Fig. 4: KO of inhibitory receptor NKG2A (KLRC1) boosts anti-AML activity of CAR33-KLRC1^ko-NK cells in vitro.

a Short-term (4 h) and long-term (24 h) flow cytometry-based killing assay of NK cells against CD33⁺/HLA-E⁺ OCI-AML2 cells at indicated E:T ratios (n = 5). Mean ± SD. b Dynamic monitoring of CAR33-KLRC1^ko-NK-mediated killing using an IncuCyte-S3 imager. CAR33-KLRC1^ko-NK cells were co-cultured with GFP⁺ OCI-AML2 cells for 26.5 h and the fluorescence emission was measured over time (OCI-AML2 only condition n = 3; rest n = 4). Median + range. c Representative images taken after 24.5 h of IncuCyte analysis of NT-NK, KLRC1^ko-NK, CAR33-NK and CAR33-KLRC1^ko-NK cells co-cultured with GFP⁺ OCI-AML2 cells (E:T = 0.5:1). Viable tumor cells are shown in green based on their GFP expression. Apoptotic tumor cells are labeled red by Annexin V staining. For imaging, the IncuCyteS3 platform was used. d Caspase-cleavage in survived and sorted OCI-AML2 cells following 2 h NK cell co-culture was analyzed using western blot (one representative experiment of 3 is shown; “clvd” = “cleaved”, “ctrl” = “control”). e qPCR gene expression analysis of NT-, KLRC1^ko-, CAR33- and CAR33-KLRC1^ko-NK cells following 2 h co-cultured with OCI-AML2 cells (E:T = 3:1) (n = 5). Mean of technical triplicates ± SD. Statistical analysis was performed by two-way ANOVA (a), paired Student’s t test (b), paired Wilcoxon (e). The entity of n is biological replicates (from different healthy donors) (a, b, e).