IL-21 Exacerbates Autoimmune Myositis by Enhancing the Accumulation of GM-CSF−Producing Δγ T Cells in the Muscle

IL-21 Exacerbates Autoimmune Myositis by Enhancing the Accumulation of GM-CSF−Producing δγ T Cells in the Muscle

Takahiro Kageyama, Akira Suto, Taro Iwamoto, Shigeru Tanaka, Kenichi Suehiro, Yusuke Downloaded from Yokoyama, Aiko Saku, Shunsuke Furuta, Kei Ikeda, Kotaro Suzuki, Koichi Hirose and Hiroshi Nakajima

ImmunoHorizons 2017, 1 (8) 176-187 doi: https://doi.org/10.4049/immunohorizons.1700053 http://www.immunohorizons.org/content/1/8/176 http://www.immunohorizons.org/ This information is current as of September 26, 2021.

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Adaptive Immunity

IL-21 Exacerbates Autoimmune Myositis by Enhancing the Accumulation of GM-CSF–Producing gd T Cells in the Muscle

Takahiro Kageyama,*,1 Akira Suto,*,†,1 Taro Iwamoto,* Shigeru Tanaka,* Kenichi Suehiro,* Yusuke Yokoyama,* Aiko Saku,* Shunsuke Furuta,* Kei Ikeda,* Kotaro Suzuki,* Koichi Hirose,* and Hiroshi Nakajima* *Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; and †Institute for Global Downloaded from Prominent Research, Chiba University, Chiba 260-8670, Japan

ABSTRACT http://www.immunohorizons.org/ IL-21 is suggested to be involved in the development of some autoimmune diseases; however, the role of IL-21 in autoimmune inflammatory myopathies (IMs) remains unknown. In this study, we found that serum levels of IL-21 were significantly elevated in a subset of IM patients. Upon the induction of experimental autoimmune myositis (EAM), IL-21 was produced by CD4+ T cells in the muscle, and muscle weakness and muscle inflammation were less obvious in IL-21–deficient (IL-212/2) mice compared with those in wild-type (WT) mice. Analysis of inflammatory cytokine production from draining lymph node cells of EAM-induced mice revealed that GM-CSF production was significantly decreased in IL-212/2 mice. Importantly, GM-CSF production from gdT cells, but not CD4+ T cells, was significantly reduced in EAM-induced IL-212/2 mice. In addition, the severity of EAM was attenuated by GM-CSF neutralization in WT mice or gdT cell deficiency. The majority of muscle-infiltrating GM-CSF–producing gdT cells expressed Vg4+Vd4+ TCR, and the number of Vg4+Vd4+ cells in the muscle was significantly decreased in EAM-induced IL-212/2 mice as compared with that in EAM-induced WT mice. Moreover, muscle-infiltrating Vg4+Vd4+ cells exhibited CX3CR1high phenotype, and the induction of by guest on September 26, 2021 Cx3cl1, a ligand for CX3CR1, in the muscle was reduced in EAM-induced IL-212/2 mice. Furthermore, reporter assays revealed that IL-21 activated the promoter of Cx3cl1. Consistent with these findings, serum levels of CX3CL1 were correlated with the levels of IL-21 in IM patients. Taken together, these results suggest that IL-21 facilitates autoimmune myositis through the accumulation of GM-CSF–producing Vg4+Vd4+ cells in the muscle possibly via CX3CR1-CX3CL1 pathways. ImmunoHorizons, 2017, 1: 176–187.

INTRODUCTION of IL-23 and produce IL-17A, IL-17F, IL-21, and GM-CSF (1, 2). IL- 17–producing gdT cells are also activated by IL-23 and implicated Autoimmune diseases are caused by a breakdown of tolerance to in the pathogenesis of autoimmune diseases (3, 4). Because the self-antigens and are characterized by the activation of T cells, blocking of IL-17 signaling is less eﬀective than that of IL-23 in including pathogenic IL-17–producing Th cells (Th17 cells). autoimmune disease models such as experimental autoimmune Pathogenic Th17 cells are eventually diﬀerentiated in the presence encephalomyelitis (EAE) (5, 6), Th17 cell–related cytokines other

Received for publication October 3, 2017. Accepted for publication October 9, 2017. Address correspondence and reprint requests to: Dr. Akira Suto and Dr. Hiroshi Nakajima, Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba City, Chiba 260-8670, Japan. E-mail addresses: [email protected] (A. Suto) and [email protected] (H.N.) ORCID: 0000-0002-2593-565X (A. Suto). 1T.K. and A. Suto contributed equally to this work. This work was supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government, the Leading Graduate School Program at Chiba University, and the Institute for Global Prominent Research, Chiba University, Japan. Abbreviations used in this article: CADM, clinically amyopathic DM; DM, dermatomyositis; EAE, experimental autoimmune encephalomyelitis; EAM, experimental autoimmune myositis; GC, germinal center; gf, gram-force; IL-212/2, IL-21–deficient; IL-21R2/2, IL-21R–deficient; IM, inflammatory myopathy; LN, lymph node; Mac, macrophage; PD-1, programmed death-1; PM, polymyositis; qPCR, quantitative PCR; RORgt, retinoid-related orphan receptor g t; SLEC, short-lived effector CD8+ T cell; TCRd2/2, TCRd-deficient; Tfh, follicular B Th; WT, wild-type. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2017 The Authors

