Figure 4. A_2AR^–/– CD8⁺ CAR T cells exhibit enhanced cytokine production in vivo when combined with PD-1 blockade.

C57BL/6-HER2 mice were injected with 1 × 10⁵ E0771-HER2 tumor cells and treated per Figure 2. On day 6 after T cell injection, TILs and splenocytes were isolated. (A) CD45.2⁺CD3⁺CD8⁺ T cells were FACS sorted 6 days after injection and lysed. IFN-g mRNA was determined by qPCR. Data are expressed relative to IFN-g mRNA content of splenic CD8⁺ T cells. Samples from 3–5 mice were pooled, and qPCR was run in triplicate. (B) TILs were restimulated with PMA (20 ng/ml) and ionomycin (1 μg/ml) for 3 hours in the presence of GolgiPlug/GolgiStop. (B–D) Proportion of CD3⁺CD8⁺ (B and C) or CD3⁺CD4⁺ (D) cells that were IFN-γ⁺, TNF-α⁺, or IFN-γ⁺TNF-α⁺. ICS, intracellular cytokine staining. (C) Representative FACS plots gated on CD3⁺CD8⁺ TILs. (E and F) Proportion (E) and absolute number (F) of splenic CD8⁺ or CD4⁺ cells expressing CD44. (B, D, E, and F) Data are shown as the mean ± SEM for at least 5 mice. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA.