Abstract
Two major phospholipase D (PLD) isozymes in mammals, PLD1 and PLD2, hydrolyze the membrane phospholipid phosphatidylcholine to choline and the lipid messenger phosphatidic acid. Although their roles in cancer cells have been well studied, their functions in tumor microenvironment have not yet been clarified. Here, we demonstrate that PLD2 in cytotoxic CD8+ T cells plays a crucial role in anti-tumor immunity by regulating their cell proliferation. We found that growth of tumors formed by subcutaneously transplanted cancer cells is enhanced in Pld2-knockout mice. Interestingly, this phenotype was found to be at least in part attributable to the ablation of Pld2 from bone marrow cells. The number of CD8+ T cells, which induce cancer cell death, significantly decreased in the tumor produced in Pld2-knockout mice. In addition, CD3/CD28-stimulated proliferation of primary cultured splenic CD8+ T cells is markedly suppressed by Pld2 ablation. Finally, CD3/CD28-dependent activation of Erk1/2 and Ras is inhibited in Pld2-deleted CD8+ T cells. Collectively, these results indicate that PLD2 in CD8+ T cells plays a key role in their proliferation through activation of the Ras/Erk signaling pathway, thereby regulating anti-tumor immunity.
Highlights
The tumor microenvironment is comprised of cancer cells and numerous components such as blood vessels, fibroblasts, bone marrow-derived inflammatory cells, the extracellular matrix, and immune cells[1]
The results obtained in this study provide evidence that PLD2 in CD8+ T lymphocytes plays an important role in the T cell receptor (TCR)-mediated proliferation of these cells in the spleen by regulating the Ras-Erk signaling pathway and IL-2 production, thereby at least in part contributing to the anti-tumor immunity
It was our concern that a partial peptide is produced in the Pld2−/− mice, which have been generated by replacing exons 14 and 15 of the Pld[2] gene, and exerts an off-target effect(s)
Summary
Ablation of Pld[2] promotes the tumor growth. To explore the pathological roles of PLD2, we analyzed growth of tumors produced by implantation of B16 melanoma and Lewis lung carcinoma (LLC) cells into Pld2knockout (Pld2−/−) mice, which had been generated in our laboratory[17]. Consistent with the result obtained with Pld2−/− mice (Fig. 1), Pld2−/−/Cas[9] mice implanted with B16 melanoma and LLC cells enhanced tumor growth (Supplementary Fig. S3), confirming that ablation of Pld[2] promotes the tumor growth. When B16 melanoma cells stably expressing the fluorescent protein mCherry were implanted, apoptosis of these cells in the tumor formed in Pld2−/− mice was significantly suppressed (Fig. 2c), which was consistent with the result of immunostaining of cleaved caspase 3, a specific marker for apoptotic cells (Fig. 2d). These results indicate that death of tumor cells is inhibited in Pld2−/− mice. PLD2 appears to be required for TCR-mediated activation of the Ras-Erk pathway and IL-2 production, which are essential cell events for TCR-stimulated proliferation of splenic CD8+ T cells
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