25% islet destruction; 3, .25% islet destruction; and 4, complete islet destruction. The result is presented as the relative proportion of each score. More than 200 islets were scored in each recipient group. (D) T1D incidence study of NOD.Rag12/2.NY8.3 female recipients of 3 3 106 CD4 T cells from 6- to 8-wk-old NOD and NOD.Tnfrsf92/2 female mice. Recipients were infused with purified CD4 T cells at 6–8 wk old and followed for the development of T1D every week over the course of 22 wk posttransfer. *p , 0.05, ***p , 0.005, ****p , 0.001 by log-rank test. 4 DUAL ROLES OF CD137 IN TYPE 1 DIABETES

T cell reconstitution, spleens from a subset of recipients were transfer experiment. In this system, CD4 T cells from either NOD analyzed for the presence of CD4 and CD8 T cells at diabetes onset or NOD.Tnfrsf92/2 donors were transferred into NOD.Rag12/2. or at the end of the incidence study. The results do not support the NY8.3 recipients. b cell–autoreactive CD8 T cells of the NY8.3 possibility that differential T cell repopulation was the underlying clonotype require help from CD4 T cells to fully exert their dia- reason of the observed T1D incidence (Supplemental Fig. 1). His- betogenic activity (32). Thus, the diabetogenic function of CD4 tological analysis was performed at the end of the diabetes inci- T cells can be tested by transferring them into NOD.Rag12/2. dence study to determine the level of islet infiltrations in NY8.3 recipients (33). Consistent with previous studies, T1D de- nondiabetic NOD.Rag12/2 recipients. As shown in Fig. 1C, in the velopment was further enhanced in NOD.Rag12/2.NY8.3 mice in two transfer groups that did not develop diabetes, NOD.Rag12/2 the presence of adoptively transferred NOD CD4 T cells recipients of NOD.Tnfrsf92/2 CD8 and NOD.Tnfrsf92/2 CD4 (Fig. 1D). Compared with NOD CD4 T cells, those isolated T cells had worse insulitis (increased scores 3 and 4) compared with from the NOD.Tnfrsf92/2 strain further accelerated diabetes de- recipients of NOD.Tnfrsf92/2 CD8 and NOD CD4 T cells that had velopment in the NOD.Rag12/2.NY8.3 recipients (Fig. 1D). less severe insulitis (increased scores 0 and 1). Thus, even though Collectively, the results from the T cell adoptive transfer experi- neither group of NOD.Tnfrsf92/2 CD8 T cell recipients developed ments indicated that CD137 expression in CD8 T cells was critical diabetes, insulitis was more advanced in the presence of NOD. for the progression of T1D in NOD mice; conversely, CD137 had Tnfrsf92/2 CD4 T cells. The lack of T1D in recipients of knockout a diabetes-protective role in CD4 T cells. CD4 and CD8 cells was surprising given the fact that NOD. Tnfrsf92/2 mice still develop T1D, albeit at a later time point. One CD137 deficiency leads to proportionally reduced explanation for this result is a possible role for CD137 on APCs. islet-infiltrating CD8 T cells Downloaded from CD137 is also expressed in non-T cells (1), so its expression in We previously showed that insulitis was reduced in the NOD. other cell populations could contribute to diabetes development in Tnfrsf92/2 stock compared with wild-type NOD mice (24). NOD mice. Particularly, CD137 has a regulatory role in dendritic However, it was not known whether intraislet accumulation of all cells, an APC subset important for T1D development in NOD mice leukocyte populations was similarly affected or certain immune (29–31). To initially test this possibility, we transferred T cells cell subsets were preferentially altered. Immunofluorescence 2/2 2/2 isolated from NOD mice into NOD.Rag1 or NOD.Tnfrsf9 . staining was performed on pancreatic sections of NOD and NOD. http://www.jimmunol.org/ Rag12/2 recipients. As shown in Supplemental Fig. 2, diabetes Tnfrsf92/2 mice to directly determine CD4, CD8, and B cell development did not significantly differ between these two recipient populations surrounding and infiltrating the islets. Although we groups, indicating that CD137 expression in non-T and non-B cells did not find a difference in the localization of these lymphocyte did not play an important role in T1D. Therefore, the lack of T1D in populations relative to the insulin-producing b cells, accumulation mice receiving NOD.Tnfrsf92/2 CD8 T cells likely reflects differ- of all three cell types was reduced in NOD.Tnfrsf92/2 islets ences in b cell Ag-stimulated survival/expansion in vivo or accu- compared with those in NOD mice (Fig. 2). In general, there was mulation in the islet (see later). concordance of infiltration among B cells, CD4 T cells, and CD8 To further confirm a diabetes-protective role of CD137 when T cells. This indicated that the increase of lymphocyte infiltrations expressed in CD4 T cells, we conducted a different adoptive found in NOD islets is independent of the cell type. To further by guest on September 26, 2021

