Apoptosis Induction in T Cell Subsets

For apoptosis induction in primary cells, cryopreserved PBMCs from healthy donors were used. Unstimulated PBMCs were cocultured with HeLa cells transfected with either SHIV KB9 or 89.6 Env for 48h. The suspension cells were collected and stained with an apoptosis panel comprising of the following antibodies: CD3-Cy7, CD4-Tx Red, CD8-APC (Beckman Coulter) along with CaspACE FITC-VAD-FMK (Promega, Madison, WI, USA) as described previously [16 (link)]. Stained cells were washed and fixed using IOTest 3 Fixative Solution (Beckman Coulter) and assayed by flow cytometry. At least 20,000 events for each sample were acquired. Data was analyzed using FlowJo software (Tree Star). Cells were first gated on the CD3+ population and apoptosis in CD4+ and CD8+ T cell subsets was determined along with the CD4:CD8 ratio. Apoptosis induction in Rhesus PBMCs was measured as above and human antibodies with cross reactivity to Rhesus CD4 (BD Biosciences 562402) and CD3 antigens (BD Biosciences 557749) were used.