Figure 4.

In vivo anti-tumor activity and phenotype of ARI and AWARI CAR-T cells

(A) Experimental design of in vivo anti-tumor activity and phenotypic studies of 5 × 10⁶ (purple dots, 25.1% of ARI⁺ cells and 22.3% of AWARI CAR-T cells: ∼1.26 and 1.12 × 10⁶ CAR-T cells, respectively; VCN of ARI = 17.8 c/c and AWARI 13.8 c/c) and 10 × 10⁶ (blue dots, 14.7% of ARI⁺ cells and 13.0% of AWARI⁺ cells: ∼1.47 and −1.3 × 10⁶ CAR-T cells; VCN of ARI = 3.6 c/c and AWARI = 5.8c/c) of T cells transduced with ARI and AWARI-LVs. Rechallenge (a new dose of Namalwa-GFP-Nluc was inoculated) is indicated with an arrow at day 13 in the 10 × 10⁶ dose experiment. Flow cytometry (FACS) analysis was performed after sacrifice at day 13 for the 5 × 10⁶ experiment and at day 28 for 10 × 10⁶ experiment. (B) Tumor bioluminescence (BLI) progression with 5 × 10⁶ (left) and 10 × 10⁶ (right) T cells doses at different days post-Namalwa inoculation is shown. “New PBS” indicated new untreated NSG mice inoculated with Namalwa as controls of the rechallenge. Two-way ANOVA-Tukey’s multiple comparisons are shown. ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.001. In the 10 × 10⁶ graph, ∗p < 0.001 NT vs ARI and #p <0.001 NT vs AWARI. (C) Percentage of surviving Namalwa-GFP-Nluc cells in bone marrow (BM), spleen, blood, liver, and blood determined by FACS after the sacrifice with the low (left panels) and high (right panels) doses is shown. This value is analyzed in the “singlets human cells” gate (see Materials and methods). One-tailed Mann-Whitney t test; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. N (mice) of low T dose: PBS = 3, NT = 3, ARI = 3, and AWARI = 3; and high T dose: PBS = 2, NT = 3, ARI = 8, and AWARI = 5. (D) Representative dot plots of CAR expression and phenotype of CAR⁺ cells of gated hCD3 from the spleen of ARI and AWARI mice after sacrifice of the high-dose experiment are shown. (E) Graphical representation of proportions of T_N/T_SCM (CD62L⁺CD45RA⁺) (green), T_CM (CD62L⁺CD45RA⁻) (blue), T_EM (CD62L⁻CD45RA⁻) (gray), and T_EF (CD62L⁻CD45RA⁺) (red) subpopulations in ARI (top) and AWARI (bottom) CAR⁺ cells in the indicated organs after sacrifice of challenged (outer circle) and rechallenged (inner circle) mice is shown. ∗p < 0.05 when comparing the T cells subsets of rechallenged ARI and AWARI treated mice. Mann-Whitney one-tail is shown. ++p < 0.01 and +++p < 0.001 of two-way ANOVA, Bonferroni post-test of all the population of the total mice ARI = 8 (3+5R); AWARI = 5 (2+3R). (F) Percentage of T_N/T_SCM and T_CM after sacrifice of ARI and AWARI hCD3⁺CAR⁺ cells is shown (ARI = 8; AWARI = 5; one-tailed T test). (G) Spleens from treated mice were isolated, and cell suspensions were cultured with 200 IU/mL of IL-2 in TexMACS medium and 2% P/S during 48 h. (H) Representative dot plots of CD45RA/CD62L and PD1/LAG3 populations from spleens of ARI and AWARI mice are shown. (I) Percentage of T_N/T_SCM and T_CM populations in the CD3⁺ population after 48 h in culture is shown. Two-tailed t test is shown (n = 4). (J) LAG-3 MeFI of the CD3⁺Tim3⁺PD-1⁺ cells is shown. One-tailed t test is shown (n = 4).