FIGURE 2. Accumulation of B cells, CD4 T cells, and CD8 T cells in islets is reduced in the NOD.Tnfrsf92/2 strain compared with NOD mice. Pancreata from 10-wk-old NOD and NOD.Tnfrsf92/2 females were processed for immunofluorescence staining to determine the numbers of islet-infiltrating B cells, CD4 T cells, and CD8 T cells as described in Materials and Methods.(A) Representative immunofluorescence staining of islets from NOD and NOD.Tnfrsf92/2 female mice (B220, CD4, and CD8, green; insulin, red; DAPI, blue). (B) Numbers of B cells, CD4 T cells, or CD8 T cells per 100 mm2 of islet area. Each symbol represents one islet. Islets from the same mouse are color coded. Results are pooled from four NOD and five NOD.Tnfrsf92/2 mice. Statistical analysis was carried out by Mann–Whitney U test. The Journal of Immunology 5 determine whether intraislet accumulation of certain cell types these b cell–autoreactive CD8 T cells accumulated in NOD than was proportionally altered in the absence of CD137, we isolated in NOD.Tnfrsf92/2 islets (Fig. 3B, 3C). CD137 deficiency therefore islet-associated leukocytes and analyzed them by flow cytometry. preferentially affected intraislet accumulation of b cell–autoreactive There was a decreased percentage of T cells in NOD.Tnfrsf92/2 CD8 T cells. islets compared with those in NOD mice, because of a reduced b frequency of CD8, but not CD4, T cells (Fig. 3A). In contrast, CD137 intrinsically promotes the accumulation of cell– there were no significant differences in the percentages of B cells, autoreactive CD8 T cells by enhancing their intraislet myeloid cells, and NK cells (Fig. 3A). NOD CD8 T cells accu- proliferation mulate in pancreatic islets in an Ag-specific manner (34). Thus, We next determined whether b cell–autoreactive CD8 T cells are these results indicate that CD137 is important for the accumula- systemically reduced in NOD.Tnfrsf92/2 mice. We evaluated the tion of b cell–autoreactive CD8 T cells in islets. We also analyzed presence of IGRP206–214–reactive CD8 T cells in the spleen, PLN, islet-associated IGRP206–214–reactive CD8 T cells in NOD and and BM of 10- to 14-wk-old mice by tetramer staining. Both NOD.Tnfrsf92/2 mice by MHC class I tetramer staining. More of the percentage and the number of CD44high effector/memory Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Intraislet accumulation of CD8 T cells is proportionally reduced in the NOD.Tnfrsf92/2 strain compared with NOD mice. (A) Flow cytometric analysis of various islet-infiltrating leukocyte populations. Islets were isolated from 9- to 12-wk-old NOD and NOD.Tnfrsf92/2 female mice and dissociated into single-cell suspension. Leukocytes were identified by CD45.1 expression, and the frequencies of different cell populations, including total T cells (CD3+), CD4 T cells (CD3+ CD4+), CD8 T cells (CD3+ CD8+), B cells (CD19+), NK cells (CD32 DX5+), and myeloid cells (CD11b+ CD11c2, CD11b+ CD11c+, and CD11b2 CD11c+) were calculated as percentages of total CD45.1+ cells. Each symbol represents cells pooled from two to seven 2/2 mice. (B and C) The frequencies of islet-associated IGRP2062214-reactive CD8 T cells in NOD and NOD.Tnfrsf9 female mice by MHC class I tetramer staining. Islet cells pooled from two to five mice (10–12 wk old) were stained with Abs against CD45.1, CD3, CD4, and CD8, as well as the IGRP206–214 MHC class I tetramer. (B) One representative experiment is shown. (C) Summarized results from five independent experiments. *p , 0.05, **p , 0.01 by Mann–Whitney U test. 6 DUAL ROLES OF CD137 IN TYPE 1 DIABETES

