Isolation and culture of allogeneic human T cells

Human T cells were isolated from leukapheresis products (Leukopaks, HemaCare) by positive selection using CD4 and CD8 MicroBeads (Miltenyi,130045101 and 130045201). T cells were frozen at 25–50 × 10^6 cells per ml of Cryostor CS10 (Stemcell Technologies, 1001061).

For editing experiments, T cells were thawed in a water bath at 37 °C and then allowed to rest overnight in ImmunoCult-XF T Cell Expansion Medium containing (Stemcell Technologies, 10981) 5% CTS Immune Cell SR, Glutamax, 10 mM HEPES, 1% Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122). The next day, T cells were activated using 25 µl of ImmunoCult Human CD3/CD28/CD2 T Cell Activator (Stemcell Technologies, 10970) per ml of cells at 1 × 10^6 cells per ml plus 300 IU/ml of IL-2 (CellGenix, 1420050). Fresh IL-2 was added to T cells every 2–3 d. T cells were cultured at 37 °C and 5% CO2.

Electroporation of human T cells

T cells were transfected 72 h after activation. Cells were resuspended in P3 Primary Cell Nucleofector Solution containing Supplement 1 (Lonza, V4SP-3960). 1 × 10^6 T cells were edited with 1 µg of synthetic sgRNA (IDT) and 2 µg of editor mRNA in a total volume of 20 µl using P3 96-well Nucleocuvette kit (Lonza, V4SP-3960). The three sgRNAs used are as follows: B2M Exon 2 (B2M Ex.2), pmSTOP C6, CD247 pomSTOP C7 and PD-1 Ex.1 SA C7 are specified in Supplementary Table 3. T cells were electroporated with the 4D-Nucleofector system (Lonza, AAF-1003B and AAF-1003S) using program DH-102. All experiments were performed with two independent T cell donors. For NGS analysis, 1×10^5 T cells per condition at each timepoint were pelleted, supernatant was removed and pellets were resuspended in 50 ml of QuickExtract DNA Extraction buffer (Lucigen, QE09050) and transferred to a PCR plate for targeted amplicon sequencing.