The role of the endoplasmic reticulum in antigen processing: N-glycosylation of influenza hemagglutinin abrogates CD4⁺ cytotoxic T cell recognition of endogenously processed antigen

Evidence is presented for an endogenous route of Ag processing for CD4⁺ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-A^d-restricted T cell clones that recognize synthetic peptides corre-sponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II⁺ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HAl 63 Asp → Asn that introduces an oligosaccharide attachment site: ASp₆₃Cys₆₄Thr₆₅. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4⁺ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp → Asn has been introduced by site directed mutagenesis.