PROGNOSTIC ROLE OF PD-L2 AND TRPV2 IN HUMAN ENDOMETRIAL CANCER AND NOVEL INTEGRATIVE THERAPIES

Programmed death-1 (PD-1, CD279) receptor and its ligands, programmed death ligand 1 (PD-L1, CD274, B7-H1) and programmed death ligand 2 (PD-L2, CD273, B7- DC), play crucial roles in one of the immune checkpoint pathways responsible for the inhibition of T- cell activation (1). PD-1 receptor belongs to the CD28 family and is mainly expressed on the cellular surface of activated T and B cells, monocytes, natural killer (NK), and dendritic cells (DCs), with a role in the induction and maintenance of peripheral tolerance and for the maintenance of the stability and the integrity of T cells (2–5). PD-1 ligands are glycoproteins, members of the B7 family, with 40% homology in amino acids sequence, but have quite distinct expression patterns, being expressed by a wide variety of immune and non-immune cells (1, 3, 4). PD-L1 is a type I transmembrane glycoprotein with a single N-terminal immunoglobulin variable (IgV)-like domain sharing 21–33% sequence identity with CTLA-4, CD28, and ICOS, about 20 amino acids that separate the IgV domain from the plasma membrane, a transmembrane domain and a cytoplasmic tail (4). It is constitutively expressed on activated T and B cells, DCs, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells (4, 6). Additionally, it is expressed on a wide variety of non-hematopoietic cells including the vascular endothelium, fibroblastic reticular cells, keratinocytes, lung, non- parenchymal cells of the liver, mesenchymal stem cells, pancreatic islet cells, astrocytes, and neurons (4, 5, 7). PD-L1 expression on human T cells is induced by common γ chain cytokines (IL-2, IL-7, and IL-15), whereas PD-L1 expression on B cells is stimulated by IL- 21 (4). In cancer cells, PD-L1 expression is regulated by the MAPK and PI3K/AKT pathways, as well as by HIF-1α, STAT-3, NF-κB and epigenetic mechanisms via microRNAs (8). PD-L1 also exists in a soluble form (sPD-L1) that originates from the cleavage of membrane-bound PD-L1 by matrix metalloproteinases. Such PD-L1 soluble isoform, mainly produced by myeloid-derived cells, retains the IgV-like domain, necessary for the interaction with PD-1, and it is able to suppress T-cell activation. However, its physiological role is still unknown. Interestingly, sPD-L1 has been found in several human 2 cancer cell lines, including H1299 non-small cell lung cancer cells, U-937 lymphoma cells, HO8910 ovarian carcinoma cells, SPCA-1 lung adenocarcinoma cells and U251 glioblastoma cells. In addition, high plasma levels of sPD-L1 have been associated with metastasis and poor prognosis in breast cancer and diffuse large B-cell lymphoma (8). PD-L2 is a type I transmembrane protein containing an IgV-like domain and an immunoglobulin constant (IgC)-like domain in its extracellular region (9). PD-L2 expression is mainly restricted to antigen-presenting cells (APCs), including macrophages and myeloid DCs (6, 7), and non-hematopoietic tissues, such as the lung (10), human umbilical vein endothelial cells, and fibroblasts (1, 5). Three isoforms of PD-L2 have been described that might influence the outcome of the immune response (9). The most common splice variant contains all 6 exons. In humans, an alternative variant with a spliced-out exon 3, resulting in a protein lacks the IgC-like domain and with a shorter—extracellular region has been reported. A third isoform misses the transmembrane domain, because exon 3 is spliced out to an alternative acceptor site within exon 4, and the protein is secreted as a soluble form. This evidence underscores the importance of post-transcriptional regulation in the expression and function of PD-L2. He et al. suggested that isoforms II and III should be able to interact with PD-1, but further confirmation is needed (9). Exposure to IL-4, Interferon-gamma (IFN-γ), IL-2, IL-7, IL-15, IL-21, and toll-like receptor ligands induces PD-L2 upregulation in DCs and macrophages (1). Additionally, IL-4, in the presence of respiratory syncytial virus infection, stimulates PD-L2 expression in alveolar epithelial cells (1, 10). Stimulation by tumor necrosis factor alpha (TNF-α) and IFN-γ enhances the constitutive expression of PD-L2 on endothelial cells from human umbilical vein in vitro (1). The NF-κB and the STAT-6 pathways are two major signaling reported to regulate PD-L2 expression (1). Different molecular mechanisms dictate PD-Ls binding to PD-1, as demonstrated by the crystallographic structures of the complexes, showing that PD-Ls cross-compete and that the concurrent presence of both ligands might modify the functional outcome of the binding (11). Specifically, PD-L1 binding to PD-1 requires complex conformational changes of the ligand, while PD-L2 directly interacts with PD-1, explaining its reported 2 to 6-fold higher affinity for the receptor (1). Consequently, when both ligands are expressed at similar levels, PD-L2 would be expected to outcompete PD-L1 for binding to PD-1. However, PD-L2 is generally expressed at lower levels in physiological conditions, such as during maturation of DCs by LPS, when PD-L1 acts as the main ligand of PD-1. A known exception is Th2 responses, where PD-L2 is predominant (1, 11). Regarding the PD-1/PD-L1 and PD-1/PD-L2 pathways involved in T cell immune evasion, different reports have been published, mainly regarding the biochemical signaling regulated by the PD-1/PD-L1. It was reported that the binding of PD-L1 to PD-1 may cause T cell apoptosis, anergy, exhaustion, and interleukin-10 (IL-10) expression, suggesting that PD-L1 can act as a defender for PD-L1+ cancer cells from CD8+ T cell–mediated lysis (12, 13) Regarding the PD-L2/PD-1 signaling pathways, it may not be biologically identical, since Repulsive Guidance Molecule B (RGMb) is also a binding partner for PD-L2 (14). Thus, the PD-L2 blockade may evocate different cellular responses, depending on the binding partner interaction, which can lead to potential varied biological outcomes. Up to now, in human anti-tumor immunity, the relationship between PD-1, PD-L1, and PD-L2 in their cellular expression profile and regulation, potential interactions and biological is considered not completely defined.