Peptide-receptive class I major histocompatibility complex molecules on TAP-deficient and wild-type cells and their roles in the processing of exogenous antigens

These studies addressed the nature and origin of peptide-receptive class I major histocompatibility complex (MHC-I) molecules used to present exogenous antigens. Peptide-receptive Kb molecules in transporter for antigen presentation (TAP)1(-/-) and TAP1(+/+) macrophages were quantitated by exposing cells to exogenous ovalbumin (OVA)(257-264) peptide and then measuring OVA(257-264):Kb complexes with a T hybridoma assay or flow cytometry (using a complex-specific antibody). Relative to TAP1(+/+) cells, TAP1(-/-) cells had decreased levels of pre-existing cell-surface peptide-receptive MHC-I molecules at 37 degrees. With continued exposure of viable cells to peptide, however, TAP1(-/-) and TAP1(+/+) cells formed similar levels of OVA(257-264):K-b complexes, suggesting that nascent labile MHC-I molecules were captured and stabilized by exogenous peptide. Brefeldin A inhibited generation of OVA(257-264) : K-b complexes on TAP1(-/-) (but not TAP1(+/+)) cells at 37 degrees, confirming the importance of a flux of unstable nascent MHC-I molecules in TAP1(-/-) cells at 37 degrees. In contrast, at 26 degrees both TAP1(-/-) and TAP1(+/+) cells expressed brefeldin A-resistant, peptide-receptive MHC-I molecules at similar levels. Alternate MHC-I processing of exogenous particulate antigen correlated with ability to present exogenous peptide. Thus, processing was brefeldin A-sensitive with TAP1(-/-) macrophages at 370, but brefeldin A-resistant with TAP1(+/+) cells at 37 degrees, as well as with TAP1(+/+) or TAP1(-/-) cells at 26 degrees. We conclude that alternate MHC-I antigen processing normally utilizes pre-existing MHC-I molecules, but TAP1(-/-) cells at 37 degrees mainly use nascent MHC-I molecules, because of a lack of pre-existing, stable, peptide-receptive MHC-I molecules. The results support a vacuolar processing mechanism with binding of peptides to MHC-I molecules in post-Golgi compartments or on the cell surface.