•PD-L1 is primarily regulated by interferon gamma signaling in melanoma cells
•PD-L2 is regulated by both interferon beta and gamma signaling
•Regulation of PD-1 ligands works mainly through the JAK1/2-STAT1/3-IRF1 axis
PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.
The signaling pathway resulting in adaptive expression of PD-L1 and PD-L2 upon exposure to interferons is of high importance for the clinical development of PD-1 blockade therapies for cancer. Upon tumor antigen recognition by T cells, the released interferons trigger the inducible expression of PD-L1 by cancer cells or other tumor microenvironment cells, thereby inhibiting the antitumor immune response in a process known as adaptive immune resistance. Adaptive immune resistance allows the specific inhibition of T cell recognition of cancer while it spares the rest of the immune responses to other antigens, avoiding a systemic immune-suppressive state (Pardoll, 2012, Ribas, 2015). Interferons were first described in the 1950s as agents that interfere with viral replication (Isaacs and Lindenmann, 1957), and signaling from the interferon receptors has been well characterized (Domanski and Colamonici, 1996, Novick et al., 1994, Velazquez et al., 1992). Janus kinase (JAK) and signal transducer and activators of transcription (STAT) are the main signaling pathways mediating interferon-induced gene expression (Darnell et al., 1994, Velazquez et al., 1992) and resulting in the activation of interferon-stimulated response elements (ISREs) (Darnell et al., 1994, Kessler et al., 1988) and gamma interferon activation sites (GASs) (Decker et al., 1991, Lew et al., 1991). There is a renewed interest in interferon signaling given its key role in regulating PD-1 ligand expression and emerging evidence of its role in primary and acquired resistance to immune checkpoint blockade therapy for cancer (Shin et al., 2017, Gao et al., 2016, Zaretsky et al., 2016, Pardoll, 2012).
Type I interferons (alpha, beta, and omega) bind to interferon receptor type 1, which is composed of two subunits, IFNAR1 and IFNAR2, and they signal through JAK1 and TYK2, which phosphorylate STAT1, STAT2, and STAT3, as well as other STAT family members, depending on the cellular context. Activated phosphorylated STAT1 (pSTAT1) typically dimerizes with pSTAT2 to form the ISGF3 complex together with the interferon regulatory factor 9 (IRF9) (Smith et al., 2005). This complex binds at the genomic level to the ISRE sequences to control a long list of interferon-induced genes (Friedman and Stark, 1985). Type I interferons can also trigger phosphorylation and subsequent activation of homo- or hetero-dimers of STAT1, STAT3, STAT4, STAT5, and STAT6.
Type II interferon gamma binds to the interferon gamma receptor, leading to phosphorylation of JAK1 and JAK2, with receptor phosphorylation followed by receptor attachment and phosphorylation of STAT1 in most cells and STAT3 in some cells. The activated dimers then accumulate in the nucleus to act as transcription factors (Schroder et al., 2004, Aaronson and Horvath, 2002). There, they bind to the GAS elements present in most interferon gamma inducible genes, such as the IRF1 gene (Platanias, 2005). Negative regulators of interferon signaling, such as the suppressor of cytokine signaling protein family (SOCS; mostly SOCS1 and SOCS3) are involved in negative feedback regulation of cytokines that signal mainly through JAK2 binding, thereby modulating the activity of both STAT1 and STAT3 (Qing and Stark, 2004).
PD-1 has two known ligands, PD-L1 (CD274 or B7-H1) and PD-L2 (CD273 or B7-DC), and both have been reported to be expressed on cell surfaces upon exposure to interferons, in particular interferon gamma (Kim et al., 2005, Dong et al., 2002, Tseng et al., 2001). Evidence has been generated for the role of STAT1 and STAT3, as well as the downstream transcription factor IRF1, in regulating the surface expression of PD-L1 upon interferon gamma exposure (Lee et al., 2006, Loke and Allison, 2003). However, there has not been a systematic analysis of the molecules involved in this signal transduction pathway. Given the importance of this process, we undertook a detailed analysis of molecules responsible for interferon receptor signaling and mapping of the PD-L1 and PD-L2 promoters to define the specific signaling that regulates their expression. Our studies demonstrate the key roles of signaling through the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis, resulting in binding of the IRF1 transcription factor to the PD-L1 promoter and weaker binding to the PD-L2 promoter, which is also regulated by STAT3 in melanoma cells.