176 https://doi.org/10.4049/immunohorizons.1700053

ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons IL-21–Vg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS 177 than IL-17 seem to be important for the development of certain malignancy were excluded. We retrospectively assessed the autoimmune diseases. baseline patient characteristics, including age, sex, diagnosis, In the initiation phase of EAE, GM-CSF, a representative and presence of malignancy. The study was approved by the cytokine that induces the differentiation of neutrophils and Ethics Committees of Chiba University and was performed in macrophages (Macs), is produced by Th17 cells and serves a accordance with the principles expressed in the Declaration of nonredundant function (1, 2). In addition, it has been shown that Helsinki. IL-7–induced Stat5 activation promotes the development of GM- CSF–producing CD4+ T cells and causes severe EAE (7). More- ELISA over, GM-CSF is shown to be produced by gdT cells during the Serum levels of IL-21 and GM-GSF were analyzed by a human development of EAE (8). In humans, blocking of GM-CSF IL-21 ELISA MAX kit and a human GM-GSF ELISA MAX kit, signaling by a neutralizing Ab has shown promising results for respectively (BioLegend, San Diego, CA). Serum levels of human rheumatoid arthritis in early-phase clinical trials (9). These CX3CL1 and murine IL-21 were analyzed by a human CX3CL1/ findings suggest that GM-CSF is involved in the development of Fractalkine Quantikine ELISA Kit and a mouse IL-21 DuoSet some autoimmune diseases. ELISA, respectively (R&D Systems, Minneapolis, MN). Detection Recent studies have shown that IL-21 exhibits pleiotropic limits are as follows: human IL-21 .31.3 pg/ml, human GM-CSF Downloaded from effects on a variety of immune responses (10). IL-21 is produced by .7.8 pg/ml, human CX3CL1 .0.63 ng/ml, and murine IL-21 many types of CD4+ T cells such as Th17 cells (11), follicular B Th .62.5 pg/ml. (Tfh) cells (12, 13), IL-6– or IL-21–stimulated CD4+ T cells (14), CCR9+ Th cells (15), and “peripheral Th” cells (16). In addition, Mice IL-21 has been shown to be involved in the development of C57BL/6 mice were purchased from Charles River Laboratories http://www.immunohorizons.org/ autoimmune disease models, including type 1 diabetes in NOD (Atsugi, Kanagawa, Japan). IL-21–deficient (IL-212/2)mice mice (17), murine models of rheumatoid arthritis (18), and lupus (B6.129S-Il21tm1Lex/Mmucd) were obtained from Mutant Mouse (19, 20). Moreover, we have shown that IL-21–producing CD4+ Resource Research Centers. IL-21R–deficient (IL-21R2/2)mice T cells are increased in Foxp3 mutant scurfy mice, and that (27) were backcrossed over eight generations onto C57BL/6 mice. abrogation of IL-21 signaling in scurfy mice significantly prolongs TCRd-deficient (TCRd2/2) mice were obtained from the Jackson survival presumably by reducing autoimmune inflammation of Laboratory (Bar Harbor, ME). All mice were housed in micro- lung through the suppression of short-lived effector CD8+ T cells isolator cages under specific pathogen-free conditions. The Chiba (SLECs) (21). University Animal Care and Use Committee approved animal Polymyositis (PM) and dermatomyositis (DM) are the major procedures used in this study. subgroups of idiopathic inflammatory myopathy (IM), which is by guest on September 26, 2021 characterized by infiltration of inflammatory cells into the affected Experimental autoimmune myositis muscle (22, 23). Regarding the relationship between IL-21 and IM Experimental autoimmune myositis (EAM) was induced as in humans, it has been reported that CXCR32 CXCR5+ Th cells, described previously (28, 29). In brief, myosin was purified which are known to produce IL-21, are increased in peripheral from the muscle of C57BL/6 mice and emulsified with CFA with blood in juvenile DM patients who have skin rash and muscular 2mg/mlMycobacterium tuberculosis (CFA) (Chondrex, Redmond, weakness (24). However, the role of IL-21 in the pathogenesis of WA). Mice were immunized three times at 1-wk intervals with IM remains unknown. 1 mg of myosin in CFA on bilateral sides of hind foot pads (day 0), In this study, we addressed this issue by using a murine model tail base (day 7), and flanks (day 14) as described previously (29). of autoimmune myositis. Our results indicate that IL-21 is involved PBS was injected as controls. Pertussis toxin (500 ng; Sigma) was in the development of autoimmune myositis possibly by enhancing injected i.p. at day 1. At day 24, after mice were perfused with neutrophil recruitment into the muscle through the accumula- 50 ml of ice-cold PBS to wash out blood cells, right hind-limb tion of GM-CSF–producing Vg4+Vd4+ CD272 cells via a CX3CR1- muscle was excised for histological analysis, left hind-limb CX3CL1 pathway. muscle for FACS analysis, and triceps for quantitative PCR (qPCR) analysis. For neutralization of GM-CSF, anti–GM-CSF mAb (300 mg/mouse, clone MP1-22E9; BioLegend) or purified MATERIALS AND METHODS rat IgG2a (BioLegend) as a control was injected i.p. on days 2, 9, and 16. Patients We reviewed the medical records of patients who were newly Evaluation of muscle strength diagnosed with IM and were admitted to Department of Allergy Muscle strength was measured by using a grip strength meter for and Clinical Immunology, Chiba University Hospital from small animal (GBM-100B) (Melquest, Toyama, Japan) at day 23. September of 2009 to June of 2016. We identified 51 sequential All tests were performed in a blinded fashion. Muscle weakness patients who fulfilled the criteria of Bohan and Peter (25) for was defined as the decrease of muscle strength of EAM-induced PM/DM or those of Sontheimer and colleagues (26) for clinically mice that was calculated by subtracting from the average of muscle amyopathic DM (CADM). Patients who were complicated by strength in age-matched PBS-injected mice.

https://doi.org/10.4049/immunohorizons.1700053 178 IL-21–Vg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS ImmunoHorizons

Histological analysis of skeletal muscle Determination of inflammatory cytokine profile Paraffin sections of quadriceps were examined histologically for At day 24 of EAM, draining lymph node (LN) cells were stimulated the presence of inflammatory cell infiltratesand necrosis of muscle with PMA plus ionomycin for 5 h in RPMI 1640 medium fibers. Sections (5 mm thick) were cut at ;200-mm distance and supplemented with 10% heat-inactivated FCS, 50 mM2-ME,and stained with H&E. Inflammatory changes of skeletal muscles were 2mML-glutamine (complete RPMI 1640 medium). Inflammatory quantified in a blinded manner by using a scoring system as cytokine profiles of culture supernatants were analyzed by a described previously (30): grade 1, single or ,5musclefibers LEGENDplex mouse inflammation panel (BioLegend) according involvement; grade 2, a lesion involving 5–30 muscle fibers; grade to the manufacturer’s instruction. Serum levels of inflammatory 3, a lesion involving a muscle fasciculus; grade 4, a diffuse extensive cytokines were analyzed by the same system at day 24 of EAM. lesion. When multiple lesions were found in one muscle block, 0.5 point was added to the indicated score. Intracellular cytokine and transcription factor staining For intracellular cytokine staining, cells were stimulated with Reagents PMA plus ionomycin for 3 h in complete RPMI 1640 medium in the presence of Golgi plug (BD), stained with fixable viability dye Abs to CD3 (145-2C11), CD28 (37.51), CD45 (30-F11), B220 (RA3- Downloaded from fi 6B2), CD4 (RM4-5), CD8 (53-6.7), Vd4(GL2),CD11b(M1/70), (Invitrogen or BioLegend) and cell surface markers, and then xed ff CXCR5 (2G8), Siglec-F (E50-2440), Fas (Jo2), IL-4 (11B11), IFN-g and permeabilized with Perm/Wash bu er (BD Biosciences) ’ (XMG1.2), and IL-17A (TC11-18H10) were purchased from BD according to the manufacturer s instructions. Intracellular cyto- Biosciences(SanDiego,CA).AbstoTCRgd (UC7-13D5 and GL3), kine staining of IL-21 was performed as described previously (14, – TCR Vg1(2.11),TCRVg4 (UC3-10A6), TCR Vg5 (536), Ly-6C 31 33). For transcription factor staining, cells were stained with

