Female 6-7-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan).

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1 Supplementary materials Materials and Methods Mice Female 6-7-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). All mice were maintained under specific pathogen free conditions, and used at 7-8 weeks of age. All procedures were reviewed and approved by the Animal Care and Use Committee of the Tokyo Medical and Dental University. Monoclonal antibodies (mabs) and flow cytometry mabs against CD3 (145-2C11), CD4 (GK1.5), CD8 ( ), CD25 (PC61), CD86 (PO3), CD207 (ebiol31), CD273 (B7-DC, TY25), CD45R (B220, RA3-6B2), CD11c (N418), Foxp3 (FJK-16s), and IFN- (XMG1.2) were used. All FITC-, PE-, PerCp-Cy5.5-, APC-eFluor 450-, APC-, efluor 780-, V500-conjugated or biotinylated mabs and isotype control Igs were obtained from ebioscience (San Diego, CA) or BD-Pharmingen (San Diego, CA). The VISTA mab, MIH63, rat IgG2a was generated as described previously (Kondo et al., 2016). Biotinylation of anti-vista mab was performed using a standard protocol. PE- or efluor 450-streptavidin was used to detect biotinylated mabs. Multicolor staining of intracellular cytokines and cell-surface Ags was performed as described previously (Sakurai et al., 2000). To detect IFN- expression, dln cells were incubated with PMA (50 ng/ml, Sigma, St. Louis, MO), ionomycin (0.5 g/ml, Sigma) and Breferdin A (5 g/ml, Sigma) for 6 h. Culture supernatant from a 2.4G2 hybridoma (anti-cd16/cd32 mab) was used to block non-specific binding. Stained cells were analyzed using the FACSCalibur running CellQuest-,

2 or FACSVerse running FACSuite-software (BD Biosciences). All data were analyzed using FlowJo software (Tree Star, Ashland, OR). Induction of contact hypersensitivity and mab treatment CH to 2,4-dinitro-1-fluorobenzene (DNFB, Sigma) was induced as described previously (Nuriya et al., 1996; Nuriya et al., 2001;Kamimura et al., 2009). Briefly, 10 l of 0.5 % (w/v) DNFB dissolved in an acetone: olive oil solution (4:1) were painted onto shaved abdominal skin on days 0 and 1 (sensitization), and then ear skin was challenged with 10 l 0.2 % (w/v) DNFB on day 5. For mab treatment, each group of mice received intraperitpneal injections of either 200 g control rat IgG (MP Biomedicals, Irvine, CA) or anti-vista mab (MIH63, rat IgG2a) (Kondo et al., 2016) 2 h before each sensitization or challenge. To deplete CD4 + T cells in vivo, anti-cd4 mab (GK1.5, 0.5 mg/mouse) was administrated intraperitoneally on days -3 and -1 before sensitization. The proportions of CD4 + cells within LNs and splenocytes were less than 2% on day 2, as confirmed by flow cytometry in a parallel study. Assessment of IFN- and TNF- in the challenged ear skin Ear skin samples 48 h after challenge were excised and supernatants from homogenized and sonicated samples were collected as described previously (Ferguson et al., 1994). ELISA for IFN- and TNF- was performed using ELISA kits (Ready-SET-Go!, ebioscience, San Diego, CA) according to the protocols recommended by the manufacturer. Isolation of draining LN cells Draining (cervical, axillary, and inguinal) LNs (dlns) were collected from sensitized and mab-treated mice on day 1 (to prepare whole cells and the APC fraction) and day 5 (to

3 prepare whole cells and the T cell fraction). Single-cell suspensions of dln cells were prepared using type I collagenase and EDTA, as described previously (Aramaki et al., 2011). To isolate T cell and APC-fractions, negative selection was performed using a biotinylated mab cocktail containing either anti-cd45r (B220), MHC class II (M5/114), CD11b (M1/70), CD49b (DX5), and Gr-1 (RB6-8C5) mabs or anti-cd90 (Thy1.2) and CD45R (B220) mabs, followed by application of Streptavidin Particles Plus-DM using the BD IMag system (BD Biosciences). The T cell fraction contained over 95% CD3 + cells and the APC fraction over 90% CD3 - CD45R - cells, as confirmed by flow cytometry. To identify migrating skin DCs in the dlns, mice were sensitized with 0.5% (w/v) FITC (20 l/mouse) and treated with control Ig or anti-vista mab as described both above and previously (Nuriya et al., 2001). Hapten-specific T cell proliferation assays Isolated dln-t cells ( /well) from sensitized mice treated with either control or anti-vista mab at day 5 served as responder cells. Whole dln cells or T cell- and B cell -depleted APC-fractions prepared from mice 1 day after DNFB-sensitization served as DNFB-pulsed APC cells. DNFB-pulsed APC cells were pre-treated with mitomycin C (50 g/ml, Sigma) for 20 min at 37 C. Cultures were pulsed with 1.0 Ci/well [ 3 H]-thymidine for the final 18 h of the 72 h culture period, and then the incorporated radioactivity was measured as described previously (Igarashi et al., 2008). Note Information of VISTA is described below. Mouse Official Gene Symbol/Vsir/

