A Novel α9 Ligand, XCL1/Lymphotactin, Is Involved in the Development of Murine Models of Autoimmune Diseases This information is current as of September 25, 2021. Naoki Matsumoto, Shigeyuki Kon, Takuya Nakatsuru, Tomoe Miyashita, Kyosuke Inui, Kodai Saitoh, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Toshimitsu Uede and Tadashi Matsuda

J Immunol 2017; 199:82-90; Prepublished online 26 May Downloaded from 2017; doi: 10.4049/jimmunol.1601329 http://www.jimmunol.org/content/199/1/82 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel a9 Integrin Ligand, XCL1/Lymphotactin, Is Involved in the Development of Murine Models of Autoimmune Diseases

Naoki Matsumoto,*,1 Shigeyuki Kon,*,†,1 Takuya Nakatsuru,* Tomoe Miyashita,* Kyosuke Inui,* Kodai Saitoh,* Yuichi Kitai,* Ryuta Muromoto,* Jun-ichi Kashiwakura,* Toshimitsu Uede,‡ and Tadashi Matsuda*

The integrin a9b1 is a key receptor involved in the development of autoimmune diseases. However, the detailed mechanism for the association of a9b1 integrin with its ligands remains unclear. In this study, we introduce XCL1/lymphotactin, a member of the family, as a novel ligand for a9 integrin. Using a9 integrin–overexpressing NIH3T3 cells and endogenously a9 integrin–expressing human rhabdomyosarcoma cells, the interaction between XCL1 and a9 integrin was confirmed by pull- Downloaded from down assays. XCL1 enhanced a9 integrin–dependent cell migration of these cells, thus acting on a9 integrin as a chemoattractant. We also analyzed the in vivo function of XCL1 in the development of anti-type II collagen Ab–induced inflammatory arthritis (CAIA) in BALB/c mice and experimental autoimmune encephalomyelitis in C57BL/6 mice, because a9 integrin is involved in these autoimmune disease models. In CAIA, recombinant XCL1 aggravated the disease and this exacerbation was inhibited by an anti-a9 integrin Ab. An XCL1-neutralizing Ab produced in this study also ameliorated CAIA. Furthermore, the XCL1- neutralizing Ab abrogated the disease progression in experimental autoimmune encephalomyelitis. Therefore, to our knowledge http://www.jimmunol.org/ this study provides the first in vitro and in vivo evidence that the interaction between XCL1 and a9 integrin has an important role for autoimmune diseases. The Journal of Immunology, 2017, 199: 82–90.

ntegrins are transmembrane receptors involved in a wide cephalomyelitis (EAE) (11). However, inhibition or knockout of OPN range of cellular processes, including cell adhesion, migra- has lesser effects than the use of the anti-a9 integrin Ab (12–14), and I tion, differentiation, proliferation, and apoptosis, and cancer the involvement of other a9 integrin ligands in CAIA remains un- metastasis (1–5). a9 integrin binds to various extracellular matrix known, indicating that there may be other players in the development , including (OPN), tenascin-C (TN-C), and vas- of autoimmune diseases. Therefore, we searched for a candidate a9 by guest on September 25, 2021 cular endothelial C and D, and is associated with integrin ligand by liquid chromatography tandem-mass spectrometry autoimmune diseases such as arthritis (6–9). An anti-a9 integrin– (LC-MS/MS) analysis and identified XCL1/lymphotactin. blocking Ab was reported to ameliorate the disease scores in collagen In this study, we demonstrate that XCL1 functions in autoim- Ab–induced arthritis (CAIA) (10) and experimental autoimmune en- mune diseases as a novel a9 integrin ligand. A physiological interaction between a9 integrin and XCL1 was confirmed by pull- down assays. XCL1 belongs to the C-class of , and its *Department of Immunology, Faculty of Pharmaceutical Sciences, Hokkaido Uni- only known receptor is XCR1 (15). We found that XCL1 stimu- versity, Sapporo 060-0815, Japan; †Department of Molecular Immunology, Faculty of lation enhanced cell migration in a9 integrin–expressing cells, and Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-0292, Japan; and ‡Division of Molecular Immunology, Institute for Genetic Medicine, Hokkaido Uni- that this XCL1-dependent cell migration was suppressed by a versity, Sapporo 060-0817, Japan function-blocking anti-a9 integrin Ab. To further investigate the 1N.M. and S.K. contributed equally to this work. role of XCL1 in vivo, we generated an XCL1-neutralizing mAb, ORCID: 0000-0001-5410-8429 (S.K.). 1A3A. The 1A3A Ab successfully protected mice against CAIA, Received for publication August 3, 2016. Accepted for publication April 27, 2017. indicating that XCL1 is involved in the development of this dis- This work was supported in part by Japan Society for the Promotion of Science ease. We also administered the Ab to mice with EAE, and ob- KAKENHI Grants JP24590072 and JP16K08221, the Fugaku Trust for Medical served amelioration of the disease. Recently, XCL1 expression Research, the Research Foundation for Pharmaceutical Sciences, The Nakatomi was demonstrated to be elevated in patients with rheumatoid ar- Foundation, the Japan Rheumatism Foundation, Pfeizer Academic Contributions, and the Pharmacological Research Foundation. thritis (RA) (16), whereas a9 integrin expression was reported to Address correspondence and reprint requests to Prof. Shigeyuki Kon, Department of be increased in the RA synovium (9). Taken together, it is sug- Molecular Immunology, Faculty of Pharmaceutical Sciences, Fukuyama University, gested that the interaction between XCL1 and a9 integrin can be a 985-1 Azasanzo, Higashimura-cho, Fukuyama, Hiroshima 729-0292, Japan. E-mail new therapeutic target for autoimmune diseases. address: [email protected] The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: CAIA, collagen Ab–induced arthritis; CHO, Chinese hamster ovary; EAE, experimental autoimmune encephalomyelitis; FLS, Cell culture and reagents fibroblast-like synoviocyte; HPRT, hypoxanthine-guanine phosphoribosyltransfer- NIH3T3 cells, Chinese hamster ovary (CHO) cells, HEK293T cells, Plat-GP ase; Kir, inward-rectifier potassium; LC-MS/MS, liquid chromatography tandem- mass spectrometry; MOG, myelin oligodendrocyte glycoprotein; OPN, osteopontin; cells, rhabdomyosarcoma (RD) cells (derived from a human rhabdomyo- qPCR, quantitative PCR; RA, ; RD, rhabdomyosarcoma; TN-C, sarcoma), and fibroblast-like synoviocytes (FLS) were cultured in DMEM tenascin-C. containing 10% FBS (HyClone, Logan, UT). Ba/F3, an IL-3–dependent murine pro-B cell line, was maintained in RPMI 1640 medium supple- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 mented with 10% FCS and 10% WEHI-3B conditioned medium as a www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601329 The Journal of Immunology 83 source of IL-3. HRP-conjugated anti-human IgG (Jackson Immuno- generation of various His-SUMO-tagged mouse XCL1 variants, the target Research, West Grove, PA) was used for ELISA and Western blot analysis. were PCR amplified using the following primers: 59-TATTGAGGCT- Anti-rat IgG (Jackson ImmunoResearch) was used for ELISA. Anti-His- CATCGCGAACAGATTGGAGGTGTGGGGACTGAAGTCCTA-39 (sense) tag Ab OGHis (MBL, Nagoya, Japan) and anti-FLAG-tag Ab 1E6 and 59-ATGCCTGCAGGTCGACTTACCCAGTCAGGGTTATCGCTG-39 (an- (WAKO, Osaka, Japan) were used for Western blot analyses. Anti-human tisense) for full, 59-TATTGAGGCTCATCGCGAACAGATTGGAGGTGTGG- a9 integrin mAb Y9A2 (Chemicon, Temecula, CA) was used for flow GGACTGAAGTCCTA-39 (sense) and 59-ATGCCTGCAGGTCGACTTAAG- cytometry and cell migration assays. An anti-a9 integrin polyclonal Ab for TCTTGATCGCTGCTTTCA-39 (antisense) for 22–83, 59-TATTGAGGCT- Western blot analyses was generated by immunizing a rabbit with a pep- CATCGCGAACAGATTGGAGGTGGGGCCATGAGAGCTGTA-39 (sense) tide derived from the C-terminal domain of human a9 integrin, and 59-ATGCCTGCAGGTCGACTTACCCAGTCAGGGTTATCGCTG-39 CEAEKNRKENEDSWDWVQKNQ. IgG from human serum (Sigma- (antisense) for 53–114, 59-TATTGAGGCTCATCGCGAACAGATTGGA- Aldrich, St. Louis, MO), and recombinant His-tagged human XCL1 pro- GGTATTTGTGCTGATCCAGAA-39 (sense) and 59-ATGCCTGCAG- tein (hXCL1-His) (PeproTech, Rocky Hill, NJ) were used for cell migra- GTCGACTTACCCAGTCAGGGTTATCGCTG-39 (antisense) for 68–114, tion assays. 59-TATTGAGGCTCATCGCGAACAGATTGGAGGTGTGGATGGCAGGG- CCAGT-39 (sense) and 59-ATGCCTGCAGGTCGACTTACCCAGTCAGG- Generation of stable cell lines GTTATCGCTG-39 (antisense) for 84–114. The PCR products were subcloned into the NruI- and SalI-digested pMsec SUMOstar vector (LifeSensors, Mal- CHO cells expressing a9 integrin or a4 integrin, and NIH3T3 cells vern, PA), a mammalian expression vector containing a His-SUMO tag, using expressing a9 integrin or SFa9, or coexpressing a9 integrin and SFa9 the In-Fusion system. HEK293T cells were transfected with the individual were generated as previously described (17). Briefly, a9-FLAG-pBabepuro, expression vectors, and the supernatants were collected at 48 h after trans- a4-FLAG-pBabepuro, a9-pWZLBlast2, or SFa9-FLAG-pBabepuro was fection. Secreted recombinant XCL1 variants were confirmed by Western blot. cotransfected with pCMV-VSV-G into Plat-GP packaging cells using Lipo- fectamine 2000 (Invitrogen, Carlsbad, CA). At 3 d after transfection, virus- Pull-down assays and Western blot analyses

