e n i c i d e M l a t n e m i r e p x E f o l a n r u o J e h T CORRESPONDENCE Brian A. Cobb: brian.cobb@case.edu Abbreviations used: ME, -mercaptoethanol; BMDC, BM-derived DC; CS, castano- spermine; GlyAg, glycoantigen; GM-CSF, granulocyte- macrophage colony-stimulating factor; IBD, inflammatory bowel disease; KF, kifunensine; MBPp, myelin basic protein peptide; PHA-L, Phaseolus vul- garis leucoagglutinin; PSA, pol-y saccharide A; TLR, Toll-like receptor; UNT, untreated. A r t i c l e MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation Sean O. Ryan, Jason A. Bonomo, Fan Zhao, and Brian A. Cobb Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106 N-linked glycans are thought to protect class II major histocompatibility complex (MHC) molecules (MHCII) from proteolytic cleavage and assist in arranging proteins within the immune synapse, but were not thought to directly participate in antigen presentation. Here, we report that antigen-presenting cells (APCs) lacking native complex N-glycans showed reduced MHCII binding and presentation of the T cell activating glycoantigen (GlyAg) polysaccharide A from Bacteroides fragilis but not conventional peptides. APCs lacking native N-glycans also failed to mediate GlyAg-driven T cell activation but activated T cells normally with protein antigen. Mice treated with the mannosidase inhibitor kifunen- sine to prevent the formation of complex N-glycans were unable to expand GlyAg-specific T cells in vivo upon immunization, yet adoptive transfer of normally glycosylated APCs into these animals overcame this defect. Our findings reveal that MHCII N-glycosylation directly impacts binding and presentation of at least one class of T cell–dependent antigen. Nearly all mammalian immune system mo-le (DRA, DRB1*1501), there are three known cules found on the cell surface are glycosylated, glycosylated sites:N78,N118,and N103 (Fig.1 A; including Toll-like receptors (TLRs), class I and II Gauthier et al., 1998). Published findings dem- MHC proteins (MHCI and MHCII), CD1, onstrate that these glycans do not play a direct and  TCRs. Complex asparagine (N)-linked role in antigen binding or presentation, which glycans can reach 30 Å in length (Rudd et al., is elegantly illustrated by crystal structures of 1999), which is comparable to the size of a nonglycosylated MHCII–peptide complexes e-x protein Ig domain, and many of these surface pressed in bacteria (Frayser et al., 1999; Li et al., glycoproteins have multiple sites of glycosy-l 2000, 2001). ation. For molecules of the adaptive immune Recently, a new class of T cell–dependent response (e.g., MHCII and TCRs), these glycans antigens has been described that includes are thought to play three primary roles: a check capsular polysaccharides from types 5 and 8 point for proper protein folding and traffick- Staphylococcus aureus (Tzianabos et al., 2001), ing to the cell surface (Bergeron et al., 1998; type I Streptococcus pneumoniae (Sp1;Tzianabos Trombetta and Helenius, 1998; Saito et al., 1999), et al., 1993; Stingele et al., 2004; Matsuda and protection of the protein backbone from pro- Cepko., 2007;Velez et al., 2009), pneumococcal teolysis once on the cell surface to maximize the C-substance (Tzianabos et al., 1993), and polysac- lifetime of cell communication events (e.g.,T cell charide A (PSA) and polysaccharide B (Tzianabos activation; Wormald and Dwek, 1999), and pro- et al., 1992, 1993, 1995) from the gut commen- motion of the appropriate geometric spacing of sal bacteriaBacteroides fragilis. Both Sp1 and PSA receptors and other molecules on the cell surface associate with MHCII molecules in professional for optimized cell–cell attachment and co m- APCs and are recognized by TCRs on CD4+ munication (e.g., the immune synapse; Dustin T cells (Tzianabos et al., 1993; Cobb et al., 2004; et al., 1997). Cobb and Kasper, 2008; Velez et al., 2009). A central player in the adaptive immune re- Sp1 and PSA both form highly charged right- sponse is MHCII, which presents exogenous handed helices with two repeating units per peptide antigens to TCRs on CD4+ T cells for recognition and T cell activation. The MHCII © 2011 Ryan et al. This article is distributed under the terms of an Attribution– allele families carry two glycosylated asparagine tNhoencpoumblmicaetricoianl–dSahtaere(sAeleikhe–ttNpo://Mwiwrrwo.rruSpitreesssli.coerngs/teefromrst)h.eAfftiresrt ssiixx mmoonntthhssaiftteisr residues that are highly conserved across species. available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ For the human MHCII molecule HLA-DR2 by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 208 No. 5 1041-1053 www.jem.org/cgi/doi/10.1084/jem.20100508 1041