Figure 4.

Schematic of the proliferation and cytotoxicity assay of recalled memory HIV-specific CD8⁺ T cells. CellTrace dye labeled PBMCs were stimulated with cognate HIV epitope peptides. Tetramer⁺ HIV-specific CD8⁺ T cell phenotype and cell number were analyzed by flow cytometry after 6 and 12 days of stimulation. Autologous PBMCs were concurrently expanded with PHA and rhIL-2 for 12 days in the presence of ARVs. CD4⁺ T cells were purified from the PHA expanded PBMCs by magnetic negative selection. Autologous CD4⁺ T cells were labeled with low levels of CSFE and pulsed with HIV peptide or labeled with high levels of CFSE and not loaded with peptide. Cytotoxic capacity of CD8⁺ T cells was measured by co-culturing the peptide stimulated CD8⁺ T cells at day 13 post-stimulation at an E/T ratio of 2 with the peptide-pulsed and non-pulsed CFSE labeled autologous CD4⁺ T cells mixed 50/50 as targets.