T Cells in the MuscleδγProducing minusAccumulation of GM-CSF IL-21 Exacerbates Autoimmune Myositis by Enhancing the

NakajimaYokoyama Aiko Saku Shunsuke Furuta Kei Ikeda Kotaro Suzuki Koichi Hirose and Hiroshi Takahiro Kageyama Akira Suto Taro Iwamoto Shigeru Tanaka Kenichi Suehiro Yusuke

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2017 1 (8) 176-187ImmunoHorizons

This information is current as of October 12 2021

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IL-21 Exacerbates Autoimmune Myositis by Enhancing theAccumulation of GM-CSFndashProducing gd T Cells in the Muscle

Takahiro Kageyama1 Akira Sutodagger1 Taro Iwamoto Shigeru Tanaka Kenichi Suehiro Yusuke Yokoyama Aiko SakuShunsuke Furuta Kei Ikeda Kotaro Suzuki Koichi Hirose and Hiroshi NakajimaDepartment of Allergy and Clinical Immunology Graduate School of Medicine Chiba University Chiba 260-8670 Japan and daggerInstitute for Global

Prominent Research Chiba University Chiba 260-8670 Japan

ABSTRACT

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-21ndashdeficient (IL-2122) 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-2122 mice Importantly GM-CSF production from gdT cells but not CD4+

T cells was significantly reduced in EAM-induced IL-2122 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-CSFndashproducing gdT cells expressed Vg4+Vd4+

TCR and the number of Vg4+Vd4+ cells in the muscle was significantly decreased in EAM-induced IL-2122 mice as compared with

that in EAM-induced WT mice Moreover muscle-infiltrating Vg4+Vd4+ cells exhibited CX3CR1high phenotype and the induction of

Cx3cl1 a ligand for CX3CR1 in the muscle was reduced in EAM-induced IL-2122 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-CSFndashproducing Vg4+Vd4+ cells in the muscle possibly via CX3CR1-CX3CL1 pathways ImmunoHorizons 2017 1 176ndash187

INTRODUCTION

Autoimmune diseases are caused by a breakdown of tolerance toself-antigens and are characterized by the activation of T cellsincluding pathogenic IL-17ndashproducing Th cells (Th17 cells)Pathogenic Th17 cells are eventually differentiated in the presence

of IL-23 andproduce IL-17A IL-17F IL-21 andGM-CSF (1 2) IL-17ndashproducing gdT cells are also activated by IL-23 and implicatedin the pathogenesis of autoimmune diseases (3 4) Because theblocking of IL-17 signaling is less effective than that of IL-23 inautoimmune disease models such as experimental autoimmuneencephalomyelitis (EAE) (5 6) Th17 cellndashrelated 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 ofMedicine Chiba University 1-8-1 Inohana Chiba City Chiba 260-8670 Japan E-mail addresses suakifacultychiba-ujp (A Suto) and nakajimhfacultychiba-ujp(HN)

ORCID 0000-0002-2593-565X (A Suto)1TK 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 Governmentthe 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 experimentalautoimmune myositis GC germinal center gf gram-force IL-2122 IL-21ndashdeficient IL-21R22 IL-21Rndashdeficient IM inflammatory myopathy LN lymph node Macmacrophage PD-1 programmed death-1 PM polymyositis qPCR quantitative PCR RORgt retinoid-related orphan receptor g t SLEC short-lived effector CD8+

T cell TCRd22 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 40 Unported license

Copyright copy 2017 The Authors

176 httpsdoiorg104049immunohorizons1700053

RESEARCH ARTICLE

Adaptive Immunity

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than IL-17 seem to be important for the development of certainautoimmune diseases

In the initiation phase of EAE GM-CSF a representativecytokine that induces the differentiation of neutrophils andmacrophages (Macs) is produced by Th17 cells and serves anonredundant function (1 2) In addition it has been shown thatIL-7ndashinduced Stat5 activation promotes the development of GM-CSFndashproducing CD4+ T cells and causes severe EAE (7) More-over GM-CSF is shown to be produced by gdT cells during thedevelopment of EAE (8) In humans blocking of GM-CSFsignaling by a neutralizing Ab has shown promising results forrheumatoid arthritis in early-phase clinical trials (9) Thesefindings suggest that GM-CSF is involved in the development ofsome autoimmune diseases

Recent studies have shown that IL-21 exhibits pleiotropiceffectsonavariety of immune responses (10) IL-21 isproducedbymany types of CD4+ T cells such as Th17 cells (11) follicular B Th(Tfh) cells (12 13) IL-6ndash or IL-21ndashstimulated CD4+ T cells (14)CCR9+ Th cells (15) and ldquoperipheral Thrdquo cells (16) In additionIL-21 has been shown to be involved in the development ofautoimmune disease models including type 1 diabetes in NODmice (17) murine models of rheumatoid arthritis (18) and lupus(19 20) Moreover we have shown that IL-21ndashproducing CD4+

T cells are increased in Foxp3 mutant scurfy mice and thatabrogation of IL-21 signaling in scurfymice significantly prolongssurvival presumably by reducing autoimmune inflammation oflung through the suppression of short-lived effector CD8+ T cells(SLECs) (21)

Polymyositis (PM) and dermatomyositis (DM) are the majorsubgroups of idiopathic inflammatory myopathy (IM) which ischaracterizedby infiltration of inflammatory cells into the affectedmuscle (22 23) Regarding the relationship between IL-21 and IMin humans it has been reported that CXCR32 CXCR5+ Th cellswhich are known to produce IL-21 are increased in peripheralblood in juvenile DM patients who have skin rash and muscularweakness (24) However the role of IL-21 in the pathogenesis ofIM remains unknown

In this study we addressed this issue by using a murine modelof autoimmunemyositis Our results indicate that IL-21 is involvedin thedevelopment of autoimmunemyositis possibly by enhancingneutrophil recruitment into the muscle through the accumula-tion ofGM-CSFndashproducingVg4+Vd4+CD272 cells via aCX3CR1-CX3CL1 pathway

MATERIALS AND METHODS

PatientsWe reviewed the medical records of patients who were newlydiagnosed with IM and were admitted to Department of Allergyand Clinical Immunology Chiba University Hospital fromSeptember of 2009 to June of 2016 We identified 51 sequentialpatients who fulfilled the criteria of Bohan and Peter (25) forPMDM or those of Sontheimer and colleagues (26) for clinicallyamyopathic DM (CADM) Patients who were complicated by

malignancy were excluded We retrospectively assessed thebaseline patient characteristics including age sex diagnosisand presence of malignancy The study was approved by theEthics Committees of Chiba University and was performed inaccordance with the principles expressed in the Declaration ofHelsinki

ELISASerum levels of IL-21 and GM-GSF were analyzed by a humanIL-21 ELISA MAX kit and a human GM-GSF ELISA MAX kitrespectively (BioLegend San Diego CA) Serum levels of humanCX3CL1 and murine IL-21 were analyzed by a human CX3CL1Fractalkine Quantikine ELISA Kit and a mouse IL-21 DuoSetELISA respectively (RampD Systems Minneapolis MN) Detectionlimits are as follows human IL-21 313 pgml human GM-CSF78 pgml human CX3CL1 063 ngml and murine IL-21625 pgml

