Role of the proteasome and non proteasomal proteases in the generation of CTL epitopes

We have previously shown that cells lacking the MHC encoded proteasome subunits LMP2 and LMP7 have a severe defect in the generation of the HLA-A2 restricted influenza Matrix epitope 58-66. We have now extended this observation to other viral epitopes and demonstrated that the antigen presentation block can be restored by transfecting LMP7. We also show that the LMP7 dependent presentation of the Matrix epitope 58-66 can be overcome by expressing fragments of the influenza Matrix, up to 100 amino acid long These results demonstrate that a single proteasome subunit is critical for the generation of defined CTL epitopes and highlight the possibility that either the length or folding of cytosolic proteins may influence the processing pathway leading to the generation of CTL epitopes. Consistent with the latter possibility we demonstrate that processing of a rapidly degraded CTL target protein is redirected at 42°C from the proteasomes to non-proteasomal cytosolic proteases. Heat shock dependent protein degradation has a profound effect on the repertoire of MHC class I bound antigenic peptides, as cells incubated at 42°C lose the ability of generating a defined CTL epitope but presentation of another epitope is significantly enhanced. The results demonstrate that antigenic peptides can be generated by different processing pathways and highlight the effect of heat shock on processing and presentation of CTL proteins.