Figure 1.

CRISPR-Cas9-mediated TIM-3, LAG-3 or 2B4 disruption does not alter the expansion capacity nor the differentiation phenotype of TCR_ED T cells. (A) Schematic representation of the protocol for the generation of TIM-3-, LAG-3, or 2B4-disrupted TCR-edited (TCR_ED) T cells. (B) Percentages of CD3^negLAG-3^neg (green bar; N=5) or CD3^negTIM-3^neg (blue bar; N=3) live cells measured by flow cytometry in T cells electroporated with CRISPR/Cas9 targeting LAG-3 or TIM-3 in combination with CRISPR/Cas9 targeting TRAC. Unmanipulated cells (UT, black bars) are shown as control. (C) Percentages of NHEJ at LAG-3 (green bar; N=4), TIM-3 (blue bar; N=3/5), and 2B4 (light orange bar; N=3) loci in UT cells (black bar; N=3/5) and in T cells electroporated with CRISPR/Cas9 targeting the specific inhibitory receptor and the TRAC locus. (D) Frequency of cells redirected against NY-ESO-1 upon lentiviral transduction of unmanipulated cells (UT, black bar; N=8) or in T cells electroporated with CRISPR/Cas9 targeting TRAC alone (TCR_ED-IR_COMP, red bar; N=8) or in combination with gRNAs targeting LAG-3 (TCR_ED-LAG-3_KO, green bar; N=5), TIM3 (TCR_ED-TIM-3_KO, blue bar; N=3) or 2B4 (TCR_ED-2B4_KO, light orange bar; N=3). Transduction efficiency was measured by flow cytometry upon HLA-A*02:01-NY-ESO-1_157-165 dextramer staining. Fold increase (E) and memory phenotype (F) of inhibitory receptor (IR) competent (TCR_ED-IR_COMP; N=5), TCR_ED-LAG-3_KO (N=4), TCR_ED-TIM-3_KO (N=3) and TCR_ED-2B4_KO (N=3) T cells at day +21. g1, g3, g4, g9 refer to gRNAs designed to disrupt the indicated genes. (G) Meta-cluster composition of TCR_ED or TCR_ED-IR_KO T cells (left). Arrows colors indicate the number of IR co-expressed in each cluster according to the color code indicated on the right. Percentages of cells co-expressing from 2 to 9 of the IRs marked with an asterisk, in TCR_ED-IR_COMP (N=3) or TCR_ED-IR_KO (N=3) cells are shown on the right. Data are shown as mean ± SEM of 3-8 biological replicates; ns= not statistically significant.