Fig 2: Anti-PD-L1 and PBF-509 restore immune responsiveness of TILs. Four different resected NSCLC tumors were disaggregated and cultured with or without anti-PD-L1 (aPDL1; 10 mg/ml), anti-PD-1 (aPD1; 10 µg/ml), or the A2aR antagonist PBF-509 (1 µM). After 3 days in culture, the supernatants were assayed for the presence of ?-interferon using an ELISA.

Fig 3: PBF-509 is specific for the A2aR. (A) Dose response curves of PBF-509 versus 4 adenosine receptors in transfected CHO (hA1), HeLa (hA2a and hA3), and HEK-293 (hA2b) cells. (B) Representative dose–response curves of PBF-509 in a binding assay against the human A2aR in buffer (median Ki = 12 nM) and in human plasma (median Ki = 32 nM).

Fig 4: Human primary tumor, CAF, and TIL cell lines express CD73 and A2aR. Tumor (Tm), cancer-associated fibroblast (CAF), and tumor infiltrating lymphocyte (TIL) cell lines were established from 12 resected NSCLC tumors. (A) Tumor cells or CAFs were stained with anti-CD73 and subjected to flow cytometry. Most cells from nearly all of the cell lines (both tumor cells and CAFs) expressed high levels of CD73. (B) Representative experiment of tumor cells stained with anti-CD73 (blue) or isotype control (green). (C) CD4+ or CD8+ TIL cells were stained with anti-A2aR and subjected to flow cytometric analysis showing that high levels of A2aR were being expressed in CD4+ cells, with variable expression in CD8+ cells.