fi fi http://www.immunohorizons.org/ (HK1.4), Ly-6G (1A8), CD64 (X54-5/7.1), KLRG1 (2F1), IL-7Ra xable viability dye and cell surface markers, then xed and ff (SB/199), CX3CR1 (SA011F11), CCR4 (2G12), CCR5 (HM-CCR5), permeabilized with a Foxp3/transcription factor staining bu er ’ CCR6 (29-2L17), CCR8 (SA214G2), CXCR3 (CXCR3-173), CXCR4 set (eBioscience) according to the manufacturer s instructions. (L276F12), GM-CSF (MP1-22E9), and IL-17A (TC11-18H10.1) were purchased from BioLegend. Abs to GL7 (GL-7), programmed Cell culture death-1 (PD-1; RMP1-30), CD27 (LG.7F9), CCR7 (4B12), CCR9 CD4+ T cells were stimulated with plate-bound anti-CD3e mAb (1 (eBioCW-1.2), Foxp3 (FJK-16s), and retinoid-related orphan mg/ml) in the presence of anti-CD28 mAb (1 mg/ml) (anti-CD3/ receptor g t(RORgt) (AFKJS-9) were purchased from eBioscience CD28 mAb) in complete RPMI 1640 medium in either neutral (San Diego, CA). Murine IL-21 was purchased from BioLegend. conditions (without exogenous cytokines), GM-CSF–producing Human TGF-b, mouse IL-21R–Fc chimera, and Abs to CCR2 conditions [anti–IFN-g mAb (10 mg/ml) and IL-7 (2 ng/ml)], or

(FAB5538P), CCR3 (FAB729B), and CCR10 (FAB2815C) were Th17-polarizing conditions [IL-6 (0.5 ng/ml), TGF-b (1 ng/ml), by guest on September 26, 2021 purchased from R&D Systems. anti–IL-4 mAb (10 mg/ml), and anti–IFN-g mAb (10 mg/ml)] for 3 d. In the case of Th17 cell induction, cells were reactivated with anti-CD3/CD28 mAb in the presence of IL-1b (10 ng/ml) and IL- Cell isolation and flow cytometry analysis 23 (10 ng/ml) for another 3 d. gdT cells were stimulated with plate- Naive CD4+ T cells were isolated by using a mouse naive CD4+ bound anti-CD3e mAb (5 mg/ml) in complete RPMI 1640 medium. T cell isolation kit (STEMCELL Technologies, Tukwila, WA). Where indicated, 100 ng/ml IL-21 was added to the culture. gdT cells were isolated by using a mouse TCRg/d Tcell isolation kit (Miltenyi Biotec). Muscle-infiltrating cells were prepared from lower hind-limb muscle by using gentleMACS qPCR analysis fi Octo Dissociator (Miltenyi Biotec) with Liberase (0.2 mg/ml; Total RNA was puri ed from triceps by using Isogen II (Nippon Roche, Basel, Switzerland) and DNase I (0.1 mg/ml; Roche). Gene, Toyama, Japan) and BioMashaer II (Nippi, Tokyo, Japan). Single-cell suspensions of muscle-infiltrating cells were then Reverse transcription and qPCR analysis were performed as obtained by sequential filtration with 100 and 30 mmofcell described previously (33). PCR primers for Cx3cl1 were as follows: strainers (BD). Viable cells were collected from the cell forward, 59-ACG AAA TGC GAA ATC ATG TGC-39;reverse,59- suspensions using Lympholyte-M (Cedarlane Laboratories, CTG TGT CGT CTC CAG GAC AA-39. PCR primers for hypoxan- Burlington, ON, Canada) according to the manufacturer’s thine phosphoribosyltransferase were as follows: forward, 59-TCA instructions. Cells were stained and analyzed by FACS Canto II GTC AAC GGG GGA CAT AAA-39; reverse, 59-GGG GCT GTA (BD) using FlowJo software (Tree Star, Ashland, OR). Neutro- CTG CTT AAC CAG-39. The levels of Cx3cl1 were normalized to phils are defined as CD11b+ Ly6G+ cells, Macs as CD11b+ Ly6G2 the levels of hypoxanthine phosphoribosyltransferase. Siglec-F2 CD64+ cells, M2 Macs as CD11b+ Ly6G2 Siglec-F2 CD64+ Ly6Chi cells, M1 Macs as CD11b+ Ly6G2 Siglec-F2 Luciferase reporter assay CD64+ Ly6Clo cells,germinalcenter(GC)BcellsasKLRG1+ The fragment of mouse Cx3lc1 promoter (2500 to +30) was Fas+ B220+ cells, follicular Th cells as CXCR5+ PD-1+ CD4+ amplifiedbyPCRwithmousegenomicDNAasatemplateand T cells, Th17 cells as RORgt+ CD4+ T cells, and SLECs as KLRG1+ subcloned into pGL4.10 luciferase vector (Promega Biotech, IL-7Ra2 CD8+ Tcells. Madison, WI) (Cx3cl1-Luci). M12.4.5 cells (5 3 104 cells) were