IGRP206–214–reactive CD8 T cells in the spleen, PLN, and BM we generated control chimeras using BM cells from NOD were higher in NOD than in NOD.Tnfrsf92/2 mice (Fig. 4A). (expressing CD45.1) and NOD.CD45.2 donors. The percentages high Interestingly, IGRP206–214–reactive CD8 T cells were enriched in of CD44 IGRP206–214-reactive CD8 T cells from both origins the BM relative to the spleen and PLN of NOD mice (Fig. 4B, were found to be comparable in the spleens of the recipients 4C), consistent with the previous observation that BM harbored (Fig. 5B, 5D), further confirming that CD137 intrinsically pro- proportionally more b cell–autoreactive T cells than did the spleen moted the accumulation of IGRP206–214-reactive CD8 T cells. (35). To further determine whether CD137 intrinsically promoted CD137 may promote the accumulation of b cell–autoreactive the accumulation of IGRP206–214–reactive CD8 T cells, we gen- CD8 T cells by enhancing their activation in PLNs and/or sub- erated mixed BM chimeras. BM cells from NOD.CD45.2 and sequent proliferation in the islets when they re-encounter the NOD.Tnfrsf92/2 (expressing CD45.1) donors were transferred in cognate Ags. To test the former possibility, we transferred CD137- a 1:1 ratio into (NOD 3 NOD.CD45.2) F1 recipients. This ex- sufficient or -deficient NY8.3 CD8 T cells into NOD mice and perimental design allowed us to simultaneously identify the ori- analyzed their in vivo activation (proliferation and CD44/CD62L gins of donor cells, as well as radiation-resistant host cells. At expression) in the spleen and PLN. We did not observe differences 10–12 wk after BM reconstitution, we analyzed the frequency of in proliferation or the expression levels of CD44 and CD62L high CD44 IGRP206–214-reactive CD8 T cells in the spleens. The between CD137-sufficient and -deficient NY8.3 CD8 T cells results showed that NOD.CD45.2 BM gave rise to a significantly (Fig. 6A, 6B) in either location. We also did not find a differ- high higher percentage of CD44 IGRP206–214-reactive CD8 T cells ence when the percentages of cells in each division were com- compared with those of the NOD.Tnfrsf92/2 origin (Fig. 5A, 5C). pared between CD137-sufficient and -deficient NY8.3 CD8 T cells

To rule out the possibility that different CD45 alleles could have (data not shown). We next determined whether proliferation of b Downloaded from affected the accumulation of IGRP206–214-reactive CD8 T cells, cell–autoreactive CD8 T cells in pancreatic islets is reduced in the http://www.jimmunol.org/ by guest on September 26, 2021

2/2 FIGURE 4. The frequencies and numbers of IGRP206–214-reactive CD8 T cells in the spleen, PLN, and BM are significantly lower in NOD.Tnfrsf9 than in NOD mice. (A) Cells were isolated from the spleen, PLN, and BM, and stained with Abs against CD8 and CD44, as well as IGRP206–214-loaded MHC class I tetramers. Representative flow cytometry profiles are shown in the left panels. The percentages and numbers of IGRP206–214 MHC class I tetramer staining of the spleen, PLN, and BM from 10- to 14-wk-old NOD and NOD.Tnfrsf92/2 female mice from four independent experiments are summarized, respectively, in the middle and right panels. In one experiment, BM from two mice of each strain was not analyzed. *p , 0.05, ***p , 0.005 by Mann–Whitney U test. (B and C) The frequency of IGRP206–214-reactive CD8 T cells is higher in the BM than in the spleen (B) and PLN (C). Paired analysis of IGRP206–214 MHC class I tetramer staining for BM versus spleen and BM versus PLN in NOD mice. ***p , 0.005 by Wilcoxon matched-pairs signed rank test. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. CD137 intrinsically promotes the accumulation of IGRP206–214-reactive CD8 T cells. Lethally irradiated (NOD 3 NOD.CD45.2) F1 mice were infused with equal number of T cell–depleted BM cells (2.5 3 106 per donor strain) from NOD.CD45.2 and NOD.Tnfrsf92/2 (A and C) or NOD. high CD45.2 and NOD mice (B and D). The frequencies of CD44 IGRP206–214-reactive CD8 T cells were analyzed in the spleens of the F1 recipients by MHC class I tetramer staining at 10–12 wk after BM reconstitution. The origin of the splenocytes was determined by CD45.1 and CD45.2 expression. (A and B) The gating strategy is shown. (C and D) The summarized results of two independent experiments are shown. **p , 0.01 by Wilcoxon matched-pairs signed rank test.

