Cell death due to apoptosis plays a critical role in thymocyte ontogeny. Several intracellular molecular mechanisms have been identified that regulate apoptosis in thymocytes. Expression of the proto-oncogene bcl-2 is believed to protect against apoptosis. The small fraction of thymocytes that undergo positive selection at the CD4+CD8+ double positive stage appear to express higher levels of intracellular bcl-2, which provides a protective mechanism against negative selection or deletion via apoptosis. More recently bcl-x, bax and R-ras have been proposed as additional regulatory proteins in apoptosis, although their precise role in T cell development has yet to be determined. Surface expression of the fas antigen has also been implicated in the induction of apoptosis in peripheral T cells, but its role in thymocytes remains unclear. 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. A multiparameter flow cytometry assay was developed to determine the role of fas and bcl-2 in apoptosis of thymocytes as a result of SIV infection. This assay facilitates the simultaneous determination of cell phenotype together with fas and bcl-2 expression. In addition, apoptosis is detected via terminal deoxynucleotide transferase (TdT) incorporation or Hoechst 33342 uptake. With this technique we have determined that retroviral infection of thymocytes results in selective thymocyte depletion, increased apoptosis accompanied by an increase in surface fas expression and an increase in intracellular bcl-2. Interestingly, bcl-2 levels do not appear to protect from apoptosis induced by SIV infection.
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