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. 2022 May 18;25:335–349. doi: 10.1016/j.omto.2022.05.003

Figure 3.

Figure 3

AWARI CAR-T cells maintain improved properties after elimination of CD19+ cells in vitro

(A) Work flow of the in vitro cytotoxic evaluation of NT, ARI, and AWARI CAR-T cells. The analyses were performed using T cells transduced at similar efficiency (%CAR+: ARI = 27.8% ± 3.9% and AWARI = 23.6% ± 2.7%; VCN: ARI = 2.1 ± 0.8 c/c and AWARI = 5.4 ± 1c/c). (B) Levels of secreted TNF-α (left) and IFN-ɣ (right) produced by NT, ARI, and AWARI CAR-T cells after 24 h of stimulation with Namalwa at ratio 1:1 are shown (four independent donors, n ≥ 4 for TNF-α and three independent donors, n ≥ 3 for IFN-ɣ). Mann-Whitney t test is shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Lysis of CD19+ cells (Nalm6 and Namalwa) at different ratios of co-culture with ARI and AWARI CAR-T cells during 48 h is shown. Percentage of lysis is related to NT-driven, non-specific CD19+ lysis (n = 5). Non-significance in two-way ANOVA test is shown. (D) (Left) Representative CD45RA/CD62L dot plots of different ARI and AWARI CAR-T cells generated from three different donors after 48 h of co-culture with Namalwa cells at ratio 1:1 are shown. (Right) Graphical representation of the different subpopulations (TSCM in green) in bulk AWARI CAR-T cells compared with ARI CAR-T cells after 48 h in the presence of Namalwa cells is shown. Two-tailed paired T test. ns, not significant. (E) Relative expression levels of LAG-3, PD-1, and Tim3 of ARI (red) and AWARI (green) CAR-T cells relative to NT (blue) 48 h after co-culture with Namalwa cells are shown. Data represent MeFI of each marker in ARI or AWARI CAR-T cells divided by the MeFI in NT cells. One-tailed paired t test is shown (n ≥ 5). (F) Diagram of the experiment performed with the CD19+ pancreatic cellular model is shown (MiaPaCa2 CD19+). NT, ARI, and AWARI CAR-T cells were challenged with MiaPaCa2 CD19+ three times at an initial ratio of 5:1 (CAR-T:target cells) every 2 days. (G) Specific lysis of MiaPaca2 CD19+ with ARI and AWARI CAR-T cells at each challenge is shown (two independent donors; n = 4 one-tailed paired t test). (H) Percentage of TSCM + TCM + TEM of ARI (black) and AWARI (red) after the different challenges with MiaPaCa2 is shown. Multiple Tukey’s comparison is shown.