Tumor-associated macrophages (TAMs) express programmed cell death ligand 1 (PD-L1) and contribute to the immune-suppressive tumor microenvironment. Although the role of the PD-L1 and PD-1 interaction to regulate T-cell suppression is established, less is known about PD-L1 signaling in macrophages and how these signals may affect the function of TAMs. We used and models to investigate PD-L1 signaling in macrophages and the effects of PD-L1 antibody treatment on TAM responses. Treatment of mouse and human macrophages with PD-L1 antibodies increased spontaneous macrophage proliferation, survival, and activation (costimulatory molecule expression, cytokine production). Similar changes were observed in macrophages incubated with soluble CD80 and soluble PD-1, and in PD-L1 macrophages. Macrophage treatment with PD-L1 antibodies upregulated mTOR pathway activity, and RNAseq analysis revealed upregulation of multiple macrophage inflammatory pathways. treatment with PD-L1 antibody resulted in increased tumor infiltration with activated macrophages. In tumor-bearing RAG mice, upregulated costimulatory molecule expression by TAMs and reduced tumor growth were observed. Combined PD-1/ PD-L1 antibody treatment of animals with established B16 melanomas cured half of the treated mice, whereas treatment with single antibodies had little therapeutic effect. These findings indicate that PD-L1 delivers a constitutive negative signal to macrophages, resulting in an immune-suppressive cell phenotype. Treatment with PD-L1 antibodies reverses this phenotype and triggers macrophage-mediated antitumor activity, suggesting a distinct effect of PD-L1, but not PD-1, antibody treatment. .

Expression of programmed cell death receptor ligand 1 (PD-L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD-L1 expression by tumor cells and tumor-infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD-L1 expression and for responsiveness to immune stimuli. We found that PD-L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD-L1 expression by all tumor cell lines was observed following IFN-γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte-derived macrophages did not express PD-L1 constitutively, but did significantly upregulate expression following treatment with IFN-γ. These findings suggest that most canine tumors express PD-L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD-L1 expression. Therefore the upregulation of PD-L1 expression by tumor cells and by tumor-infiltrating macrophages in response to cytokines such as IFN-γ may represent an important mechanism of tumor-mediated T-cell suppression in dogs as well as in humans.

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 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 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. We demonstrated that tumor-conditioned media strongly induced increased PD-L1 expression by bone marrow-derived monocytes and TNF-α to be a cytokine that causes an upregulation of PD-L1 expression by the monocytes. Furthermore, we found production of TNF-α by the monocytes themselves to be a TLR2-dependent response to versican secreted by tumor cells. Thus, PD-L1 expression by tumor macrophages appears to be regulated in a different manner than by tumor cells themselves.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)–expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti–PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti–PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti–PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD-1) protein were shown to be effective for treatment of non-Hodgkin lymphoma in people, but little is known about the expression of PD-1 or its ligand PD-L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD-1 and PD-L1 in canine lymphoma, using fine-needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine-needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD-1 and PD-L1 was assessed in vitro. These studies revealed increased expression of PD-L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD-1 and PD-L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD-1 and PD-L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy-resistant BCL and TCL cell lines exhibited increases in both PD-1 and PD-L1 expression, compared to non-chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.