4 Official Full Name: V-set immunoregulatory receptor Aliases: N05Rik, Dies1, PD-1H, VISTA Human Official Gene Symbol/VSIR/ Official Full Name: V-set immunoregulatory receptor Aliases: B7H5, GI24, B7-H5, PD-1H, SISP1, VISTA, PP2135, C10orf54, DD1alpha References Aramaki O, Chalermsarp N, Otsuki M, Tagami J, Azuma M (2011) Differential expression of co-signal molecules and migratory properties in four distinct subsets of migratory dendritic cells from the oral mucosa. Biochem Biophys Res Commun 413: Igarashi H, Cao Y, Iwai H, Piao J, Kamimura Y, Hashiguchi M, et al. (2008) GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells. Biochem Biophys Res Commun 369: Ferguson TA, Dube P, Griffith TS (1994) Regulation of contact hypersensitivity by interleukin 10. J Exp Med 179: Kamimura Y, Iwai H, Piao J, Hashiguchi M, Azuma M (2009) The glucocorticoid-induced TNF receptor-related protein (GITR)-GITR ligand pathway acts as a mediator of cutaneous dendritic cell migration and promotes T cell-mediated acquired immunity. J Immunol 182:

5 Kondo Y, Ohno T, Nishii N, Harada K, Yagita H, Azuma M (2016) Differential contribution of three immune checkpoint (VISTA, CTLA-4, PD-1) pathways to antitumor responses against squamous cell carcinoma. Oral Oncol 57: Nuriya S, Yagita H, Okumura K, Azuma M (1996) The differential role of CD86 and CD80 co-stimulatory molecules in the induction and the effector phases of contact hypersensitivity. Int Immunol 8: Nuriya S, Enomoto S, Azuma M (2001) The role of CTLA-4 in murine contact hypersensitivity. J Invest Dermatol 116: Sakurai J, Ohata J, Saito K, Miyajima H, Hirano T, Kohsaka T, et al. (2000) Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory CD8 + T cells. J Immunol 164:

6 sensitized sensitized intact cont-ig anti-vista 76.8± ± ± ±1.4 * * ± ±1.4, CD8 + T CD62L 1.8± ±0.3 * 7.1±1.5 *, 74.7± ± ± ±0.8 * 72.2± ±1.2 * CD4 + T CD62L CD44 3.2±1.2 CD44 6.2±0.3 * 6.3±0.3 * CD44 Supplementary Figure S1. Draining LN (dln) cells from intact and sensitized mice treated with control Ig or anti-vista mab 4 days after the final sensitization were stained with APC-anti-CD3, V450-anti-CD4, PerCP-Cy5.5-anti-CD8, FITC-anti-CD44 and PE-anti-CD62L mabs. An electronic gate was placed on CD8 + CD3 + or CD4 + CD3 + lymphocytes, and the expression profiles of CD44 and CD62L are shown as pseudo color dotted plots. Data shown are representative from each group of four mice. The values in each fraction are mean percentages ± SD. Statistically different from the *intact and control Ig group (p < 0.05).

7 a Treg Tconv CD8+ T 2359± ± ± ± ± ±87 intact sensitized VISTA VISTA b VISTA FITC+CD11c+ gated FITC CD11c+ FITC CD11c + CD11chi CD207CD11cint/lo CD207- CD11c CD207 CD11chiCD207- CD11cint/loCD207- CD11chiCD207- CD11cint/loCD ± ± ± ± ± ± ± ±5420 cont Ig anti-vista CD86 CD86 B7-DC B7-DC Supplementary Figure S2. (a) dln cells from unsensitized or sensitized mice prepared 4 days after the final sensitization were stained with APC-eFluor780-anti-CD3, PerCP-Cy5.5-anti-CD4, V500-anti-CD8, APC-anti-Foxp3, and biotinylated anti-vista mabs, followed by PE-streptavidin. An electronic gate was placed on CD8+CD3+, Foxp3-CD4+CD3+ and Foxp3+CD4+CD3+ lymphocytes to identify CD8+ T, conventional CD4+ T (Tconv), and Foxp3+ regulatory T cells (Treg), and the VISTA expression levels in these cells were determined. Representative histogram profiles before and after sensitization for each subset are shown, together with control (PE-streptavidin alone) histograms (open histograms). The values are mean fluorescence intensities (MFIs) ± SD from three mice. No significant difference was noted post-sensitization. (b) dlns from FITC-painted and control Ig- or anti-vista mab-treated mice were collected 2 days after the initial painting. Cells were stained with PerCp-Cy5.5-anti-CD11c, PE-anti-CD207, and biotinylated-anti-cd86 or anti-b7-dc mabs, followed by V450-streptavidin,. An electronic gate was placed on FITC+CD11c+ cells to detect migrating DCs from the skin, and the mean MFIs for CD86 and B7-DC expression on CD11chiCD207- and CD11cint/loCD207- cells were calculated. Representative histogram profiles in the control Ig- or anti-vista mab-treated groups are shown, together with control histograms (open histograms). The values are mean MFIs ± SD for each group of three mice. No significant difference was evident between the control and anti-vista mab-treated group.