until analysis. Lipocalin-2 levels were determined in the supernatants using the duoset murine Lcn-2 ELISA kit(R&D Systems, Minneapolis, MN) following the manufacturers instructions FITC Dextran Assay to assess intestinal permeability. mice were injected with either one dose or two doses of anti-CTLA4 or isotype control mAbs(administration as detailed above) and were water-starved overnight, 2 days following their last i.p. mAb administration. The following day, mice were orally administered with 0.44 mgg body weight of a 100 mg/ml solution of FITC-dextran(FD4, Sigma)in PBS (pH 7.4). Four hrs later, blood was collected from each mouse by cardiac puncture. Blood was allowed to clot overnight at 4C, then subsequently centrifuged at 3,000 rpm for 20 minutes to collect the serum. Dilutions of FITC dextran in PBS, and separately in pooled mouse serum were used as a standard curve, with serum from mice not administered FITC-dextran used to determine the background. absorbance of 100ul serum(diluted in PBS) was measured by microplate reader with excitation and emission filters set at 485 nm(20 nm band width) and 528 nm(20 nm band width), respectively(19) Experiments were performed at least twice, independently, with each read performed in duplicate Histology of gut tissue. The whole small intestine(duodenum, jejunum and ileum) and the colon were removed, cleaned from feces and fixed in 4% PFA for 2h. Rehydratation of the tissue was performed in 15% sucrose for Ih and in 30% sucrose overnight. Small intestines or colons were cut longitudinally, with the resulting ribbons rolled, then embedded in optimum cutting temperature(OCT) compound(Sakura), snap frozen, and longitudinal 6 um sections were prepared. For histological analysis, longitudinal sections were counterstained with hematoxilin and eosin. For histological quantitative analysis, inflammatory foci, appearance of the submucosa, length of villi, and the thickness of lamina propria were scored for each section by a pathologist. Score distribution between the groups were compared by proportional odds logistic regression using r software Intestinal MUC2 and Ki67 staining and evaluation. Intestinal tissue was fixed in freshly prepared Methacarn solution, subsequently incubated in methanol and toluene and embedded in paraffin For immunohistochemistry, 5 um-thick tissue sections were placed on Superfrost Plus
8 until analysis. Lipocalin-2 levels were determined in the supernatants using the Duoset murine Lcn-2 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. FITC Dextran Assay to assess intestinal permeability. Mice were injected with either one dose or two doses of anti-CTLA4 or isotype control mAbs (administration as detailed above), and were water-starved overnight, 2 days following their last i.p. mAb administration. The following day, mice were orally administered with 0.44 mg/g body weight of a 100 mg/ml solution of FITC-dextran (FD4, Sigma) in PBS (pH 7.4). Four hrs later, blood was collected from each mouse by cardiac puncture. Blood was allowed to clot overnight at 4°C, then subsequently centrifuged at 3,000 rpm for 20 minutes to collect the serum. Dilutions of FITCdextran in PBS, and separately in pooled mouse serum were used as a standard curve, with serum from mice not administered FITC-dextran used to determine the background. Absorbance of 100ȝl serum (diluted in PBS) was measured by microplate reader with excitation and emission filters set at 485 nm (20 nm band width) and 528 nm (20 nm band width), respectively (ͷͿ). Experiments were performed at least twice, independently, with each read performed in duplicate. Histology of gut tissue. The whole small intestine (duodenum, jejunum and ileum) and the colon were removed, cleaned from feces and fixed in 4% PFA for 2h. Rehydratation of the tissue was performed in 15% sucrose for 1h and in 30% sucrose overnight. Small intestines or colons were cut longitudinally, with the resulting ribbons rolled, then embedded in optimum cutting temperature (OCT) compound (Sakura), snap frozen, and longitudinal 6 μm sections were prepared. For histological analysis, longitudinal sections were counterstained with hematoxilin and eosin. For histological quantitative analysis, inflammatory foci, appearance of the submucosa, length of villi, and the thickness of lamina propria were scored for each section by a pathologist. Score distribution between the groups were compared by proportional odds logistic regression using R software. Intestinal MUC2 and Ki67 staining and evaluation. Intestinal tissue was fixed in freshly prepared Methacarn solution, subsequently incubated in methanol and toluene and embedded in paraffin. For immunohistochemistry, 5 μm-thick tissue sections were placed on Superfrost Plus
slides(Thermo Scientific), incubated for 10 min at 60%C and rehydrated through a series of graded alcohols and distilled water. Endogenous peroxidases were blocked by 3% hydrogen peroxide for 10 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6) by steaming sections in a microwave oven for 20 min. Tissue sections were blocked with 5% BSA/PBS for 30 min at RT and primary antibodies against MUC2(1: 200, sc15334, Santa Cruz Biotechnology) Ki67(1: 100, ab15580, Abcam) and Cleaved Caspase 3(1: 50, #9661, Cell Signaling Technology), were directly applied and incubated for I h at RT or overnight at 4C, espectively. Slides were washed 3x in PBs and secondary antibody (1: 200, UP511380 nterchim Optima) was applied for I hour at RT. Targeted antigens were visualized by using 3.3-diaminobenzidine solution (bd Pharmingen) followed by nuclear counterstain with hematoxylin. For immunofluorescence staining, staining procedure was done according to the protocol provided by Cell Signaling Technology. Primary antibody against MUC2 was used as indicated. Secondary antibody(1: 200, Al1008, Life Technologies) was applied for 1 hr at RT followed by nuclear counterstain with DAPI. Microscopic analyses were performed by using the Zeiss Axioplan 2 imaging microscope, Axio Imager ZI microscope, Axiovision software(all from Zeiss, Oberkochen, Germany), and ImageJ software(20). Evaluation of MUC2-and Ki67- positive signals was performed by counting MUC2-and Ki-67-positive epithelial cells in all intact villi and/or crypts per tissue sections. Caspase 3 cleavage was assessed by calculating ositively stained epithelial cells per mm mucousal area. Thickness of pre-epithelial mucus lyer in distal colon was obtained by calculating mean values of 10 distinct measurement points per tissue section Fluorescent in situ hybridization. Methacarn-fixed, paraffin-embedded colonic tissue sections (7 um) were deparaffinized in toluene, washed in 95 ethanol, and air-dried at RT, Tissue sections were incubated overnight(45C)in hybridization buffer(20 mM Tris-HCl, 0.9 M NaC 0. 1 SDS, pH 7.4) containing Cy3-labeled bacterial probes EUB338 (5 GCTGCCTCCCGTAGGAGT-3) and Bfra602 (5-GAGCCGCAAACTTTCACAA- espectively, in a concentration of 5 ng/ul(21-23). Non-specific binding of probes was removed by subsequent incubation of slides in pre-warmed hybridization buffer and washing buffer(20 mM Tris-HCl, 0.9 M NaCl, pH 7.4), both for 15 min at 37C. DAPI was used for nuclear counterstain and air-dried tissue sections were covered by using ProLongR Gold Antifade
9 slides (Thermo Scientific), incubated for 10 min at 60° C and rehydrated through a series of graded alcohols and distilled water. Endogenous peroxidases were blocked by 3% hydrogen peroxide for 10 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6) by steaming sections in a microwave oven for 20 min. Tissue sections were blocked with 5% BSA/PBS for 30 min at RT and primary antibodies against MUC2 (1:200, sc15334, Santa Cruz Biotechnology) Ki67 (1:100, ab15580, Abcam) and Cleaved Caspase 3 (1:50, #9661, Cell Signaling Technology), were directly applied and incubated for 1 h at RT or overnight at 4°C, respectively. Slides were washed 3x in PBS and secondary antibody (1:200, UP511380, Interchim Uptima) was applied for 1 hour at RT. Targeted