absence of CD137 by Ki-67 staining (36, 37). Ki-67 expression number of these pathogenic effectors is artificially increased. We used was analyzed in islet-associated T cells isolated from NOD and NOD mice transgenically expressing the CD8 TCR of the NY8.3 NOD.Tnfrsf92/2 mice. We observed a higher percentage of islet- clonotype to test this possibility. The proportions of CD4/CD8 double- infiltrating Ki-67+ CD8 T cells in NOD than in NOD.Tnfrsf92/2 negative, double-positive (DP), and single-positive (SP) thymocytes did mice (Fig. 6C). In contrast, no difference was found in CD4 not significantly differ between 6- and 7-wk-old NOD.NY8.3 and T cells (Supplemental Fig. 3). These results indicated that CD137 NOD.Tnfrsf92/2.NY8.3 femalemice(Fig.7A).WealsousedMHC promoted accumulation of b cell–autoreactive CD8 T cells, at class I tetramers to specifically identify DP and CD8 SP thymocytes d least in part, through enhancing their proliferation in the islets. recognizing K -restricted IGRP206–214 peptide. NY8.3 CD8 T cells were found to develop normally in the thymus in the absence of CD137 deficiency does not impact T1D development in NY8.3 CD137, albeit their average number was reduced in the CD8 SP TCR-transgenic NOD mice compartment in the NOD.Tnfrsf92/2.NY8.3 strain compared with We reasoned that if the T1D-promoting role of CD137 is through NOD.NY8.3 mice (Fig. 7A). This was largely due to lower total thy- sustaining the expansion and accumulation of b cell–autoreactive CD8 mocyte numbers in some of the NOD.Tnfrsf92/2.NY8.3 than in NOD. T cells, its impact on diabetes development would be reduced when the NY8.3 females (data not shown). However, the numerical difference of 8 DUAL ROLES OF CD137 IN TYPE 1 DIABETES Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Regulation of autoreactive CD8 T cell proliferation by CD137. (A and B) CD137 is not required for initial activation of b cell–autoreactive CD8 T cells in PLNs. CD8 T cells purified from NOD.NY8.3 and NOD.Tnfrsf92/2.NY8.3 mice were labeled with eFluor670 cell proliferation dye and transferred into NOD recipients. Six days later, proliferation of adoptively transferred NY8.3 CD8 T cells was analyzed in the spleen and PLN. (A) Representative flow cytometry profiles of cell division are shown on the left. The results of cell proliferation (middle panels) and the recovered numbers (right panels) of adoptively transferred NY8.3 CD8 T cells are summarized from two independent experiments. Each symbol represents one recipient mouse. (B) Representative flow cytometry profiles of CD44 and CD62L expression on the adoptively transferred NY8.3 CD8 T cells. (C) Intraislet proliferation of CD8 T cells is reduced in the NOD.Tnfrsf92/2 strain compared with the NOD mice. Ki-67 expression was analyzed by flow cytometry in islet-infiltrating CD8 T cells from 10- to 14-wk-old NOD and NOD.Tnfrsf92/2 female mice. Representative flow cytometry profiles of the percentages of Ki-67+ CD8 T cells are shown (left). The results are summarized from five independent experiments (right). Each symbol represents islet cells pooled from three to five mice. *p , 0.05 by Mann–Whitney U test.

thymic IGRP206–214-reactive CD8 T cells between NOD.NY8.3 and duced soluble CD137, which could suppress the proliferation of NOD.Tnfrsf92/2.NY8.3 mice was not observed in a separate analysis CD4 T cells; however, we had not tested the effect of soluble where 7-wk-old males were compared (data not shown). Analyses of CD137 on CD8 T cells. We purified CD8 T cells and stimulated spleens in NOD.NY8.3 and NOD.Tnfrsf92/2.NY8.3 females did not them with or without recombinant soluble CD137. Soluble reveal a numerical difference in IGRP206–214-reactive CD8 T cells CD137 clearly suppressed proliferation of CD8 T cells (Fig. 8). (Fig. 7B). Importantly, T1D development was comparable between We previously published that suppression of CD4 proliferation these two NY8.3 TCR transgenic strains (Fig. 7C). These results by soluble CD137 was decreased by coculture with anti-CD137L suggested that the function of CD137 in b cell–autoreactive CD8 Ab (22). We used the same approach and demonstrated that T cells became less critical when the precursor frequency of these suppression of CD8 cell proliferation by soluble CD137 was pathogenic T cells was greatly increased in the TCR-transgenic mice. blocked by addition of anti-CD137L Ab (Supplemental Fig. 4). The result indicates that binding of soluble CD137–CD137L CD8 T cells are regulated by soluble CD137 expressed on CD8 T cells is required for the suppressive activity. Overall, our results indicated that CD137 promoted the pathogenicity This suggests that one mechanism of enhanced pathogenicity in of CD8 T cells but also enhanced the protective capacity of CD4 the transfer of CD137-deficient CD4 T cells is due to decreased T cells. We previously showed that CD137+ CD4+ Tregs pro- regulation of CD8 cells via soluble CD137. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. CD137 deficiency does not alter diabetes development in NOD mice transgenically expressing TCRs of the NY8.3 clonal type. (A) Thymi 2/2 from 6- to 7-wk-old NOD.NY8.3 and NOD.Tnfrsf9 .NY8.3 female mice were analyzed for IGRP206–214-specific DP and CD8 SP thymocytes. Repre- sentative flow cytometry profiles for each strain are shown (left and middle panels). The results are summarized in the right panels. Each symbol represents one mouse. **p , 0.01 by Mann–Whitney U test. (B) Spleens from 6- to 7-wk-old NOD.NY8.3 and NOD.Tnfrsf92/2.NY8.3 female mice were analyzed for