https://doi.org/10.4049/immunohorizons.1700053 ImmunoHorizons IL-21–Vg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS 179 resuspended with 0.5 mgofpGL4.1vector(Cx3cl1-Luci or pGL4.1 that IL-21 is associated with muscle involvement in a subset of empty vector) and 5 ng of pGL4.74 vector in resuspension buffer R patients with IM. and electroporated by using Neon transfection system (Invitrogen) at 1500 V/20 ms per 1 pulse. Cells were rested for 6 h and then left IL-21 is involved in the pathogenesis of EAM unstimulated (PBS) or stimulated with IL-21 (100 ng/ml) for 18 h. To further address the roles of IL-21 in the pathogenesis of IM, we Relative light units were assessed with a dual luciferase assay used EAM (28), which is induced by the immunization with system (Promega). myosin. When EAM was induced in wild-type (WT) mice, IL-21 was undetectable in sera at day 24 of EAM. However, when EAM Data analysis was induced in IL-21–deficient (IL-212/2) mice, muscle weakness Data are shown as mean 6 SEM. Statistical analyses of the results waslessevidentinIL-212/2 mice than that in WT mice (WT mice were performed by the unpaired t test, Mann–Whitney U test, 104.6 6 12.0 gram-force [gf ] versus IL-212/2 mice 65.4 6 11.8 gf, p Kruskal–Wallis test, and Dunn multiple comparisons test or , 0.05) (Fig. 2A). Consistently, histological analyses revealed that Spearman rank correlation as appropriate. The p values ,0.05 inflammatory changes in the muscle were less obvious in IL-212/2 were considered significant. mice than those in WT mice (Fig. 2A). These results indicate that IL-21 is involved in the development of EAM. Downloaded from To explore the mechanisms underlying the attenuated muscle RESULTS weakness in EAM-induced IL-212/2 mice, we next examined the numbers of inflammatory cells in the skeletal muscle in EAM- Serum levels of IL-21 are elevated in a subset of patients induced WT mice and IL-212/2 mice. Consistent with a previous with IM report (28), the number of CD45+ hematopoietic cells in the http://www.immunohorizons.org/ To investigate the possible involvement of IL-21 in the pathogen- muscle was significantly increased in WT mice upon EAM esis of IM, we first examined serum levels of IL-21 in patients with induction (Fig. 2B). Importantly, the infiltration of CD11b+ IM (n = 51) and healthy donors (n = 20). We found that serum IL-21 myeloid cells in the muscle was less evident in EAM-induced was detected in 17 patients with IM (33%) and 2 healthy donors IL-212/2 mice than that in EAM-induced WT mice (n =12mice, (10%). The mean serum level of IL-21 was significantly elevated in p , 0.05). Among CD11b+ myeloid cells, Ly6G+ neutrophils were patients with IM as compared with healthy donors (68.6 6 33.9 significantly decreased in the muscle in EAM-induced IL-212/2 versus 2.6 6 1.8 pg/ml, mean 6 SEM, p , 0.05) (Fig. 1A). Among mice (Fig. 2B), whereas the numbers of M1 Macs, M2 Macs, thepatientswithIM,serumIL-21wassignificantly elevated in eosinophils, and mast cells were similar between EAM-induced PM/DM patients, but not in CADM patients, as compared with IL-212/2 mice and WT mice (Fig. 2B, data not shown). As those in healthy donors (83.9 6 41.9 versus 6.0 6 4.2 versus 2.6 6 for lymphocytes, the numbers of CD4+ Tcells,CD8+ Tcells,and by guest on September 26, 2021 1.8 pg/ml, mean 6 SEM, p , 0.05) (Fig. 1B). These results suggest gdT cells in the muscle were comparable between EAM-induced IL-212/2 mice and WT mice (Fig. 2B). Because CD8+ T cells infiltrate into the muscle in patients with IM (22), we further analyzed the subtypes of CD8+ T cells in the muscle in EAM- induced mice. Consistent with our previous finding that IL-21 induces the accumulation of SLECs into the lung in scurfy mice (21), the number of SLECs in the muscle was significantly decreased in EAM-induced IL-212/2 mice as compared with that in EAM-induced WT mice (Fig. 2B). Because IL-21 is involved in the development of Tfh cells, Th17 cells, and GC B cells (34), we also analyzed the development of these cell populations in the draining LNs of EAM-induced mice. As expected, GC B cells were significantly decreased in the draining LNs in EAM-induced IL-212/2 mice(Fig.2C).Incontrast, FIGURE 1. Serum levels of IL-21 are elevated in a subset of patients the numbers of Tfh cells and Th17 cells were not significantly 2 2 with IM. different between EAM-induced IL-21 / mice and WT mice, (A) Serum levels of IL-21 in patients with IM (n = 51) and healthy donors suggesting the redundant roles of IL-21 in the differentiation of (HD) (n = 20) were analyzed by ELISA. Statistical analyses of the results Tfh and Th17 cells in EAM-induced mice (Fig. 2C). were performed by Mann–Whitney U test. *p , 0.05. (B) Serum levels of IL-21 in patients with PM/DM (n = 41), CADM (n = 10), and HD (n = CD4+ T cells are the major producer of IL-21 in EAM 20). The cohort used in (B) is identical to (A). Serum IL-21 was detected We next analyzed cell types that produce IL-21 in the muscle in in 15 of 41 in PM/DM patients, 2 of 10 in CADM patients, and 2 of 20 in EAM-induced mice. Intracellular IL-21 staining together with cell HD. Because y-axis is expressed on a logarithmic scale, only values .0 surface phenotyping revealed that most of the IL-21–producing were plotted. Statistical analyses were performed by Kruskal–Wallis test cells in the muscle were CD4+ T cells (Fig. 2D). We also analyzed and Dunn multiple comparisons test. *p , 0.05. n.s., not significant. surface markers of IL-21–producing CD4+ T cells in the muscle

https://doi.org/10.4049/immunohorizons.1700053 180 IL-21–Vg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS ImmunoHorizons Downloaded from http://www.immunohorizons.org/

FIGURE 2. IL-21 is involved in the pathogenesis of EAM. EAM was induced in IL-212/2 mice and WT mice. (A) Upper panel, Data are mean 6 SEM of muscle weakness of EAM-induced mice. n = 5 mice. Middle panels, Representative photomicrographs of H&E staining of the section of quadriceps. Scale bars, 100 mm. Lower panel, Mean 6 SEM of muscle histological scores. n = 11 (WT mice) and 8 (IL-212/2 mice). (B)Mean6 SEM of the numbers of indicated cells harvested from the muscle. n =12forcell populations except for SLECs. n = 5 (WT mice) and 7 (IL-212/2 mice) for SLECs. (C) The frequencies of GC B cells, Tfh cells, and Th17 cells in draining 2/2 + 2 LNs at day 15. n = 5 (WT mice) and 6 (IL-21 mice). (D) Upper panel, Representative FACS profiles of CD4 versus IL-21 of CD45 CD11b lymphocytes by guest on September 26, 2021 in the muscle of indicated mice. n = 3. Lower panel, Representative FACS profiles of CXCR5 versus either PD-1, CCR2, CCR5, CCR9, or CCR6 on IL-21–producing CD4+ T cells in the draining LNs and muscle. Data are representative of four independent experiments. *p , 0.05. and draining LNs of EAM-induced WT mice. As shown in Fig. 2D, inflammatory cytokines/chemokines, as well as the production of the majority of IL-21–producing CD4+ Tcellsinthemuscle these cytokines/chemokines from draining LN cells upon stimula- exhibited PD-1+ CXCR52 phenotype. In draining LNs, ;20% of tion with PMA plus ionomycin in EAM-induced IL-212/2 mice and IL-21–producing CD4+ T cells were PD-1+ CXCR5+ conventional WT mice. Among 13 cytokines/chemokines, the level of IL-23 was Tfh cells, which did not express CCR2, CCR5, or CCR9. In lower in EAM-induced IL-212/2 mice than that in EAM-induced contrast, PD-1+ CXCR5+ IL-21–producing CD4+ T cells in the WT mice (Supplemental Fig. 2), consistent with the reduced muscle simultaneously expressed CCR2, CCR5, and CCR9, but not neutrophilic inflammation in EAM-induced IL-212/2 mice (Fig. CCR6. These results suggest that IL-21–producing CD4+ T cells in 2B). Meanwhile, upon the activation of draining LN cells, IL-10 the muscle of EAM mice seem to be different from conventional production was significantly decreased in EAM-induced IL-212/2 Tfh cells. mice as compared with that in EAM-induced WT mice (Fig. 3A), consistent with a previous finding that IL-21 induces IL-10 IL-21 enhances GM-CSF production from gdT cells production (36). In addition, GM-CSF levels were significantly Given that murine neutrophils did not express IL-21R (35), it is decreased in EAM-induced IL-212/2 mice (Fig. 3A). In contrast, the suggested that IL-21 indirectly induces the infiltration of neutro- levels of IL-1a,IL-1b, IL-6, IL-12p70, IL-17A, IL-23, IL-27, IFN-b, phils into the muscle in EAM. To identify the factors that induce IFN-g,TNF-a, and CCL2 were not significantly different between neutrophil infiltration into the muscle in EAM-induced mice, we EAM-induced IL-212/2 mice and WT mice (Fig. 3A). first analyzed the expression of chemokines that induce the Intracellular cytokine staining confirmed that the numbers of recruitment of neutrophils in the muscle and found that GM-CSF–producing cells were decreased in the draining LNs in mRNA expression of CXCL1, CXCL2, and CXCL5 was comparable EAM-induced IL-212/2 mice as compared with those in EAM- between EAM-induced IL-212/2 mice and WT mice (Supplemental induced WT mice (Fig. 3B). Importantly, .50% of GM-CSF– Fig. 1). We then comprehensively analyzed the serum levels of producing cells in the draining LNs in EAM-induced WT mice

