Cell death due to apoptosis plays a critical role in the development of T cells in the thymus. Several intracellular molecular mechanisms have been identified that regulate apoptosis in thymocytes, including bcl-2, bcl-x, bax, R-ras and fas. Retroviral infection of the thymus with HIV-1 or SIV results in selective thymocyte depletion, and apoptosis has been shown to contribute to this increase in cell death. The precise mechanism whereby lentiviruses induce apoptosis remains controversial. Using a novel system of in vitro T cell differentiation, we developed a multiparameter flow cytometry assay to determine the role of fas, fas-ligand and bcl-2 in apoptosis of thymocytes as a result of SIV infection. We detected a 3-fold increase of apoptosis (detected by Tdt staining) in in vitro cultures 48 post infection with SIV239. Furthermore, the increase in Tdt+ cells is accompanied by a decrease in bcl-2 (2- 3-fold) expression and an increase in surface fas expression (4-5 fold) as determined by flow cytometry. Fas ligand was found to be increased in single positive thymocytes in vivo, but not in the in vitro culture system. This data clearly demonstrates that both the bcl-2 and fas pathways are involved in SIV induced apoptosis of thymocytes and that lentiviral infection leads to thymocyte apoptosis. Using FACS we demonstrated that SIV infection results in an initial 15-20% decrease of surface fas expression in CD4+CD8+ and CD4+CD8- cells, and not CD4-CD8+ cells. However, by day 7 post-infection, we observed a profound reversal of this phenomenon, with a dramatic increase (6 fold) in fas expression in CD4+CD8+ and CD4+CD8- cells, as compared to uninfected cultures. This correlates with data generated from in vivo studies in collaboration with Dr. Andrew Lackner, in the department of Pathology at the New England Regional Primate Research Center. These studies will continue to address the mechanisms associated with thymocyte depletion as a result of retroviral infection. A better understanding of the role of these apoptotic pathways in contributing to impaired T cell production in AIDS may lead to novel approaches to reconstitute immune function in HIV-infected people.
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