Expansion of Epitope-Specific CD8 T Cells

Epitope-specific CD8 T-cell lines were expanded in vitro as previously described [14 (link)]. Briefly, cryopreserved PBMCs (obtained from chronically HIV-1 infected patients) were thawed and plated in a 48-well plate at 1.2×10⁶ cells/ml in serum free RPMI media. Plates were incubated at 37C/5% CO₂ for two hours, after which media containing non-adherent cells was removed. Adherent cells were irradiated at 3,000 rad and pulsed with the appropriate peptide at 10 μM for 2 h. Autologous CD8 T cells were isolated from the same PBMC sample using the CD8 untouched isolation kit (MACS Miltenyi Biotec). CD8 T cells were then plated at 0.5×10⁶ cells/well onto the peptide-pulsed monocytes in the presence of complete media (RPMI+10% FBS) containing IL-7 (25 ng/ml). IL-2 (50U/ml) was added to the culture on the second day. The culture was then maintained by replacing half the media with freshly made media containing IL-2 (50U/ml) every three days, and CD8 T cells were re-stimulated on day seven (and weekly thereafter) with peptide-pulsed monocytes. CD8 T cell clone (SL9) was a gift from Dr. June Kan-Mitchell.