Differential requirements for endosomal reduction in the presentation of two H2-E(d)-restricted epitopes from influenza hemagglutinin.

We examined the role of reduction in the presentation of two H2-E(d)-restricted epitopes (site 1 epitope (S1) and site 3 epitope (S3)) occupying distinct domains of the influenza hemagglutinin major subunit that contains four intrachain disulfide bonds and is connected to the virion by one interchain bond. S3 is situated within the stalk region that unfolds in response to mild acidification, and loads onto recycling H2-E(d) in the early endosome, while S1, located in the structurally constrained globular domain, loads onto nascent H2-E(d) in the late endosome. Predicting dependence upon reduction for either epitope seemed plausible but the results from several approaches were clear: presentation of S1 but not S3 is reduction dependent. Surprisingly, IFN-gamma-inducible lysosomal thiol reductase (GILT), the only reductase thus far known to be involved in MHC class II-restricted processing, is not necessary for the generation of S1. However, GILT is necessary for presentation of either epitope when the virus is pretreated with a reducible cross-linker. The results suggest that unfolding of the Ag, perhaps a prerequisite for proteolytic processing in many cases, proceeds either spontaneously in the early endosome or via reduction in a later endosome. They further imply mechanisms for GILT-independent reduction in the late endosome, with GILT perhaps being reserved for more intractable Ags.