containing supernatants were harvested, passed through a 0.45-mmfilter, Downloaded from added to 40% confluent CHO cells or NIH3T3 cells in the presence of 8 mg/ml NIH3T3, CHO, and RD cells were lysed with CelLytic M by gentle rotation polybrene, and cultured for 1 d. The virus-containing medium was at 4˚C for 15 min. The lysates were clarified by centrifugation at 15,000 3 g removed and the cells were cultured in 10% FBS/DMEM supplemented with for 15 min at 4˚C, and then incubated with recombinant human Fc-G1 (Bio 10 mg/ml puromycin (Sigma-Aldrich) or 5 mg/ml blasticidin (Invitrogen) for X Cell, West Lebanon, NH), mXCL1-Ig, hXCL1-Ig, or hXCL2-Ig (5 mg/tube) NIH3T3 cells and CHO cells. For generation of cells coexpressing a9 for 30 min at 4˚C. A-Sepharose (50% slurry, 12 ml/tube) was integrin and SFa9, retroviruses were generated by transfection of SFa9- applied to each tube and incubated for another 1 h at 4˚C. After three FLAG-pBabepuro, and then applied to a9/NIH3T3 cells, followed by se- washes of the beads with TBS containing 0.05% Tween-20, the precipitated lection with 10% FBS/DMEM supplemented with 10 mg/ml puromycin and polypeptides were extracted in SDS sample buffer (93.75 mM Tris–HCl pH http://www.jimmunol.org/ 5 mg/ml blasticidin. Cells expressing a9 integrin and/or SFa9 were identified 6.8, 30% glycerol, 15% 2-ME, 7.5% SDS), separated by SDS–PAGE under by flow cytometry with anti-a9 Ab Y9A2, and Western blot with the rabbit reducing conditions, transferred to polyvinylidene fluoride membrane, pro- anti-a9 integrin polyclonal Ab. For a9-FLAG/CHO and a4-FLAG/CHO bed with the anti-a9 integrin Ab or anti-FLAG Ab 1E6, and detected by cells, expression was confirmed by Western blot with anti-FLAG Ab 1E6. Plus-ECL (PerkinElmer, Waltham, MA). Stable Ba/F3 transformants were generated as follows. A cDNA for human aL integrin was PCR-amplified from human B lymphoblast cell line SKW6- Flow cytometry CL4 with following primers: 59-GGCGCCGGCCGGATCCGCCACCAT- For adherent cells, PBS containing 0.5 mM EDTA was used to dissociate cells GAAGGATTCCTGCATCAC-39 (sense) and 59-ATTCCACAGGGTCGACT- from the culture dish, followed by blocking with FBS, and then sequentially TACTTGTCATCGTCATCCTTGTAGTCCTTGCCACCACCACTC-39 incubated with anti-a9 integrin Ab Y9A2 and PE-labeled goat anti-mouse Ab (antisense). Amplicon was cloned into BamHI (Takara, Kusatsu, Japan)- and (Jackson ImmunoResearch) for human a9 integrin expression, APC-labeled by guest on September 25, 2021 SalI (Takara)-digested pBabepuro vector using the In-Fusion system (Takara) anti-a9integrinAb55A2Cformurinea9 integrin expression (10), and PE- to construct aL-FLAG-pBabepuro. a9-FLAG-pBabepuro, a4-FLAG- labeled mouse anti-XCR1 Ab ZET (BioLegend, San Diego, CA) for XCR1 pBabepuro, and aL-FLAG-pBabepuro were then transfected to Ba/F3 by expression before flow cytometry analysis. Ba/F3 cells were washed with electroporation using Pulser II (Bio-Rad, Richmond, CA), followed by PBS, blocked by FBS, then incubated with PE-labeled anti-aLintegrin selection with Ba/F3 maintaining medium supplemented with 2 mg/ml (BioLegend), PE-labeled anti-a4integrin9F10(BioLegend),andanti-a9 puromycin. integrin Ab Y9A2 and PE-labeled goat anti-mouse Ab. LC-MS/MS analysis Generation of Abs NIH3T3 cells expressing SFa9-FLAG-His were exposed to hypotonic Anti-XCL1 Abs (clones 1A3A and 3P11R) were generated in Sprague- buffer (25 mM NaCl, 0.5 mM CaCl2, 18 mM Tris–HCl pH 8) and the Dawley rats immunized with recombinant XCL1 emulsified in Freund’s membrane fraction was collected by centrifugation at 4000 3 g for 10 min adjuvant. Their splenocytes were fused with 363-Ag8-653 mouse mye- at 4˚C. After removal of the supernatant, the membrane fraction was lysed loma cells as described previously (18). The resulting hybridoma cells with CelLytic M (Sigma-Aldrich) containing a protease inhibitor mixture were screened by ELISA. For characterization of the cloned Abs, ELISA 13 Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzer- and Western blot analyses were performed using Ig-tagged or His-tagged land), followed by centrifugation at 15,000 3 g for 10 min at 4˚C. The mouse and human XCL1, and His-SUMO-tagged XCL1 variants. supernatant was applied to Anti-FLAG M2 Affinity Gel (Sigma-Aldrich). After washing with TBS, the bound proteins were eluted with TBS con- ELISA taining a FLAG peptide. The eluted proteins were checked by Western blot, and then concentrated by 20-fold using a Vivaspin (Sartorius, Go¨t- Nunc-immuno plates (Thermo Fisher Scientific, Roskilde, Denmark) were tingen, Germany) for LC-MS/MS analysis. coated with either 1 mg/ml recombinant mouse integrin a9b1 (R&D Systems, Minneapolis, MN) or 10 mg/ml XCL1 proteins overnight at 4˚C Generation of recombinant XCL1 proteins in 0.1 M carbonate-bicarbonate buffer (pH 9.2). After PBS wash, a9b1- and XCL1-coated plates were blocked with 5% skim milk and 0.1% BSA XCL1-Ig fusion proteins were generated as follows. A cDNA for mouse XCL1 in PBS respectively for 1 h at room temperature. Plates were then washed was PCR-amplified from the thymi of C57BL/6 mice using the following once with washing buffer (PBS containing 0.05% Tween-20), followed by primers: 59-AGTGAATTCGCCACCATGAGACTTCTCCTCCTGA-39 (sense) application of 5 mg/ml recombinant human Fc-G1 and XCL1-Ig to a9b1- and 59-TGTGGATCCCCAGTCAGGGTTATCGCTGT-39 (antisense). cDNAs coated plate and 10 mg/ml anti-mouse XCL1 Abs 1A3A and 3P11R to for human XCL1 and human XCL2 were purchased from Open Biosystems, XCL1-coated plates. After incubation for 1 h at room temperature, plates and PCR amplified using the following primers: 59-GGTGAATTCGCCAC- were washed three times with washing buffer, then HRP-conjugated anti- CATGAGACTTCTCATCCTGG-39 (sense) and 59-ATGGGATCCCCAGT- human IgG and HRP-conjugated anti-rat IgG were applied to a9b1-coated CAGAGTCACAGCTGTA-39 (antisense). The PCR products were digested plates and XCL1-coated plates respectively for another 30 min incubation with EcoRI (Takara) and BamHI and inserted into an EcoRI- and BamHI- at room temperature. Plates were then washed four times with washing digested mammalian expression vector containing the Fc portion of human buffer and the substrate 3,39,5,59-tetramethylbenzidine (KPL, Gaithers- IgG1. The resulting expression vectors were transfected into HEK293T cells, burg, MD) was added. After 15 min of incubation at room temperature, an and recombinant Ig-tagged mouse XCL1 (mXCL1-Ig), human XCL1 (hXCL1- equal amount of 1 N sulfuric acid was applied to stop the reaction. Ab- Ig), and human XCL2 (hXCL2-Ig) proteins were purified from the supernatants sorbance was measured at 450 nm (reference wavelength of 620 nm) using with protein A-Sepharose beads (GE Healthcare, Little Chalfont, U.K.). For the an iMark microplate reader (Bio-Rad). 84 XCL1–a9 INTEGRIN AXIS IS INVOLVED IN AUTOIMMUNE DISEASES

