HE4 regulates anticancer immune evasion by upregulating PD-L1 expression on macrophages through the IFN-?R-STAT3 axis [LLC tumors, scRNA-seq]

Current immune checkpoint inhibitors (ICIs) targeting PD-L1/PD-1 have achieved great success in clinical cancer treatment. However, it still faces great challenges, such as a low overall response rate and immune-related adverse events (irAEs). PD-L1 is transcriptionally induced by immune cell-secreted cytokines, such as IFN-?, in the tumor microenvironment (TME), but whether the expression of PD-L1 is regulated by cancer cell-derived molecules is still unclear. Here, we show that HE4 is another critical transcriptional regulator of PD-L1 on macrophages in the TME through the IFN-?R1/IFN-?R2-JAK-STAT3 signaling pathway and that blocking mouse or human HE4 with monoclonal neutralizing antibodies has a significant therapeutic effect on multiple mouse or human cancers by downregulating PD-L1 expression on macrophages and promoting CD8+ T-cell activation in the TME. Furthermore, we showed that the expression level of HE4 is high in multiple human cancer samples and that the level of HE4 is correlated with the efficiency of anti-PD-1 immunotherapy in human adenocarcinoma patients. Together, we not only identify a critical molecule and mechanism regulating the expression level of PD-L1 on myeloid cells in the TME but also identify a cancer-derived therapeutic target upstream of PD-L1 for immunotherapy, which may be more specific and has fewer irAEs. Overall design: Subcutaneous LLC tumors were harvested on the day after the last administration of the mAbs. Tissues were minced on ice and then digested at 37°C with a shaking speed of 50 rpm or 30 min. Cell suspensions were filtered through a 40-µm strainer, and the cell pellet was resuspended in red blood cell lysis solution and incubated on ice for 5 min. The cell pellet was washed and resuspended in PBS containing 0.5% FBS, followed by the evaluation of cell viability. The samples were then sent to Majorbio Biopharm Technology Co., Ltd. (Shanghai, China), for further sequencing and bioinformatic analysis. Briefly, library construction was performed according to the manufacturer's protocol (DNBelab C TaiM4 V3.0) and quantified via a high-sensitivity DNA chip on a Bioanalyzer 2100 and a Qubit high-sensitivity DNA assay (Thermo Fisher Scientific). Libraries were sequenced on the MGI DNBSEQ-T7 platform (MGI-Tech, Shenzhen, China).