MiceC57BL6 mice were purchased from Charles River Laboratories(Atsugi Kanagawa Japan) IL-21ndashdeficient (IL-2122) mice(B6129S-Il21tm1LexMmucd) were obtained from Mutant MouseResource Research Centers IL-21Rndashdeficient (IL-21R22) mice(27) were backcrossed over eight generations onto C57BL6miceTCRd-deficient (TCRd22) mice were obtained from the JacksonLaboratory (Bar Harbor ME) All mice were housed in micro-isolator cages under specific pathogen-free conditions The ChibaUniversity Animal Care and Use Committee approved animalprocedures used in this study

Experimental autoimmune myositisExperimental autoimmune myositis (EAM) was induced asdescribed previously (28 29) In brief myosin was purifiedfrom themuscle of C57BL6mice and emulsifiedwith CFAwith2mgmlMycobacteriumtuberculosis (CFA) (ChondrexRedmondWA) Mice were immunized three times at 1-wk intervals with1mg ofmyosin inCFAon bilateral sides of hind foot pads (day 0)tail base (day 7) and flanks (day 14) as described previously (29)PBSwas injected as controls Pertussis toxin (500ng Sigma)wasinjected ip at day 1 At day 24 after mice were perfused with50 ml of ice-cold PBS to wash out blood cells right hind-limbmuscle was excised for histological analysis left hind-limbmuscle for FACS analysis and triceps for quantitative PCR(qPCR) analysis For neutralization of GM-CSF antindashGM-CSFmAb (300 mgmouse clone MP1-22E9 BioLegend) or purifiedrat IgG2a (BioLegend) as a control was injected ip on days 2 9and 16

Evaluation of muscle strengthMuscle strength was measured by using a grip strength meter forsmall animal (GBM-100B) (Melquest Toyama Japan) at day 23All tests were performed in a blinded fashion Muscle weaknesswas defined as the decrease of muscle strength of EAM-inducedmice thatwas calculatedby subtracting from the average ofmusclestrength in age-matched PBS-injected mice

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Histological analysis of skeletal muscleParaffin sections of quadriceps were examined histologically forthepresenceof inflammatorycell infiltratesandnecrosis ofmusclefibers Sections (5 mm thick) were cut at 200-mm distance andstainedwithHampE Inflammatory changes of skeletalmuscleswerequantified in a blinded manner by using a scoring system asdescribed previously (30) grade 1 single or 5 muscle fibersinvolvement grade 2 a lesion involving 5ndash30muscle fibers grade3 a lesion involving amuscle fasciculus grade4 adiffuse extensivelesionWhenmultiple lesionswere found in onemuscle block 05point was added to the indicated score

ReagentsAbs to CD3 (145-2C11) CD28 (3751) CD45 (30-F11) B220 (RA3-6B2) CD4 (RM4-5) CD8 (53-67) Vd4 (GL2) CD11b (M170)CXCR5 (2G8) Siglec-F (E50-2440) Fas (Jo2) IL-4 (11B11) IFN-g(XMG12) and IL-17A (TC11-18H10) were purchased from BDBiosciences (San Diego CA) Abs to TCR gd (UC7-13D5 and GL3)TCR Vg1 (211) TCR Vg4 (UC3-10A6) TCR Vg5 (536) Ly-6C(HK14) Ly-6G (1A8) CD64 (X54-571) KLRG1 (2F1) IL-7Ra(SB199) CX3CR1 (SA011F11) CCR4 (2G12) CCR5 (HM-CCR5)CCR6 (29-2L17) CCR8 (SA214G2) CXCR3 (CXCR3-173) CXCR4(L276F12)GM-CSF(MP1-22E9) andIL-17A (TC11-18H101)werepurchased from BioLegend Abs to GL7 (GL-7) programmeddeath-1 (PD-1 RMP1-30) CD27 (LG7F9) CCR7 (4B12) CCR9(eBioCW-12) Foxp3 (FJK-16s) and retinoid-related orphanreceptorg t (RORgt) (AFKJS-9)werepurchased fromeBioscience(San Diego CA) Murine IL-21 was purchased from BioLegendHuman TGF-b mouse IL-21RndashFc chimera and Abs to CCR2(FAB5538P) CCR3 (FAB729B) and CCR10 (FAB2815C) werepurchased from RampD Systems

Cell isolation and flow cytometry analysisNaive CD4+ T cells were isolated by using a mouse naive CD4+

T cell isolation kit (STEMCELL Technologies Tukwila WA)gdT cells were isolated by using a mouse TCRgd T cellisolation kit (Miltenyi Biotec) Muscle-infiltrating cells wereprepared from lower hind-limb muscle by using gentleMACSOcto Dissociator (Miltenyi Biotec) with Liberase (02 mgmlRoche Basel Switzerland) and DNase I (01 mgml Roche)Single-cell suspensions of muscle-infiltrating cells were thenobtained by sequential filtration with 100 and 30 mm of cellstrainers (BD) Viable cells were collected from the cellsuspensions using Lympholyte-M (Cedarlane LaboratoriesBurlington ON Canada) according to the manufacturerrsquosinstructions Cells were stained and analyzed by FACSCanto II(BD) using FlowJo software (Tree Star Ashland OR) Neutro-phils are defined as CD11b+ Ly6G+ cells Macs as CD11b+ Ly6G2

Siglec-F2 CD64+ cells M2 Macs as CD11b+ Ly6G2 Siglec-F2

CD64+ Ly6Chi cells M1 Macs as CD11b+ Ly6G2 Siglec-F2

CD64+ Ly6Clo cells germinal center (GC) B cells as KLRG1+

Fas+ B220+ cells follicular Th cells as CXCR5+ PD-1+ CD4+

T cells Th17 cells as RORgt+ CD4+ T cells and SLECs as KLRG1+

IL-7Ra2 CD8+ T cells

Determination of inflammatory cytokine profileAtday24ofEAMdraining lymphnode (LN)cellswere stimulatedwith PMA plus ionomycin for 5 h in RPMI 1640 mediumsupplemented with 10 heat-inactivated FCS 50 mM 2-ME and2mM L-glutamine (complete RPMI 1640medium) Inflammatorycytokine profiles of culture supernatants were analyzed by aLEGENDplex mouse inflammation panel (BioLegend) accordingto the manufacturerrsquos instruction Serum levels of inflammatorycytokines were analyzed by the same system at day 24 of EAM

Intracellular cytokine and transcription factor stainingFor intracellular cytokine staining cells were stimulated withPMA plus ionomycin for 3 h in complete RPMI 1640 medium inthe presence of Golgi plug (BD) stained with fixable viability dye(InvitrogenorBioLegend) andcell surfacemarkers and thenfixedand permeabilized with PermWash buffer (BD Biosciences)according to the manufacturerrsquos instructions Intracellular cyto-kine staining of IL-21 was performed as described previously (1431ndash33) For transcription factor staining cells were stained withfixable viability dye and cell surface markers then fixed andpermeabilized with a Foxp3transcription factor staining bufferset (eBioscience) according to the manufacturerrsquos instructions

