performed after a 6-h stimulation with 10 ng/ml PMA and 500 nM ion- omycin. Analyses of FACS data were performed using FCS Express (De Novo Software). Confocal microscopy. Raji B cells were incubated in the presence or absence of 10 µg/ml KF or 175 µM CS for 3 d, with fresh drug and media added daily. On the final day, Alexa Fluor 594–conjugated PSA was added to the cells. 24 h later, cells were harvested, washed, and fixed in 4% paraf-or maldehyde for 30 min on ice. The cells were washed and incubated with Alexa Fluor 488–conjugated L243 mAb for 30 min on ice. The cells were then washed and mounted on coverslips for viewing by confocal microscopy using an SP5 laser scanning confocal microscope (63× oil-immersion objective; Leica). Cross-sectional histograms of the pixel intensities were determined from high-resolution confocal images n( > 10 per sample) using the Leica Applica- tion Suite software, and then used to calculate the average pixel intensities found within the borders of each cell (vesicular) and those found on the bo-r ders of the cells (surface) as described previously (Cobb et al., 2004). Statis-ti cal analyses of surface and vesicular-localized signals were performed using an unpaired analysis of variance with Bonferroni Multiple Compar-i sons post-test with GraphPad InStat software (GraphPad Software). Antigen processing assays. PSA processing was measured as previously described (Cobb et al., 2004). Raji B cells (170) treated with and without 10 µg/ml KF or 175 µM CS for 3 d before antigen exposure were cultured with 0.5 mg [3H]PSA for 15 h at 37°C to allow for endocytosis and proces-s ing. Cells were harvested and washed with PBS, and then mechanically lysed by passage through a 27-gauge needle in 250 mM sucrose buffered with 25 mM Tris-HCl, pH 7.5. Microsomes were isolated for analysis of endocytosed glycoantigen by centrifugation at 100,000 g for 1 h. The pellet was resu-s pended in 25 mM Tris, pH 7.5, and 0.5 mg/ml Pronase to eliminate any in- teractions between PSA and resident proteins for 2 h at 37°C.The resulting sample was then passed through a Superose 12 molecular sieve column fitted to an Äkta Purifier10 HPLC system (GE Healthcare) while collecting fractions. The fractions were analyzed by liquid scintillation counting and graphed by elution volume to determine the molecular size of the glycoantigen fr-ag ments isolated from the vesicular compartments of Raji B cells. Antigen presentation assays. 107 Raji B cells untreated or treated with 10 µg/ml KF or 175 µM CS for 3 d before antigen exposure were cultured with 0.25 mg biotin-conjugated PSA or 0.25 mg biotin-conjugated MBPp for 24 h at 37°C to allow for presentation. Cells were harvested, washed, and digested with papain (0.1 mg/ml papain; 10 mM Tris-HCL, pH 7.5; 1 mM EDTA; 1% ME) to release surface-localized MHCII proteins and their inte-r acting antigens (Wiman et al., 1982). Protease inhibitor cocktail (Sigma-Aldrich) was added and the samples incubated in wells of ELISA plates precoated with either 25 µg HLA-DR–specific L243 mAb or IgG2a isotype control over- night at 4°C.The wells were washed and incubated with europium-conjugated streptavidin (Eu-SA; PerkinElmer) for 45 min.The wells were washed again and the biotinylated PSA or MBPp present quantified by time-resolved fluo- rescence (TRF) on a Victor3V Multilabel Counter using dissociation enhanced fluorescence Enhancement Solution according to the manufacturer’s protocol (PerkinElmer). Statistical comparisons between untreated and drug- treated samples with each antigen were performed with a student’s t test using InStat. Cell viability assays. 105 Raji B cells untreated or treated with 10 µg/ml KF or 175 µM CS for 3 d were cultured in 200 µl of media in wells of a 96-well plate in quadruplicate. At the end of day 3, 50 µl of culture sup-er natant was removed from each culture well and combined with 50 µl of ATPlite reagent (PerkinElmer) for 5 min. ATP concentration was measured by luminescence on aVictor3V Multilabel Counter as a measure of cell me-ta bolic activity and viability. Binding assays. The interaction between HLA-DR2 and MBPp or PSA was measured exactly as previously described (Cobb and Kasper, 2008, JEM VOL. 208, May 9, 2011 A r t i c l e Kreisman et al., 2007). In brief, 200 ng of each purified recombinant DR2 protein (UNT-DR2, CS-DR2, or KF-DR2) was immobilized in wells of an Immulon 4HBX 384 well ELISA plate. 