Cells were then washed with PBs and incubated in complete Iscove's medium supplemented with gentamicin(50ug/ml)to kill extracellular bacteria. For Burkholderia cepacia, the antibiotic meropenem(2ug/mL, Astra Zeneca) was required. After 24h, BM-DCs were cultured together with CD4 T cells purified from the spleens of mice that had received 7 days treatment of anti CTLA4 mAb(i.e 3 anti-CTLA4 mAb injections, as detailed above)in complete RPMI medium CD4 T cells were isolated by depletion of non CD4 T cells using a cocktail of biotin- conjugated antibodies against CD8o, CDllb, CDllc, CD19, CD45R(B220), CD49b(DX5), CD105, MHC-class Il, Ter-119 and TCRyo as primary labeling reagent. The cells were then magnetically labeled with anti-biotin MicroBeads(Miltenyi Biotec, France). The magnetically labeled non-target cells were depleted by retaining them on an Automacs Separator, while the unlabeled target cells passed through the column. Co-cultures were set up at a ratio of 1 dC to 2 CD4 T cells. We confirmed by flow cytometry that over 95% of magnetic-sorted cells were CD4 T cells Co-culture supernatants were collected at 24h, and assayed for IL-10 and IFN-O using commercial ELISA. Recall of human CD T cell responses directed against commensals. Frozen PBMc before and/or after ipilimumab therapy were thawed, washed and suspended in the recommended separation medium(PBS, ImM EDTA, 2% human AB serum (Jacques Boy): STEMCELL Technologies) for magnetic bead separation. Monocytes were enriched from 2 x 10 PBMC(EasySepTM Human Monocyte Enrichment Kit, STEMCELL Technologies)and resuspended in RPMI-1640(GIBCO Invitrogen), 10% human AB'serum supplemented with 1%2 mmol/L glutamine(GIBCO Invitrogen), and GM-CSF (1000UI/mL Miltenyi), without any antibiotics. Monocytes were seeded in 96-well round bottom plates at 5 x 10 cells/well either alone, in the presence of one of the five selected bacterial strains(Table 2)at a multiplicity of infection(MOD)of 100, or with LPS as positive control(lug/mL, Sigma) plus SCD40L (lug/mL, Miltenyi) and incubated for 1 hour at 37C, 5%CO2. During incubation, the remaining autologous PBMC fractions were enriched for memory CD4 T cells(Easy SepTM Human Memory CD4 Enrichment Kit, STEMCELL Technologies). The enriche CD4 CD45RO T cells were washed, counted and resuspended at 5 x 10/well in RPMI-1640 10% human AB serum, 1% 2 mmol/L glutamine, 20UI/mL IL-2(Proleukine)either with 1% penicillin/streptomycin(PEST; GIBCO Invitrogen) and 50ug/ml of gentamycin (GIBCO Invitrogen) or with meropenem antibiotic for Burkholderia cepacia(2ug/mL, Astra Zeneca) CD4CD45ROT cells were also incubated alone or with CD3/CD28 beads (1uL/mL13 Cells were then washed with PBS and incubated in complete Iscove’s medium supplemented with gentamicin (50μg/ml) to kill extracellular bacteria. For Burkholderia cepacia, the antibiotic meropenem (2ȝg/mL, Astra Zeneca) was required. After 24h, BM-DCs were cultured together with CD4+ T cells purified from the spleens of mice that had received 7 days treatment of anti￾CTLA4 mAb (i.e 3 anti-CTLA4 mAb injections, as detailed above) in complete RPMI medium. CD4+ T cells were isolated by depletion of non CD4+ T cells using a cocktail of biotin￾conjugated antibodies against CD8α, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC-class II, Ter-119 and TCRȖį as primary labeling reagent. The cells were then magnetically labeled with anti-biotin MicroBeads (Miltenyi Biotec, France). The magnetically labeled non-target cells were depleted by retaining them on an Automacs Separator, while the unlabeled target cells passed through the column. Co-cultures were set up at a ratio of 1 DC to 2 CD4+ T cells. We confirmed by flow cytometry that over 95% of magnetic-sorted cells were CD4+ T cells. Co-culture supernatants were collected at 24h, and assayed for IL-10 and IFN- using commercial ELISA. Recall of human CD4+ T cell responses directed against commensals. Frozen PBMC before and/or after ipilimumab therapy were thawed, washed and resuspended in the recommended separation medium (PBS, 1mM EDTA, 2% human AB+ serum (Jacques Boy); STEMCELL Technologies) for magnetic bead separation. Monocytes were enriched from 2 × 106 PBMC (EasySep™ Human Monocyte Enrichment Kit, STEMCELL Technologies) and resuspended in RPMI-1640 (GIBCO Invitrogen), 10% human AB+ serum supplemented with 1% 2 mmol/L glutamine (GIBCO Invitrogen), and GM-CSF (1000UI/mL, Miltenyi), without any antibiotics. Monocytes were seeded in 96-well round bottom plates at 5 × 103 cells/well either alone, in the presence of one of the five selected bacterial strains (Table 2) at a multiplicity of infection (MOI) of 100, or with LPS as positive control (1ȝg/mL, Sigma) plus sCD40L (1ȝg/mL, Miltenyi) and incubated for 1 hour at 37°C, 5% CO2. During incubation, the remaining autologous PBMC fractions were enriched for memory CD4+ T cells (EasySep™ Human Memory CD4 Enrichment Kit, STEMCELL Technologies). The enriched CD4+ CD45RO+ T cells were washed, counted and resuspended at 5 × 104 /well in RPMI-1640, 10% human AB+ serum, 1% 2 mmol/L glutamine, 20UI/mL IL-2 (Proleukine) either with 1% penicillin/streptomycin (PEST; GIBCO Invitrogen) and 50μg/ml of gentamycin (GIBCO Invitrogen) or with meropenen antibiotic for Burkholderia cepacia (2ȝg/mL, Astra Zeneca). CD4+ CD45RO+ T cells were also incubated alone, or with CD3/CD28 beads (1ȝL/mL