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1 Supporting Information Kayama et al /pnas SI Materials and Methods Reagents. 1-methyl-L-tryptophan and nordihydroguaiaretic acid were purchased from Sigma Aldrich. N W -hydroxyl-nor-l-arginine, S-(-bronoethyl)-L-cysteine, N G -mono-methyl-l-arginine, adenosine 3, -cyclic monophosphorothioate, and Rp-isomer, triethylammonium salt were purchased from albiochem. Superoxide dismutase was purchased from nacalai tesque. Anti-mouse 1/ 3 (.G), 1 (rm-3), 13 (M9), NK-1.1 (PK13), I-A b (AF-1.1), and (RA3-) antibodies were purchased from Pharmingen. Anti-mouse 7 (FR7) and F/ 8 (M8) were purchased from eioscience. Anti-mouse (NL-1) and 8 (FA-11) were purchased from iolegend. Anti-human X 3 R1 (A9-1) was purchased from ML. Anti- R antibody (8) was purchased from Abcam. Flow ytometry. Flow cytometric analysis was performed using a FAS anto II flow cytometer ( iosciences) with FlowJo software (Tree Star). The instrumental compensation was set in each experiment using single-color, two-color, or four-color stained samples. Isolation of olonic Lamina Propria ells. Large intestinal lamina propria myeloid cells and lymphocytes were isolated using previously described protocol (1). Histopathological Analysis. Paraffin-embedded colon samples were sectioned and stained with hematoxylin and eosin. Severity of colitis was evaluated by the standard scoring system as previously described (). The scoring was performed blinded manner. Images of hematoxylin and eosin staining were taken using iozero (KYN). Proliferation Assay. Splenic + T cells were cultured with s with or without cells at a 1:1:1 ratio in number in the 9-well round-bottom plates in the presence of 1 μg/ml soluble anti-3 antibody for 7 h. At h after the start of coculture, each well was pulsed with 1 μi [ 3 H]-thymidine. The cells were harvested onto filters, and radioactivity was measured in a 1 microbeta scintillation counter (Wallac). ata were expressed in counts per min (PM). In some experiments, cells were incubated with the indicated blocking mabs for h at 8, vigorously washed with culture media, and then used for proliferation assays. For further experiments, purified T cells were labeled with FS and cultured with 1 1 s with or without 1 1 cells in the presence of 1 μg/ml soluble anti-3 antibody for 7 h. Then the intensity of FS expression was analyzed using a FAS anto II system. Real-time RT-PR. Real-time RT-PR was performed as previously described (1). Statistical Analysis. ifferences between control and experimental groups were evaluated by the Student s t test. 1. Atarashi K, et al. (8) ATP drives lamina propria T(H)17 cell differentiation. Nature (71): Kobayashi M, et al. (3) Toll-like receptor-dependent production of IL-1p causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice. J lin Invest 111(9): Kayama et al. 1of9

2 11c PM (x 1 ) b X 3 R1-P high int neg T X 3 R1 X 3 R1 lnt b neg int high X 3 R1-P F/8 X 3 R1-P X 3 R1-GFP 8 F 11c b X 3 R1-GFP NK1.1 R PM (x 1 3 ) 3 1 I-A b T GFP GFP high G MLN Spleen Thymus X 3 R1-GFP Fig. S1. (A) Flow cytometry of large intestinal lamina propria cells from 7L/J mice stained for 11b, 11c, and X 3 R1. Numbers adjacent to the bars indicate percentages of cells (Right), X 3 R1 int (enter), and X 3 R1 cells (Left) in the 11b + 11c + population. ()[ 3 H]Thymidine uptake of + T cells cultured with the indicated cells. P <.38. () Surface expression of 1, 7, 13, F/8,, 8,, NK1.1, R, and I-A b (open histogram) on,x 3 R1 int, and X 3 R1 cells. Filled histograms, isotype control. () olonic lamina propria cells were stained with May Grunwald Giemsa. () Flow cytometry of large intestinal lamina propria cells from X 3 R1 +/GFP mice. (F) olonic lamina propria cells from X 3 R1-GFP mice were stained with phycoerythrin-conjugated anti-x 3 R1 Ab. (G)[ 3 H]Thymidine incorporation by + T cells cocultured with the indicated cells from X 3 R1 +/GFP mice., not detected; P <.. (H)xpressionofX 3 R1 on 11b + 11c + cells from MLNs, spleen, and thymus (open histogram) of X 3 XR1 +/GFP mice. Filled histogram, expression of X 3 R1 on colonic lamina propria 11b + 11c + cells from X 3 R1 +/GFP mice. All data are representative of three independent experiments (means ± S of triplicate well measurements). R high R high + IL-17 IL- Foxp3 IFN-γ IFN-γ IL-1 Fig. S. Flow cytometric plots of IL-17-, IFN-γ-, IL--, and IL-1-producing or Foxp3-expressing large intestinal lamina propria + T cells in SI mice given R high + T cells with or without myeloid cells. Representative of three independent experiments. Kayama et al. of9

