Figure 1 |. CAR, OT-I, and endogenous CD8⁺ TILs isolated from B16-OVA-huCD19 tumors exhibit similar phenotypes.

(a) Experimental design to assess CAR and endogenous TILs; 1.5×10⁶ CAR-T cells were adoptively transferred into C57BL/6J mice 13 days after tumor inoculation. (b) Left, representative flow cytometry plot identifying CD45.2⁺ endogenous TILs and CD45.1⁺Thy1.1⁺ CAR-TILs (Thy1.1 encoded in the CAR retroviral vector). Right, flow cytometry plots showing PD-1 and TIM3 surface expression on CD8⁺ CAR and endogenous TILs. (c) Bar graph showing the percentage of CAR and OT-I TILs in total CD8⁺ TILs. Bars show mean values with data points for 6, 5 and 11 independent experiments for CAR, OT-I and endogenous TILs respectively. (d) Quantification of cytokine production after restimulation of CAR, OT-I and endogenous CD8⁺ TILs, compared to cultured CD8⁺ CAR-T cells stimulated with PMA/ionomycin or left unstimulated. Bars show mean values with data points for 3 independent experiments. All p-values were calculated using two-tailed unpaired t-tests with Welch’s correction, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. (e) Representative flow cytometry plots of cytokine production after restimulation.