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FIGURE 3. IL-21 enhances GM-CSF production from gdT cells. (A–C) EAM was induced in IL-212/2 mice and WT mice. (A) The levels of inflammatory cytokines and chemokines produced by draining LN cells at day 24 of EAM. n = 4 (WT mice) and 3 (IL-212/2 mice). (B) At day 15 of EAM, GM-CSF–producing cells in draining LNs were analyzed by flow cytometry. Left two panels, The frequencies of GM-CSF–producing cells and GM-CSF–producing CD4+ T cells gated on total live cells. Middle

panels, Representative profiles of TCR gd versus GM-CSF of indicated mice. Right panel, The frequency of GM-CSF–producing gdT cells. n = 6. (C) by guest on September 26, 2021 At day 24 of EAM, GM-CSF–producing cells in the muscle were analyzed. Left panels, Representative profiles of GM-CSF versus IL-17A of CD4+ T cells and gdT cells. Right panels, The numbers of IL-17A–producing CD4+ T cells, GM-CSF–producing CD4+ T cells, IL-17A–producing gdT cells, and GM-CSF–producing gdT cells. n =8.(D) Naive CD4+ T cells from WT mice were stimulated under neutral (PBS), IL-7 + anti–IFN-g, or Th17 conditions in the presence or absence of IL-21. Representative profiles of GM-CSF versus IL-17A from three independent experiments are shown. (E) gdT cells from WT and IL-21R2/2 mice were stimulated with anti-CD3 Ab for 2 d in the presence or absence of IL-21. Representative profiles of GM-CSF versus IL-17A and the frequencies of GM-CSF–producing gdT cells are shown. n =3.*p , 0.05. were gdT cells (Fig. 3B). Indeed, whereas the frequency of GM- the culture of CD4+ T cells suppressed GM-CSF production under CSF–producing CD4+ T cells was comparable between IL-212/2 IL-7 + anti–IFN-g conditions (Fig. 3D). IL-21 did not affect GM- mice and WT mice (Fig. 3B), the frequency of GM-CSF– CSF production from Th17 cells (Fig. 3D). In contrast, IL-21 producing gdT cells was significantly decreased in EAM- modestly but significantly increased GM-CSF production from induced IL-212/2 mice as compared with that in EAM-induced anti-CD3–stimulated gdT cells in WT mice, but not in IL-21R2/2 WT mice (WT mice: 0.14 6 0.05% versus IL-212/2 mice 0.04 6 mice (Fig. 3E), indicating that IL-21 enhances the production of 0.01%, p , 0.05) (Fig. 3B). Consistently, the number of GM-CSF– GM-CSF from gdTcells,butnotCD4+ T cells. producing gdT cells was decreased in the muscle in EAM-induced IL-212/2 mice (p , 0.05) (Fig. 3B). In contrast, the numbers of GM-CSF–producing gdT cells are involved in the GM-CSF–producing CD4+ T cells, IL-17A–producing CD4+ development of EAM T cells, and IL-17A–producing gdT cells in the muscle were not We next examined whether GM-CSF is involved in the develop- significantly different between EAM-induced IL-212/2 mice and ment of EAM by administering neutralizing anti–GM-CSF Ab. As WT mice (Fig. 3C). shown in Fig. 4A, weekly administration of neutralizing anti–GM- We next analyzed the effect of IL-21 on GM-CSF production CSF Ab significantly attenuated muscle weakness and histological from CD4+ T cells and gdT cells in vitro. Consistent with previous scores in EAM-induced WT mice. The number of neutrophils in reports (1, 7), CD4+ T cells stimulated under IL-7 + anti–IFN-g the muscle was also significantly decreased in mice treated with conditions produced GM-CSF (Fig. 3D). The addition of IL-21 to anti–GM-CSF Ab (Fig. 4B). In contrast, the numbers of Macs,

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FIGURE 4. GM-CSF–producing gdT cells are involved in the development of EAM. (A and B) EAM was induced in WT mice. Where indicated, 300 mgofanti–GM-CSF mAb or control IgG was injected i.p. on days 2, 7, and 16 of EAM. (A) Upper panel, Mean 6 SEM of muscle weakness. n = 3 mice. Middle panels, Representative photomicrographs of H&E staining of the quadriceps. Scale bars, 100 mm. Lower panel, Muscle histological scores. n =5(anti–GM-CSF mAb) and 6 (control IgG). (B) The numbers of the indicated cells in the muscle. n =4 (anti–GM-CSF mAb) and 6 (control IgG). *p , 0.05. (C and D) EAM was induced in TCRd2/2 miceandWTmice.(C)Upperpanel,Mean6 SEM of muscle weakness. n =3mice.Middlepanels,RepresentativephotomicrographsofH&Estainingofthequadriceps.Scalebars,100mm. Lower panel, Mean 6 SEM by guest on September 26, 2021 of muscle histological scores. n = 6 mice. (D) The numbers of indicated cells in the muscle. Mean 6 SEM. n =6(WTmice)and5(TCRd2/2 mice). *p , 0.05.

