colonization was checked by culture of feces 48h post oral gavage. Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides umiformis, Lactobacillus plantarum and Enterococcus hirae were grown on blood agar plates(Biomerieux) for 48h at 37C in anaerobic conditions. E. coli and Burkholderia cepacia were grown on blood agar plates for 24h at 37C in aerobic conditions. Bacteria were harvested from the agar plates, suspended in sterile PBS, centrifuged and washed once with PBS, then resuspended in sterile PBS at an optical density(600nm) of 1, which corresponds approximately to 1 x 10 colony-forming units (CFU)ml In cases where more than one bacteria was administered, an equal volume of each bacteria suspension was mixed to give a suspension of equal proportion of each type of bacteria to a total 1 x 10 bacteria/ml. For bacteria reconstitution experiments using mice previously treated with antibiotics, antibiotics treatment was stopped after 2-3 weeks at the first anti-CTLA4 injection, and mice were orally gavaged with 1 x 10 CFU the following day. B. distasonis, B niformis, E. hirae, and E coli isolates used in the experiments were originally isolated from feces or mesenteric lymph nodes of SPF mice treated with anti-CTLA4 and identified as described above. L. plantarum, B. fragilis and B. thetaiotaomicron were provided by the Biobank of the Institut Pasteur, Paris, France. Burkholderia cepacia was kindly provided by the IUH Mediterranee Infection, Marseille, France( Supplemental Table 2). LPS-EK and LTA-SA (Invivogen)were administrated by oral gavage, at a dose of 500ug per mouse TCR cross-linking assays. For cross-linking experiments, total cells isolated from draining or contralateral lymph nodes(after red blood cell lysis)were incubated in MaxiSorp plates(Nunc; 2 x 10 cells per well), precoated with anti-CD3 mAb(145-2Cll)(0.5 g per well; eBioscience The supernatants were assayed at 48h by ELISa for mouse IFN-Y(BD) Cytokine and antimicrobial peptide quantification. IL-12p70, IFN-Y(BD Biosciences)and IL-10(R&D Systems, Minneapolis, MN), were measured by ELisa following the manufacturers instructions. For quantification of lipocalin-2 from feces and caecum contents individual(not pooled) samples were reconstituted in PBS containing 0. 1% Tween 20(at 100 g/ml) and vortexed .20 min to get a homogenous fecal suspension. These samples were then centrifuged for 10 min at 10,000 rpm. Clear supernatants were collected and stored at -20C7 colonization was checked by culture of feces 48h post oral gavage. Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides uniformis, Lactobacillus plantarum and Enterococcus hirae were grown on blood agar plates (Biomerieux) for 48h at 37°C in anaerobic conditions. E. coli and Burkholderia cepacia were grown on blood agar plates for 24h at 37°C in aerobic conditions. Bacteria were harvested from the agar plates, suspended in sterile PBS, centrifuged and washed once with PBS, then resuspended in sterile PBS at an optical density (600nm) of 1, which corresponds approximately to 1 × 109 colony-forming units (CFU)/ml. In cases where more than one bacteria was administered, an equal volume of each bacteria suspension was mixed to give a suspension of equal proportion of each type of bacteria, to a total 1 × 109 bacteria/ml. For bacteria reconstitution experiments using mice previously treated with antibiotics, antibiotics treatment was stopped after 2-3 weeks at the first anti-CTLA4 injection, and mice were orally gavaged with 1 × 109 CFU the following day. B. distasonis, B. uniformis, E. hirae, and E.coli isolates used in the experiments were originally isolated from feces or mesenteric lymph nodes of SPF mice treated with anti-CTLA4 and identified as described above. L. plantarum, B. fragilis and B. thetaiotaomicron were provided by the Biobank of the Institut Pasteur, Paris, France. Burkholderia cepacia was kindly provided by the IUH Méditerranée Infection, Marseille, France (Supplemental Table 2). LPS-EK and LTA-SA (Invivogen) were administrated by oral gavage, at a dose of 500μg per mouse. TCR cross-linking assays. For cross-linking experiments, total cells isolated from draining or contralateral lymph nodes (after red blood cell lysis) were incubated in MaxiSorp plates (Nunc; 2 × 105 cells per well), precoated with anti-CD3 mAb (145-2C11) (0.5 g per well; eBioscience). The supernatants were assayed at 48h by ELISA for mouse IFN-γ (BD). Cytokine and antimicrobial peptide quantification. IL-12p70, IFN-γ (BD Biosciences) and IL-10 (R&D Systems, Minneapolis, MN), were measured by ELISA following the manufacturer’s instructions. For quantification of lipocalin-2 from feces and caecum contents, individual (not pooled) samples were reconstituted in PBS containing 0.1% Tween 20 (at 100 mg/ml) and vortexed for 10-20 min to get a homogenous fecal suspension. These samples were then centrifuged for 10 min at 10,000 rpm. Clear supernatants were collected and stored at -20°C