IGRP206–214-specific CD8 T cells. Representative flow cytometry profiles for each strain are shown (left and middle panels). The results are summarized in the right panels. Each symbol represents one mouse. (C) T1D incidence study of NOD.NY8.3 and NOD.Tnfrsf92/2.NY8.3 female mice. T1D development was monitored weekly using urine glucose strips with onset defined by two consecutive readings of .250 mg/dl. Diabetes development is not significantly different between NOD.NY8.3 and NOD.Tnfrsf92/2.NY8.3 mice.

Blocking CD137–CD137L interaction dominantly inhibits T1D and could enhance T1D development. In contrast, it would also progression prevent stimulation of CD137 on CD8 T cells by CD137L, which T1D is suppressed in NOD.Tnfrsf92/2 mice, albeit immune could suppress T1D. Injections of anti-CD137L significantly regulation conferred by Treg-derived soluble CD137 is also delayed the onset of diabetes in NOD mice (Fig. 9). However, lacking. We thus asked whether CD137L blockade using a diabetes suppression was transient, and T1D development in the blocking Ab (clone TKS-1) can inhibit T1D development in anti-CD137L–treated NOD mice reached the same level as in the NOD mice. The same Ab blocked the suppressive function of control group once Ab administration was ceased. Therefore, soluble CD137 on CD4 and CD8 T cell proliferation in vitro CD137L blockade dominantly suppressed T1D development in (Supplemental Fig. 4) (22). Therefore, blocking CD137L NOD mice but did not provide long-term protection from the in vivo would prevent the suppressive action of soluble CD137 disease (Fig. 10). 10 DUAL ROLES OF CD137 IN TYPE 1 DIABETES

class I tetramer analysis. In addition to islets, b cell–autoreactive CD8 T cells were also found to be reduced in the spleen, PLN, and BM as indicated by those reactive to IGRP208–214, the prevalent pathogenic T effectors in NOD mice (38–40). The results from the BM chimera experiments (Fig. 5) further demonstrate that the 2/2 reduction of IGRP206–214-reactive CD8 T cells in NOD.Tnfrsf9 mice is due to the absence of CD137 expression on these cells rather than a consequence of delayed T1D development in this strain. b cell–autoreactive CD8 T cells are activated in PLNs and can be further stimulated to undergo proliferation and differentiation when they re-encounter the cognate Ag in islets (41). To further determine the underlying mechanism of reduced b cell–autoreactive CD8 T cells in NOD.Tnfrsf92/2 mice, we asked whether their activation in PLNs or proliferation in islets was affected. Adoptive transfer of CD137-sufficient or -deficient NY8.3 CD8 T cells into NOD mice failed to reveal any difference in initial expansion and activation phenotypes (CD44 upregulation and