antigens were visualized by using 3.3’-diaminobenzidine solution (BD Pharmingen) followed by nuclear counterstain with hematoxylin. For immunofluorescence staining, staining procedure was done according to the protocol provided by Cell Signaling Technology. Primary antibody against MUC2 was used as indicated. Secondary antibody (1:200, A11008, Life Technologies) was applied for 1 hr at RT followed by nuclear counterstain with DAPI. Microscopic analyses were performed by using the Zeiss Axioplan 2 imaging microscope, Axio Imager Z1 microscope, Axiovision software (all from Zeiss, Oberkochen, Germany), and ImageJ software (20). Evaluation of MUC2- and Ki67- positive signals was performed by counting MUC2- and Ki-67-positive epithelial cells in all intact villi and/or crypts per tissue sections. Caspase 3 cleavage was assessed by calculating positively stained epithelial cells per mm2 mucousal area. Thickness of pre-epithelial mucus layer in distal colon was obtained by calculating mean values of 10 distinct measurement points per tissue section. Fluorescent in situ hybridization. Methacarn-fixed, paraffin-embedded colonic tissue sections (7 ȝm) were deparaffinized in toluene, washed in 95 % ethanol, and air-dried at RT. Tissue sections were incubated overnight (45°C) in hybridization buffer (20 mM Tris-HCl, 0.9 M NaCl, 0.1 % SDS, pH 7.4) containing Cy3-labeled bacterial probes EUB338 (5’- GCTGCCTCCCGTAGGAGT-3’) and Bfra602 (5’-GAGCCGCAAACTTTCACAA-3’), respectively, in a concentration of 5 ng/ȝl (21-23). Non-specific binding of probes was removed by subsequent incubation of slides in pre-warmed hybridization buffer and washing buffer (20 mM Tris-HCl, 0.9 M NaCl, pH 7.4), both for 15 min at 37°C. DAPI was used for nuclear counterstain and air-dried tissue sections were covered by using ProLongR Gold Antifade
reagent (Life Technologies). For combined mucus staining, anti-MUC2 antibody (1: 100 15334, Santa Cruz Biotechnology) was applied for 15 min at RT, after a first wash step in hybridization buffer. Secondary antibody incubation(1: 200, Al1008, Life Technologies including DAPI counterstain was performed for 15 min at RT. Tissue sections were washed in washing buffer for 5 min at RT and covered with antifade mounting medium. Microscopic analysis was performed by using the Axio Imager ZI microscope and Axiovision software(all Zeiss) Flow cytometry analyses of LP cell subsets. Isolation of lamina propria cells from colon. The whole colon was harvested and Peyers patches were removed, as well as all fat residues and feces. Colons were cut longitudinally and then transversally into pieces cm length. After removing the intra-epithelial lymphocytes (IELs), the colon pieces further cut into approximately Imm squares, and incubated with 0.25 mg/ml collagenase VIll and 10 U/ml DNase I for 40 min at 37C with shaking, in order to isolate lamina propria cells(LPCs). After digestion, intestinal pieces were passaged through a 100um cell strainer. For flow cytometry analysis, cell suspensions were subjected to a percoll gradient for 20 min, centrifuged at 2100 RPMI. Anti-mouse antibodies for CD45.2(104), CD3(145-2CI1), CD4(GK1.5), IFN (XMG1. 2), RORyt(AFKJS-9), anti-ICOS(7E17G9)were obtained from BioLegend, eBioscience and R&D. CTLA4 staining on lamina propria cell subsets. Large intestines longitudinally, washed from feces and incubated on ice in PBS/EDTA(25 without Ca /Mg, gibco). Epithelial cells were removed by repeated rounds of shaking in PBS Subsequently, the intestine was cut into small pieces and digested with Liberase TL(Roche)/ DNasel( Sigma) mix in DMEM(Gibco) at 37 C Tissue was disrupted by pipetting and passing over a 100 um mesh(BD). Homogenates were subjected to a 40/80 Percoll (GE Healthcare) gradient. Lymphocytes were harvested from the interphase and stained with fixable Live/Dead stain Blue(Invitrogen) according to the manufacturers instruction. Surface staining was performed with anti-CD3-PE-CF594(145-2Cll, BD), anti-CD4-Horizon V500(RM4-5, BD), anti-CD19-Brilliant Violet650 (6D5, BioLegend), anti-Thy 1. 2-PerCp-eFluor710 (30-H12 eBioscience), anti-NKp46-biotin (polycle (BioLegend) and anti-CTLA-4-APC (UC10-4B9, eBioscience) or Armenian Hamster IgG type Control APC(eBio299Arm, eBioscience) Cells were fixed with 4% PFA(Sigma)and