Cell migration assay Analysis of mRNA expression For chemotactic migration assays, 24-well Transwell plates (Corning, Total RNA from short hairpin RNA–transduced cells, FLS, and the spinal Corning, NY) and RPMI 1640 medium containing 1% FBS were used. cords of EAE mice at days 0, 7, 14, 21, and 28 was extracted with TRIzol Transwell filters (5.0-mm pore size) were equilibrated at 37˚C overnight. The (Invitrogen). Random primers were used for RT-PCR and specific primers equilibration medium was then removed, and medium containing human IgG were used for qPCR assays to amplify hypoxanthine-guanine phosphor- as an Ig control, mXCL1-Ig, or hXCL1-His (indicated concentration for ibosyltransferase (HPRT) (59-TCCTCCTCAGACCGCTTTT-39 and Fig. 2A and 300 ng/ml each for other experiments) was added to the bottom 59-CCTGGTTCATCATCGCTAATC-39), b-actin (59-TGACAGGATGCA- chambers. Next, 1 3 106 cells were added to the upper chambers and GAAGGAGA-39 and 59-CGCTCAGGAGGAGCAATG-39), XCL1 allowed to migrate for 5 h at 37˚C. After unmigrated cells were gently re- (59-AGACTTCTCCTCCTGACTTTCCT-39 and 59-GGACTTCAGTCCC- moved from the membranes with a cotton swab, the membranes were fixed CACACC-39), XCR1 (59-GCACTGGAGGAGATCAAAGG-39 and and stained with Diff-Quik (Sysmex, Kobe, Japan). The stained cells were 59-CGGGATGCAGGGATACTGAG-39), and a9 integrin (59-ATGACG- counted in four randomly chosen high-power fields per filter. For anti-a9 GGTTCCCAGATG-39 and 59-TGTAGACTGCGCCAGCAA-39). The qPCR integrin Ab treatment, cells were incubated with 10 mg/ml Y9A2 or control assays were conducted in a CFX96 Touch (Bio-Rad). The amplified cDNAs IgG prior to addition to the upper chambers. In a checkerboard type mi- was detected using SYBR Green (Kapa Biosystems, Woburn, MA) and gration assay, cells were treated with either PBS of mXCL1-Ig, then ap- standardized by the ROX dye levels. The cDNA concentrations were plied to the upper chamber and allowed to migrate toward the lower expressed as the number of cycles to threshold, and the threshold values were chamber containing media supplemented with PBS, hIgG1 Fc control, or normalized by the HPRT cDNA levels in the same samples. mXCL1-Ig. For barium treatment, barium chloride was added to cells and applied to the upper chamber. For anti-mXCL1 Ab treatment, medium Statistical analysis containing mXCL1-Ig was treated with PBS, 1A3A, or 3P11R (10 mg/ml) Data are presented as mean 6 SEM and are representative of at least three and incubated for 15 min on ice prior to addition to the bottom chambers. independent experiments. The statistical significance of differences be- Downloaded from Animals tween groups was calculated using a two-tailed Student t test or non- parametric Wilcoxon Mann–Whitney U test where applicable. Differences BALB/c and C57BL/6 mice were kept under specific pathogen-free con- were considered significant for p , 0.05 or p , 0.01. ditions and provided with food and water ad libitum. Sprague-Dawley rats were purchased from Charles River Japan (Yokohama, Japan) and kept Results under conventional conditions. All experiments were conducted in accor- XCL1 interacts with both a9 integrin and SFa9 dance with the guidelines of the Institutional Animal Care and Use

