HE4 regulates anticancer immune evasion by upregulating PD-L1 expression on macrophages through the IFN-?R-STAT3 axis

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: Fresh tumor and paired adjacent non-tumor tissues were collected from two lung adenocarcinoma patients immediately after surgical resection. Tissues were rinsed with ice-cold PBS three times to remove blood clots and then minced on ice. The tissue block was incubated with a digestion solution made of 0.25% trypsin and 10 µg/mL DNase I, at 37°C with a shaking speed of 50 rpm or 30 min. Cell suspensions were filtered using a 70-µm cell strainer and then through a 40-µm strainer again, and the filtrate was collected into a new centrifuge tube. The cell pellet was resuspended in 3 ml of red blood cell lysis solution and incubated on ice for 5 min. Then, 10 ml of precooled PBS was added and centrifuged at 250 × g for 5 min. The cell pellet was resuspended in PBS containing 0.5% FBS. Cells were stained with 0.4% AO/PI to evaluate cell viability. 10 × Genomics single-cell capture, Illumina library construction, preprocessing of scRNA-seq data, integration, dimensionality reduction and clustering were performed by Capital Bio-Tech (Beijing, China).