CFSE-based T cell proliferation assay

Overnight-rested cryopreserved blood mononuclear cells isolated from one HIV-1-infected elite controller individual (#1010) were stained with 0.25 μM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described^{49 (link)}. Cells were then exposed to RV-Empty, RV-Gag, NYVAC-Empty or NYVAC-GPN vectors at various vector concentrations (0.1, 1 and 10 pfu/cell). In addition, cells were stimulated with GAG peptide (GPNHKARVL; internal control), 200 ng/ml of SEB (positive control; Sigma-Aldrich) or left unstimulated (negative control). At day 6, cells were harvested and stained (4 °C; 20 min) using the aqua LIVE/DEAD stain kit (Invitrogen) and Abs (4 °C; 30 min) to CD3, CD4, and CD8. Cells were fixed with CellFix (BD), acquired on an LSRII SORP (4 lasers: 405, 488, 532 and 633 nm) and frequencies of proliferating CFSE^low CD8 T cells were assessed using FlowJo (version 8.8.2; Tree star Inc, Ashland, OR, USA).