Analysis of PD-L1 Transcriptional Activity by Chromatin Immunoprecipitation.

Immune checkpoint PD-L1 was originally identified as a cell surface transmembrane protein, but recent studies have demonstrated its presence also in the nucleus and suggested its role in transcriptional regulation. Interleukin-8 (IL-8, CXCL8) is a pro-angiogenic chemokine that promotes cancer progression. We have recently shown that in ovarian cancer (OC) cells, IFNγ induces nuclear accumulation of PD-L1, which is then recruited to IL-8 promoter, resulting in increased transcription of IL-8. Since the increased expression of IL-8 induces proliferation and invasion in OC cells, understanding the role of PD-L1 in transcriptional regulation of IL-8 is important for increasing the effectiveness of PD-L1 targeting immunotherapies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) followed by real time PCR to quantitatively measure PD-L1 recruitment to IL-8 promoter. The main points of the protocol are the ChIP analysis of PD-L1 recruitment to IL-8 promoter using antibody that specifically recognizes endogenous PD-L1, and quantitative real time PCR using primers for human IL-8 promoter spanning the transcription start site (TSS) and a region ~500 bp upstream of the TSS. Our results show that IFNγ significantly increases PD-L1 recruitment to TSS of IL-8 promoter and this recruitment is even higher to the ~500 bp upstream region containing multiple transcription factors binding sites, suggesting that PD-L1 associates with IL-8 promoter by binding to other transcription factors.