Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: Intracellular loading and kinetics of presentation

This study investigates the differential capacity of TAP-deficient T2 cells , TAP-competent EBV cells, and immature and mature dendritic cells to prese nt peptides to preformed CTL lines. It demonstrates that presentation of ex ogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affini ty peptides in the presence of irrelevant peptides competing for HLA bindin g sites. Under these circumstances, inhibition of protein synthesis with cy cloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous beta2-microglobulin to stabilize the MHC complex on the cell sur face. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-compe tent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity p eptides require empty MHC molecules within cells. Accordingly, very high co ncentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation again st a background of competing intracellular high affinity peptides in TAP-co mpetent cells. These findings have implications for the design of peptide a nd protein-based vaccines.