Committee of Hokkaido University. First, we determined candidates for novel ligands of a9integrinby http://www.jimmunol.org/ LC-MS/MS using a9 integrin–splicing variant SFa9, which con- Isolation of FLS tains only the b-propeller domain and thigh domain (17). We pre- Hind limbs were surgically cut from the ankle joint, followed by surgical viously reported that SFa9 does not interact with known ligands of separation of the dermal, s.c., tendinous, and muscle tissues from joints. The a9 integrin, thus SFa9 ligands remain unknown. Because SFa9 remaining soft tissues were removed from bone tissues and homogenized roughly with dissecting scissors. Homogenates were washed once with contains the b-propeller domain, which is important for ligand culture medium and treated with 3 mg/ml type II collagenase (Worthington binging of a9 integrin, it is possible that novel ligands of SFa9also Biochemical, Lakewood, NJ) in FBS-free DMEM at 37˚C by vigorous have a binding capability to native a9 integrin and regulate cellular stirring. After washing and straining with a 70 mm cell strainer (Greiner functions. Therefore, we postulated that either selective ligands of

Bio-One, Frickenhausen, Germany), cells were cultured overnight. Non- by guest on September 25, 2021 adherent cells were removed, and attached cells were regarded as FLS. SFa9 or common ligands of a9integrinandSFa9 could be novel ligands involved in autoimmune diseases by regulating a9 integrin CAIA induction activity. Among many candidates (Supplemental Fig. 1A), we fo- Arthritis was induced using an arthritogenic mAb mixture kit (Chondrex, cused on the C-class chemokine XCL1 as a functional ligand of a9 Redmond, WA). Briefly, 6–8 wk-old female BALB/c mice (Charles River) integrin. A previous study has demonstrated that secretory proteins were injected intravenously with a mixture of anti-type II collagen mAbs such as vascular endothelial growth factor C and D are the ligand of on day 0, followed by i.p. injection of 50 mg of LPS (0111:B4) on day 3. a9 integrin (8), suggesting the secretory protein XCL1 may also be FLS were obtained from the joints and expression of XCL1 was evaluated an a9 integrin ligand. Because secreted chemokines induce che- by quantitative PCR (qPCR). For XCL1 treatment, Ig-fused mouse XCL1 or Ig control was administered i.p. at a dose of 300 ng per mouse from days motaxis through binding to chemokine receptors (20), we hypoth- 21 to 9. For a9 integrin inhibition, anti-a9 integrin Abs 18R18D or esized that XCL1 may act as a chemokine for a9integrin.Toverify 55A2C (10) were i.p. administered to XCL1-treated mice on days 21 and the interactions between XCL1 and SFa9, and XCL1 and a9 2 as a control IgG and a9 integrin-neutralizing Ab, respectively. For XCL1 integrin, we generated recombinant XCL1 and XCL2 proteins fused inhibition, anti-mouse XCL1 Ab 3P11R or 1A3A was i.p. injected on days 21 and 2 as a control IgG and XCL1-blocking Ab, respectively. The with IgG1 Fc (Supplemental Fig. 1B). XCL2 is a homolog of XCL1 clinical severity of arthritis was graded for up to 16 d after Ab adminis- in humans with only 2 aa differences (21), suggesting that XCL2 tration in each of the four paws on a scale of 0–4 as follows: 0, no signs; 1, could also be a ligand of a9 integrin. In pull-down assays using slight focal swelling and/or redness in one digit; 2, moderate swelling and recombinant XCL1-Ig and XCL2-Ig, we confirmed the interactions erythema of more than two digits; 3, marked swelling and erythema of the of XCL1 with both a9integrinandSFa9incellsoverexpressinga9 limb; 4, maximal swelling, erythema, deformity, and/or ankyloses. Mice were sacrificed, and then joint sections were stained with H&E and integrin, SFa9, or both (Fig. 1A). XCL2 also bound to a9 integrin safranin-O for immunohistochemical evaluation. and SFa9, but its interactions were weaker than those of XCL1. NIH3T3 cells did not express endogenous a9 integrin (Supplemental EAE induction Fig. 1C), and showed no enhanced cell migration toward XCL1 EAE was induced in 8 wk-old C57BL/6 mice by s.c. injection with 100 mg (Fig. 2B). We also observed the interaction of XCL1 with a9 of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (MEVG- integrin in RD cells, the human rhabdomyosarcoma cell line that WYRSPFSRVVHLYRNGK) emulsified with CFA on day 0 and i.v. in- endogenously expresses a9 integrin (Fig. 1B), indicating interaction jection with 400 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) on days 0 and 2. Anti-mouse XCL1 Ab 1A3A or 3P11R, or between a9 integrin and XCL1 may occur endogenously. a9 PBS as a control, was administered at a dose of 400 mg per mouse at 1 d integrin is structurally and functionally similar to a4 integrin, and before and 2 d after MOG 35–55 immunization. The clinical scores of shares some common ligands such as OPN and propolypeptide of EAE severity were assessed daily as previously described (19): 0, no von Willebrand factor (22, 23). Therefore, we examined whether the clinical signs; 0.5, partially limp tail; 1, paralyzed tail; 2, loss of coordi- nated movement, hind limb paresis; 2.5, one hind limb paralyzed; 3, both interaction between XCL1 and a9 integrin is selective. In pull-down hind limbs paralyzed; 3.5, hind limbs paralyzed, weakness in forelimbs; 4, assays using a4 integrin– and a9 integrin–overexpressing cells, a4 forelimbs paralyzed; and 5, moribund. integrin showed no interaction with XCL1 (Fig. 1C), indicating that The Journal of Immunology 85