CD4+ Tcells,CD8+ T cells, and gdT cells in the muscle were not EAM-induced WT mice. Because it has been reported that CD272 affected by anti–GM-CSF Ab (Fig. 4B). CCR6+ gdT cells express RORgt and produce IL-17 (37–39), we We also examined whether gdT cells are required for the examined the expression of CD27 and CCR6 on gdTcellsbyflow development of EAM. As shown in Fig. 4C, muscle weakness and cytometry. As shown in Fig. 5A, gdT cells in the muscle consisted histological score of the muscle were significantly reduced in mainly of CD272 CCR62 gdT cells in EAM-induced mice, whereas EAM-induced TCRd2/2 mice as compared with those in EAM- gdT cells in the draining LNs mainly consisted of CD27+ CCR62 induced WT mice. The infiltration of neutrophils, but not of Macs, gdT cells. Importantly, more than half of gdT cells in the muscle CD4+ Tcells,orCD8+ T cells, into the muscle was significantly were positive for Vg4, and the majority of them were also positive decreased in EAM-induced TCRd2/2 mice (Fig. 4D). Serum levels for Vd4(HeiligandTonegawa’s nomenclature) in EAM-induced of GM-CSF tended to be decreased in EAM-induced TCRd2/2 mice (Fig. 5A). gdTcellsinthemusclewerenegativeforVg1or mice as compared with those in EAM-induced WT mice (WT Vg5 in EAM-induced mice (Fig. 5A), whereas ;30% of gdTcellsin mice 49.4 6 20.2 pg/ml versus IL-212/2 mice 10.6 6 5.1 pg/ml, p = the draining LNs were positive for Vg1 in EAM-induced mice. 0.12). In contrast, serum IL-21 was undetectable in both EAM- These results suggest that Vg4+Vd4+ CD272 cells preferentially induced WT mice and TCRd2/2 mice. Taken together with the accumulate in the muscle in EAM-induced mice. finding that gdT cells are the main producer of GM-CSF in EAM- It has been reported that Vg4+Vd4+ cells produce IL-17 and induced mice (Fig. 3B), these results suggest that GM-CSF– are involved in the development of collagen-induced arthritis producing gdT cells are involved in the induction of EAM. (40) and psoriasis-like skin changes (41–43). By using intracellular cytokine staining,wefoundthatthemostofGM- IL-21 induces the accumulation of GM-CSF–producing Vg4+ CSF–producing gdT cells in the draining LNs and the muscle in Vd4+ CD272 cells in the muscle in EAM EAM-induced mice were Vg4+Vd4+ cells (Fig. 5B). In contrast, Given that gdT cells seem to play a pathogenic role in EAM (Fig. 4), IL-17A production was found in both Vg4+Vd4+ cells and non- we next analyzed the characteristics of gdT cells in the muscle of Vg4+Vd4+ cells in the muscle in EAM-induced mice (Fig. 5B).

https://doi.org/10.4049/immunohorizons.1700053 ImmunoHorizons IL-21–Vg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS 183 Downloaded from http://www.immunohorizons.org/ FIGURE 5. IL-21 induces the accumulation of GM-GSF–producing Vg4+Vd4+ CD272 cells in the muscle. (A and B) EAM was induced in WT mice. (A) The expression of indicated cell surface markers on gdT cells in draining LNs and muscle at day 24. Data are representative of four independent experiments. (B) Representative profiles of either Vg4orVd4 versus either GM-CSF or IL-17A on gdT cells in the draining LNs and the muscle. Data are representative of four independent experiments. (C) EAM was induced in IL-212/2 mice and WT mice. Representative profiles of Vg4 versus either Vd4 or CD27 on gdT cells in the muscle (left panels) and the frequencies of Vg4+ Vd4+ CD272 cells (right panel). n = 6 (WT mice) and 5 (IL-212/2 mice). *p , 0.05.

Importantly, the number of Vg4+Vd4+ CD272 cells in the suggest that CX3CR1 could be involved in IL-21–mediated muscle was significantly decreased in EAM-induced IL-212/2 accumulation of Vg4+Vd4+ CD272 cells in the muscle in EAM- by guest on September 26, 2021 mice as compared with that in EAM-induced WT mice (Fig. induced mice. 5C). Taken together, these results suggest that IL-21 is involved We next analyzed the expression of CX3CL1, a CX3CR1 ligand, in the accumulation of GM-CSF–producing Vg4+Vd4+ CD272 in the muscle. We found that the expression of Cx3cl1 mRNA in the cells in the muscle in EAM-induced mice. muscle was significantly increased by EAM induction in WT mice, but not in IL-212/2 mice (Fig. 6C). Furthermore, analysis of the IL-21 induces the expression of CX3CL1 in the muscle conserved noncoding sequence of Cx3cl1 gene loci of human and To determine the mechanisms underlying the accumulation of mouse identified 100 bp of conserved noncoding sequence at the Vg4+Vd4+ CD272 cells in the muscle, we next examined the upstream of the transcription start site of the Cx3cl1 gene. Search expression of chemokine receptors on gdT cells in the draining for a potential STAT binding site within the conserved noncoding 2 2 LNsandthemuscleinEAM-inducedWTandIL-21 / mice. As sequence in Cx3cl1 loci identified several putative Stat1, Stat3, and shown in Fig. 6A, the expression of CCR3, CCR6, CCR8, CXCR4, Stat5a binding sites (Fig. 6D). Indeed, by using reporter assays, we and CX3CR1 was significantly upregulated in muscle-infiltrating found that IL-21 activated the promoter of Cx3cl1 in M12.4.5 gdT cells as compared with gdT cells in the draining LNs in cells (Fig. 6E), suggesting the direct activation of Cx3cl1 promoter EAM-induced WT mice. Importantly, the expression of CX3CR1 by IL-21. Taken together, these results suggest that the reduced in muscle-infiltrating gdT cells was significantly decreased in EAM- expression of CX3CL1 in the muscle seems to be responsible for induced IL-212/2 mice as compared with that in EAM-induced WT the reduced accumulation of Vg4+Vd4+ CD272 cells in the muscle mice (Fig. 6A). Further analysis of CX3CR1 expression in the by the absence of IL-21 in EAM. subpopulation of gdT cells in the draining LNs of EAM-induced WT We finally measured the serum levels of CX3CL1 and GM-CSF mice revealed that the expression levels of CX3CR1 on Vg4+ in patients with IM and examined the correlation with the levels of Vd4+ CD272 cells was much higher than those on CD27+ gdT cells, IL-21. Although GM-CSF was undetectable in the vast majority of Vg42Vd42 CD272 cells, or Vg4+Vd42 CD272 cells (Fig. 6B). PM/DM patients (data not shown), the serum level of CX3CL1 was Moreover, the expression of CX3CR1 on Vg4+Vd4+ CD272 cells in significantly correlated with the level of IL-21 in PM/DM patients the draining LNs and the muscle was modestly but significantly (r = 0.4334, p = 0.0046, n = 41) (Fig. 6F), suggesting that the decreased in EAM-induced IL-212/2 mice as compared with that IL-21–CX3CL1 axis might be involved in the pathogenesis of IM in in EAM-induced WT mice (Fig. 6B). Taken together, these results humans.

