Project Summary Virus infected cells express MHC-I molecules associated with virus-derived peptides that are recognized by cytotoxic CD8+ T cells. The peptides are generated by proteasomal degradation of viral proteins synthesized in the cytosol and bind to assembling MHC-I heavy chain-β2microglobulin dimers in the ER. Naïve CD8+ T-cells, however, must be primed to induce a mature cytotoxic phenotype. Priming is generally mediated by DCs that acquire antigens by endocytosis and/or phagocytosis to generate the MHC-I-bound peptides, a process called cross-presentation. The mechanisms of cross-presentation remain poorly understood, with only a few effectors identified and none are absolutely required. We propose that the reason is that there is not a single cross-presentation pathway. Literature data points to three pathways. In the first, phagolysosomal proteases (cathepsins) degrade antigens to generate the peptides in situ and these bind to recycling MHC-I by peptide exchange. We discount this as a major mechanism because it would result in a mismatch between the MHC-I-peptide complexes generated by cross-presentation and those generated by proteasomes in the ultimate cytotoxic CD8+ T cell target, the virus infected cell. In the second, phagocytosed or endocytosed antigens are transferred across phagosomal/endosomal membranes into the cytosol where, like the newly synthesized proteins in virus infected cells, they are degraded by cytosolic proteasomes to generate peptides. These are translocated into the ER (or phagosomes that have recruited ER components) by the Transporter associated with Antigenic Processing (TAP) and bind to assembling MHC-I molecules, which are then transported to the cell surface. We propose to determine the mechanism(s) of translocation from endocytic compartments to the cytosol. In the third pathway, rather than internalized antigens entering the cytosol, cytosolic proteasomes are delivered into the lumen of phagosomes and/or endosomes. The antigens are then processed in situ by the proteasomes to generate peptides that bind to recycling MHC-I. This pathway is independent of TAP transport of the antigenic peptides but is dependent on proteasome activity. We propose that the second and third routes of cross-presentation can both operate, the common denominator being the endpoint, namely antigen recognition by the CD8+ T cells. The precise mechanisms required to mount and maintain a successful CD8+ T cell response will be determined.