10 reagent (Life Technologies). For combined mucus staining, anti-MUC2 antibody (1:100, sc15334, Santa Cruz Biotechnology) was applied for 15 min at RT, after a first wash step in hybridization buffer. Secondary antibody incubation (1:200, A11008, Life Technologies) including DAPI counterstain was performed for 15 min at RT. Tissue sections were washed in washing buffer for 5 min at RT and covered with antifade mounting medium. Microscopic analysis was performed by using the Axio Imager Z1 microscope and Axiovision software (all Zeiss). Flow cytometry analyses of LP cell subsets. Isolation of lamina propria cells from colon. The whole colon was harvested and Peyer’s patches were removed, as well as all fat residues and feces. Colons were cut longitudinally and then transversally into pieces of 1-2 cm length. After removing the intra-epithelial lymphocytes (IELs), the colon pieces were further cut into approximately 1mm squares, and incubated with 0.25 mg/ml collagenase VIII and 10 U/ml DNase I for 40 min at 37 °C with shaking, in order to isolate lamina propria cells (LPCs). After digestion, intestinal pieces were passaged through a 100μm cell strainer. For flow cytometry analysis, cell suspensions were subjected to a percoll gradient for 20 min, centrifuged at 2100 RPMI. Anti-mouse antibodies for CD45.2 (104), CD3 (145-2C11), CD4 (GK1.5), Ǧγ (XMG1.2), γ (AFKJS-9), anti-ICOS (7E17G9) were obtained from BioLegend, eBioscience and R&D. CTLA4 staining on lamina propria cell subsets. Large intestines were opened longitudinally, washed from feces and incubated on ice in PBS/EDTA (25 mM, without Ca2+/Mg2+, Gibco). Epithelial cells were removed by repeated rounds of shaking in PBS. Subsequently, the intestine was cut into small pieces and digested with Liberase TL (Roche) / DNaseI (Sigma) mix in DMEM (Gibco) at 37o C. Tissue was disrupted by pipetting and passing over a 100 μm mesh (BD). Homogenates were subjected to a 40/80 % Percoll (GE Healthcare) gradient. Lymphocytes were harvested from the interphase and stained with fixable Live/Dead stain Blue (Invitrogen) according to the manufacturer’s instruction. Surface staining was performed with anti-CD3-PE-CF594 (145-2C11, BD), anti-CD4-HorizonV500 (RM4-5, BD), anti-CD19-BrilliantViolet650 (6D5, BioLegend), anti-Thy1.2-PerCp-eFluor710 (30-H12, eBioscience), anti-NKp46-biotin (polyclonal, R&D), Streptavidin-BrilliantViolet421 (BioLegend) and anti-CTLA-4-APC (UC10-4B9, eBioscience) or Armenian Hamster IgG Isotype Control APC (eBio299Arm, eBioscience). Cells were fixed with 4% PFA (Sigma) and
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. The
11 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
presence of LPS in capsular polysaccharide-enriched fractions was investigated using the HEK BlueTM TLR4 cell line(Invivogen, Toulouse, a derivative of HEK293 cells that stably expresses the human TLR4, MD2 and CD14 genes along with a NF-KB-inducible reporter system(secreted alkaline phosphatase). Cells were used according to the manufacturer's instructions. The different capsular polysaccharide-enriched fractions were added at concentrations ranging from I ng to 10 ug/ml in 96-wells plates and cells were then distributed at 5x 10 per well in 200 ul DMEM culture medium. Alkaline phosphatase activity in the culture supernatant was measured after 18h. LPS content was determined by TLR4 signaling activation by comparison with a standard curve obtained using ultrapure LPS from E coli K12 (Invivogen, Toulouse). The LPS content in the PS B. fragilis fraction was estimated at 1. 