Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit(Life TechnologiesM) Stained samples were run on a Canto II(BD Bioscience, San Jose, CA, USA) cytometer, and analyses were performed with Flow Jo software(Tree Star, Ashland, OR, USA) For cytokine staining, cells were stimulated for 4 hrs at 37C with 50ng/ml of phorbol 12 myristate 13-acetate(PMA; Calbiochem), lug/ml of ionomycin( Sigma), and BD Golgi STOP (BD Biosciences). Anti-CD45.2(104), anti-FoxP3(FJK-16s), anti-ICOS(7E17G9), anti-IFN-Y (XMG1. 2), anti-TNF-a(MP6-XT22), anti-CXCR3( CXCR3-173)and isotype controls rat IgGl (eBrGl), IgG2a(eBRG2a), IgG2b(eBRG2b) were purchased from eBioscience. Anti-CD3 (145-2C11), anti-CD25(PC61.5.3), KI67(FITC mouse anti-human KI67 set), rat IgGlK were obtained from BD Bioscience. Anti-CD4(GK1.5), anti-CD8B (YTS1567.7), Rat IgG2a (RTK2758)were purchased from Biolegend(San Diego, CA, USA). Anti-CCR6(140706)was obtained from R&D Systems, Minneapolis, MN. Eight-color flow cytometry analysis was performed with antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, phycoerythrin cyanin 7, peridinin chlorophyll protein cyanin 5.5, allophycocyanin cyanin 7, pacific blue, or allophycocyanin. All cells were analyzed on a FACS CANTO II(BD) flow cytometer with FlowJo(Tree Star) software Microbial DNA extraction, 454 pyrosequencing and bacteria identification. Fecal samples used in this study were collected before or after one injection of anti-CTLA4 (or isotype control) from mice under vancomycin regimen or water, and were kept at -80oC until further analysis Library preparation and sequencing were conducted at GATC Biotech AG(Konstanz, Germany) Bacterial isolation, culture, and identification. Fecal pellet contents were harvested and suspended in BHI+15% glycerol at 0.1g/ml. Serial dilutions of feces were plated onto sheep's blood agar plates and incubated for 48h at 37C with 5%CO2 in aerobic or anaerobic conditions After 48h, single colonies were isolated and Gram staining was performed. The identification of specific bacteria was accomplished through the combination of morphological tests and analysis by means of an Andromas MALDI-TOF mass spectrometer(Andromas, France) Gut colonization with dedicated bacterial species. For inoculation of gF mice or mice treated bad-spectrum antibiotics, colonization was performed the day following the first anti- CTLA4 injection by oral gavage with 100ul of suspension containing 1 x 10 bacteria. Efficient6 Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit (Life TechnologiesTM). Stained samples were run on a Canto II (BD Bioscience, San Jose, CA, USA) cytometer, and analyses were performed with FlowJo software (Tree Star, Ashland, OR, USA). For cytokine staining, cells were stimulated for 4 hrs at 37°C with 50ng/ml of phorbol 12- myristate 13-acetate (PMA; Calbiochem), 1μg/ml of ionomycin (Sigma), and BD Golgi STOP TM (BD Biosciences). Anti-CD45.2 (104), anti-FoxP3 (FJK-16s), anti-ICOS (7E17G9), anti-IFN-Ȗ (XMG1.2), anti-TNF-Į (MP6-XT22), anti-CXCR3 (CXCR3-173) and isotype controls rat IgG1 (eBRG1), IgG2a (eBRG2a), IgG2b (eBRG2b) were purchased from eBioscience. Anti-CD3 (145-2C11), anti-CD25 (PC61.5.3), KI67 (FITC mouse anti-human KI67 set), rat IgG1ț were obtained from BD Bioscience. Anti-CD4 (GK1.5), anti-CD8ȕ (YTS1567.7), Rat IgG2a (RTK2758) were purchased from Biolegend (San Diego, CA, USA). Anti-CCR6 (140706) was obtained from R&D Systems, Minneapolis, MN. Eight-color flow cytometry analysis was performed with antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, phycoerythrin cyanin 7, peridinin chlorophyll protein cyanin 5.5, allophycocyanin cyanin 7, pacific blue, or allophycocyanin. All cells were analyzed on a FACS CANTO II (BD) flow cytometer with FlowJo (Tree Star) software. Microbial DNA extraction, 454 pyrosequencing and bacteria identification. Fecal samples used in this study were collected before or after one injection of anti-CTLA4 (or isotype control) from mice under vancomycin regimen or water, and were kept at −80°C until further analysis. Library preparation and sequencing were conducted at GATC Biotech AG (Konstanz, Germany). Bacterial isolation, culture, and identification. Fecal pellet contents were harvested and resuspended in BHI+15% glycerol at 0.1g/ml. Serial dilutions of feces were plated onto sheep’s blood agar plates and incubated for 48h at 37°C with 5% CO2 in aerobic or anaerobic conditions. After 48h, single colonies were isolated and Gram staining was performed. The identification of specific bacteria was accomplished through the combination of morphological tests and analysis by means of an Andromas MALDI-TOF mass spectrometer (Andromas, France). Gut colonization with dedicated bacterial species. For inoculation of GF mice or mice treated with broad-spectrum antibiotics, colonization was performed the day following the first anti￾CTLA4 injection by oral gavage with 100μl of suspension containing 1 × 109 bacteria. Efficient