Figure 2.

Pdcd1 can be efficiently disrupted in CAR T cells using Cas9 ribonucleoproteins (Cas9 RNPs). (a) Schematic of protocol for combined CRISPR gene editing and lentiviral transduction of human primary T cells. (b) Efficient PD-1 deletion and CAR transduction in primary human T cells. PD-1 surface staining and CAR transduction were assessed 48 hours post editing. A >50% reduction in PD-1+ cells was routinely observed, with CAR transduction >70%. Right panel, individual dots represent independent editing experiments. (c) PD-1 edited CAR T cells are stable in culture. Resting PD-1 edited CD8+ anti-CD19 CAR T cells were re-stimulated with CD19+ K562 cells. Activation was measured by CD69 induction and percent reduction in PD-1+ cells measured by flow cytometry based on surface PD-1 expression; percent reduction of PD-1+ cells was similar to that observed 48 hours after editing (rightmost panel, pairwise plot).