FUNCTIONAL TCR ANALYSIS OF SIV SPECIFIC CTL

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have completed the study to characterize the TCR repertoire and the functional evolution of TCRs that recognize dominant and subdominant SIV-CTL epitopes that arise during immunization. We demonstrated that vaccine-elicited epitope-specific CD8+ T lymphocytes have a clonal diversity comparable to that induced by SHIV-89.6P infection, and these clonal CD8+ T lymphocyte populations can persist. Moreover, in the vaccinated monkey cohort, the clonal make-up of an epitope-specific CD8+ T lymphocyte population was almost identical in monkeys vaccinated with plasmid DNA/rMVA and in monkeys following vaccination with rAd. We have also completed the first part of the study to investigate the mechanism of CTL immunodominance. Our data suggest that in our experimental system the peptide binding affinity, efficiency of Ag presentation or processing and T cell functional differences are not likely to be the mechanism responsible for immunodominance. However, the analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-alpha and -beta chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of alpha and alpha beta chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Thus these data suggest that Ag-specific na¿ve T cell precursor frequency may be predetermined and that this dictates immunodominance of SIV-specific CD8+ T cell responses. To further understand the basic functional properties of antigen-specific CTL responses, the required optimal antigen concentration for efficient proliferation and the sensitivity of the specific functional cytotoxic activity among CTLs specific for different epitopes were compared in detail. In this study, we demonstrated that regardless of their immunodominance hierarchies, every CTL needed a low peptide concentration (between 0.01-1nM) for their optimal CTL expansion in vitro. Interestingly, the functional cytotoxic sensitivity of every CTL was lower than its sensitivity of in vitro proliferation. More importantly, the sensitivity of cytotoxic activity was very homogeneous among every CTLs studied. This homogeneous feature will allow us to distinguish the real epitope-specific CTL population from the cross-reactive ones. Finally, we have also demonstrated that in the in vitro CTL expansion system with fresh PBMC, monocyte/macrophages and B cells did not function as APCs. Only a small fraction of cells with the characteristics of DCs were capable of expanding those specific CTLs at low peptide concentration.