Background and Significance: PD-L1 is an immune checkpoint protein that has emerged as a major signaling molecule involved with tumor escape from T cell immune responses. Studies have shown that intra-tumoral expression of PD-L1 can inhibit antitumor immune responses. However, it has recently been shown in a study using human patient tumor samples that expression of PD-L1 on myeloid cells from the tumor is a stronger indicator of prognosis than tumor cell PD-L1 expression. Therefore, it is important to understand the factors that govern the regulation of PD-L1 expression on tumor-infiltrating myeloid cells. We initially investigated the role of IFN-γ in regulating macrophage PD-L1 expression using the murine B16 model and found that IFN-γ did not affect PD-L1 expression by tumor macrophages in this model. Thus, we conducted studies to identify other cytokines that may play an important role in regulating PD-L1 expression on myeloid cells in the tumor microenvironment.
Methods: In our preliminary studies, IFN-γ knockout and wild-type B57BL/6 mice were inoculated with B16 melanoma cells and tumor tissue was harvested and stained for flow cytometric analysis of PD-L1 expression on tumor and myeloid cells. In vitro cell staining experiments were conducted using immature bone marrow monocytes (CD11b+/ Ly6C+/Ly6G-), circulating blood monocytes (CD11b+/F4-80+/ Ly6C+/Ly6G-), and macrophages in tumor tissue (CD11b+/F4-80+) that were stained for baseline expression levels of PD-L1. Bone marrow-derived monocytes were then co-cultured with B16 tumor cells, B16 supernatants, or stimulated with recombinant cytokines for flow cytometric analysis to detect changes in PD-L1 expression. The supernatants from co-cultured cells were also collected for identification and quantification of cytokines by ELISA.
Results: We found that immature bone marrow monocytes in tumor-bearing mice had low levels of PD-L1 expression, while higher levels of expression were observed on monocytes in circulation. In contrast, macrophages found in tumor tissues expressed much higher levels of PD-L1 than circulating monocytes, implying upregulation by the tumor microenvironment. Using in vitro co-culture systems, we demonstrated co-culture with tumor cells strongly induced PD-L1 upregulation by bone marrow-derived monocytes, and that this effect was mediated by a soluble factor. Studies are currently in progress to identify the cytokines secreted by tumor cells that are responsible for PD-L1 upregulation.
Conclusion: Cytokines other than IFN-γ are responsible for upregulation of PD-L1 expression by tumor-associated macrophages, but in vitro and in vivo. Thus, PD-L1 expression by tumor macrophages appears to be regulated in a different manner than by tumor cells themselves.
Citation Format: Genevieve Hartley, Dan Regan, Amanda Guth, Steven Dow. Cytokine regulation of PD-L1 expression on tumor-associated myeloid cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B180.
- ©2016 American Association for Cancer Research.