FIGURE 1. Interactions of a9 integrin with XCL1 and XCL2. (A) a9 integrin and SFa9 interact with both human XCL1 and XCL2. Recombinant human Fc-G1 and Ig-fused human XCL1 and XCL2 were applied to cell lysates of various NIH3T3 cells. Protein A-Sepharose beads were then added for pull- down assays. (B) XCL1 interacts with endogenous a9 integrin expressed on RD cells. The surface expression of a9 integrin was analyzed by flow cytometry (top). Pull-down assays confirmed the interaction between XCL1 and endogenous a9 integrin (bottom). (C) XCL1 does not interact with a4 Downloaded from integrin. (D) Direct binding of recombinant a9b1 integrin and XCL1 is shown in ELISA. Data are representative of three or more independent experiments with similar results. **p , 0.01 (Student t test).

XCL1 selectively binds to a9 integrin. We also determined the se- pression was increased in CAIA mice (Fig. 3A), suggesting local lective binding of XCL1 with aL integrin. Because aLintegrinre- expression of a9integrinandXCL1maybeinvolvedinthedevel- http://www.jimmunol.org/ quires b2 integrin to be expressed on the cell surface as a heterodimer, opment of inflammatory arthritis. To examine the effect of XCL1 in we transduced aL into murine pro-B cell line Ba/F3, which CAIA, we administrated recombinant Ig-fused XCL1 consecutively expresses b2 integrin endogenously. Ba/F3 transformants express and observed significant exacerbation of arthritis in XCL1-treated these integrin heterodimers on the cell surface (Supplemental Fig. mice (Fig. 3B, 3C). H&E staining revealed severe erosion of the 1D). Pull-down assays with these cells showed selective interaction joint cartilage (Fig. 3D, box 1) and infiltration of inflammatory cells between XCL1 and a9 integrin (Supplemental Fig. 1E). Physical (Fig. 3D, box 2) in XCL1-treated mice. We then stained the joints interaction of a9b1 integrin with XCL1 was also observed in direct with safranin-O to visualize proteoglycans in the arthritic cartilage, ELISA (Fig. 1D), suggesting XCL1 is a novel a9integrinligand. and observed more extensive destruction of proteoglycans in XCL1- treated mice (Fig. 3D). From theseresults,weconfirmedthatXCL1 by guest on September 25, 2021 XCL1 enhances a9 integrin–mediated cell migration deteriorates arthritis in CAIA. XCL1 is a C-class chemokine that induces chemotaxis through its Blocking of a9 integrin was reported to ameliorate CAIA (10). known receptor XCR1 (24, 25). Therefore, we examined whether Thus, we treated XCL1-administered CAIA mice with a9-integrin XCL1 induces migration of cells expressing a9 integrin. In cells neutralizing Ab 55A2C (Fig. 3E). Inhibition of a9integrinwiththe overexpressing a9 integrin, XCL1 showed a biphasic chemotactic Ab significantly reduced the clinical score of the CAIA by XCL1 response as previously reported (21), with the best response ob- (Fig. 3F). We then stained the joint with H&E and safranin-O, and served at 300 ng/ml XCL1 concentration (Fig. 2A). XCL1 did not revealed that the erosion of the joint cartilage, infiltration of in- induce cell migration in parent cells (Fig. 2B), and NIH3T3 cells flammatory cells, and destruction of proteoglycans were improved did not express XCR1 (data not shown), indicating XCL1 acted on by blocking a9integrininXCL1-treatedCAIAmice(Fig.3G).To a9 integrin. We then treated the cells with anti-a9 integrin– further investigate the role of XCL1 in CAIA, we isolated FLS from neutralizing Ab Y9A2. As a result, XCL1-induced cell migration CAIA mice. We confirmed that FLS expressed a9integrin in a9 integrin–expressing cells was significantly inhibited (Supplemental Fig. 2A) as reported previously (10). Upon FLS (Fig. 2B). Checkerboard-type migration assay also confirmed this stimulation with XCL1, various inflammatory were up- migration is directional, indicating XCL1 induced chemotaxis on regulated (Supplemental Fig. 2C), and this response was success- a9 integrin–expressing cells (Fig. 2C). We also examined the fully inhibited by treating FLS with a9 integrin–neutralizing Ab effects of XCL1 and Y9A2 in RD cells. XCL1 successfully en- (Supplemental Fig. 2D). Therefore, it is suggested that XCL1 ag- hanced the cell migration of RD cells, and Y9A2 treatment caused gravated CAIA in an a9 integrin–dependent manner by inducing inhibition of this migration (Fig. 2C). In a previous paper, it was cell migration of a9 integrin–expressing cells as well as increasing reported that a9 integrin–dependent cell migration is dependent the mRNA expression level of inflammatory cytokines. on inward-rectifier potassium (Kir) channels and that Kir channels Anti-XCL1 mAb 1A3A neutralizes cell migration toward XCL1 can be blocked by Ba2+ (26). Therefore, we investigated whether XCL1-induced migration is dependent on a9 integrin by using To further evaluate the effects of XCL1, we generated anti-XCL1 Ba2+. We confirmed diminished cell migration toward XCL1 in mAbs that block XCL1 function. First, we characterized two of the presence of Ba2+ (Fig. 2D). These results suggest that XCL1- our cloned anti-XCL1 mAbs, 1A3A and 3P11R, for their specificity. induced migration is a9 integrin–dependent. In ELISA, both 1A3A and 3P11R recognized Ig-tagged and His- tagged mouse XCL1, but showed no binding toward human XCL1 XCL1 deteriorates inflammatory arthritis in CAIA mice (Supplemental Fig. 3A). Thus, we confirmed that the generated Abs It was previously reported that a9b1 integrin is involved in the de- were specific for mouse XCL1 and showed no cross-reactivity with velopment of CAIA (10). Therefore, we investigated the in vivo role human XCL1. We then determined the binding regions of these of XCL1 in CAIA. Real-time PCR of FLS revealed that XCL1 ex- mAbs. For this, we generated various recombinant XCL1 proteins 86 XCL1–a9 INTEGRIN AXIS IS INVOLVED IN AUTOIMMUNE DISEASES Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 2. XCL1 induces a9 integrin–dependent cell migration. (A) Dose-dependent induction of cell migration by XCL1 in a9 integrin–transfected NIH3T3. XCL1 showed a biphasic response with maximum response at 300 ng/ml. Cells on membranes were fixed and stained with Diff-Quik (top), and then counted in four randomly chosen high-power fields (bottom). Data represent mean 6 SEM. *p , 0.05 versus 0 ng/ml XCL1 (nonparametric Wilcoxon Mann–Whitney U test). (B) Migration assays of NIH3T3 and a9 integrin–transfected NIH3T3 cells treated with XCL1, assessed the same as (A). Data represent mean 6 SEM. *p , 0.05 (nonparametric Wilcoxon Mann–Whitney U test). (C) Checkerboard type migration assays of a9 integrin–transfected NIH3T3 cells, assessed the same as (A). Upper and lower indicates the condition of media in each well. Data represent mean 6 SEM. *p , 0.05 (nonparametric Wilcoxon Mann–Whitney U test). (D) Migration assays of RD cells with XCL1, assessed the same as (A). Data represent mean 6 SEM. *p , 0.05 (nonparametric Wilcoxon Mann–Whitney U test). (E) Migration assays of NIH3T3 and a9 integrin-transfected NIH3T3 cells treated with XCL1 and Ba2+, assessed the same as (A). Data represent mean 6 SEM. Data are representative of three or more independent experiments with similar results. *p , 0.05 (nonparametric Wilcoxon Mann–Whitney U test). with different lengths (Fig. 4A). When the secretion of recombinant XCL1 neutralization with mAb 1A3A ameliorates CAIA XCL1 proteins was examined by Western blot, we noticed that and EAE proteins with C-terminal region of XCL1 showed smear bands at Next, we investigated the involvement of XCL1 in CAIA using our higher m.w. than expected. The C-terminal region of XCL1 exists as anti-XCL1 mAbs. Mice were treated with 1A3A or 3P11R during an a-helix, resulting in the formation of complexes causing the CAIA induction (Fig. 5A). Because 3P11R showed no inhibitory smear bands. In ELISA, both 1A3A and 3P11R recognized the effect in migration assays (Fig. 4C), we used this clone as a control closed C-terminal region of XCL1, at least from amino acid posi- Ab. In mice administered XCL1-neutralizing Ab 1A3A, clinical tions 68–144 (Fig. 4B, Supplemental Fig. 3B). Of note, when the score and immunohistochemical staining showed that the severity of C terminus of XCL1 was shortened to aa 84–114, the two Abs did not arthritis was significantly decreased compared with that in mice detect the protein, suggesting that the epitopes of these two mAbs treated with 3P11R (Fig. 5B, 5C). Last, we determined the asso- were dependent on the conformation of the C terminus of XCL1. To ciation of XCL1 with EAE, another autoimmune disease model determine their neutralizing effects, we treated a9 integrin–expressing involving a9 integrin (11). As the disease progressed, the gene cells with 1A3A and 3P11R. The 1A3A-treated cells showed signif- expression levels of XCL1 and XCR1 were significantly increased, icant inhibition of cell migration toward XCL1, whereas the 3P11R- together with elevated a9 integrin expression in the spinal cord at treated cells exhibited no effect (Fig. 4C). We then examined whether day 7 (Fig. 5D). We then administered our anti-XCL1 mAbs to these observations arose through enhanced cell death caused by EAE mice (Fig. 5E). Treatment with a9 integrin–neutralizing Ab 1A3A. In cell viability assays, neither 1A3A nor 3P11R had any 1A3A was observed to suppress the disease progression (Fig. 5F). effect on cell viability (Fig. 4D). Therefore, we successfully estab- Taken together, these findings show XCL1 is involved in the de- lished 1A3A as an XCL1-neutralizing Ab. velopment of CAIA and EAE. The Journal of Immunology 87 Downloaded from http://www.jimmunol.org/