4 pg/ ug of Ps. The LPS residual content in the PS B fragilis fraction could not account for its activity, as confirmed by the lack of bioactivity observed by adding LPS to the polysaccharide capsula fractions of B. distasonis compared to its effect in the absence of LPS Monosaccharide analysis of the capsular polysaccharide-enriched fractions from B fragilis and B. distasonis Monosaccharides were analyzed after total hydrolysis of 10 ug of capsular polysaccharide-enriched fraction by 2N TFA at 110C for 2 hrs. They were detected by capillary electrophoresis monitored by laser-induced fluorescence(CE-LIF)using a 20 mM sodium borate buffer after re-N-acetylation and derivatization by the fluorescent probe 8-Aminopyrene-1, 3, 6- Trisulfonate(26, 27). Galacturonic acid, Fucose, Galactose and N-Acetyl-amino sugars(N- Acetyl-Glucosamine, N-Acetyl-Galactosamine and N-Acetyl-Quinovosamine) were detected, in agreement with the composition of B. fragilis capsular polysaccharides described in Pantosti et al (25)and Bauman et al (28) Assessing CD4 T cell memory responses. Murine systems. Bone marrow-derived dendritic cells(BM-DCs)were generated from femurs and tibiae of C57BL/6 mice, cultured for 7 days in Iscove's medium (Sigma-Aldrich) with J558 supernatant(final GM-CSF concentration of ng/ml), 10% FCS, 100 IU/ml penicillin/streptomycin, 2 mM L-glutamine, 50 uM 2 mercaptoethanol(Sigma-Aldrich) and split every 3-4 days. At day 7, BM-DCs were infected with the isolated bacterial strains at multiplicity of infection(MOI)of 10 or 50, for lh at 37C in complete Iscove's medium without antibiotics, onto specific low binding 6 wells plates(Sigma)
12 presence of LPS in capsular polysaccharide-enriched fractions was investigated using the HEKBlueTM TLR4 cell line (Invivogen , Toulouse), a derivative of HEK293 cells that stably expresses the human TLR4, MD2 and CD14 genes along with a NF-țB-inducible reporter system (secreted alkaline phosphatase). Cells were used according to the manufacturer’s instructions. The different capsular polysaccharide-enriched fractions were added at concentrations ranging from 1 ng to 10 μg/ml in 96-wells plates and cells were then distributed at 5 × 104 per well in 200 μl DMEM culture medium. Alkaline phosphatase activity in the culture supernatant was measured after 18h. LPS content was determined by TLR4 signaling activation by comparison with a standard curve obtained using ultrapure LPS from E. coli K12 (Invivogen, Toulouse). The LPS content in the PS B. fragilis fraction was estimated at 1.4 pg/ μg of PS. The LPS residual content in the PS B.fragilis fraction could not account for its activity, as confirmed by the lack of bioactivity observed by adding LPS to the polysaccharide capsular fractions of B. distasonis compared to its effect in the absence of LPS. Monosaccharide analysis of the capsular polysaccharide-enriched fractions from B. fragilis and B. distasonis Monosaccharides were analyzed after total hydrolysis of 10 ȝg of capsular polysaccharide-enriched fraction by 2N TFA at 110°C for 2 hrs. They were detected by capillary electrophoresis monitored by laser-induced fluorescence (CE-LIF) using a 20 mM sodium borate buffer after re-N-acetylation and derivatization by the fluorescent probe 8-Aminopyrene-1,3,6- Trisulfonate (26, 27). Galacturonic acid, Fucose, Galactose and N-Acetyl-amino sugars (NAcetyl-Glucosamine, N-Acetyl-Galactosamine and N-Acetyl-Quinovosamine) were detected, in agreement with the composition of B. fragilis capsular polysaccharides described in Pantosti et al (25) and Bauman et al (28). Assessing CD4+ T cell memory responses. Murine systems. Bone marrow-derived dendritic cells (BM-DCs) were generated from femurs and tibiae of C57BL/6 mice, cultured for 7 days in Iscove’s medium (Sigma-Aldrich) with J558 supernatant (final GM-CSF concentration of 40 ng/ml), 10% FCS, 100 IU/ml penicillin/streptomycin, 2 mM L-glutamine, 50 μM 2- mercaptoethanol (Sigma-Aldrich) and split every 3-4 days. At day 7, BM-DCs were infected with the isolated bacterial strains at multiplicity of infection (MOI) of 10 or 50, for 1h at 37°C in complete Iscove’s medium without antibiotics, onto specific low binding 6 wells plates (Sigma)