3 API API + T cell + T cell API API API + T cell + T cell + T cell API + T cell API + T cell X 3R1 high The number of + T cell (+) cell Fig. S3. Splenic naïve + T cells (3 1 ) were transferred with or without FS-labeled cells i.p. into Rag / mice. At 7 d after transfer, large intestine, MLNs, and spleen were collected. Fixed cryosections were stained with anti-mouse Ab ( iosciences) (red) and API (blue). (A and ) MLNs (A) and spleen () from mice given naïve + T cells and cells. All data are representative of eight fields from two independent experiments. ( and ) olonic lamina propria of mice given naïve + T cells with cells () or without cells (). () The number of + T cells that were present around cells (within white frame) or in a cluster (within yellow frame). ata show the average of 1 fields from independent experiments. (Original magnification,.) Images were taken using an IX71 fluorescence microscope (Olympus). P <.1. A 7L/ -week-old SI -week-old (x 1 cells) (x 1 cells) Naïve T cells.8 1. Naïve T cells + cells Fig. S. (A) Total number of cells in the colonic lamina propria 11b + 11c + population from -wk-old 7L/ and SI mice. () Number of 11b + 11c + cells in mice given naïve + T cells (3 1 ) alone or with cells for wk. P <.17 (n = per group). Kayama et al. 3of9

4 .3..1 Hpgd d13 Hmox1 d9f d9g ebpb wild-type.7 11c..3 Il1 -/- 11b X 3 R1-P Fig. S. (A) xpression of Hpgd, d13, Hmox1, 9f, 9g, and ebpb mrna in cells and X 3 R1 cells from 7L/ mice (means ± S of at least triplicate PRs on the identical sample determinations)., not detected; P <.. () Flow cytometry of large intestinal lamina propria cells from wild-type and Il1 / mice stained for 11b, 11c, and X 3 R1. Kayama et al. of9

5 PM (x1 3 ) 1 1 (+) Nor-NOHA PM (x1 3 ) 1 1 (+) PM (x1 3 ) (+) L-NMMA PM (x1 3 ) 9 3 PM (x1 3 ) F PM (x1 3 ) 3 1 1MT (+) G Foxp3. tla.1 Folr H T reg.1.8. PM (x1 ) 3 1 (+) (+) No transwell with transwell Fig. S. (A and F) + T cells were cocultured with X 3 R1 s and cells in the presence or absence of several inhibitors such as 1 μm nor-noha (A), 1 μm (), 1 μm N G -monomethyl-l-arginine (L-NMMA) (), and μm 1MT (F). ( and ) cells were treated with μm nordihydroguaiaretic acid (NGA) () or units/ml superoxide dismutase (SO) () for h and washed three times. Then, cells were cocultured with s and + T cells. (G) xpression of Foxp3, tla, and Folr mrna in cells and T reg cells. (H) Splenic + T cells and X 3 R1 s were cocultured in a 3-μm transwell chamber containing cells. ells were cultured in the presence of 1 μg/ml soluble anti-3 Ab for 7 h. [ 3 H]Thymidine was pulsed for the last 1 h, and T-cell proliferation was determined by [ 3 H] incorporation. P <.3; P <.18. ata are representative of two independent experiments (means ± S of triplicate well determinations). 1MT, 1-methyl-L-tryptophan;, S-(-boronoethyl)-L-cysteine; nor-noha, N w -hydroxy-nor-arginine. Kayama et al. of9

6 8 PM IAM-1 IAM- VAM-1 LFA-1 wild-type X 3 R1 - wild-type Il1 / Fig. S7. (A) cells were treated with the indicated blocking mabs for h and washed three times. Then, + T cells were cultured with X 3 R1 s in the presence or absence of cells at a ratio of 1:1:1. ells were cultured in the presence of soluble 1 μg/ml anti-3 Ab for 7 h, and for the last 1 h, [ 3 H]thymidine was pulsed. P <.3. ata are representative of three independent experiments (means ± S of triplicate well determinations). () xpression of IAM-1, IAM-, VAM-1, and LFA-1 on wild-type X 3 R1 (gray), (blue), and IL-1-deficient (red) cells. ata are representative of two independent experiments. Kayama et al. of9