FIGURE 3. Administration of recombinant XCL1 exacerbates inflammatory arthritis in CAIA mice. (A) Relative mRNA expression level of XCL1 in by guest on September 25, 2021 FLS from normal and CAIA BALB/c mice at day 9. The expression level was normalized by HPRT expression level. (B) Protocol for CAIA with XCL1 application. Ig control or recombinant XCL1 was injected i.p. into BALB/c mice from days 21 to 9 during CAIA induction. (C) Disease severity scores of the arthritic mice described in (B). Data represent mean 6 SEM (n = 3 per group). *p , 0.05 (Student t test). (D) Representative histological images of arthritic joints at day 16 from the mice described in (B). Sections were stained with H&E or safranin-O. For the H&E-stained sections, magnified views of the boxed areas in the left panels are shown in the middle and right panels. Arrows indicate infiltrating inflammatory cells. (E) Protocol for CAIA with XCL1 application together with control or anti-a9 integrin Ab treatment. XCL1 was injected i.p. into BALB/c mice from days 21 to 9, followed by Ab treatment on days 21 and 3 during CAIA induction. (F) Disease severity scores of the arthritic mice described in (E). Data represent mean 6 SEM (n =4 per group). *p , 0.05, **p , 0.01 (Student t test). (G) Representative histological images of arthritic joints at day 14 from the mice described in (E). Sections were stained with H&E or safranin-O. For the H&E-stained sections, magnified views of the boxed areas in the left panels are shown in the middle and right panels. Arrows indicate infiltrating inflammatory cells. Data are representative of three independent experiments with similar results.

