ATP-GATED P2X7 RECEPTOR AS A NOVEL CHECKPOINT MOLECULE IN T EFFECTOR/MEMORY CELL FUNCTION

Adenosine triphosphate is an ubiquitous extracellular messenger, which activates P2 purinergic receptors in the plasma membrane of eukaryotic cells. Activation of P2 receptors regulates many cellular functions ranging from survival and proliferation to apoptosis. The final effect of extracellular ATP on a cell depends on the composition of P2 receptor expressed on its surface. We showed that T effector/memory (TEM) cells express high levels of P2rx7 encoding for the ATP-gated ionotropic P2X7 receptor subtype. The deletion of the gene in these cells results in increased survival and cell proliferation both in vitro and in an in vivo model of homeostatic expansion. P2rx7-/- TEM cells are characterized by a bioenergetic advantage with respect to wild-type (WT) cells and morphometric analysis of mitochondria revealed that P2rx7-/- TEM cells are characterized by a fused mitochondria network, while WT TEM cells have more fissed mitochondria. Microarray gene expression analysis showed that P2rx7-/- TEM cells clustered together and separately from WT cells. Among differentially expressed genes we identified cyclin-dependent kinase inhibitor 1A (Cdkn1a), encoding for p21Waf1/Cip1, as a transcript upregulated in WT cells. P21 inhibits progression through G1 to S phase in mammalian cells and it is upregulated in senescent cells. We demonstrated that P2X7 signaling directly upregulated Cdkn1a in TEM cells and this resulted in a block in cell cycle progression. We hypothesize that P2X7 receptor could induce premature senescence in TEM cells. Accordingly, the analysis of β-galactosidase, a marker of senescence, revealed that P2X7 induces an increase in the enzyme activity in TEM cells. Moreover, WT TEM cells stimulated with a pharmacological agonist of P2X7 are characterized by increased in Trp53 and Gadd45b expression as well as p38 MAPK phosphorylation. Moreover, P2X7 stimulation induces an increase in mitochondrial ROS and an increase in histone H2AX phosphorylation as a marker of DNA damage and cellular senescence. The tumor microenvironment is reach in extracellular ATP; we hypothesized this feature could affect TEM cells survival and function. In fact, we show that lack of P2X7 promoted expansion and accumulation of both CD4 and CD8 tumor infiltrating lymphocytes in a melanoma mouse model, thus favoring the control of tumor size. Altogether our results point to P2X7 as a checkpoint for limiting CD4 TEM cells expansion in inflammatory environments that might be targeted to implement cytotoxic T cell responses in cancer immunotherapy.