acid residues are not critical for binding, yet native complex N-glycans are necessary for GlyAg but not conventional pe-p tide antigen binding to MHCII molecules. The binding results suggest that GlyAg molecules may form carbohydrate–carbohydrate interactions with MHCII N-glycans. To determine whether GlyAg can bind to glycans independently from the MHCII protein backbone,Alexa Fluor 488–conjugated PSA was screened for binding against a library of 320 glycans at the Consortium for Functional Glycomics (Fig. 4 J). We found no significant interactions above background with any of the immobilized glycans (see Table S1 for a complete list). These binding results directly demonstrate that the reduc- tion in MHCII-dependent cell surface presentation of GlyAg is caused by defects in the direct interaction between GlyAg and MHCII. Furthermore, it is clear that neither the MHCII protein backbone nor the N-glycans are independently suff-i cient for GlyAg binding. Native complex N-glycans are required for GlyAg-mediated T cell activation in vitro BM-derived DCs (BMDCs) were cultured for 3 d with and without CS or KF, as performed with the Raji and CHO cells. Fluorescent lectin staining analyzed by flow cytometry verified the loss of native complex N-glycans on CS- and KF-treated cells F(ig. 5 A) and the cells were confirmed as CD11c+ by flow cytometry (Fig. 5 B). On day 4, the cells were provided fresh drug and pulsed with 50 µg/ml PSA or 50 µg/ml OVA peptide (OVAp; residues 323–339) and 20 ng/ml LPS, 20 ng/ml LPS alone, 50 µg/ml DQ-OVA, 50 µg/ml Sp1, 50 µg/mlB. fragilis capsule containing polysac- charide B, or no antigen for 24 h. DQ-OVA is intact OVA that is nonfluorescent until cleaved to peptide, thus aliquots of the DQ-OVA–pulsed cells were analyzed by flow cytometry to verify protein uptake and processing. No difference was seen between UNT, CS-, and KF-treated BMDCs (Fig. 5 C). Figure 5. Native complex N-glycans are required for PSA-driven T cell activation in vitro. (A) BMDCs were cultured in the presence or absence of CS (red) and KF (blue) for 3 d, and then analyzed by PHA-L, PHA-E, WGA, and Con A lectin flow cytometry to detect N-glycans. Representative histo- grams and n = 4 MFI analyses are shown. (B) BMDCs were cultured for 8 d, probed with CD11c mAb, and analyzed by flow cytometry to verify proper DC cell differentiation. Representative histogram is shown. (C) BMDCs were incubated with nonfluorescent intact DQ-OVA protein for 24 h to allow uptake and processing. Cells were then analyzed for fluorescence, which is indicative of endocytosis and cleavage to peptides. Representative histograms are shown. n = 4 for MFI analyses. (D) BMDC maturation upon exposure to antigen was measured by surface staining of MHCII 24 h after stimulation with OVA peptide and LPS (O+L), LPS alone, or PSA. Representative histograms shown. (E) BMDCs grown with and without CS and KF were pulsed with either DQ-OVA (OVA), OVAp+LPS (O+L), or LPS alone for 24 h, after which untreated fresh naive CD4+ T cells isolated from OT-II animals were added. n = 3 per condition, per antigen. (F) As with OVAp, BMDCs were grown with CS and KF and pulsed with PSA, Sp1, or B. fragilis capsule (Caps) for 24 h. Next, naive WT C57BL/6 CD4+ T cells were added to allow activation to occur. n = 3 per condition. (G) TLR2 activation by PSA, a necessary step in GlyAg-mediated T cell activation (Wang et al., 2006), and a control agonist Pam3Cysk4 (P3C) was measured in untreated (UNT), CS-treated, or KF-treated TLR2+ HEK293 cells. Bars represent background-subtracted values. n = 3 per condition. All bar graphs show the mean ± SEM. 1046 MHC class II glycosylation in presentation of glycoantigens | Ryan et al.