HIV persists as a latent infection in memory CD4 T cells. Current approaches for eradication rely on a shock and kill strategy where pharmaceutical agents reactivate the latent reservoir leading to cytopathic effects or immune clearance. Immune recognition requires the presentation of HIV peptides by MHC at the cell surface following LRA-induced HIV protein expression and processing. Some of the LRA that showed promising in vitro activity but limited efficacy in clinical trials such as HDAC inhibitors and PKC agonists have not been evaluated for their effects on HIV antigen processing and presentation by MHC.
We assessed the hydrolytic activities of cytosolic peptidases in live primary CD4 T cells treated with HDACi or PKCa using a fluorometric assay. The effects on antigen processing were studied by an in vitro degradation experiment of synthetic HIV peptides in cytosolic extracts of mock- or LRA-treated CD4 T cells. Finally, we analyzed the effects of LRA treatment of PBMCs on the MHC-peptidome by direct acid elution from the cell surface and mass spectrometry analysis.
Treatment with HDACi significantly decreased the hydrolytic activities of the proteasome and cytosolic aminopeptidases in primary CD4 T cells by up to 0.8-fold, whereas treatment with PKCa increased these activities by up to 4.9-fold. Consequently, the degradation of HIV synthetic peptides in cytosolic extracts from LRA-treated primary CD4 T cells yielded altered degradation patterns with 4-8 sites significantly changed in a 35-mer and 2-4 in a 24-mer depending on the LRA. Additionally, there were changes in the generation of known epitopes as well as other potential HLA-binding peptides. Treatment with LRA partly changed the MHC-peptidome of PBMCs, showing common peptides as well as different numbers and location of peptides derived from some common source proteins.
The observed changes in the hydrolytic activities of cytosolic peptidases in primary CD4 T cells upon LRA-treatment result in altered peptide degradation and a modified MHC-peptidome. The effects are both drug- and sequence-dependent. These results suggest that productively infected and LRA-reactivated CD4 T cells might present different HIV epitopes to the immune system, thus requiring different CD8 T cells responses for effective clearance of reservoirs.