Cell cultureCD4+ T cells were stimulated with plate-bound anti-CD3emAb (1mgml) in the presence of anti-CD28 mAb (1 mgml) (anti-CD3CD28 mAb) in complete RPMI 1640 medium in either neutralconditions (without exogenous cytokines) GM-CSFndashproducingconditions [antindashIFN-g mAb (10 mgml) and IL-7 (2 ngml)] orTh17-polarizing conditions [IL-6 (05 ngml) TGF-b (1 ngml)antindashIL-4mAb (10 mgml) and antindashIFN-gmAb (10mgml)] for 3d In the case of Th17 cell induction cells were reactivated withanti-CD3CD28 mAb in the presence of IL-1b (10 ngml) and IL-23 (10ngml) for another3dgdTcellswere stimulatedwithplate-bound anti-CD3emAb (5mgml) in completeRPMI 1640mediumWhere indicated 100 ngml IL-21 was added to the culture

qPCR analysisTotal RNA was purified from triceps by using Isogen II (NipponGene Toyama Japan) and BioMashaer II (Nippi Tokyo Japan)Reverse transcription and qPCR analysis were performed asdescribed previously (33) PCR primers for Cx3cl1were as followsforward 59-ACG AAA TGC GAA ATC ATG TGC-39 reverse 59-CTG TGT CGT CTC CAGGACAA-39 PCR primers for hypoxan-thine phosphoribosyltransferase were as follows forward 59-TCAGTC AAC GGG GGA CAT AAA-39 reverse 59-GGG GCT GTACTG CTT AAC CAG-39 The levels of Cx3cl1 were normalized tothe levels of hypoxanthine phosphoribosyltransferase

Luciferase reporter assayThe fragment of mouse Cx3lc1 promoter (2500 to +30) wasamplified by PCR with mouse genomic DNA as a template andsubcloned into pGL410 luciferase vector (Promega BiotechMadison WI) (Cx3cl1-Luci) M1245 cells (5 3 104 cells) were

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resuspended with 05 mg of pGL41 vector (Cx3cl1-Luci or pGL41empty vector) and 5 ng of pGL474 vector in resuspension buffer RandelectroporatedbyusingNeon transfectionsystem(Invitrogen)at 1500V20ms per 1 pulse Cells were rested for 6 h and then leftunstimulated (PBS) or stimulated with IL-21 (100 ngml) for 18 hRelative light units were assessed with a dual luciferase assaysystem (Promega)

Data analysisData are shown as mean6 SEM Statistical analyses of the resultswere performed by the unpaired t test MannndashWhitney U testKruskalndashWallis test and Dunn multiple comparisons test orSpearman rank correlation as appropriate The p values 005were considered significant

RESULTS

Serum levels of IL-21 are elevated in a subset of patientswith IMTo investigate the possible involvement of IL-21 in the pathogen-esis of IMwefirst examined serum levels of IL-21 in patientswithIM(n =51) andhealthydonors (n =20)We found that serumIL-21was detected in 17 patients with IM (33) and 2 healthy donors(10) Themean serum level of IL-21was significantly elevated inpatients with IM as compared with healthy donors (686 6 339versus 266 18 pgml mean6 SEM p 005) (Fig 1A) Amongthe patients with IM serum IL-21 was significantly elevated inPMDM patients but not in CADM patients as compared withthose in healthy donors (8396 419 versus 606 42 versus 26618 pgml mean6 SEM p 005) (Fig 1B) These results suggest

that IL-21 is associated with muscle involvement in a subset ofpatients with IM

IL-21 is involved in the pathogenesis of EAMTo further address the roles of IL-21 in the pathogenesis of IMweused EAM (28) which is induced by the immunization withmyosin When EAM was induced in wild-type (WT) mice IL-21was undetectable in sera at day 24 of EAM However when EAMwas induced in IL-21ndashdeficient (IL-2122) mice muscle weaknesswas less evident in IL-2122mice than that inWTmice (WTmice10466 120 gram-force [gf ] versus IL-2122mice 6546 118 gf p 005) (Fig 2A) Consistently histological analyses revealed thatinflammatory changes in themusclewere less obvious in IL-2122

mice than those in WTmice (Fig 2A) These results indicate thatIL-21 is involved in the development of EAM

To explore themechanisms underlying the attenuatedmuscleweakness in EAM-induced IL-2122mice we next examined thenumbers of inflammatory cells in the skeletal muscle in EAM-inducedWTmice and IL-2122mice Consistent with a previousreport (28) the number of CD45+ hematopoietic cells in themuscle was significantly increased in WT mice upon EAMinduction (Fig 2B) Importantly the infiltration of CD11b+

myeloid cells in the muscle was less evident in EAM-inducedIL-2122 mice than that in EAM-inducedWTmice (n = 12 micep 005) Among CD11b+ myeloid cells Ly6G+ neutrophils weresignificantly decreased in the muscle in EAM-induced IL-2122

mice (Fig 2B) whereas the numbers of M1 Macs M2 Macseosinophils and mast cells were similar between EAM-inducedIL-2122 mice and WT mice (Fig 2B data not shown) Asfor lymphocytes the numbers of CD4+ T cells CD8+ T cells andgdT cells in the muscle were comparable between EAM-inducedIL-2122 mice and WT mice (Fig 2B) Because CD8+ T cellsinfiltrate into the muscle in patients with IM (22) we furtheranalyzed the subtypes of CD8+ T cells in the muscle in EAM-induced mice Consistent with our previous finding that IL-21induces the accumulation of SLECs into the lung in scurfy mice(21) the number of SLECs in the muscle was significantlydecreased in EAM-induced IL-2122mice as comparedwith thatin EAM-induced WTmice (Fig 2B)

Because IL-21 is involved in the development of Tfh cells Th17cells and GC B cells (34) we also analyzed the development ofthese cell populations in the draining LNs of EAM-induced miceAs expected GC B cells were significantly decreased in thedrainingLNs inEAM-inducedIL-2122mice (Fig 2C) Incontrastthe numbers of Tfh cells and Th17 cells were not significantlydifferent between EAM-induced IL-2122 mice and WT micesuggesting the redundant roles of IL-21 in the differentiation ofTfh and Th17 cells in EAM-induced mice (Fig 2C)

CD4+ T cells are the major producer of IL-21 in EAMWe next analyzed cell types that produce IL-21 in the muscle inEAM-inducedmice Intracellular IL-21 staining togetherwith cellsurface phenotyping revealed that most of the IL-21ndashproducingcells in the muscle were CD4+ T cells (Fig 2D) We also analyzedsurface markers of IL-21ndashproducing CD4+ T cells in the muscle

FIGURE 1 Serum levels of IL-21 are elevated in a subset of patients

with IM

(A) Serum levels of IL-21 in patients with IM (n = 51) and healthy donors

(HD) (n = 20) were analyzed by ELISA Statistical analyses of the results

were performed by MannndashWhitney U test p 005 (B) Serum levels

of IL-21 in patients with PMDM (n = 41) CADM (n = 10) and HD (n =

20) The cohort used in (B) is identical to (A) Serum IL-21 was detected

in 15 of 41 in PMDM patients 2 of 10 in CADM patients and 2 of 20 in

HD Because y-axis is expressed on a logarithmic scale only values 0

were plotted Statistical analyses were performed by KruskalndashWallis test

and Dunn multiple comparisons test p 005 ns not significant

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and draining LNs of EAM-inducedWTmice As shown in Fig 2Dthe majority of IL-21ndashproducing CD4+ T cells in the muscleexhibited PD-1+ CXCR52 phenotype In draining LNs 20 ofIL-21ndashproducing CD4+ T cells were PD-1+ CXCR5+ conventionalTfh cells which did not express CCR2 CCR5 or CCR9 Incontrast PD-1+ CXCR5+ IL-21ndashproducing CD4+ T cells in themuscle simultaneously expressedCCR2CCR5 andCCR9 but notCCR6 These results suggest that IL-21ndashproducing CD4+ T cells inthe muscle of EAM mice seem to be different from conventionalTfh cells

