Tumor cell-intrinsic programmed death protein 1 expression and induction in human cancer cell lines

Abstract

Abstract The programmed death protein 1 (PD-1) and ligand (PD-L1) axis inhibits T cell activation and reduces anti-tumor immunity. Most studies of the PD-1/PD-L1 axis test extrinsic, immune-dependent effects. However, tumor cell-intrinsic PD-L1 regulates immune-independent tumor mTOR signals, autophagy, and tumor initiating cell generation. Melanoma cell-intrinsic PD-1 regulates melanoma growth and mTOR. We tested PD-1 expression and induction in multiple human tumor cell lines. We detected PD-1 in human cell lines in vitro from ES2 ovarian cancer, VMM122 melanoma, and RT4 bladder cancer by flow cytometry, Western blot and RT-PCR. All lines were negative for the other PD-L1 receptor, CD80, by flow cytometry. Incubating lines with IFNα or IFNγ for 48 hours further augmented PD-1 expression as detected by flow, Western blot and RT-PCR. αPD-1 antibody (50 μg/ml) reduced proliferation of RT4 bladder cancer cells in vitro, demonstrating a direct PD-1 signal effect in these cells, and αPD-L1 similarly reduced in vitro proliferation. Interestingly, anti-PD-1 effects on reducing proliferation were similar: 41.5% reduction; p<0.0001. We used CRISPR/Cas9 to generate PD-L1KO RT4 cells. αPD-L1 did not reduce in vitro proliferation as expected, but unexpectedly, αPD-1 also had no anti-proliferative effect on PD-L1KO RT4 cells, suggesting a PD-L1/PD-1 interaction. In murine B16 melanoma, we generated PD-L1KO and PD-1KO cells using CRISPR/Cas9. Cell-intrinsic PD-L1 and PD-1 downregulate chemokine production. Among chemokines analyzed, PD-L1 suppressed CCL2 and CCL5, PD-1 suppressed CCL4 and CCL7, and both suppressed CCL3 and CXCL2. Understanding intrinsic PD-1 and PD-L1 effects on tumor cells can be exploited to improve cancer immunotherapy