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 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-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 (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 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)