7 . 1 8 Il1 mrna (A) () (A) () X 3 R1 - IL-1 ()ng/ml 8 LPS (+) (+) PM (x 1 3 ) X 3 R1 - (wild-type) 7-H HVM P-L1 P-L PM 1 1 (wild-type) (Stat3 / ) F 3 wild-type 3 G P-1 Tg H PM (x1 3 ) 1 1 PM (x1 3 ) 3 1 PM (x1 3 ) X 3 R1 (+) (+) X 3 R1 high high (+) control IgG 7 Ab (MIH) wild-type P-1 / Fig. S8. (A) Total RNA isolated from wild-type cells and X 3 R1 cells was reverse-transcribed. xpression of Il1 mrna was analyzed by real-time RT-PR in cells and X 3 R1 cells. All data were normalized to the level of Gapdh expression, and the fold difference relative to Gapdh is shown. ata are means ± S of triplicate PRs on the identical sample determinations. () cells and X 3 R1 cells were isolated from the colonic lamina propria and cultured with or without 1 ng/ml LPS. After 8 h, the supernatants were assayed for production of IL-1 by LISA. All of the graphs show mean values ± S of duplicate well measurements. () + T cells were cultured with s in the presence or absence of wild-type cells at a 1:1 ratio in the presence of 1 μg/ml soluble anti-3 mab for 7 h. ocultured cells were treated with control IgG or 1 mg/ml Abs neutralizing IL-1 and the IL-1 receptor. P <.8. () Wild-type and Stat3 / cells were preincubated with or without 1 ng/ml IL-1 for 7 h. Then, the cells were analyzed for the suppressive activity of T-cell proliferation. P <.38. Mean values ± S of two independent experiments are shown. () Surface expression of coinhibitory molecules such as 7-H, HVM, P-L1, and P-L on cells and s from wild-type or LysM-cre; Stat3 fl/fl mice. losed histograms, isotype control. (F) + T cells from wild-type or P-1 Tg mice were cocultured with wild-type X 3 R1 s and cells at the same ratios in the presence or absence of 1 μg/ml anti-7 (MIH) Ab or control IgG. P <.13. (G) + T cells from wild-type or P-1-deficient mice were cocultured with wild-type X 3 R1 s and cells. P <.13. (H) cells were pretreated with 1 μg/ml anti-mouse 7-H (HMH-G1) Ab or control IgG for h at º. After washing, cells were added to the mixture of + T cells and X 3 R1 s. P <.3. All data are representative of at least of two independent experiments. Kayama et al. 7of9

8 wild-type Stat3 / 8 8 IFN-γ (pg/ml) 8 3 IL-17 (ng/ml) 1 1 X 3 R1 int 11b + 11c + PM (x1 ) 1 8 Hpgd d13 Hmox1 d9f d9g Arg1 Nos ybb S1a8 cell MS S1a cell MS Fig. S9. (A) Wild-type and Stat3 / cells were analyzed for surface expression of 8, 8, and by flowcytometry. () Splenic naïve + T cells were co-cultured with,x 3 R1 int, or 11b + 11c + cells at a 1:1 ratio in the presence of anti-3 antibody. After 7 h, + T cells cocultured as above were harvested and restimulated with plate-bound anti-3 antibody for h, and the supernatants were assayed for production of IFN-γ and IL-17 by LISA.,not detected. P <.1, P <.8, P <.. () Splenic + T cells were cultured with wild-type, Il1 / or Stat3 - / large intestinal 11b + 11c + cells at a 1:1 ratio in the presence of 1 μg/ml soluble anti-3 antibody for 7 h. [ 3 H]-thymidine was pulsed for the last 1 h, and [ 3 H] incorporation was measured. All the graphs show mean values ± S of triplicate well measurements. P <.3, P <.1. ata are representative of two independent experiments (A and ). ( and ) To obtain MS, 3 x 1 L- tumor cells were injected i.p. into 7L/ mice as described (1). After 3 wk, cells from tumor sites were collected, and then Gr b + cells were isolated as MS by FAS Aria ( iosciences). RNA was extracted from MS and colonic lamina propria 11b + 11c + cells. xpression of -related genes in intestinal cells and MS were analyzed by real-time RT-PR (). xpression of Arg1, Nos, ybb, S1a8, and S1a9 in MS and intestinal cells were analyzed using real-time RT-PR (). 1. orzo A, et al. (1) HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. J xp Med 7:39 3. Kayama et al. 8of9

9 8 IFN-γ (pg/ml) 3 IL-17 (ng/ml) 1 PM (x1 ) X 3 R1 int 11b + 11c + Hpgd d13 Hmox1 d9f d9g cell MS Arg1 Nos ybb S1a8 S1a cell MS Fig. S1. (A) Splenic naïve + T cells were cocultured with,x 3 R1 int, or 11b + 11c + cells at a 1:1 ratio in the presence of anti-3 Ab. After 7 h, + T cells cocultured as above were harvested and restimulated with plate-bound anti-3 Ab for h, and the supernatants were assayed for production of IFN-γ and IL-17 by LISA., not detected; P <.1; P <.8; P <.. () Splenic + T cells were cultured with wild-type, Il1 /,or Stat3 / large intestinal 11b + 11c + cells at a 1:1 ratio in the presence of 1 μg/ml soluble anti-3 Ab for 7 h. [ 3 H]Thymidine was pulsed for the last 1 h, and [ 3 H] incorporation was measured. All of the graphs show mean values ± S of triplicate well measurements. P <.3; P <.1. ata are representative of two independent experiments (A and ). ( and ) To obtain MS, 3 1 L- tumor cells were injected i.p. into 7L/ mice as described (1). After 3 wk, cells from tumor sites were collected, and then Gr b + cells were isolated as MSs by FASAria ( iosciences). RNA was extracted from MSs and colonic lamina propria 11b + 11c + cells. xpression of -related genes in intestinal cells and MS were analyzed by real-time RT-PR (). xpression of Arg1, Nos, ybb, S1a8, and S1a9 in MS and intestinal cells were analyzed using real-time RT-PR (). 1. orzo A, et al. (1) HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. J xp Med 7:39 3. Kayama et al. 9of9