Fig 2: Combination of PD-L1 blockade and CAR T cell therapy depletes M2 macrophages via IFN-γ signaling. (A, B) Analysis of M2 macrophages in the prostate cancer immune-suppression assay in the presence or absence of PD-L1 blockade. (C, D) Analysis of M2 macrophages stimulated with PSCA-CAR T cell-derived CM in the presence or absence of PD-1 or PD-L1 blockade. (E–G) Representative brightfield images and analysis of M2 macrophage stimulated with PSCA-CAR T cell-derived CM in the presence or absence of PD-L1 and/or IFN-γR1 blockade. The number of total viable M2 macrophages (A, C, F) and the frequency and number of CD163+ M2 macrophages (B, D, G) were evaluated by flow cytometry. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; CM, conditioned media; IFN, interferon; PD-L1, programmed death ligand-1; PD-1, programmed cell death protein-1; PSCA, prostate stem cell antigen; UTD, untransduced.

Fig 3: PD-L1 blockade restores CAR T cell function in the presence of suppressive M2 macrophages. CAR T cell function was evaluated in the prostate cancer immune-suppression assay in the presence of PD-L1 blockade. (A) Flow cytometry plots indicating the number of viable DU145-PSCA tumor cells in each condition in the presence or absence of anti-PD-L1 antibody, atezolizumab (Atezo). (B–D) Quantification of PSCA-CAR T cell-mediated killing of DU145-PSCA tumor cells (B), T cell activation (C), and IFN-γ secretion (D). (E) DU145-PSCA tumor cell killing of CAR T cells in the presence or absence of two clinically approved anti-PD-L1 antibodies, Atezo and avelumab (Ave), and Ave that lacks CH2 domain (Ave (ΔCH2)). Tumor killing and T cell activation were evaluated by flow cytometry, and IFN-γ secretion was evaluated by ELISA. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; IFN, interferon; PD-L1, programmed death ligand-1; PSCA, prostate stem cell antigen; UTD, untransduced.