IL-21 enhances GM-CSF production from gdT cellsGiven that murine neutrophils did not express IL-21R (35) it issuggested that IL-21 indirectly induces the infiltration of neutro-phils into the muscle in EAM To identify the factors that induceneutrophil infiltration into the muscle in EAM-induced mice wefirst analyzed the expression of chemokines that induce therecruitment of neutrophils in the muscle and found thatmRNAexpression ofCXCL1CXCL2 andCXCL5was comparablebetweenEAM-induced IL-2122miceandWTmice (SupplementalFig 1) We then comprehensively analyzed the serum levels of

inflammatory cytokineschemokines as well as the production ofthese cytokineschemokines from draining LN cells upon stimula-tionwith PMAplus ionomycin in EAM-induced IL-2122mice andWTmice Among 13 cytokineschemokines the level of IL-23 waslower in EAM-induced IL-2122 mice than that in EAM-inducedWT mice (Supplemental Fig 2) consistent with the reducedneutrophilic inflammation in EAM-induced IL-2122 mice (Fig2B) Meanwhile upon the activation of draining LN cells IL-10production was significantly decreased in EAM-induced IL-2122

mice as compared with that in EAM-induced WT mice (Fig 3A)consistent with a previous finding that IL-21 induces IL-10production (36) In addition GM-CSF levels were significantlydecreased inEAM-induced IL-2122mice (Fig 3A) In contrast thelevels of IL-1a IL-1b IL-6 IL-12p70 IL-17A IL-23 IL-27 IFN-bIFN-g TNF-a and CCL2 were not significantly different betweenEAM-induced IL-2122mice andWTmice (Fig 3A)

Intracellular cytokine staining confirmed that the numbers ofGM-CSFndashproducing cells were decreased in the draining LNs inEAM-induced IL-2122 mice as compared with those in EAM-induced WT mice (Fig 3B) Importantly 50 of GM-CSFndashproducing cells in the draining LNs in EAM-induced WT mice

FIGURE 2 IL-21 is involved in the pathogenesis of EAM

EAMwas induced in IL-2122 mice and WTmice (A) Upper panel Data are mean6 SEM of muscle weakness of EAM-induced mice n = 5 mice Middle

panels Representative photomicrographs of HampE 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-2122 mice) (B) Mean6 SEM of the numbers of indicated cells harvested from the muscle n = 12 for cell

populations except for SLECs n = 5 (WT mice) and 7 (IL-2122 mice) for SLECs (C) The frequencies of GC B cells Tfh cells and Th17 cells in draining

LNs at day 15 n = 5 (WT mice) and 6 (IL-2122mice) (D) Upper panel Representative FACS profiles of CD4 versus IL-21 of CD45+ CD11b2 lymphocytes

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-21ndashproducing CD4+ T cells in the draining LNs and muscle Data are representative of four independent experiments p 005

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were gdT cells (Fig 3B) Indeed whereas the frequency of GM-CSFndashproducing CD4+ T cells was comparable between IL-2122

mice and WT mice (Fig 3B) the frequency of GM-CSFndashproducing gdT cells was significantly decreased in EAM-induced IL-2122 mice as compared with that in EAM-inducedWT mice (WT mice 014 6 005 versus IL-2122 mice 0046001 p 005) (Fig 3B) Consistently the number of GM-CSFndashproducinggdTcellswasdecreased in themuscle inEAM-inducedIL-2122 mice (p 005) (Fig 3B) In contrast the numbers ofGM-CSFndashproducing CD4+ T cells IL-17Andashproducing CD4+

T cells and IL-17Andashproducing gdT cells in the muscle were notsignificantly different between EAM-induced IL-2122 mice andWT mice (Fig 3C)

We next analyzed the effect of IL-21 on GM-CSF productionfromCD4+ T cells and gdT cells in vitro Consistent with previousreports (1 7) CD4+ T cells stimulated under IL-7 + antindashIFN-gconditions produced GM-CSF (Fig 3D) The addition of IL-21 to

the culture of CD4+ T cells suppressedGM-CSF production underIL-7 + antindashIFN-g conditions (Fig 3D) IL-21 did not affect GM-CSF production from Th17 cells (Fig 3D) In contrast IL-21modestly but significantly increased GM-CSF production fromanti-CD3ndashstimulated gdT cells in WT mice but not in IL-21R22

mice (Fig 3E) indicating that IL-21 enhances the production ofGM-CSF from gdT cells but not CD4+ T cells

GM-CSFndashproducing gdT cells are involved in thedevelopment of EAMWe next examined whether GM-CSF is involved in the develop-ment of EAMby administering neutralizing antindashGM-CSFAb Asshown inFig 4Aweekly administration ofneutralizing antindashGM-CSFAb significantly attenuatedmuscleweakness andhistologicalscores in EAM-induced WT mice The number of neutrophils inthe muscle was also significantly decreased in mice treated withantindashGM-CSF Ab (Fig 4B) In contrast the numbers of Macs

FIGURE 3 IL-21 enhances GM-CSF production from gdT cells

(AndashC) EAM was induced in IL-2122 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-2122 mice) (B) At day 15 of EAM GM-CSFndashproducing cells in draining LNs were analyzed by flow

cytometry Left two panels The frequencies of GM-CSFndashproducing cells and GM-CSFndashproducing 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-CSFndashproducing gdT cells n = 6 (C)

At day 24 of EAM GM-CSFndashproducing 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-17Andashproducing CD4+ T cells GM-CSFndashproducing CD4+ T cells IL-17Andashproducing gdT cells

and GM-CSFndashproducing gdT cells n = 8 (D) Naive CD4+ T cells from WT mice were stimulated under neutral (PBS) IL-7 + antindashIFN-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-21R22 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-CSFndashproducing gdT cells are shown n = 3 p 005

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CD4+ T cells CD8+ T cells and gdT cells in the muscle were notaffected by antindashGM-CSF Ab (Fig 4B)

We also examined whether gdT cells are required for thedevelopment of EAM As shown in Fig 4C muscle weakness andhistological score of the muscle were significantly reduced inEAM-induced TCRd22 mice as compared with those in EAM-inducedWTmice The infiltration of neutrophils but not ofMacsCD4+ T cells or CD8+ T cells into the muscle was significantlydecreased in EAM-inducedTCRd22mice (Fig 4D) Serum levelsof GM-CSF tended to be decreased in EAM-induced TCRd22

mice as compared with those in EAM-induced WT mice (WTmice 4946 202 pgml versus IL-2122mice 1066 51 pgml p =012) In contrast serum IL-21 was undetectable in both EAM-induced WT mice and TCRd22 mice Taken together with thefinding that gdT cells are the main producer of GM-CSF in EAM-induced mice (Fig 3B) these results suggest that GM-CSFndashproducing gdT cells are involved in the induction of EAM

IL-21 induces the accumulation of GM-CSFndashproducing Vg4+

Vd4+ CD272 cells in the muscle in EAMGiven thatgdTcells seemtoplayapathogenic role inEAM(Fig 4)we next analyzed the characteristics of gdT cells in the muscle of

EAM-inducedWTmice Because it has been reported that CD272

CCR6+ gdT cells express RORgt and produce IL-17 (37ndash39) weexamined the expression of CD27 and CCR6 on gdT cells by flowcytometry As shown in Fig 5A gdT cells in the muscle consistedmainly ofCD272CCR62gdTcells inEAM-inducedmicewhereasgdT cells in the draining LNs mainly consisted of CD27+ CCR62

gdT cells Importantly more than half of gdT cells in the musclewere positive for Vg4 and themajority of themwere also positivefor Vd4 (Heilig and Tonegawarsquos nomenclature) in EAM-inducedmice (Fig 5A) gdT cells in the muscle were negative for Vg1 orVg5 inEAM-inducedmice (Fig 5A)whereas30ofgdTcells inthe draining LNs were positive for Vg1 in EAM-induced miceThese results suggest that Vg4+Vd4+ CD272 cells preferentiallyaccumulate in the muscle in EAM-induced mice

It has been reported that Vg4+Vd4+ cells produce IL-17 andare involved in the development of collagen-induced arthritis(40) and psoriasis-like skin changes (41ndash43) By using in-tracellular cytokine staining we found that the most of GM-CSFndashproducing gdT cells in the draining LNs and themuscle inEAM-induced mice were Vg4+Vd4+ cells (Fig 5B) In contrastIL-17A production was found in both Vg4+Vd4+ cells and non-Vg4+Vd4+ cells in the muscle in EAM-induced mice (Fig 5B)

FIGURE 4 GM-CSFndashproducing gdT cells are involved in the development of EAM

(A and B) EAM was induced in WT mice Where indicated 300 mg of antindashGM-CSF mAb or control IgG was injected ip on days 2 7 and 16 of EAM (A)

Upper panel Mean6 SEM of muscle weakness n = 3mice Middle panels Representative photomicrographs of HampE staining of the quadriceps Scale bars

100 mm Lower panel Muscle histological scores n = 5 (antindashGM-CSFmAb) and 6 (control IgG) (B) The numbers of the indicated cells in the muscle n = 4

(antindashGM-CSF mAb) and 6 (control IgG) p 005 (C and D) EAM was induced in TCRd22 mice and WT mice (C) Upper panel Mean 6 SEM of muscle

weakness n = 3 mice Middle panels Representative photomicrographs of HampE staining of the quadriceps Scale bars 100 mm Lower panel Mean6 SEM

of muscle histological scores n = 6mice (D) The numbers of indicated cells in themuscle Mean6 SEM n = 6 (WTmice) and 5 (TCRd22mice) p 005

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Importantly the number of Vg4+Vd4+ CD272 cells in themuscle was significantly decreased in EAM-induced IL-2122

mice as compared with that in EAM-induced WT mice (Fig5C) Taken together these results suggest that IL-21 is involvedin the accumulation of GM-CSFndashproducing Vg4+Vd4+ CD272

cells in the muscle in EAM-induced mice

IL-21 induces the expression of CX3CL1 in the muscleTo determine the mechanisms underlying the accumulation ofVg4+Vd4+ CD272 cells in the muscle we next examined theexpression of chemokine receptors on gdT cells in the drainingLNs and the muscle in EAM-induced WT and IL-2122 mice Asshown in Fig 6A the expression of CCR3 CCR6 CCR8 CXCR4and CX3CR1 was significantly upregulated in muscle-infiltratinggdT cells as compared with gdT cells in the draining LNs inEAM-induced WT mice Importantly the expression of CX3CR1inmuscle-infiltratinggdTcellswas significantlydecreased inEAM-induced IL-2122mice as comparedwith that inEAM-inducedWTmice (Fig 6A) Further analysis of CX3CR1 expression in thesubpopulationofgdTcells in thedrainingLNsofEAM-inducedWTmice revealed that the expression levels of CX3CR1 on Vg4+

Vd4+CD272cellswasmuchhigher than those onCD27+gdTcellsVg42Vd42 CD272 cells or Vg4+Vd42 CD272 cells (Fig 6B)Moreover the expression of CX3CR1 on Vg4+Vd4+ CD272 cells inthe draining LNs and the muscle was modestly but significantlydecreased in EAM-induced IL-2122mice as compared with thatin EAM-inducedWTmice (Fig 6B) Taken together these results

suggest that CX3CR1 could be involved in IL-21ndashmediatedaccumulation of Vg4+Vd4+ CD272 cells in the muscle in EAM-induced mice

Wenext analyzed the expressionofCX3CL1 aCX3CR1 ligandin themuscleWe found that the expressionofCx3cl1mRNAin themusclewas significantly increased byEAM induction inWTmicebut not in IL-2122 mice (Fig 6C) Furthermore analysis of theconserved noncoding sequence of Cx3cl1 gene loci of human andmouse identified 100 bp of conserved noncoding sequence at theupstream of the transcription start site of the Cx3cl1 gene Searchfor a potential STAT binding site within the conserved noncodingsequence in Cx3cl1 loci identified several putative Stat1 Stat3 andStat5a binding sites (Fig 6D) Indeed by using reporter assays wefound that IL-21 activated the promoter of Cx3cl1 in M1245cells (Fig 6E) suggesting the direct activation of Cx3cl1 promoterby IL-21 Taken together these results suggest that the reducedexpression of CX3CL1 in the muscle seems to be responsible forthe reduced accumulation of Vg4+Vd4+ CD272 cells in themuscleby the absence of IL-21 in EAM

Wefinallymeasured the serum levels of CX3CL1 andGM-CSFinpatientswith IMandexamined the correlationwith the levels ofIL-21 Although GM-CSFwas undetectable in the vast majority ofPMDMpatients (datanot shown) the serum levelofCX3CL1wassignificantly correlated with the level of IL-21 in PMDMpatients(r = 04334 p = 00046 n = 41) (Fig 6F) suggesting that theIL-21ndashCX3CL1 axismight be involved in the pathogenesis of IM inhumans

FIGURE 5 IL-21 induces the accumulation of GM-GSFndashproducing 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 Vg4 or Vd4 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-2122 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-2122 mice) p 005

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DISCUSSION

In this study we show that IL-21 is involved in the pathogenesis ofautoimmune myositis We found that serum levels of IL-21 wereincreased in a subset of patientswith IMas comparedwith those inhealthy controls (Fig 1) By using EAM a murine model of IM wefound that the severity of EAM was diminished in IL-2122 mice(Fig 2) GM-CSF production from gdT cells but not Th17 celldifferentiationwas significantly reduced inEAM-inducedIL-2122

mice (Fig 3) Importantly the neutralization of GM-CSF or thedeficiency ofgdTcells significantly improvedmuscleweakness andmuscle inflammation in EAM-inducedmice (Fig 4)We also foundthat the major GM-CSF producers in the muscle in EAM-inducedmice were Vg4+Vd4+ CD272 cells (Fig 5B) and that Vg4+Vd4+

CD272cellsweresignificantlydecreased inEAM-inducedIL-2122

mice (Fig 5C) Intriguingly Vg4+Vd4+ CD272 cells expressedhigher levels of CX3CR1 (Fig 6B) In addition CX3CL1 a ligand ofCX3CR1 was induced in the muscle upon EAM induction in WTmice but not in IL-2122 mice (Fig 6C) Taken together theseresults suggest that IL-21 enhances autoimmune myositis throughthe accumulation of GM-CSFndashproducing Vg4+Vd4+ CD272 cells inthe muscle possibly via a CX3CR1-CX3CL1 pathway

We show that IL-21 plays a pathogenic role in IM Previousstudies have shown that IL-21 is involved in the development ofseveral autoimmune disease models including type 1 diabetes inNOD mice (17) murine models of rheumatoid arthritis (18) andlupus (19 20)We found that serum levelsof IL-21were elevated ina subset of patients with IM (Fig 1) consistent with a previousfinding that CXCR32CXCR5+ Th cells which are capable of pro-ducing IL-21 are increased in peripheral blood in patients withjuvenile DM (24) We also found that EAM was significantlyattenuated in IL-2122 mice (Fig 2) consistent with a previousfinding that im injectionof lymphocytesofFoxp3-deficient scurfymice in which IL-21ndashproducing CD4+ T cells are significantlyincreased (21) into RAG-deficient mice causes IM-like patholog-ical changes (44 45) Taken together these findings suggest thatIL-21 plays a pathogenic role in IM in mice and possibly inhumans and could be a therapeutic target for IM

Regarding the mechanism underlying IL-21ndashmediated muscleinflammation we found that the accumulation of GM-CSFndashproducinggdTcells in themusclewas less severe inEAM-inducedIL-2122 mice than EAM-induced WT mice (Fig 3B) We alsofound that the neutralization of GM-CSF significantly improvedmuscleweakness andmuscle inflammation in EAM-inducedmice

FIGURE 6 IL-21 induces the expression of CX3CL1 in the muscle in EAM

(AndashC) EAM was induced in IL-2122 mice and WT mice (A) Mean fluorescent intensity (MFI) of indicated chemokine receptors on gdT cells in the

draining LNs and the muscle n = 3ndash6 mice (B) Upper panels Representative profiles of CD27 versus TCRgd on gdT cells and Vd4 versus Vg4 on

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 005 p 001 (C) The expression of Cx3cl1mRNA in the muscle was assessed by qPCR

at day 24 of EAM Mean 6 SEM n = 7 (EAM) and 3 (PBS) p 005 (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 005 (F) The

correlation between IL-21 levels and CX3CL1 levels in serum of PMDM patients (n = 41) Spearman rank correlation coefficient was used

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(Fig 4) In addition muscle weakness and muscle inflammationupon EAM induction were reduced in TCRd22 mice (Fig 4)Taken together these results suggest that the reduction of GM-CSFndashproducing gdT cells in the muscle might be responsible forthemild phenotype of EAM in IL-2122mice The analysis ofmicespecifically lacking GM-CSF production in gdT cells is required toconfirm this notion

In contrast we found that the numbers of IL-17AndashproducingCD4+ T cells and IL-17Andashproducing gdT cells were not signifi-cantly different between EAM-induced IL-2122 mice and WTmice (Fig 3C) In this regard it has been reported that serum levelsof IL-17A are increased in patientswith IM (46)Moreover Zhanget al (47) have recently reported that the level of IL-17A in themuscle tissue is positively correlated with the degree of muscleinflammation and that antindashIL-17A Ab treatment attenuatesmuscle inflammation in EAM These findings indicate that IL-17A is also involved in the pathogenesis of EAM and suggest thatthemaincellular sourcesof IL-17AinEAMmightbedifferent fromCD4+T cells andgdTcells Further studies are required to addressthe cellular andmolecular relationship between IL-17A and IL-21in EAM patients as well as patients with IM

Regarding gd T cells in the pathogenesis of IM in humans arare case of PM characterized by massive infiltration of gd T cellsinto the muscle has been reported (48) In this case clonallyexpanded gd T cells recognize histidyl-tRNA synthetase (49) Incontrast the frequency of gd T cells among muscle-infiltratingcells is3 in the vastmajority of IMpatients (48) indicating thatthe patient with massive infiltration of gd T cells is not arepresentative of IM patients and that the role of gd T cells inpatientswith IMremains largely unknown In this studywe foundthat gd T cells which account for 1 of muscle-infiltratinghematopoietic cells play an important role in EAM (Fig 4)Although further studies are required our findings raise thepossibility that a small number of muscle-infiltrating gd T cellsmay play a role in certain populations of patients with IM

Among gdT cells we show that Vg4+Vd4+ CD272 cells are themajor population in the muscle in EAM-induced mice and thatthe accumulation of Vg4+Vd4+ CD272 cells in the muscle issignificantly reduced in EAM-induced IL-2122 mice (Fig 5)Vg4+Vd4+ cells have been discovered in draining LNs in collagen-induced arthritis models (40) Subsequently Vg4+Vd4+ cells havebeen shown to be pathogenic in psoriasis models (41 43) We alsofound that Vg4+Vd4+ CD272 cells are the major producer of GM-CSF in the muscle in EAM (Fig 5B) These results suggest thatsimilar to psoriasis models Vg4+Vd4+ cells seem to be pathogenicin EAM although specific deletion of cells expressing Vg4 or Vd4is needed to prove the role of Vg4+Vd4+ cells in EAM

Inpsoriasismodels Vg4+Vd4+ cellsmigrate fromdrainingLNsto the inflamed skin via a CCR2-CCL2 pathway (41 43) Incontrast we found that Vg4+Vd4+ CD272 cells expressed higherlevels of CX3CR1 than Vg42Vd42 cells (Fig 6B) and that the ex-pression of CX3CL1 was induced in the muscle in WTmice uponEAM induction (Fig 6C) suggesting that Vg4+Vd4+ CD272 cellsinfiltrate into the muscle via a CX3CR1-CX3CL1 pathway Thisnotion is consistent with previous findings that the inhibition of

CX3CR1 improves the severity of experimental myositis in SJLJmice (50) and that serum level of CX3CL1 is correlated withdisease activity inpatientswithPMDM(51) In contrast althoughCX3CR1 has been shown to be expressed on muscle-infiltratingCD4+ cells CD8+ cells and Macs in EAM-induced mice (50) thenumbers of these cells in the muscle were not significantlydifferent between EAM-induced IL-2122 mice and WT mice(Fig 2B) These results suggest that other chemokinechemokinereceptor systems may compensate the recruitment of CD4+ cellsCD8+ cells and Macs into the muscle in EAM-induced mice

Analysis ofCx3cl1 gene loci of human andmouse identified 100bp of conserved noncoding sequence at the upstream of thetranscriptional start site of Cx3cl1 (Fig 6D) Further search for apotential Stat binding site within the conserved noncodingsequence identifiedseveral putativeStat1 Stat3 andStat5abindingsites (Fig 6D) Consistent with these in silico analyses weconfirmed that IL-21 activatedCx3cl1 promoter by reporter assays(Fig 6E) Moreover the expression of Cx3cl1 was elevated in themuscle uponEAMinduction inWTmice but not in IL-2122mice(Fig 6C) Thesefindings suggest that IL-21 directly inducesCx3cl1expression through the activation of the promoter in the musclealthough the cell type that expresses Cx3cl1 in EAM-inducedmuscle remains unknown The positive correlation betweenserum levels of IL-21 and CX3CL1 in patients with PMDM (Fig6F) also supports this notion

We found that the accumulation of SLECs in the muscle isreduced in EAM-induced IL-2122 mice (Fig 2B) Because it hasbeen shown that IL-21 induces the expression ofCX3CR1 onCD8+

T cells (52) it is possible that IL-21 facilitates the infiltration ofSLECs into themuscle through the induction of CX3CR1 on CD8+

T cells These findings underscore the notion that the neutraliza-tion of IL-21 may be more beneficial for CD8+ T cellndashmediatedautoimmune diseases such as PM (22) We also found that GCB cells are significantly decreased in the draining LNs of EAM-induced IL-2122mice Because antindashB cell therapy has beneficialeffects on patients with PMDM (53) the reduction of GC B cellsmay also be involved in the attenuated muscle weakness inEAM-induced IL-2122 mice In contrast the implication for thereduced numbers of neutrophils in EAM-induced IL-2122 mice(Fig 2B) remains obscure Further studies are needed to addressthis point