CD62L downregulation) in PLNs. This was not due to lack of Downloaded from FIGURE 8. Soluble CD137 (sCD137) suppresses CD8 T cell activation. CD137 expression on wild-type NY8.3 cells in PLNs upon Ag CD8 T cells purified from 8- to 10-wk-old female NOD mice were labeled stimulation. Indeed, we observed CD137 upregulation on adop- by CFSE (final concentration: 0.5 mM), stimulated by 25,000 CD3/CD28 Dynabeads, and cultured with or without recombinant sCD137 (30 mg/ml) tively transferred wild-type NY8.3 cells that have been stimulated on 96-well U-bottom culture plate. After 3 d of culture, CFSE-dilution and but have not undergone cell division in PLNs (data not shown). 7-AAD+ population were quantified on FACSCalibur. (A) One represen- Expression of CD137 was transient because it was not seen on tative of four experiments for CFSE dilution without sCD137 (left) and transferred NY8.3 CD8 T cells that have divided at least once http://www.jimmunol.org/ with sCD137 (right) is shown. (B) sCD137 significantly increases the (data not shown). Our observation is consistent with a previous number of undivided CD8 T cells and decreases the number of divided study where CD137 was shown to be transiently upregulated on CD8 T cells. Mean percentage of divided and undivided cells is shown (n = OT-I CD8 T cells after in vivo immunization with OVA (42). We C 4 experiments). ( ) 7-aminoactinomycin D staining shows no difference in subsequently found that proliferation of islet-infiltrating CD8 CD8 cell death after 3 d of culture with or without sCD137 (mean of n =4 T cells was significantly lower in NOD.Tnfrsf92/2 than in wild- experiments). **p , 0.01, ***p , 0.005 by unpaired t test. type NOD mice, indicating that CD137 is important to promote secondary expansion of b cell–autoreactive CD8 T cells in the target tissue. Our observation is reminiscent of the role of CD137 Discussion signaling in the secondary CD8 T cell response to influenza in- by guest on September 26, 2021 An array of costimulatory and coinhibitory molecules modulate the fection (43, 44). It remains to be determined whether transient function of T cells, but their individual roles in T1D have not been expression of CD137 during the initial activation of b cell–auto- completely dissected (11). Soluble CD137 produced by Tregs is reactive CD8 T cells is essential and sufficient to sustain their immunosuppressive, but CD137-deficient NOD mice are more subsequent proliferation in the islets. resistant to T1D than the wild-type control (21, 24). To reconcile The function of CD137–CD137L interaction in CD8 T cells has these seemingly inconsistent results, we asked in which cell types been studied in several viral infection models as reviewed by CD137 exerts its diabetogenic function. Using a T cell adoptive Wortzman et al.(45). In one study, CD8 T cells in CD137L- transfer approach, we demonstrate in this article that the diabe- deficient mice have reduced cytotoxic function in response to togenic activity of CD8 T cells was significantly reduced in the murine gammaherpesvirus-68 infection (46). Therefore, CD137 absence of CD137, providing an explanation for T1D suppression observed in NOD.Tnfrsf92/2 mice. Consistent with a role of CD137 in Treg-suppressive function, we also showed that CD4 T cells lacking this molecule were more diabetogenic than the wild-type counterparts. Our results also indicate that a balance exists between the diabetes-protective and -stimulatory roles of CD137 in NOD mice, and the combined effects of its expression (or its absence) in CD4 and CD8 T cells dictate the outcome of diabetes progression. When pancreatic sections were stained for the presence of B cells, CD4 T cells, and CD8 T cells, lower numbers of all three lymphocyte populations were found in NOD.Tnfrsf92/2 islets compared with those from NOD mice (Fig. 2). However, as a proportion of total islet-infiltrating leukocytes (CD45+), only CD8 T cells were reduced in NOD.Tnfrsf92/2 mice (Fig. 3). These results indicate that accumulation of CD8 T cells is preferentially 2/2 FIGURE 9. CD137L blockage delays T1D onset in NOD mice. Starting affected in NOD.Tnfrsf9 mice. Previous studies indicate that at 9 wk of age, NOD female mice were injected every other week for a accumulation of CD8 T cells in the islet is an Ag-dependent total of five injections with 250 mg of anti-CD137L (TKS-1) each treat- process (34). Thus, our results indicate that CD137 deficiency ment or an isotype control Ab. T1D development was monitored weekly reduces the number of islet-infiltrating b cell–autoreactive CD8 using urine glucose strips with onset defined by two consecutive readings . , T cells, an interpretation also supported by the IGRP208–214 MHC of 250 mg/dl. *p 0.05 by log-rank test. The Journal of Immunology 11

