Deanabeads T-Activator, InVitrogen) as a positive control. Monocyte-bacteria/ CD4 CD45RO T cell co-cultures were incubated for 48 hrs at 37 C, 5%CO2 Monocytes and memory CD4 CD45RO T cell enrichment were confirmed as being >98% purity by flow cytometry. Supernatants were harvested, cleared by centrifugation(1200rpm, 5 min) and stored at-20C for determination of IFN-y or IL-10, as measured by commercial ELISA or Luminex MagPix technology(Biorad) Adoptive cell transfer. CD4 T cells were isolated from co-culture with DCs that had been pulsed with bacteria at a MOI of 10, as described above. 1 x 10" CD4 T cells were adoptively transferred iv into GF or ACS treated mice, one day after the first anti-CTLA4 mAb injection Mice were injected with anti-CTLA4 mAb and tumor sizes were monitored as detailed previously. After 24h of coculture with bacteria as described above, BM-DCs were removed from low binding plates, washed, and counted. Alternatively to bacteria infection, BM-DC wer pulsed with peptides(KDho synthesized by Smartox Biotechnology(Gillonay, France) or with capsular polysaccharide-enriched fractions(final concentration of 10ug/ml). One million DC were adoptively transferred i v into antibiotics -treated mice one day after the first anti-CTLA4 mAb injection. Mice were administered with anti-CTLA4 mAb and tumor sizes were monitored as detailed above Small intestine crypt isolation and organoid culture. Crypt isolation and organoid culture was performed as previously described (29)with the following modifications. Briefly, the ileum of 10-13 week old mice were cut longitudinally and scraped with a cover slip to remove villi. The intestine was cut transversely into 2-4 mm pieces and washed 4 times with cold PBS. Fragments were then incubated in 2 mM EDTA in PBs for 30 minutes on ice. Following the removal of EDTA medium, fragments were vigorously resuspended in PBS containing 10% FCS(Gibco) and passed through a 70 uM strainer(BD Bioscience). This step was repeated 3 times. Isolated were pelleted and washed in Advanced DMEM/F12(ADF)(Invitrogen). Crypts were then resuspended in ImL of Matrigel growth factor reduced basement membrane matrix( Corning) uL drops were placed into pre-warmed 24-well plates. Following Matrigel polymerisation, crypts were overlayed with ADF supplemented with 100 U/mL penicillin G sodium, 100 ug/mL streptomycin sulphate, 2 mM L-glutamine, 10 mM HEPES, Ix N214 Deanabeads T-Activator, InVitrogen) as a positive control. Monocyte-bacteria/ CD4+ CD45RO+ T cell co-cultures were incubated for 48 hrs at 37°C, 5% CO2. Monocytes and memory CD4+ CD45RO+ T cell enrichment were confirmed as being >98% purity by flow cytometry. Supernatants were harvested, cleared by centrifugation (1200rpm, 5 min) and stored at -20°C for determination of IFN-Ȗ or IL-10, as measured by commercial ELISA or Luminex MagPix technology (Biorad). Adoptive cell transfer. CD4+ T cells were isolated from co-culture with DCs that had been pulsed with bacteria at a MOI of 10, as described above. 1 × 106 CD4+ T cells were adoptively transferred i.v into GF or ACS treated mice, one day after the first anti-CTLA4 mAb injection. Mice were injected with anti-CTLA4 mAb and tumor sizes were monitored as detailed previously. After 24h of coculture with bacteria as described above, BM-DCs were removed from low binding plates, washed, and counted. Alternatively to bacteria infection, BM-DC were pulsed with peptides (KD)20 synthesized by Smartox Biotechnology (Gillonay, France) or with capsular polysaccharide-enriched fractions (final concentration of 10μg/ml). One million DC were adoptively transferred i.v into antibiotics -treated mice one day after the first anti-CTLA4 mAb injection. Mice were administered with anti-CTLA4 mAb and tumor sizes were monitored as detailed above. Small intestine crypt isolation and organoid culture. Crypt isolation and organoid culture was performed as previously described (29) with the following modifications. Briefly, the ileum of 10-13 week old mice were cut longitudinally and scraped with a cover slip to remove villi. The intestine was cut transversely into 2-4 mm pieces and washed 4 times with cold PBS. Fragments were then incubated in 2 mM EDTA in PBS for 30 minutes on ice. Following the removal of EDTA medium, fragments were vigorously resuspended in PBS containing 10% FCS (Gibco) and passed through a 70 μM strainer (BD Bioscience). This step was repeated 3 times. Isolated crypts were pelleted and washed in Advanced DMEM/F12 (ADF) (Invitrogen). Crypts were then resuspended in 1mL of Matrigel growth factor reduced basement membrane matrix (Corning) and 50 μL drops were placed into pre-warmed 24-well plates. Following Matrigel polymerisation, crypts were overlayed with ADF supplemented with 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulphate, 2 mM L-glutamine, 10 mM HEPES, 1x N2