Discussion and medullary thymic epithelial cells (20, 25, 32–36). It acts on a9 integrin is expressed on various cells including airway epithelial the known receptor XCR1, which is mainly expressed on CD8a+ cells and smooth muscle cells (27), as well as cancer cells such as dendritic cells to induce cell migration (37). Of note, XCL2, an- melanoma and breast tumor cells (28, 29). The known ligands for a9 other member of the C-class chemokines that differs from XCL1 integrin are OPN, TN-C, propolypeptide of von Willebrand factor, by only 2 aa (38), also bound to a9 integrin and SFa9. Compared fibronectin-EIIIA, and polydom (6, 7, 23, 30, 31). a9integrinisin- with XCL1, the interactions of XCL2 with a9 integrin and SFa9 volved in autoimmune diseases such as RA, and in mouse models of were weaker, suggesting the 2 aa difference between XCL1 and inflammatory arthritis and EAE (9–11). A neutralizing Ab for a9 XCL2 may alter their functions. Considering that XCL2 showed a integrin was reported to inhibit the disease scores in CAIA and EAE weaker interaction with a9 integrin than XCL1, it is possible that (10, 11). However, investigations of CAIA using OPN-knockout mice the cell migration of a9 integrin–expressing cells toward XCL2 or anti-OPN Ab treatment showed lesser effects compared with anti- may be diminished. More detailed experiments are necessary to a9 integrin Ab treatment (12–14). Therefore, we hypothesized that elucidate the functional differences between XCL1 and XCL2. We other ligands of a9 integrin are also involved in autoimmune diseases. also confirmed that XCL1 could directly interact with a9 integrin To search for novel a9 integrin ligands, we performed LC-MS/ using ELISA. To our knowledge, our result is the first to dem- MS analyses using SFa9 and identified XCL1/lymphotactin as onstrate the direct binding of secretory chemokine to the integrin one of the candidates for an SFa9-binding partner. We showed receptor. We found that XCL1 enhanced the cell migration of a9 that XCL1 selectively interacted with a9 integrin and SFa9by integrin–expressing cells, and that XCL1-induced cell migration pull-down assays. XCL1 is a C-class chemokine secreted by was inhibited by an a9 integrin–neutralizing Ab. a9 integrin– various immune cells including CD8+ T, CD4+ T, gd T, NK, NKT, dependent migration involves Kir channels, which can be blocked 88 XCL1–a9 INTEGRIN AXIS IS INVOLVED IN AUTOIMMUNE DISEASES Downloaded from http://www.jimmunol.org/

FIGURE 4. Functional analysis of the generated anti-mouse XCL1 mAbs. (A) Secretion of the various generated His-SUMO-tagged mouse XCL1 variants was confirmed by Western blot with an anti-SUMO Ab. (B) ELISA data for the XCL1 variants with anti-mouse XCL1 Abs. Data represent mean 6 by guest on September 25, 2021 SEM in quadruplicate wells. (C) Migration assays of a9 integrin–transfected NIH3T3 cells treated with XCL1 and the anti-mouse XCL1 mAbs. Cells on membranes were fixed and stained with Diff-Quik, and then counted in four randomly chosen high-power fields. Data represent mean 6 SEM. *p , 0.05 (nonparametric Wilcoxon Mann–Whitney U test). (D) Cell viability assays using a Cell Counting Kit-8 (Dojindo) showed that neither 1A3A nor 3P11R had cell toxicity. Data represent mean 6 SEM in triplicate wells. Data are representative of three or more independent experiments with similar results. by Ba2+ with a consequent decrease in the migration ability of a9 reported (10), FLS isolated from CAIA mice expressed a9 integrin–expressing cells (26). We successfully showed that the en- integrin, but not XCR1 (Supplemental Fig. 2A). Stimulation of hanced migration of a9 integrin–expressing cells induced by XCL1 FLS with XCL1 enhanced mRNA expression of inflammatory was blocked by Ba2+. These results suggested that the XCL1-induced cytokines such as TNF-a, TGF-b, IL-6, and others (Supplemental cellmigrationoccurredinana9 integrin–dependent manner. Real- Fig. 2C). Because FLS expressed no XCR1, it was suggested that time qPCR and flow cytometry analyses revealed there was little ex- XCL1 interaction with a9 integrin upregulated the expression of pression of XCR1 on NIH3T3 cells used in the migration assays, these cytokines. A previous study showed extracellular matrix indicating that XCL1 directly interacted with a9 integrin to induce cell proteins such as OPN and TN-C, which are the known ligands of migration. We further knocked down XCR1 by a short hairpin RNA a9 integrin, induced mRNA expression of IL-6 and IL-1a in FLS (Supplemental Fig. 4A, 4B) and found that cell migration toward (10). This suggests that XCL1 bound to a9 integrin as a ligand and XCL1 was not altered (Supplemental Fig. 4C), thereby supporting the induced mRNA expression of inflammatory cytokines in a similar notion that the enhanced cell migration was independent of XCR1. manner to the previous report. In addition, neutralizing a9 integrin In our in vivo experiments, we examined whether XCL1 is Ab 55A2C inhibited the XCL1 stimulation on FLS (Supplemental associated with CAIA and found that continuous administration of Fig. 2D), supporting that XCL1 is a novel ligand involved in the recombinant XCL1 during the development of CAIA exacerbated development of autoimmune disease. It has also been reported that the disease. In a previous report, a9 integrin expressed on synovial a9 integrin is associated with RA (9), and that XCL1 is elevated in fibroblasts was found to interact with locally produced OPN and plasma from RA patients (16). These studies suggest that XCL1 TN-C, followed by secretion of various chemokines that resulted could be involved in the development of RA in an a9 integrin– in the induction of inflammatory arthritis (10). Based on our re- dependent manner. However, we could not determine the in- sults, it is suggested that XCL1 possibly acted on synovial fibro- volvement of XCL1-XCR1 interaction in vivo. Further study using blasts and caused local infiltration of cells. In fact, we showed a either mutant XCL1, which has no binding capability to a9 blockade of a9 integrin by Ab treatment ameliorated the disease integrin, or an Ab that specifically blocks interaction between a9 severity in XCL1-treated CAIA mice. We determined our hy- integrin and XCL1 will clarify the detailed mechanism. pothesis that FLS could be one of the cells where XCL1-a9 We further examined whether aggravation of CAIA could be integrin interaction occurs by ex vivo analysis. As previously treated by the blockade of XCL1. However, there were no The Journal of Immunology 89 Downloaded from http://www.jimmunol.org/