This study has some limitations First we did not assesspatients with other diseases that causemyopathy The presence ofa disease control group could have clarified the specificity of ourfindings in PMDMpatients Especially because lymphocytes alsoinfiltrate into the muscle in patients with sporadic inclusion bodymyositis but immunohistological findings as well as the prognosisof the diseases are quite different between PMDM and inclusionbody myositis (23) the comparison of serum levels of IL-21between these diseases might be intriguing Second our studylacked the analysis of muscle biopsy samples in PMDM patientsBecause we found that IL-21 was detected in muscles and drain-ing LNs (Fig 2) but not in sera in EAM the analysis of musclebiopsy samples of PMDM patients would provide more detailedinformation on the production of IL-21 Third serum levels of

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IL-21 were under the detection limits in 60 of PMDMpatients A relatively small sample size did not allow forstratification by background information such as the diseaseactivity and the presence of autoantibody Studies with a largecohort of PMDM patients by a more sensitive measure of IL-21are desired

In conclusion we have shown that IL-21 is involved in thedevelopment of autoimmune myopathy presumably by inducingthe accumulation of GM-CSFndashproducing Vg4+Vd4+ CD272 cellsin the muscle Although further studies are needed our resultsshould add a new insight into the pathogenesis of IM and suggestthat the neutralization of IL-21 or GM-CSF could be a therapeuticstrategy for the treatment of IM

DISCLOSURES

The authors have no financial conflicts of interest

ACKNOWLEDGMENTS

We thank Dr M Grusby for IL-21R22 mice We thank J Iwata andK Nemoto for excellent technical assistance

REFERENCES

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2 Codarri L G Gyulveszi V Tosevski L Hesske A FontanaL Magnenat T Suter and B Becher 2011 RORgt drives productionof the cytokine GM-CSF in helper T cells which is essential for theeffector phase of autoimmune neuroinflammation Nat Immunol 12560ndash567

3 Bonneville M R L OrsquoBrien and W K Born 2010 GammadeltaT cell effector functions a blend of innate programming and acquiredplasticity Nat Rev Immunol 10 467ndash478

4 Vantourout P and A Hayday 2013 Six-of-the-best unique contri-butions of gd T cells to immunology Nat Rev Immunol 13 88ndash100

5 Haak S A L Croxford K Kreymborg F L Heppner S PoulyB Becher and A Waisman 2009 IL-17A and IL-17F do not con-tribute vitally to autoimmune neuro-inflammation in mice J ClinInvest 119 61ndash69

6 Cua D J J Sherlock Y Chen C A Murphy B Joyce B SeymourL Lucian W To S Kwan T Churakova et al 2003 Interleukin-23rather than interleukin-12 is the critical cytokine for autoimmuneinflammation of the brain Nature 421 744ndash748

7 Sheng W F Yang Y Zhou H Yang P Y Low D M KemenyP Tan A Moh M H Kaplan Y Zhang and X-Y Fu 2014 STAT5programs a distinct subset of GM-CSF-producing T helper cells that isessential for autoimmune neuroinflammation Cell Res 24 1387ndash1402

8 Lukens J R M J Barr D D Chaplin H Chi and T-D Kanneganti2012 Inflammasome-derived IL-1b regulates the production of GM-CSF by CD4+ T cells and gd T cells J Immunol 188 3107ndash3115

9 Wicks I P and A W Roberts 2016 Targeting GM-CSF in in-flammatory diseases Nat Rev Rheumatol 12 37ndash48

10 Spolski R and W J Leonard 2014 Interleukin-21 a double-edgedsword with therapeutic potential Nat Rev Drug Discov 13 379ndash395

11 Korn T E Bettelli M Oukka and V K Kuchroo 2009 IL-17 andTh17 cells Annu Rev Immunol 27 485ndash517

12 King C S G Tangye and C R Mackay 2008 T follicular helper(TFH) cells in normal and dysregulated immune responses AnnuRev Immunol 26 741ndash766

13 Crotty S 2011 Follicular helper CD4 T cells (TFH) Annu RevImmunol 29 621ndash663

14 Suto A D Kashiwakuma S Kagami K Hirose N WatanabeK Yokote Y Saito T Nakayama M J Grusby I Iwamoto andH Nakajima 2008 Development and characterization of IL-21-producing CD4+ T cells J Exp Med 205 1369ndash1379

15 McGuire H M A Vogelzang C S Ma W E Hughes P A SilveiraS G Tangye D Christ D Fulcher M Falcone and C King 2011 Asubset of interleukin-21+ chemokine receptor CCR9+ T helper cellstarget accessory organs of the digestive system in autoimmunityImmunity 34 602ndash615

16 Rao D A M F Gurish J L Marshall K Slowikowski C Y FonsekaY Liu L T Donlin L A Henderson K Wei F Mizoguchi et al2017 Pathologically expanded peripheral T helper cell subset drivesB cells in rheumatoid arthritis Nature 542 110ndash114

17 Sutherland A P R T Van Belle A L Wurster A Suto M MichaudD Zhang M J Grusby and M von Herrath 2009 Interleukin-21 isrequired for the development of type 1 diabetes in NOD mice Di-abetes 58 1144ndash1155

18 Young D A M Hegen H L M Ma M J Whitters L M AlbertL Lowe M Senices P W Wu B Sibley Y Leathurby et al 2007Blockade of the interleukin-21interleukin-21 receptor pathwayameliorates disease in animal models of rheumatoid arthritis ArthritisRheum 56 1152ndash1163

19 Vinuesa C G M C Cook C Angelucci V Athanasopoulos L RuiK M Hill D Yu H Domaschenz B Whittle T Lambe et al 2005 ARING-type ubiquitin ligase family member required to repress fol-licular helper T cells and autoimmunity Nature 435 452ndash458

20 Herber D T P Brown S Liang D A Young M Collins andK Dunussi-Joannopoulos 2007 IL-21 has a pathogenic role in a lupus-prone mouse model and its blockade with IL-21RFc reduces diseaseprogression J Immunol 178 3822ndash3830

21 Iwamoto T A Suto S Tanaka H Takatori K Suzuki I Iwamotoand H Nakajima 2014 Interleukin-21-producing c-Maf-expressingCD4+ T cells induce effector CD8+ T cells and enhance autoimmuneinflammation in scurfy mice Arthritis Rheumatol 66 2079ndash2090

22 Dalakas M C and R Hohlfeld 2003 Polymyositis and dermato-myositis Lancet 362 971ndash982

23 Simon J-P I Marie F Jouen O Boyer and J Martinet 2016Autoimmune myopathies where do we stand Front Immunol 7 234

24 Morita R N Schmitt S-E Bentebibel R Ranganathan L BourderyG Zurawski E Foucat M Dullaers S Oh N Sabzghabaei et al 2011Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicularcells and contain specific subsets that differentially support antibodysecretion Immunity 34 108ndash121

25 Bohan A and J B Peter 1975 Polymyositis and dermatomyositis(second of two parts) N Engl J Med 292 403ndash407

26 Gerami P J M Schope L McDonald H W Walling andR D Sontheimer 2006 A systematic review of adult-onset clinicallyamyopathic dermatomyositis (dermatomyositis sine myositis) a missinglink within the spectrum of the idiopathic inflammatory myopathiesJ Am Acad Dermatol 54 597ndash613