earlier studies, we showed that treating NOD mice at 6 wk of age with agonistic anti-CD137 Ab suppressed T1D (20). Similar anti-CD137 treatment accelerated T1D development in NOD-scid recipients of CD4 and CD8 T cells isolated from diabetic NOD donors (20). The age-dependent effects of OX40 and CD137 stimulations likely resulted from differentially targeting of Tregs versus pathogenic T cells whose balance changes during diabetes progression. Unlike the results of our previous study that a short-term treatment of 6- to 8-wk-old NOD females with recombinant soluble CD137 was sufficient to confer long-term protection from T1D (22), we show in this study that anti-CD137L only FIGURE 10. Schematic diagram illustrating the functions of CD137– temporarily suppressed diabetes development. These results CD137L interactions in the regulation of b cell–autoreactive T cells. Treg- indicate that the in vivo T1D-suppressive activity of soluble derived soluble CD137 (sCD137) binds to CD137L expressed on T cells CD137 is not solely by interfering with the costimulatory in- and suppresses their activation. CD137L expressed by APC binds to teraction between APCs and T cells. The T1D-inhibitory effect membrane CD137 (mCD137) on b cell–autoreactive CD8 T cells to of soluble CD137 most likely involves its ability to actively promote their expansion and accumulation. For therapeutic purposes, suppress T cells by eliciting CD137L reverse signaling (22, 56), recombinant sCD137 can suppress activation of b cell–autoreactive T cells a possibility that remains to be tested. through direct binding to CD137L expressed on their surface, as well as by T1D development in NOD mice can be suppressed not only by Downloaded from blocking the interaction between mCD137 on CD8 T cells and CD137L on CD137 deficiency but also by introducing the B10-derived Idd9.3 APC. CD137L blocking Ab inhibits interaction between mCD137 on CD8 T cells and CD137L on APC. CD137L blocking Ab also binds to CD137L congenic region. The apparent contradiction arises because of the on T cells without inducing an inhibitory effect, but it prevents the sup- fact that the B10-derived Idd9.3 region contains a more functional pressive function of sCD137. Tnfrsf9 allele (13). This may be explained by the dual role of CD137 in Tregs and CD8 T cells to respectively suppress and

promote T1D in NOD mice (Fig. 10). The collective results de- http://www.jimmunol.org/ scribed in this article and published previously suggest that the signaling may also enhance the cytotoxic activity of b cell–auto- Tnfrsf9 allele (null, NOD, or B10) influences the balance of Tregs reactive CD8 T cells. Nevertheless, our results suggest that CD137 and b cell–autoreactive T cells, which in turn modulates T1D plays a relatively less critical role in the cytotoxic function of b progression. Complete CD137 deficiency inhibits accumulation of cell–autoreactive CD8 T cells in NOD mice. This interpretation is b cell–autoreactive CD8 T cells and tips the balance to favor im- based on our observation that T1D development was comparable mune regulation and T1D suppression. A more functional B10 between NOD.NY8.3 and NOD.NY8.3.Tnfrsf92/2 mice, where CD137 molecule promotes the accumulation of CD137+ Tregs and NY8.3 b cell–autoreactive CD8 T cells represent the main patho- the level of soluble CD137, which also leads to T1D suppression. In genic effectors. If CD137 is important for the cytotoxic function of contrast, a hypofunctional NOD CD137 molecule does not affect by guest on September 26, 2021 NY8.3 CD8 T cells, a delay in T1D development in NOD.NY8.3. the accumulation of b cell–autoreactive CD8 T cells but reduces Tnfrsf92/2 mice would have been observed. In contrast, the sig- CD137+ Tregs and the level of soluble CD137, which in turn pro- nificantly increased precursor frequency of b cell–autoreactive CD8 motes T1D development. In conclusion, our studies provide a better T cells in NOD.NY8.3 and NOD.NY8.3.Tnfrsf92/2 likely obviates understanding of how CD137 regulates T1D progression in NOD the need for molecules important for peripheral expansion and ac- mice and may facilitate the development of clinical therapeutics cumulation of these pathogenic effectors. Thus, in the condition aimed at either blocking the stimulatory function of CD137 or where the number of NY8.3 CD8 T cells is artificially inflated, enhancing its suppressive capacity. CD137 plays a less discernible role in the development of T1D. Previous studies suggest that islet-infiltrating autoreactive T cells can exit the site of inflammation to recirculate and later re-enter the Acknowledgments pancreas for subsequent b cell destruction (47). These observations We thank Shamim Khaja and Kevin Mueller for maintaining NOD and re- lated mouse strains, and Christine Duris at the Children’s Research Institute and our findings support a model in which CD137 expression on b histology core for excellent technical assistance. We thank the National cell–autoreactive CD8 T cells contributes to their increased prolif- Institutes of Health Tetramer Core Facility for providing MHC class I b eration and accumulation upon successive rounds of exposure to tetramers. cell Ag in PLNs and islets. Our previous and current findings that CD137 plays roles in both Disclosures Tregs and b cell–autoreactive T cells are not unique to this costim- The authors have no financial conflicts of interest. ulatory pathway. Previous studies have shown dual roles of CD28- and ICOS-ICOSL pathways in T1D progression in the NOD mouse model. Although CD28 signaling is considered important for T cell References activation, NOD mice deficient in this molecule develop rapid onset of 1. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cell re- diabetes because of largely reduced Tregs (48). ICOS-deficient NOD sponses. Annu. Rev. Immunol. 23: 23–68. mice are resistant to diabetes development, but blocking ICOS– 2. Arch, R. H., and C. B. Thompson. 1998. 4-1BB and Ox40 are members of a (TNF)-nerve growth factor receptor subfamily that bind ICOSL interaction in the BDC2.5 TCR-transgenic model causes TNF receptor-associated factors and activate nuclear factor kappaB. Mol. Cell. accelerated T1D onset as a result of impaired Treg function (49–53). Biol. 18: 558–565. Costimulation signals can regulate T1D in NOD mice in an age- 3. Jang, I. K., Z. H. Lee, Y. J. Kim, S. H. Kim, and B. S. Kwon. 1998. Human dependent manner. Blocking OX40-OX40L at 12 wk, but not at 6, 4-1BB (CD137) signals are mediated by TRAF2 and activate nuclear factor- kappa B. Biochem. Biophys. Res. Commun. 242: 613–620. 9, or 15 wk, of age protected NOD mice from T1D (54). Conversely, 4. Cannons, J. L., K. P. Hoeflich, J. R. Woodgett, and T. H. Watts. 1999. Role of the soluble OX40L treatment rapidly induced diabetes development in stress kinase pathway in signaling via the T cell costimulatory receptor 4-1BB. J. NOD mice when injected at 12 wk, but not at 6 wk, of age (55). In our Immunol. 163: 2990–2998. 12 DUAL ROLES OF CD137 IN TYPE 1 DIABETES

