injected 5 times at 3-day intervals with 9D9, and tumor size was routinely monitored by means of a caliper. In order to evaluate the synergistic effect of vancomycin and anti-CTLA4, a prophylactic setting was established. In figure S21, anti-CTLA4 treatment started two days after umor inoculation. In experiments using anti-IL-12p40 mAb(clone C17.8, 500ug per mouse)or anti-Ly6G mAb(clone 1A8, 200ug per mouse), mAbs(or their isotype controls, clone 2A3 in both cases) were injected i.p. every 2 days starting from day 0 until the final anti-CTLA-4 injection. Anti-ICOS mAb(clone 17G9)or isotype control mAb(clone LTF-2)were injected at 250ug per mouse, at the same times as for anti-CTLA4 mAb. In order to deplete CD4+ and CD8+ cells, clone GK1.5 and clone 53.72. 1. or isotype control mAb were injected ip at 200ug/mice 4 days before anti-CTLA4 treatment. All mAbs for in vivo use were obtained from BioXcell(West Lebanon, NH, USA), using the recommended isotype control mAbs. The inhibitor of iNOS, N-monomethyl-L-arginine (L-NMMA; Sigma), was administered in PBS daily to mice i.p. at a dose of 2mg per mouse from the start of anti-CTLA4 treatment Antibiotic treatments. Mice were treated with antibiotics 2-3 weeks before tumor implantation and continued on antibiotics until the end of the experiment. A mix of ampicillin(1 mg/ml) streptomycin (5 mg/ml), and colistin(1 mg/ml)(Sigma-Aldrich), or vancomycin alone(0.25 mg/ml), or imipenem alone(0.25 mg/ml), or colistin alone(2.10U/ml)were added in sterile drinking water. Solutions and bottles were changed 2-3 times a week, or daily for experiments with imipenem. Antibiotic activity was confirmed by macroscopic changes observed at the level of caecum(dilatation) and by cultivating the fecal pellets resuspended in BHI+15% glycerol at 0.1g/ml on blood agar plates for 48h at 37C with in aerobic or anaerobic conditions Flow cytometry. Tumors, spleens and lymph nodes were harvested two days after the third injection of anti-CTLA-4 for antibiotics experiments or at the end of tumor growth(around day 20)for germ-free experiments. Excised tumors were cut into small pieces and digested in RPMI medium containing Liberase at ml(roche) and DNasel at 150UI/ml(Roche)for minutes at 37C. The mixture was uently passaged through a 100um cell strainer. 2 x 10 splenocytes(after red blood cells lysis), lymph nodes or tumor cells were preincubated with purified anti-mouse CD16/CD32(clone 93; eBioscience) for 15 minutes at 4C, before membrane staining. For intracellular staining, the FoxP3 staining kit(eBioscience) was used5 injected 5 times at 3-day intervals with 9D9, and tumor size was routinely monitored by means of a caliper. In order to evaluate the synergistic effect of vancomycin and anti-CTLA4, a prophylactic setting was established. In figure S21, anti-CTLA4 treatment started two days after tumor inoculation. In experiments using anti-IL-12p40 mAb (clone C17.8, 500μg per mouse) or anti-Ly6G mAb (clone 1A8, 200μg per mouse), mAbs (or their isotype controls, clone 2A3 in both cases) were injected i.p. every 2 days starting from day 0 until the final anti-CTLA-4 injection. Anti-ICOS mAb (clone 17G9) or isotype control mAb (clone LTF-2) were injected at 250μg per mouse, at the same times as for anti-CTLA4 mAb. In order to deplete CD4+ and CD8+ cells, clone GK1.5 and clone 53.72.1. or isotype control mAb were injected ip at 200μg/mice 4 days before anti-CTLA4 treatment. All mAbs for in vivo use were obtained from BioXcell (West Lebanon, NH, USA), using the recommended isotype control mAbs. The inhibitor of iNOS, NG-monomethyl-L-arginine (L-NMMA; Sigma), was administered in PBS daily to mice i.p. at a dose of 2mg per mouse from the start of anti-CTLA4 treatment. Antibiotic treatments. Mice were treated with antibiotics 2-3 weeks before tumor implantation and continued on antibiotics until the end of the experiment. A mix of ampicillin (1 mg/ml), streptomycin (5 mg/ml), and colistin (1 mg/ml) (Sigma-Aldrich), or vancomycin alone (0.25 mg/ml), or imipenem alone (0.25 mg/ml), or colistin alone (2.103 U/ml) were added in sterile drinking water. Solutions and bottles were changed 2-3 times a week, or daily for experiments with imipenem. Antibiotic activity was confirmed by macroscopic changes observed at the level of caecum (dilatation) and by cultivating the fecal pellets resuspended in BHI+15% glycerol at 0.1g/ml on blood agar plates for 48h at 37°C with in aerobic or anaerobic conditions. Flow cytometry. Tumors, spleens and lymph nodes were harvested two days after the third injection of anti-CTLA-4 for antibiotics experiments or at the end of tumor growth (around day 20) for germ-free experiments. Excised tumors were cut into small pieces and digested in RPMI medium containing LiberaseTM at 25μg/ml (Roche) and DNase1 at 150UI/ml (Roche) for 30 minutes at 37°C. The mixture was subsequently passaged through a 100μm cell strainer. 2 × 106 splenocytes (after red blood cells lysis), lymph nodes or tumor cells were preincubated with purified anti-mouse CD16/CD32 (clone 93; eBioscience) for 15 minutes at 4°C, before membrane staining. For intracellular staining, the FoxP3 staining kit (eBioscience) was used