Panoply™ Human PLA2G4A Over-expressing Stable Cell Line

γδ T cells are promising candidate cells for tumor immunotherapy. However, the underlying mechanism by which γδ T cells polarize into CD39+γδ Treg cells after induction in colorectal cancer (CRC) remains unclear. This study shows that the frequency of CD39+γδ Treg cells is significantly higher in right-sided colorectal cancer (RSCRC) than in left-sided colorectal cancer (LSCRC), and is positively correlated with poor prognosis. Interestingly, CD39+γδ Treg cells in RSCRC exhibit a stronger immunosuppressive phenotype and function than those in LSCRC. Furthermore, quantitative mass spectrometry analysis indicates that the polarization of CD39+γδ Treg cells is associated with abnormal activation of the phospholipase α2-IVa/arachidonic acid (PLA2G4A/AA) metabolic pathway in RSCRC. Researchers used an in vitro co-culture system and an orthotopic mouse colorectal cancer model to find that overexpression of Pla2G4a in CT26 cells can induce the production of CD39+γδ regulatory T cells (Tregs), thereby suppressing anti-tumor immune responses. Furthermore, they found that the overall survival of the PLA2G4Ahi group was significantly shortened compared with PLA2G4Alo RSCRC, while the survival of LSCRC showed the opposite. In conclusion, abnormal PLA2G4A expression in RSCRC can induce γδ T cell differentiation into CD39+γδ Tregs, thereby promoting tumor progression and metastasis.

To elucidate whether PLA2G4A overexpression in cancer cells activates the adenosine pathway in γδ T cells, researchers constructed a Pla2g4a-overexpressing CT26 cell line and validated this using quantitative PCR (qPCR) and Western blot. Pla2g4a-overexpressing CT26 cells and control cells were then co-cultured with intestinal intraepithelial lymphocytes (IELs) derived from the ileum (IL-IELs), colon (CO-IELs), or spleen of BALB/c mice. Cell status was observed using fluorescence microscopy at 12 and 36 hours in the co-culture system. The results showed that both IELs and spleen cells significantly damaged control cells, while Pla2g4a-overexpressing CT26 cells suppressed the anti-tumor immune response (Figure 1A-D). Furthermore, lymphocytes were collected at 12 and 36 hours, and CD39 expression on γδ T cells was detected by flow cytometry. As expected, the vast majority of γδ T cells co-cultured with CT26-Pla2g4a were CD39 positive (Figure 1E-G). Therefore, the frequency of CD39+γδ Tregs increased 3.8-fold at 12 hours compared to the control group (Figure 1H). Similarly, researchers observed similar results at 36 hours.

Figure 1. Overexpression of Pla2g4a in CT26-induced CD39+γδ Tregs in vitro. (Zhan Y, et al., 2021)