Isolation and Activation of Immune Cells

HEK293T human embryonic kidney cells and P815 mouse lymphoblast-like mastocytoma cells (obtained from ATCC) were grown as previously described (39 (link), 65 (link)). Primary human peripheral blood mononuclear cells (PBMCs), CD4⁺ T cells, and NK cells were isolated, activated, and cultured as previously described (66 (link), 67 (link)). Briefly, PBMCs were obtained by Ficoll density gradient from whole-blood samples obtained from 9 different HIV-1-negative donors. CD4⁺ T lymphocytes and NK cells were purified from resting PBMCs by negative selection using immunomagnetic beads per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). CD4⁺ T cells were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin-2 (100 U/ml; R&D Systems) (see Fig. S1 in the supplemental material). NK cells were isolated and rested overnight in RPMI 1640 complete medium on the day prior to the redirection and killing assays.