FACTORS LIMITING ANTI HIV RESPONSES OF CYTOTOXIC T CELLS

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It is becoming increasingly evident that along with the level of cognate peptide-MHC (pMHC) complexes on target cells their distribution at the cell surface could substantially influence the magnitude of a T cell response. We have demonstrated that MHC class I molecules are co- clustered with adhesion molecules (ICAM-1) in membrane rafts and that disruption of the raft integrity on target cells decreases the potency of presentation of viral peptides. Based on these data we hypothesize that along with the level of cognate pMHC complexes on target cells their distribution at the cell surface might also influence the efficacy of the antigen presentation. To test this hypothesis we propose to vary the distribution of HLA-A2 molecules containing an immunodominant peptide epitope SLYNTVATL (SL9) from HIV p17 gag protein on the surface on target cells using different pathways on SL9 delivery and to determine whether variations in the distribution of SL9-HLA-A2 complexes correlates with the intensity of gag-specific CTL responses. This will be accomplished in 4 specific aims: (i) to produce soluble oligomeric TCR (from the CTL clone D3) specific to SL9-HLA-A2 complexes on target cells; (ii) to mimic the physiological pathway of SL9-HLA-A2 presentation delivering SL9 into the target cell using colloidal gold particles of membrane-translocating carriers; (iii) to compare the distribution of SL9-HLA-A2 complexes delivered to the cell surface through "physiological pathway" or generated with synthetic SL9 peptide added to the extracellular medium and to determine how variations in the distribution of the pHMCs affect the sensitivity of gag- specific CTL responses; (iv) using oligomeric D3 TCR to measure the fraction of peripheral blood mononuclear cells (PBMC) presenting SL9 derived from HLA-A2+ HIV-infected patients in earlier and late stages of the disease and to evaluate the effect of therapeutic intervention on the presentation of SL9 on PBMC isolated from patients undergoing treatment. The data produced are expected to improve our understanding of the mechanisms of presentation of viral peptides and to aid the development of new approaches to monitor the expression of viral epitopes on the infected cells during infection, treatment, and vaccination.