Human PDCD1LG2 Knockout Cell Line-786-O

The role of the PD-1/PD-L1 axis in immune escape in multiple cancers has been intensively studied. However, the potential functions of programmed cell death receptor 1 ligand 2 (PDCD1LG2, also known as PD-L2, B7-DC) remain unclear. Here, researchers demonstrate that PD-L2 is primarily expressed in exosomes localized on the surface of clear cell renal cell carcinoma (ccRCC) cells. Tumor cell-derived exosomal PD-L2 (TDE-PD-L2) exhibits higher expression than TDE-PD-L1 in multiple cancers. In the absence of adaptive immunity, TDE-PD-L2 inhibits tumor growth and metastasis. In the presence of normal immune function, TDE-PD-L2 is hijacked by immune cells in a PD-1-dependent manner to systematically dampen the function of T cells via the increased proportion of regulatory T cells and the decreased proportion of cytotoxic CD8+ T cells in both tumor-infiltrating T cells and spleen. Antibodies targeting PD-L2 can restore the effects of TDE-PD-L2 on tumors. In conclusion, these studies demonstrate that the PD-1/TDE-PD-L2 axis systemically suppresses T-cell function, which represents a potential therapeutic strategy for ccRCC.

Here, the researchers used immunohistochemistry (IHC) to detect PD-L2 expression in ccRCC tissue chips and found that the expression pattern of PD-L2 had unexpected changes, with increased extracellular expression of PD-L2 in cancer tissues and higher cellular localization of PD-L2 in paracancerous tissues (Figure 1A), indicating extracellular PD-L2 may regulates progression of ccRCC. Then, they used CRISPR-Cas9 technology to construct two independent PD-L2 knockout (KO) cell lines, which were further verified by immunoblotting (Figure 1B). Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) showed that there were no significant differences in exosome secretion and morphology between wild-type (WT) and KO cells (Figure 1C). The researchers used enzyme-linked immunosorbent assay (ELISA) to detect the content of TDE-PD-L2 and found that the expression of PD-L2 on the surface of exosomes originated from WT cells, not PD-L2 KO cells (Figure 1D). In addition, immunofluorescence (IHF) staining of PD-L2 under NTA fluorescence field confirmed the expression of PD-L2 on the surface of exosomes (Figure 1E). Ultrafiltration combined with iodixanol density gradient centrifugation further confirmed that PD-L2 was mainly associated with the exosome fraction, rather than the free fraction and MV fraction (Figure 1F). In addition, INF-γ and IL-4 upregulated the expression of cellular and exosomal PD-L2 in cancer cells. These data indicate that TDE-PD-L2 has the same membrane orientation as cell surface PD-L2, and its extracellular domain is exposed on the outer surface of exosomes.

Figure 1. PD-L2 is expressed on exosome surface. (Liu T, et al., 2024)