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FIGURE 6. IL-21 induces the expression of CX3CL1 in the muscle in EAM. (A–C) EAM was induced in IL-212/2 mice and WT mice. (A) Mean fluorescent intensity (MFI) of indicated chemokine receptors on gdT cells in the draining LNs and the muscle. n =3–6 mice. (B) Upper panels, Representative profiles of CD27 versus TCRgd on gdT cells and Vd4 versus Vg4on CD272 gdT cells. Middle panels, Representative histograms of CX3CR1 on indicated subsets of gdT cells. Lower panel, Mean 6 SEM of MFI of CX3CR1 in indicated subsets of gdT cells. n = 6 mice. *p , 0.05, **p , 0.01. (C) The expression of Cx3cl1 mRNA in the muscle was assessed by qPCR at day 24 of EAM. Mean 6 SEM. n = 7 (EAM) and 3 (PBS). *p , 0.05. (D) VISTA plot between human and mouse Cx3cl1 promoter region and putative

Stat1, Stat3, and Stat5a binding sites of conserved region (green line). (E) Mean 6 SEM of fold induction of Cx3cl1-Luc (n = 4). *p , 0.05. (F) The by guest on September 26, 2021 correlation between IL-21 levels and CX3CL1 levels in serum of PM/DM patients (n = 41). Spearman rank correlation coefficient was used.

DISCUSSION We show that IL-21 plays a pathogenic role in IM. Previous studies have shown that IL-21 is involved in the development of In this study, we show that IL-21 is involved in the pathogenesis of several autoimmune disease models, including type 1 diabetes in autoimmune myositis. We found that serum levels of IL-21 were NOD mice (17), murine models of rheumatoid arthritis (18), and increased in a subset of patients with IM as compared with those in lupus (19, 20). We found that serum levelsofIL-21 were elevated in healthy controls (Fig. 1). By using EAM, a murine model of IM, we a subset of patients with IM (Fig. 1), consistent with a previous 2 found that the severity of EAM was diminished in IL-212/2 mice finding that CXCR3 CXCR5+ Th cells, which are capable of pro- (Fig. 2). GM-CSF production from gdT cells, but not Th17 cell ducing IL-21, are increased in peripheral blood in patients with differentiation, was significantly reduced in EAM-induced IL-212/2 juvenile DM (24). We also found that EAM was significantly 2 2 mice (Fig. 3). Importantly, the neutralization of GM-CSF or the attenuated in IL-21 / mice (Fig. 2), consistent with a previous deficiency of gdT cells significantly improved muscle weakness and finding that i.m. injection of lymphocytes of Foxp3-deficient scurfy muscle inflammation in EAM-induced mice (Fig. 4). We also found mice, in which IL-21–producing CD4+ Tcellsaresignificantly that the major GM-CSF producers in the muscle in EAM-induced increased (21), into RAG-deficient mice causes IM-like patholog- mice were Vg4+Vd4+ CD272 cells (Fig. 5B), and that Vg4+Vd4+ ical changes (44, 45). Taken together, these findings suggest that CD272 cells were significantly decreased in EAM-induced IL-212/2 IL-21 plays a pathogenic role in IM in mice, and possibly in mice(Fig.5C).Intriguingly,Vg4+Vd4+ CD272 cells expressed humans, and could be a therapeutic target for IM. higher levels of CX3CR1 (Fig. 6B). In addition, CX3CL1, a ligand of Regarding the mechanism underlying IL-21–mediated muscle CX3CR1, was induced in the muscle upon EAM induction in WT inflammation, we found that the accumulation of GM-CSF– mice, but not in IL-212/2 mice (Fig. 6C). Taken together, these producing gdT cells in the muscle was less severe in EAM-induced 2 2 results suggest that IL-21 enhances autoimmune myositis through IL-21 / mice than EAM-induced WT mice (Fig. 3B). We also the accumulation of GM-CSF–producing Vg4+Vd4+ CD272 cells in found that the neutralization of GM-CSF significantly improved themusclepossiblyviaaCX3CR1-CX3CL1pathway. muscle weakness and muscle inflammation in EAM-induced mice