2.0 µg of either biotinylated, low molecular weight PSA or biotinylated MBPp was incubated in the DR2 or negative control FBS-blocked wells for 24 h at 37°C. For the MBPp binding, the binding was performed in Tris-buffered saline, pH 7.5, while PSA bind- ing was performed in citrate-buffered saline, pH 4.5 as previously reported (Cobb and Kasper, 2008). Bound biotinylated ligand was quantified as de- scribed for the coimmunoprecipitation presentation experiments using Eu-SA and TRF on a Victor3V counter. Statistical analyses of the resulting data were performed using a Student’s t test in InStat. Lectin ELISA. The nature of N-glycans on CHO-expressed UNT-DR2, CS-DR2, and KF-DR2 proteins was determined by ELISA with several biotinylated lectins (indicated in Fig. 4, D and E;Vector Laboratories). 2 µg of each protein was immobilized in wells of an Immulon 4HBX 96-well ELISA plate and probed with 5 µg/ml of each lectin. Each protein was done in at least triplicate for each of the lectins. Comparisons were made by normal-iz ing the signal to unity for the UNT-DR2 and then calculating the fold change in lectin binding for each of the CS-DR2 and KF-DR2. Glycan-binding array. High-throughput glycan-binding array screening was performed as described previously (Paulson et al., 2006; von Gunten et al., 2009) by the Core H of the National Institutes of Health–funded Consortium for Functional Glycomics, except Alexa Fluor 488–conjugated PSA was used as the potential glycan binding molecule. The glycan targets immobilized on the array is presented inTable S1. TLR2 activation. HEK293 cells expressing recombinant TLR2 were grown in 6-well plates in the presence and absence of 10 µg/ml KF or 175 µM CS for 3 d. On day 4, cells were transferred to a 96-well plate at 2 × 140cells per well. Cells were stimulated for 12 h with either 50 µg/ml PSA, as shown p-re viously (Wang et al., 2006), or 15 nM Pam3Cysk4. Culture supernatants were then analyzed for IL-8 production by sandwich ELISA according to the manufacturer’s protocol (BioLegend). T cell activation (inhibitors). Mouse BM of WT C57BL/6 mice (The Jackson Laboratory) were differentiated into BMDCs by growing them in RPMI 1640 containing 10% FBS, ME, pen/strep, 15 ng/ml GM-CSF (Invitrogen) for 10 d. On days 7–9, BMDCs were treated with 10 µg/ml KF or 175 µM CS. On day 10, BMDCs were collected and verified by flow cytom- etry using CD11c-specific mAb and cultured for 24 h with and without fresh drugs and antigen (see Results section). On day 11, fresh naive CD4+ T cells were isolated from the spleens of untreated C57BL/6 mice (or OT-II mice for OVAp assays; The Jackson Laboratory) by positive selection using CD4+ magnetic beads (Miltenyi Biotec) and verified by flow cytometry using CD4-specific mAb. CD4+ T cells were then added to the BMDC cultures at a 10:1 ratio (i.e., 10 T cells per BMDC) and the samples were incubated for 3 d (DQ-OVA and OVAp assays) or 4 d (GlyAg assays) without the addition of more CS or KF. Culture supernatants were then analyzed for IFN- by sandwich ELISA according to the manufacturer’s protocol (BioLegend). Sta- tistical analyses were performed using an unpaired analysis of variance with Bonferroni Multiple Comparisons post-test. BMDC maturation. As described in the previous section for T cell activa- tion, BMDCs were cultured for 3 d with and without CS and KF. Antigen- induced maturation after 24 h stimulation was measured by cell surface staining and flow cytometry using FITC-conjugated mouse MHCII-specific mAb (clone 25–9-17; BioLegend). MGat2 experiments. BM cells collected from WT C57BL/6 (MGatw2t) and B6.129-MGat2tm1Jxm/J (MGat2flox) mice (The Jackson Laboratory) were depleted of erythrocytes using Red Blood Cell Lysing Buffer (Sigma-Aldrich). Using the Neon Transfection System and the Neon 100 µl kit (Invitrogen), 5 × 106 cells were electroporated with 6 µg pCAG-Cre (Matsuda and Cepko, 2007) 1051