HIV-1 infected individuals accumulate a reservoir of treatment-resistant, latently infected resting CD4+ T cells, even when successful combination therapy clears their viremia. A newly developed technique, polychromatic flow cytometry (PFC), has revealed that the body's population of resting T cells comprises a complex mixture of cells with a variety of different proteins on their surfaces, presumably all specialized for different tasks and in a range of activation states. Thus, T cell activation is far from an """"""""all or none"""""""" process. We have begun to investigate the role(s) that the growing number of resting T cell subtypes and states of partial activation may play in the development of viral reservoirs. Using PFC and a quantitative assay for HIV-1 integration we plan to dissect the activation requirements for establishment of stable HIV-1 latency (versus complete productive infection) and the resting T cell subtypes comprising the latent reservoir in infected individuals. A number of pertinent variables will be examined, including: 1. The relative efficiency of integration vs production in naive, central and effector memory resting CD4+ T cells exposed to virus in vitro without activation. 2. The effect of antigen independent intercellular interactions on integration vs production in resting CD4+ T cells. 3. The effects of distinct stimuli alone and in combinations on the efficiency of integration vs production. Stimuli to be tested include individual cytokines and costimulators (e.g., CTLA-4) immobilized on artificial immunostimulatory beads 4. The relative frequency of integrated HIV-1 proviral DNA in memory and naive CD4+ T cells isolated from the blood of HIV-1 infected individuals, compared to the same populations purified from uninfected mononuclear cells and exposed to HIV-1 in vitro. Long term Objectives: Because latently infected resting T cells are the barrier to curing HIV disease, we hope to better define the mechanisms underlying latency, including the cellular activation requirements for its efficient establishment. We will then apply what we learn from our in vitro studies to define more precisely the phenotype(s) of the CD4+ resting T cells that are latently infected in patients.