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(Fig. 4). In addition, muscle weakness and muscle inflammation CX3CR1 improves the severity of experimental myositis in SJL/J upon EAM induction were reduced in TCRd2/2 mice (Fig. 4). mice (50), and that serum level of CX3CL1 is correlated with Taken together, these results suggest that the reduction of GM- disease activity in patients with PM/DM (51). In contrast, although CSF–producing gdT cells in the muscle might be responsible for CX3CR1 has been shown to be expressed on muscle-infiltrating the mild phenotype of EAM in IL-212/2 mice. The analysis of mice CD4+ cells, CD8+ cells, and Macs in EAM-induced mice (50), the specifically lacking GM-CSF production in gdTcellsisrequiredto numbers of these cells in the muscle were not significantly confirm this notion. different between EAM-induced IL-212/2 mice and WT mice In contrast, we found that the numbers of IL-17A–producing (Fig. 2B). These results suggest that other chemokine/chemokine CD4+ T cells and IL-17A–producing gdT cells were not signifi- receptor systems may compensate the recruitment of CD4+ cells, cantly different between EAM-induced IL-212/2 mice and WT CD8+ cells, and Macs into the muscle in EAM-induced mice. mice (Fig. 3C). In this regard, it has been reported that serum levels Analysis of Cx3cl1 gene loci of human and mouse identified 100 of IL-17A are increased in patients with IM (46). Moreover, Zhang bp of conserved noncoding sequence at the upstream of the et al. (47) have recently reported that the level of IL-17A in the transcriptional start site of Cx3cl1 (Fig.6D).Furthersearchfora muscle tissue is positively correlated with the degree of muscle potential Stat binding site within the conserved noncoding inflammation, and that anti–IL-17A Ab treatment attenuates sequence identified several putative Stat1, Stat3, and Stat5a binding Downloaded from muscle inflammation in EAM. These findings indicate that IL- sites (Fig. 6D). Consistent with these in silico analyses, we 17A is also involved in the pathogenesis of EAM and suggest that confirmed that IL-21 activated Cx3cl1 promoter by reporter assays the main cellular sourcesofIL-17Ain EAM might be different from (Fig. 6E). Moreover, the expression of Cx3cl1 was elevated in the CD4+ T cells and gdT cells. Further studies are required to address muscle upon EAM induction in WT mice, but not in IL-212/2 mice the cellular and molecular relationship between IL-17A and IL-21 (Fig.6C).Thesefindings suggest that IL-21 directly induces Cx3cl1 http://www.immunohorizons.org/ in EAM patients, as well as patients with IM. expression through the activation of the promoter in the muscle, Regarding gd T cells in the pathogenesis of IM in humans, a although the cell type that expresses Cx3cl1 in EAM-induced rare case of PM characterized by massive infiltration of gd Tcells muscle remains unknown. The positive correlation between into the muscle has been reported (48). In this case, clonally serum levels of IL-21 and CX3CL1 in patients with PM/DM (Fig. expanded gd T cells recognize histidyl-tRNA synthetase (49). In 6F) also supports this notion. contrast, the frequency of gd T cells among muscle-infiltrating We found that the accumulation of SLECs in the muscle is cells is ,3% in the vast majority of IM patients (48), indicating that reduced in EAM-induced IL-212/2 mice (Fig. 2B). Because it has the patient with massive infiltration of gd T cells is not a been shown that IL-21 induces the expression of CX3CR1 on CD8+ representative of IM patients, and that the role of gd T cells in T cells (52), it is possible that IL-21 facilitates the infiltration of patients with IM remains largely unknown. In this study, we found SLECs into the muscle through the induction of CX3CR1 on CD8+ by guest on September 26, 2021 that gd T cells, which account for ;1% of muscle-infiltrating Tcells.Thesefindings underscore the notion that the neutraliza- hematopoietic cells, play an important role in EAM (Fig. 4). tion of IL-21 may be more beneficial for CD8+ Tcell–mediated Although further studies are required, our findings raise the autoimmune diseases such as PM (22). We also found that GC possibility that a small number of muscle-infiltrating gd Tcells B cells are significantly decreased in the draining LNs of EAM- may play a role in certain populations of patients with IM. induced IL-212/2 mice. Because anti–B cell therapy has beneficial Among gdT cells, we show that Vg4+Vd4+ CD272 cells are the effects on patients with PM/DM (53), the reduction of GC B cells major population in the muscle in EAM-induced mice, and that may also be involved in the attenuated muscle weakness in the accumulation of Vg4+Vd4+ CD272 cells in the muscle is EAM-induced IL-212/2 mice. In contrast, the implication for the significantly reduced in EAM-induced IL-212/2 mice (Fig. 5). reduced numbers of neutrophils in EAM-induced IL-212/2 mice Vg4+Vd4+ cells have been discovered in draining LNs in collagen- (Fig. 2B) remains obscure. Further studies are needed to address induced arthritis models (40). Subsequently, Vg4+Vd4+ cells have this point. beenshowntobepathogenicinpsoriasismodels(41,43).Wealso This study has some limitations. First, we did not assess found that Vg4+Vd4+ CD272 cells are the major producer of GM- patients with other diseases that cause myopathy. The presence of CSF in the muscle in EAM (Fig. 5B). These results suggest that a disease control group could have clarified the specificity of our similar to psoriasis models, Vg4+Vd4+ cells seem to be pathogenic findings in PM/DM patients. Especially because lymphocytes also in EAM, although specific deletion of cells expressing Vg4orVd4 infiltrate into the muscle in patients with sporadic inclusion body is needed to prove the role of Vg4+Vd4+ cells in EAM. myositis, but immunohistological findings as well as the prognosis In psoriasis models, Vg4+Vd4+ cells migrate from draining LNs of the diseases are quite different between PM/DM and inclusion to the inflamed skin via a CCR2-CCL2 pathway (41, 43). In body myositis (23), the comparison of serum levels of IL-21 contrast, we found that Vg4+Vd4+ CD272 cells expressed higher between these diseases might be intriguing. Second, our study levels of CX3CR1 than Vg42Vd42 cells (Fig. 6B), and that the ex- lacked the analysis of muscle biopsy samples in PM/DM patients. pression of CX3CL1 was induced in the muscle in WT mice upon Because we found that IL-21 was detected in muscles and drain- EAM induction (Fig. 6C), suggesting that Vg4+Vd4+ CD272 cells ing LNs (Fig. 2), but not in sera in EAM, the analysis of muscle infiltrate into the muscle via a CX3CR1-CX3CL1 pathway. This biopsy samples of PM/DM patients would provide more detailed notion is consistent with previous findings that the inhibition of information on the production of IL-21. Third, serum levels of

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IL-21 were under the detection limits in ;60% of PM/DM 11. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and patients. A relatively small sample size did not allow for Th17 cells. Annu. Rev. Immunol. 27: 485–517. stratiﬁcation by background information such as the disease 12. King, C., S. G. Tangye, and C. R. Mackay. 2008. T follicular helper (TFH) cells in normal and dysregulated immune responses. Annu. activity and the presence of autoantibody. Studies with a large Rev. Immunol. 26: 741–766. cohort of PM/DM patients by a more sensitive measure of IL-21 13. Crotty, S. 2011. Follicular helper CD4 T cells (TFH). Annu. Rev. are desired. Immunol. 29: 621–663. In conclusion, we have shown that IL-21 is involved in the 14. Suto, A., D. Kashiwakuma, S. Kagami, K. Hirose, N. Watanabe, development of autoimmune myopathy presumably by inducing K. Yokote, Y. Saito, T. Nakayama, M. J. Grusby, I. Iwamoto, and the accumulation of GM-CSF–producing Vg4+Vd4+ CD272 cells H. Nakajima. 2008. Development and characterization of IL-21- producing CD4+ T cells. J. Exp. Med. 205: 1369–1379. in the muscle. Although further studies are needed, our results 15. McGuire, H. M., A. Vogelzang, C. S. Ma, W. E. Hughes, P. A. Silveira, should add a new insight into the pathogenesis of IM and suggest S. G. Tangye, D. Christ, D. Fulcher, M. Falcone, and C. King. 2011. A that the neutralization of IL-21 or GM-CSF could be a therapeutic subset of interleukin-21+ chemokine receptor CCR9+ T helper cells strategy for the treatment of IM. target accessory organs of the digestive system in autoimmunity. Immunity 34: 602–615.

16. Rao, D. A., M. F. Gurish, J. L. Marshall, K. Slowikowski, C. Y. Fonseka, Downloaded from DISCLOSURES Y. Liu, L. T. Donlin, L. A. Henderson, K. Wei, F. Mizoguchi, et al. 2017. Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature 542: 110–114. The authors have no ﬁnancial conﬂicts of interest. 17. Sutherland, A. P. R., T. Van Belle, A. L. Wurster, A. Suto, M. Michaud, D. Zhang, M. J. Grusby, and M. von Herrath. 2009. Interleukin-21 is required for the development of type 1 diabetes in NOD mice. Di-

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