In vitro expansion of epitope-specific CD8+ T-cell lines was performed as described previously by our group87 (link). Briefly, freshly thawed cryopreserved autologous PBMCs were plated in a 48 well plate at 1.2 × 106 cells/ml in serum free RPMI media. Supernatants containing non-adherent cells were removed after a two-hour incubation at 37°C. Adherent cells, mainly monocytes, were irradiated (3,300 rad, 45 min) and pulsed with the appropriate autologous peptide (10 μM) for 2 hrs. CD8+ T cells were isolated from the non-adherent cells using the CD8+ untouched isolation kit (MACS Miltenyi Biotec) and were plated onto peptide-pulsed monocytes in the presence of complete media (RPMI+10% Hyclone serum) containing IL-7 (25 ng/ml). The CD8+ T-cell culture was maintained by adding IL-2 (50 U/ml) every 2 to 3 days and re-stimulating the CD8+ T cells with peptide-pulsed monocytes as described above on day 7. On day 13, T-cell lines were tested for cytokine responses to the cognate HIV peptide in a 6 hr ICS assay as outlined above.