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1´10 7

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D8 D8 C C - - D CD8 -/ -/ O 9 9 NOD CD8 rsf rsf + Tnf Tnf D4 C D. - O D CD4 + N -/ O 9 N rsf D4 + NOD. Tnf C - D. D CD4 + N -/ O O 9 N N rsf Tnf D. O N

8´10 6

6´10 6

4´10 6

2´10 6 ubro D cells T CD8 of Number 0

D8 D8 C C - - -/ -/ OD CD8 sf9 sf9 fr fr Tn Tn D4 + N C - OD. -/ OD CD4 + NOD CD8 N rsf9 f D4 + N Tn C - -/ OD. OD CD4 + NOD. N N sf9 fr Tn OD. N

Supplementary Figure 1. NOD.Tnfrsf9-/- T cells do not have a defect to repopulate NOD.Rag1-/- recipients. Splenic CD4 and CD8 T cells from a subset of recipietns were enumerated at diabetes onset or at the end of the incidence study. Each symbol represents one recipient mouse. NOD.Rag1-/- (n=10) NOD.Tnfrsf9-/-.Rag1-/- (n=6) 100

50 ecn diabeticPercent 0 0 5 10 15 20 25 Weeks post transfer

Supplementary Figure 2. CD137 expression in non-T and non-B cells does not influence T1D development. Purified NOD T cells (5x106) were injected intravenously into the indicated recipients. T1D development was followed for 25 weeks post-transfer. Diabetes incidence is not different between the two recipient groups. 40 g n s o l

l 30 m e a c + T 20 7 4 6 - i D K C 10 % 0 NOD NOD.Tnfrsf9-/-

Supplementary Figure 3. Intra-islet proliferation of CD4 T cells is comparable between NOD and NOD.Tnfrsf9-/- strain mice. Ki-67 expression was analyzed by flow cytometry in islet infiltrating CD4 T cells from 10-14 week-old NOD and NOD.Tnfrsf9-/- female mice. The results are summarized from 5 independent experiments. Each symbol represents islet cells pooled from 3-5 mice. 2 .0  1 0 5 **

**

1 .5  1 0 5 M 5

P 1 .0  1 0 C

5 .0  1 0 4

0

soluble CD137 + + + CD137L block + Isotype block +

Supplementary Figure 4. Soluble CD137 suppresses CD8 T cell proliferation in a CD137 ligand dependent manner. NOD splenic CD8 T cells were cultured with CD3/CD28 beads and soluble CD137. In some wells as indicated, anti-CD137L blocking antibodies or IgG2a isotype control antibodies were added. Cell proliferation was measured by thymidine incorporation. CD137L blocking eliminated suppression of CD8 cell proliferation by soluble CD137 while isotype IgG2a did not (n=3 experiments). P values were calculated by unpaired T-test using Graphpad Prism. **: p<0.001