Abstract
BackgroundThe PD-1/PD-L1 checkpoint is a central mediator of immunosuppression in the tumor immune microenvironment (TME) and is primarily associated with IFN-g signaling. To characterize other factors regulating PD-L1 expression on tumor and/or immune cells, we investigated TME-resident cytokines and the role of transcription factors in constitutive and cytokine-induced PD-L1 expression.MethodsThirty-four cultured human tumor lines [18 melanomas (MEL), 12 renal cell carcinomas (RCC), 3 squamous cell carcinomas of the head and neck (SCCHN), and 1 non-small-cell lung carcinoma (NSCLC)] and peripheral blood monocytes (Monos) were treated with cytokines that we detected in the PD-L1+ TME by gene expression profiling, including IFN-g, IL-1a, IL-10, IL-27 and IL-32g. PD-L1 cell surface protein expression was detected by flow cytometry, and mRNA by quantitative real-time PCR. Total and phosphorylated STAT1, STAT3, and p65 proteins were detected by Western blotting, and the genes encoding these proteins were knocked down with siRNAs. Additionally, the proximal promoter region of PDL1 (CD274) was sequenced in 33 cultured tumors.ResultsPD-L1 was constitutively expressed on 1/17 cultured MELs, 8/11 RCCs, 3/3 SCCHNs, and on Monos. Brief IFN-g exposure rapidly induced PD-L1 on all tumor cell lines and Monos regardless of constitutive PD-L1 expression. PD-L1 mRNA levels were associated with protein expression, which was diminished by exposure to transcriptional inhibitors. siRNA knockdown of STAT1 but not STAT3 reduced IFN-g- and IL-27-induced PD-L1 protein expression on tumor cells. In contrast, STAT3 knockdown in Monos reduced IL-10-induced PD-L1 protein expression, and p65 knockdown in tumor cells reduced IL-1a-induced PD-L1 expression. Notably, constitutive PD-L1 expression was not affected by knocking down STAT1, STAT3, or p65. Differential effects of IFN-g, IL-1a, and IL-27 on individual tumor cell lines were not due to PDL1 promoter polymorphisms.ConclusionsMultiple cytokines found in an immune-reactive TME may induce PD-L1 expression on tumor and/or immune cells through distinct signaling mechanisms. Factors driving constitutive PD-L1 expression were not identified in this study. Understanding complex mechanisms underlying PD-L1 display in the TME may allow treatment approaches mitigating expression of this immunosuppressive ligand, to enhance the impact of PD-1 blockade.
Highlights
The PD-1/programmed death-ligand 1 (PD-L1) checkpoint is a central mediator of immunosuppression in the tumor immune microenvironment (TME) and is primarily associated with IFN-g signaling
Activation of ALK/STAT3 in T cell lymphoma [5], AP-1/JAK/signal transducer and activator of transcription (STAT) in classical Hodgkin lymphoma [6], the microRNA-200/ZEB1 axis in non-small-cell lung cancer (NSCLC) [7], c-jun/STAT3 in BRAF inhibitorresistant melanoma [8], and PI3K in glioma [9] have each been reported to upregulate PD-L1 expression on tumor cells
STAT1 and STAT3 play distinct roles in cytokine-induced PD-L1 expression on monocytes We have previously reported that IL-1a, IL-10, IL-27 and IL-32 g each increase PD-L1 protein expression on normal human Monos in short-term culture [13, 14]
Summary
The PD-1/PD-L1 checkpoint is a central mediator of immunosuppression in the tumor immune microenvironment (TME) and is primarily associated with IFN-g signaling. To characterize other factors regulating PD-L1 expression on tumor and/or immune cells, we investigated TME-resident cytokines and the role of transcription factors in constitutive and cytokine-induced PD-L1 expression. Understanding how TME-resident cytokines and signaling pathways regulate PD-L1 expression may provide therapeutic opportunities to mitigate PD-L1-induced intratumoral immunosuppression [3]. Innate resistance refers to constitutive PDL1 expression on tumor cells, resulting from PDL1 gene amplification or aberrant activation of oncogenic signaling pathways. Myc has been shown to regulate constitutive PD-L1 expression at the mRNA level in multiple tumors, such as T cell acute lymphoblastic leukemia, melanoma and NSCLC [10]. Post-transcriptional regulation of PD-L1 has attracted attention, with reports that cyclin-dependent kinase-4 (CDK4) and glycogen synthase kinase 3 beta (GSK3B) can promote PD-L1 protein degradation in cultured tumors [11, 12]
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