permeabilized and stained with lx Perm buffer(eBioscience)and anti-Foxp3-PE(eBioscience) anti-GFP-Alexa488 (polyclonal, Molecular Probes) and CTLA-4-APC (UC10-4B9 eBioscience). Nonspecific binding was blocked with purified CD16/32(93, eBioscience) and rat IgG(Dianova). Flow cytometry was performed on a Fortessa(BD)and data was analysed with FlowJo(TreeStar). Analyses of Dc subsets in anti-CTLA4 antibody-treated intestines Mesenteric lymph nodes were prepared by digestion with collagenase and DNase for 60 min and subsequently strained through a 70 um mesh Colonic lymphocytes were isolated as previously described(24). In brief, colons were digested in PBS containing 5 mM EDTA and 2 mM DTT with shaking at 37C. After initial digestion, colonic tissue pieces were digested in collagenase/DNAse containing RPMI medium for 30 min. Tissue pieces were further strained through a 70 um mesh. For flow cytometry analyses, cell suspensions were stained with antibodies against the following surface markers: CDllc(N4 18), CDllb(M1/70), MHC class II (M5/114152),CD24(M1/69)CD317(ebio927),CD45(30-F11),CD86(GL1),CD40(1C10) DAPI was used for dead cell exclusion. Antibodies were purchased from bIosciences, BD Biosciences or BioLegend respectively. Cell populations were gated as follows: CD103 DC (CD45 CDllc MHC-Il CD103 CD24), CDllb DC (CD45 CDllc MHC-I CD11b CD24), plasmacytoid DC (CD45* CDIIc' MHC-II" CD317), mesenteric LNs(migratory fraction): CD103+ DC(CD45 CDllc MHC-Il CD103 CD24),CD11b CD103 DC (CD45 CDIIc MHC-Il CD103 CDIIb CD24), CDIIb DC(CD45 CDIIc MHC-ll CDIIb CD24), plasmacytoid DC(CD45* CDllc* MHC-II CD317), mesenteric LNs (resident fraction ) CD8a DC( CD45 CDI lc MHC-IT CD24 CD1 1b), CDllb DC(CD45 CDllc MHC-II CD11b) Preparation of capsular polysaccharide-enriched fractions. Fractions containing capsular polysaccharides were prepared as previously described(25). Briefly, bacteria were extracted twice by a hot 75% phenol/water mixture for Ih at 80oC. After centrifugation at 1000 g for 20 mIn. phases were pooled and extracted with an equivalent volume of ether. Water phases were extensively dialyzed against water and lyophilized. Extracted compounds were subsequently submitted to digestion by DNAase, RNAase, a-chymotrypsin, Streptomyces griseus proteases and trypsin. The digested solutions were dialysed against water and dried, resulting in fractions enriched in capsular polysaccharides. Quantification of LPS content. The11 permeabilized and stained with 1x Perm buffer (eBioscience) and anti-Foxp3-PE (eBioscience), anti-GFP-Alexa488 (polyclonal, Molecular Probes) and CTLA-4-APC (UC10-4B9, eBioscience). Nonspecific binding was blocked with purified CD16/32 (93, eBioscience) and rat IgG (Dianova). Flow cytometry was performed on a Fortessa (BD) and data was analysed with FlowJo (TreeStar). Analyses of DC subsets in anti-CTLA4 antibody-treated intestines. Mesenteric lymph nodes were prepared by digestion with collagenase and DNase for 60 min and subsequently strained through a 70 μm mesh. Colonic lymphocytes were isolated as previously described (24). In brief, colons were digested in PBS containing 5 mM EDTA and 2 mM DTT, with shaking at 37°C. After initial digestion, colonic tissue pieces were digested in collagenase/DNAse containing RPMI medium for 30 min. Tissue pieces were further strained through a 70 ȝm mesh. For flow cytometry analyses, cell suspensions were stained with antibodies against the following surface markers: CD11c (N418), CD11b (M1/70), MHC class II (M5/114.15.2), CD24 (M1/69), CD317 (ebio927), CD45 (30-F11), CD86 (GL1), CD40 (1C10). DAPI was used for dead cell exclusion. Antibodies were purchased from eBiosciences, BD Biosciences or BioLegend respectively. Cell populations were gated as follows: CD103+ DC (CD45+ CD11c+ MHC-II+ CD103+ CD24+ ), CD11b+ DC (CD45+ CD11c+ MHC-II+ CD11b+ CD24+ ), plasmacytoid DC (CD45+ CD11c+ MHC-II+ CD317+ ), mesenteric LNs (migratory fraction): CD103+ DC (CD45+ CD11c+ MHC-II++ CD103+ CD24+ ), CD11b+ CD103+ DC (CD45+ CD11c+ MHC-II++ CD103+ CD11b+ CD24+ ), CD11b+ DC (CD45+ CD11c+ MHC-II++ CD11b+ CD24+ ), plasmacytoid DC (CD45+ CD11c+ MHC-II+ CD317+ ), mesenteric LNs (resident fraction): CD8α+ DC (CD45+ CD11c+ MHC-II+ CD24+ CD11b- ), CD11b+ DC (CD45+ CD11c+ MHC-II+ CD11b+ ). Preparation of capsular polysaccharide-enriched fractions. Fractions containing capsular polysaccharides were prepared as previously described (͸ͻ). Briefly, bacteria were extracted twice by a hot 75% phenol/water mixture for 1h at 80°C. After centrifugation at 1000 g for 20 min, water phases were pooled and extracted with an equivalent volume of ether. Water phases were then extensively dialyzed against water and lyophilized. Extracted compounds were subsequently submitted to digestion by DNAase, RNAase, ȽǦchymotrypsin, Streptomyces griseus proteases and trypsin. The digested solutions were dialysed against water and dried, resulting in fractions enriched in capsular polysaccharides. Quantification of LPS content. The