FIGURE 5. XCL1 neutralization by generated anti-mouse XCL1 mAb 1A3A ameliorates CAIA and EAE. (A) Protocol for CAIA with anti-XCL1 Ab treatment. The Ab was administered into BALB/c mice on days 21 and 3 during CAIA induction. (B) Disease severity scores of the arthritic mice described in (A). Data represent means 6 SEM (n = 4 per group). *p , 0.05 (Student t test). (C) Representative histological images of arthritic joints at day 15 from the mice described in (A). Sections were stained with H&E or safranin-O. For the H&E-stained sections, magnified views of the boxed areas in the left panels are shown in the middle and right panels. Arrows indicate infiltrating inflammatory cells. (D) Relative mRNA expression levels of XCL1, XCR1, and a9 integrin in the spinal cords of C57BL/6 by guest on September 25, 2021 EAE mice on the indicated days after MOG immunization. The expression levels were normalized by the HPRT expression levels. Data represent mean 6 SEM (n = 4 per group). *p , 0.05 versus day 0 (nonparametric Wilcoxon Mann–Whitney U test). (E) Procedure for EAE with anti-XCL1 Ab treatment. The Ab was administered to C57BL/6 mice on days 21 and 2 during EAE induction. (F) Disease severity scores of the EAE mice described in (E). The clinical scores of EAE severity were assessed daily as previously described (19): 0, no clinical signs; 0.5, partially limp tail; 1, paralyzed tail; 2, loss of coordinated movement, hind limb paresis; 2.5, one hind limb paralyzed; 3, both hind limbs paralyzed; 3.5, hind limbs paralyzed, weakness in forelimbs; 4, forelimbs paralyzed; 5, moribund. Data represent mean 6 SEM. Data are representative of three independent experiments with similar results. *p , 0.05, 1A3A versus 3P11R (Student t test). available functional mAbs against murine XCL1. Therefore, we investigations using XCR1-neutralizing Abs or XCR1-deficient mice generated Abs against XCL1, and established two clones, 1A3A are required to fully elucidate the relationships between a9integrin, and 3P11R. We examined the epitopes of these Abs, and found XCL1, and XCR1 in various autoimmune diseases. that both Abs recognized the C terminus of XCL1. Further in- In conclusion, we have shown the novel interaction of a9 vestigations are required to fully identify the epitopes of these integrin and XCL1 and XCL1 involvement in the development of generated Abs. We also confirmed that 1A3A had a neutralizing autoimmune diseases. In general, integrins interact with various ability against XCL1 in migration assays using a9 integrin– matricellular proteins such as fibronectin, OPN, and TN-C to expressing NIH3T3, and thus used 1A3A and 3P11R for in vivo promote various cellular functions. Chemokines induce integrin- analyses as a functional XCL1-blocking Ab and its control Ab, mediated signals through indirect mechanisms, wherein they bind respectively. Using the two generated Abs, we showed that to chemokine receptors and transduce their signals intracellularly XCL1-neutralizing Ab 1A3A diminished the severity of arthritis to activate integrins, known as inside-out activation (39, 40). To in CAIA mice. Our results indicated that XCL1 is involved in the our knowledge, our study is the first to demonstrate a direct in- development of CAIA. However, this study could not clarify the teraction between an integrin and a chemokine, and to suggest that involvement of XCR1 in CAIA development. XCL1 is a functional ligand of a9 integrin in autoimmune dis- We further demonstrated that anti-XCL1 Ab 1A3A inhibited the eases. Therefore, the XCL1–a9 integrin axis may be a potential progression of EAE, another autoimmune disease model. In EAE, therapeutic target for treating autoimmune diseases. activated a9 integrin expressed on lymphatic endothelial cells en- hances the egress of by interacting with TN-C (11). Acknowledgments XCL1 could play a role in this process by binding to a9integrinasa We thank D. Sheppard (University of California, San Francisco) for provid- novel ligand and inducing the cell migration of lymphocytes. It is ing the pBabepuro and pWZL-blast2 vectors. also possible that local elevation of XCL1 could directly enhance the infiltration of a9 integrin–expressing lymphocytes such as dendritic Disclosures cells and , resulting in the aggravation of EAE. Further The authors have no financial conflicts of interest. 90 XCL1–a9 INTEGRIN AXIS IS INVOLVED IN AUTOIMMUNE DISEASES

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Active induction of experimental 110: 673–687. allergic encephalomyelitis. Nat. Protoc. 1: 1810–1819. 40. Peled, A., O. Kollet, T. Ponomaryov, I. Petit, S. Franitza, V. Grabovsky, 20. Kelner, G. S., J. Kennedy, K. B. Bacon, S. Kleyensteuber, D. A. Largaespada, M. M. Slav, A. Nagler, O. Lider, R. Alon, et al. 2000. The chemokine SDF-1 N. A. Jenkins, N. G. Copeland, J. F. Bazan, K. W. Moore, T. J. Schall, and activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) A. Zlotnik. 1994. Lymphotactin: a cytokine that represents a new class of che- cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mokine. Science 266: 1395–1399. mice. Blood 95: 3289–3296. Supplemental Fig. 1 A B

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Supplemental Figure 1. Assessment of XCL1 proteins and transformants used for in vitro analyses (A) Result of LC-MS/MS indicates XCL1/lymphotactin (No.17, in bold) is one of the candidates of binding protein. (B) CBB staining and western blotting analyses revealed that the recombinant XCL1 proteins were successfully generated. Note that the bands for Ig-tagged XCL1 and XCL2 were shifted under non-reducing conditions, indicating that the Ig-fused proteins existed as dimers. (C) NIH3T3 cells and α9 integrin- overexpressing NIH3T3 cells were analyzed by flow cytometry using APC-conjugated antibody 18R18D for surface expression of α9 integrin. (D) Integrin expression of Ba/F3 transformants were analyzed by flow cytometry. (E) XCL1 does not interact with αL integrin. Data are representative of three or more independent experiments with similar result. Supplemental Fig. 2 B A

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Supplemental Figure 2. Ex-vivo assessment of α9 integrin and XCL1 interaction using FLS isolated from CAIA mice (A) Surface expression of XCR1 and α9 integrin in FLS was analyzed by flow cytometry. (B) Primer sequences used for qPCR analysis of mouse gene. (C) Relative mRNA expression levels of various inflammatory cytokines in FLS by XCL1 stimulation. The expression levels were normalized by the β-actin expression levels. Data represent means ± SEM (n = 3 per group). *p < 0.05, **p < 0.01 (Student’s t-test). (D) Relative mRNA expression levels of various inflammatory cytokines in XCL1-stimulated FLS with anti-α9 integrin antibody, assessed same as (C). Data represent means ± SEM (n = 3 per group). *p < 0.05, **p < 0.01 (Student’s t-test). Data are representative of three or more independent experiments with similar result. FLS were used between passage 4 to 7. Supplemental Fig. 3 A B

Supplemental Figure 3. Two anti-XCL1 monoclonal antibodies, 1A3A and 3P11R, are specific for the C-terminal region of mouse XCL1 (A) ELISA confirmed the reactivity of both antibodies toward mouse XCL1, but not toward human XCL1. (B) Western blot analyses revealed that both antibodies bound to the C-terminal region of XCL1. Supplemental Fig. 4

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Supplemental Figure 4. Knockdown of XCR1 in α9 integrin-expressing NIH3T3 cells has no influence on XCL1- induced cell migration (A) Knockdown efficiency of shXCR1 (target sequence: 5′-CCACAAAGTGATCTCCTTAAA-3′) in α9 integrin- expressing NIH3T3 cells was confirmed by real-time qPCR. Expression of α9 integrin was not affected. Data represent means ± SEM (n = 3 per group). *p < 0.05 (Student’s t-test). (B) The surface expression of XCR1 and α9 integrin in XCR1-knockdown cells was analyzed by flow cytometry. (C) The migration ability of shXCR1-knockdown cells toward XCL1 was assessed in migration assays. Cells on membranes were fixed and stained with Diff-Quik (top), and then counted in four randomly-chosen high-power fields (bottom). Data represent means ± SEM. Data are representative of three or more independent experiments with similar result.