K562 cells (ATCC, CCL-243) were transduced with a lentivirus vector encoding the extracellular and transmembrane domain of the SIVmac239 Env (amino acids 1–720) or a V1V2 domain region of the SIV Env (amino acids 78–232). Envelope expression was evaluated using flow cytometry and staining with a biotinylated ITS06.01 antibody followed by BV-421 streptavidin and/or an anti-RQR8/CD34 (Qbend/10, R&D Systems) for the K562-V1V2 Env. Transduced K562 cells were isolated using magnetic-activated cell sorting (MACS) (Miltenyi Biotec) following the manufacturer’s instructions after incubation with a biotinylated ITS06.01 antibody.
To assess CAR T cell proliferation in response to stimulators, CellTrace Violet (Thermo Fisher)-labeled CAR T cells or EGFR T cells were cocultured at a ratio of 1 stimulator:1 CAR or EGFR T cell with mitomycin C-treated K562-Env or mitomycin C-treated SIV-infected CD4+ T cells or their respective negative control K562 or CD4+ T cells for 4 days at 37°C. Cells were harvested and stained with anti-EGFR, and proliferation was assessed by determining the percentage of EGFR+ cells present in peaks 3–7 of decreased CellTrace Violet fluorescence intensity after gating on lymphocytes, single cells, and EGFR+ cells using the FlowJo software.
To assess CAR T cell proliferation in response to stimulators, CellTrace Violet (Thermo Fisher)-labeled CAR T cells or EGFR T cells were cocultured at a ratio of 1 stimulator:1 CAR or EGFR T cell with mitomycin C-treated K562-Env or mitomycin C-treated SIV-infected CD4+ T cells or their respective negative control K562 or CD4+ T cells for 4 days at 37°C. Cells were harvested and stained with anti-EGFR, and proliferation was assessed by determining the percentage of EGFR+ cells present in peaks 3–7 of decreased CellTrace Violet fluorescence intensity after gating on lymphocytes, single cells, and EGFR+ cells using the FlowJo software.