A r t i c l e with the pCRE but not in mock controls. Using biotinylated adhesion molecules is even thought to contribute to the PHA-L, the PHA-L BMDCs were then isolated by mag- stability and avidity of the immune synapse formed between netic bead negative selection, reanalyzed for PHA-L staining, APCs and T cells, however, the fundamental binding prope-r and compared with pCRE-electroporated WT BMDCs ties of MHC for peptides and TCRs for the MHC-peptide (Fig. 7 C).The purity of each population was consistently >90% complex are essentially unaffected by the presence or absence for both PHA-Lneg and PHA-Lpos BMDCs. mRNA was of complex N-glycans. In our studies, we even found that with isolated from the two populations, and the lack ofMGat2 tran- bacteria-expressed nonglycosylated MHCII, peptide binding script in the PHA-Lneg population was confirmed by RT-PCR was increased over the glycosylated form. Although the (Fig. 7 C). As before, the expression levels of CD11c and mechanism for this increase remains unclear, many biochemi- MHCII were verified and no significant differences were ob- cal and structural studies on MHC and TCRs have used bacte- served (Fig. 7 D). Finally, the MGat2pos and MGat2neg BMDCs rially expressed protein (Frayser et al., 1999; Li et al., 2000) or were used in T cell activation assays with either OT-II CD4+ baculovirus/insect cell–expressed molecules (Brown et al., T cells (OVA assays) or WT CD4+ T cells (PSA assays). As 1995; Gauthier et al., 1998) that are characterized by regular, seen with the CS- and KF-treated BMDCs (Fig. 5), the pro- but limited, mannose core structures that lack mammalian tein antigen response was unaffected by the loss of MGat2, yet complexity and heterogeneity (Altmann et al., 1999). In the GlyAg response was nearly eliminated when native com- stark contrast to the wealth of information on conventional plex N-glycans were missing in the BMDCs (Fig. 7 E).These peptide antigens, our findings point to a fundamental yet data validate the inhibitor-based observations and confirm poorly understood difference in the mechanism of interaction that PSA presentation and downstream T cell activation relies between carbohydrate antigens and MHCII in that the native upon native complex N-glycosylation of MHCII proteins. complex N-glycans appear to play a critical functional role during association. DISCUSSION Another function of N-linked glycans is a check on pro- Most immune cell surface receptors and accessory molecules tein folding within the endoplasmic reticulum. The addition are glycoproteins, including CD4, CD8, T cell receptors, of the core GlcNAc2Man9Glc3 N-glycan to asparagine resi- and both class I and class II MHC. MHCII-dependent antigen dues within the Asn-X-Ser/Thr consensus sequence occurs presentation is a central event for adaptive immune responses in the ER. After the initial cleavage of two terminal glucose against extracellular fungi, bacteria, and other pathogens. In residues, these structures bind to calnexin and calreticulin, which this study, modification of MHCII N-glycosylation with are thought to be an important check on folding and facilitate N-glycosylation processing enzyme inhibitors (CS or KF) or MHCII trafficking out of the ER into the Golgi apparatus through CRE-mediated ablation of theMGat2 gene resulted (Trombetta and Helenius, 1998). However, at least one study in the elimination of native complex N-glycans on APCs, reported that treatment of APCs with the N-glycosylation which significantly reduced binding, presentation, and T cell inhibitor tunicamycin failed to alter the plasma membrane activation by the glycoantigen PSA isolated from the capsule concentration of MHCII (Hart, 1982), suggesting that MHCII of the commensal bacteriaB. fragilis.The reduction in presen- trafficking to the cell surface is not particularly sensitive to tation translated into a reduction in GlyAg-mediated CD4+ N-linked glycosylation. Indeed, our results support this inte-r T cell activation in vitro and in vivo.These findings demon- pretation in that modification of the N-glycans on MHCII strate for the first time that MHCII-mediated binding and by blocking early steps in the glycosylation pathway did not presentation of at least one class of antigens depends heavily alter MHCII surface staining or peptide presentation, yet upon native complex N-glycans decorating the MHCII pro- GlyAg presentation was significantly reduced. Unaltered pe-p tein backbone. tide presentation and T cell activation in vitro demonstrate Complex N-glycans are large and highly flexible str-uc that the MHCII on the cell surface is properly folded and can tures that can extend to 30 Å, roughly corresponding to the be normally recognized by cognate TCRs on opposing CD4+ size of an immunoglobulin domain (Rudd et al., 1999, 2001). T cells. In vitro binding experiments with MHCII lacking These molecules are thought to contribute to the functiona-l native N-glycans reveal no reduction in peptide binding, but ity of the underlying protein in several indirect ways. For near ablation of GlyAg binding. Confocal microscopy further example, the N-linked glycans attached to the adhesion mol-e demonstrates vesicular colocalization of GlyAg and MHCII, cule CD2 near the plasma membrane are thought to structurally confirming that antigen endocytosis and trafficking to the restrict the protein conformation such that trans-interactions normal MHCII compartment was unaffected by the glyco- between CD2 and its ligand CD48 on opposing cells are sylation changes. Finally, structural studies using bacterial promoted over cis-interactions between CD2 and CD48 ex- (Frayser et al., 1999; Li et al., 2000) and insect cell expressed pressed on the same cell (Dustin et al., 1996). This general MHCII (Brown et al., 1995; Gauthier et al., 1998) show no principle is thought to apply to many of these surface glyc-o significant differences in overall structure. We conclude that proteins including MHC and TCRs, whereby the glycans can although modification of N-glycans directly impacts GlyAg- prevent lateral nonspecific aggregation and provide a regular mediated responses and APC surface localization of GlyAg, structural lattice for glycoprotein packing on the cell surface this alteration in MHCII antigen presentation activity is not (Rudd et al., 1999, 2001). Ordering of surface receptors and caused by changes in endocytosis, vesicular trafficking, surface JEM VOL. 208, May 9, 2011 1049