turn and a pitch of 20 Å (Wang et al., 2000; Choi et al., 2002), GlyAg-mediated T cell recognition and activation despite no whereas initial recognition of these “glycoantigens” (GlyAg) detectable defects in peptide and intact protein antigen con- leads to cytokine production (e.g., IFN-) and T cell pro- trols. In contrast to the current model, our findings reveal that liferation (Brubaker et al., 1999; Stingele et al., 2004; Stephen the nature of the N-glycans found on MHCII can directly et al., 2005) with an overall phenotype indistinguishable from modulate antigen binding and presentation. BecauseT cell re-c T helper type I (TH1) cells. GlyAg association with HLA-DR2 ognition of GlyAg is critical for gut immune homeostasis (DR2) shows high affinity (Kd = 315 nM; Cobb and Kasper, (Mazmanian et al., 2005, 2008), these results raise intriguing 2008), is dependent on positive and negative charges found questions about the variability of MHCII glycosylation in the within the polysaccharide (Kreisman et al., 2007; Cobb and gut (Barrera et al., 2002) and how inflammation-associated Kasper, 2008; Velez et al., 2009), and requires processing via variability could modulate antigen presentation and T cell nitric oxide to fragments in the 5–10 kD size range (Cobb et al., responses against the commensal microbiota. 2004;Velez et al., 2009). Although GlyAg–MHCII binding is competitive with peptide antigens and requires the expression RESULTS of HLA-DM in APCs for presentation (Cobb and Kasper, Native MHCII N-glycans are required for GlyAg presentation 2008;Velez et al., 2009), the manner in which GlyAgs interact Although MHCII glycosylation does not appear to play a direct with MHCII remains unknown. role in conventional peptide antigen binding (Frayser et al., Here, we present a study in which it was discovered that 1999; Li et al., 2000), we sought to determine the functional bacterially expressed DR2 failed to associate with GlyAg despite role of these glycan structures during commensal-derived being properly folded and fully capable of binding to an-ti GlyAg presentation. Human Raji B cells, which have been genic peptide. We also show that elimination of native com- previously demonstrated to present GlyAg via MHCII bind- plex N-linked glycans (i.e., unaltered mammalian complex ing (Cobb et al., 2004), were cultured in the presence and ab- N-glycans) on MHCII reduced presentation of GlyAg in sence of the glucosidase inhibitor castanospermine (CS; 175 µM) live APCs, nearly eliminates GlyAg binding to recombinant or the mannosidase inhibitor kifunensine (KF; 10 µg/ml) for MHCII in vitro, and significantly limits in vitro and in vivo 3 d to reduce the presence of hybrid and native complex Figure 1. HLA-DR2 glycosylation sites and mechanism. (A) HLA-DR2 (DRA; DRB1*1501), shown with myelin basic protein peptide (green; space-filling), contains three N-glycosylation sites. The N78 position is highly conserved across species and is located at one end of the canonical peptide-binding groove, which is comprised of -helices (blue) and a -sheet foundation (red). Coordinates are from Gauthier et al., 1998. (B) Simplified N-glycosylation pathway in mammalian cells, highlighting the sites of CS (glucosidase inhibitor) and KF (mannosidase inhibitor) action and the representative structures expected. The asterisk highlights a complex N-glycan example from public data via the Consortium for Functional Glycomics. Specifically, this structure was found expressed by murine RAW macrophages, has a mass of 5035.3 Daltons, and is available in the dataset for A. Merrill request #1689 (http://www.functionalglycomics.org/ glycomics/publicdata/glycoprofiling-new.jsp). 1042 MHC class II glycosylation in presentation of glycoantigens | Ryan et al.