27 Kasaian M T M J Whitters L L Carter L D Lowe J M JussifB Deng K A Johnson J S Witek M Senices R F Konz et al 2002IL-21 limits NK cell responses and promotes antigen-specific T cellactivation a mediator of the transition from innate to adaptive im-munity Immunity 16 559ndash569

28 Allenbach Y S Solly S Gregoire O Dubourg B Salomon G Butler-Browne L Musset S Herson D Klatzmann and O Benveniste

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186 IL-21ndashVg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS ImmunoHorizons

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2009 Role of regulatory T cells in a new mouse model of experi-mental autoimmune myositis Am J Pathol 174 989ndash998

29 Prevel N Y Allenbach D Klatzmann B Salomon and O Benveniste2013 Beneficial role of rapamycin in experimental autoimmunemyositis [Published erratum appears in 2014 PLoS One 9] PLoS One8 e74450

30 Kojima T N Tanuma Y Aikawa T Shin A Sasaki and Y Matsumoto1997 Myosin-induced autoimmune polymyositis in the rat J Neurol Sci151 141ndash148

31 Kashiwakuma D A Suto Y Hiramatsu K Ikeda H TakatoriK Suzuki S Kagami K Hirose N Watanabe I Iwamoto andH Nakajima 2010 B and T lymphocyte attenuator suppresses IL-21production from follicular Th cells and subsequent humoral immuneresponses J Immunol 185 2730ndash2736

32 Hiramatsu Y A Suto D Kashiwakuma H Kanari S KagamiK Ikeda K Hirose N Watanabe M J Grusby I Iwamoto andH Nakajima 2010 c-Maf activates the promoter and enhancer of theIL-21 gene and TGF-b inhibits c-Maf-induced IL-21 production inCD4+ T cells J Leukoc Biol 87 703ndash712

33 Tanaka S A Suto T Iwamoto D Kashiwakuma S KagamiK Suzuki H Takatori T Tamachi K Hirose A Onodera et al 2014Sox5 and c-Maf cooperatively induce Th17 cell differentiation viaRORgt induction as downstream targets of Stat3 J Exp Med 2111857ndash1874

34 Vogelzang A H M McGuire D Yu J Sprent C R Mackay andC King 2008 A fundamental role for interleukin-21 in the generationof T follicular helper cells Immunity 29 127ndash137

35 Pelletier M A Bouchard and D Girard 2004 In vivo and in vitroroles of IL-21 in inflammation J Immunol 173 7521ndash7530

36 Spolski R H-P Kim W Zhu D E Levy and W J Leonard 2009IL-21 mediates suppressive effects via its induction of IL-10 JImmunol 182 2859ndash2867

37 Haas J D F H M Gonzalez S Schmitz V Chennupati L FohseE Kremmer R Forster and I Prinz 2009 CCR6 and NK11 distin-guish between IL-17A and IFN-g-producing gammadelta effectorT cells Eur J Immunol 39 3488ndash3497

38 Ribot J C A deBarros D J Pang J F Neves V PeperzakS J Roberts M Girardi J Borst A C Hayday D J Pennington andB Silva-Santos 2009 CD27 is a thymic determinant of the balancebetween interferon-g- and interleukin 17-producing gammadeltaT cell subsets Nat Immunol 10 427ndash436

39 Martin B K Hirota D J Cua B Stockinger and M Veldhoen 2009Interleukin-17-producing gammadelta T cells selectively expand inresponse to pathogen products and environmental signals Immunity31 321ndash330

40 Roark C L J D French M A Taylor A M Bendele W K Bornand R L OrsquoBrien 2007 Exacerbation of collagen-induced arthritis byoligoclonal IL-17-producing g d T cells J Immunol 179 5576ndash5583

41 Gray E E F Ramırez-Valle Y Xu S Wu Z Wu K E Karjalainenand J G Cyster 2013 Deficiency in IL-17-committed Vg4(+) gd

T cells in a spontaneous Sox13-mutant CD451(+) congenic mouse

substrain provides protection from dermatitis Nat Immunol 14584ndash592

42 Hartwig T S Pantelyushin A L Croxford P Kulig and B Becher2015 Dermal IL-17-producing gd T cells establish long-lived memoryin the skin Eur J Immunol 45 3022ndash3033

43 Ramırez-Valle F E E Gray and J G Cyster 2015 Inflammationinduces dermal Vg4+ gdT17 memory-like cells that travel to distantskin and accelerate secondary IL-17-driven responses Proc NatlAcad Sci USA 112 8046ndash8051

44 Sharma R W N Jarjour L Zheng F Gaskin S M Fu and S-T Ju2007 Large functional repertoire of regulatory T-cell suppressibleautoimmune T cells in scurfy mice J Autoimmun 29 10ndash19

45 Young N A R Sharma A K Friedman B H Kaffenberger B Bolonand W N Jarjour 2013 Aberrant muscle antigen exposure in mice issufficient to cause myositis in a Treg cell-deficient milieu ArthritisRheum 65 3259ndash3270

46 Szodoray P P Alex N Knowlton M Centola I Dozmorov I CsipoA T Nagy T Constantin A Ponyi B Nakken and K Danko 2010Idiopathic inflammatory myopathies signified by distinctive periph-eral cytokines chemokines and the TNF family members B-cell ac-tivating factor and a proliferation inducing ligand Rheumatology 491867ndash1877

47 Zhang H F He M Shi W Wang X Tian J Kang W Han R WuL Zhou M Hu et al 2017 Toll-like receptor 4ndashmyeloid differenti-ation primary response gene 88 pathway is involved in the in-flammatory development of polymyositis by mediating interferon-gand interleukin-17A in humans and experimental autoimmune myo-sitis mouse model Front Neurol 8 132 httpsdoi 103389ndashfneur201700132

48 Hohlfeld R A G Engel K Ii and M C Harper 1991 Polymyositismediated by T lymphocytes that express the gd receptor N Engl JMed 324 877ndash881

49 Bruder J K Siewert B Obermeier J Malotka P ScheinertJ Kellermann T Ueda R Hohlfeld and K Dornmair 2012 Targetspecificity of an autoreactive pathogenic human gd-T cell receptor inmyositis J Biol Chem 287 20986ndash20995

50 Suzuki F T Nanki T Imai H Kikuchi S Hirohata H Kohsaka andN Miyasaka 2005 Inhibition of CX3CL1 (fractalkine) improves ex-perimental autoimmune myositis in SJLJ mice J Immunol 1756987ndash6996

51 Suzuki F T Kubota Y Miyazaki K Ishikawa M Ebisawa S HirohataT Ogura H Mizusawa T Imai N Miyasaka and T Nanki 2012Serum level of soluble CX3CL1fractalkine is elevated in patients withpolymyositis and dermatomyositis which is correlated with diseaseactivity Arthritis Res Ther 14 R48

52 Tian Y M A Cox S M Kahan J T Ingram R K Bakshi andA J Zajac 2016 A context-dependent role for IL-21 in modulatingthe differentiation distribution and abundance of effector and mem-ory CD8 T cell subsets J Immunol 196 2153ndash2166

53 Faurschou M and D R W Jayne 2014 Anti-B cell antibody ther-apies for inflammatory rheumatic diseases Annu Rev Med 65263ndash278

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ImmunoHorizons IL-21ndashVg4+Vd4+ CELL AXIS IN AUTOIMMUNE MYOSITIS 187

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