Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly g enerated and loaded in endolysosomal compartments, but the major pathways r esponsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules pre sent peptides from proteins such as OVA or conalbumin introduced into the c ytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to th eir presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the pr esentation of exogenous Ags taken up by endocytosis is not. Inhibitors of n onproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC cla ss I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ece,,, yields an epitope that is similar to the one generated during constitutive presentat ion of I-Ea as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II -mediated presentation of the endogenous transmembrane protein I-Ea is also specifically inhibited over time by inhibitors of cytosolic proteolysis. T hus, Ag processing in the cytoplasm appears to be essential for the efficie nt presentation of endogenous proteins, even transmembrane ones, on MHC cla ss H, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.