PT  - JOURNAL ARTICLE
AU  - Shuming Chen
AU  - George A. Crabill
AU  - Theresa S. Pritchard
AU  - Tracee L. McMiller
AU  - Ping Wei
AU  - Drew M. Pardoll
AU  - Fan Pan
AU  - Suzanne L. Topalian
TI  - Mechanisms regulating PD-L1 expression on tumor and immune cells
AID  - 10.1186/s40425-019-0770-2
DP  - 2019 Dec 01
TA  - Journal for ImmunoTherapy of Cancer
PG  - 305
VI  - 7
IP  - 1
4099  - http://jitc.bmj.com/content/7/1/305.short
4100  - http://jitc.bmj.com/content/7/1/305.full
SO  - J Immunother Cancer2019 Dec 01; 7
AB  - Background 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.Methods Thirty-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.Results PD-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.Conclusions Multiple 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.George A. Crabill and Theresa S. Pritchard contributed equally to this work.Presented in part at the SITC 33rd Annual Meeting, Washington DC, November 10, 2018Abbreviations:ActDactinomycin DcHLclassical Hodgkin lymphomaCHXcycloheximideEBVEpstein-Barr virusMELmelanomaMonosmonocytesNSCLCnon-small-cell lung carcinomaPD-L1programmed death-ligand 1qRT-PCRquantitative reverse transcriptase polymerase chain reactionRCCrenal cell carcinomaSCCHNsquamous cell carcinoma of head and neckSTATSignal transducer and activator of transcriptionTMEtumor microenvironment