Abstract A173: The extracellular ATP receptor P2RX7 is required for CD8+ T-cells to maintain and respond to chronic virus and melanoma tumors

T-cell exhaustion is an acquired state of T-cell dysfunction induced by sustained antigen stimulation (such as in chronic viral infections and cancer). Recently, it was described that exhausted CD8+ T-cells are heterogeneous, with some being preferentially rescued by checkpoint blockade therapy. These cells typically express the chemokine receptor CXCR5 and depend on the transcription factor TCF1 for their development. However, little is known about the extracellular signals required for the generation and function of this subset. Our group has been working with the “danger signal” extracellular ATP (eATP), which sense cellular damage and is recognized by purinergic receptors in mammals. Notably, we recently discovered that the eATP purinergic receptor P2RX7 is crucial for the generation and maintenance of long-lived memory CD8+ T-cells in a cell-intrinsic way, by controlling the mitochondrial function and viability of these cells during acute viral infections. Here, we show that P2RX7 is also required, in a cell-intrinsic way, for CD8+ T-cells to survive and maintain in the presence of chronic antigen. In response to chronic lymphocytic choriomeningitis virus (LCMV Clone 13) infection, we tracked WT vs P2RX7KO LCMV-specific CD8+ T-cells, using specific tetramer staining in germline knock-out vs WT mice, or co-adoptive transfer of antigen-specific (P14) cells into CD4-depleted recipient mice. In response to LCMV-Clone 13, P2RX7KO CD8+ T-cells initially expand but fail to maintain after the first week of infection. This is accompanied by almost no expansion of the CXCR5+ subset – which express higher surface P2RX7 levels. The few CXCR5+ cells that form in the absence of P2RX7 are aberrant in their phenotype, with higher expression of PD1 and Tim3 and lower expression of TCF1. During chronic LCMV, P2RX7KO CD8+ T-cells have increased cell death as early as one week after infection, but no proliferation defects. Correlating with this, both mitochondrial mass and membrane potential (a readout of mitochondrial functionality) of P2RX7KO CD8+ T-cells are severely impaired. Consequently, germline P2RX7KO mice have defective control of viral titers in comparison to WT counterparts. Despite having higher PD1 expression, anti-PD1 therapy with do not induce expansion of adoptively transferred P2RX7KO P14 cells in comparison to WT P14 cells, in pair with the absence of the CXCR5+ subset. Importantly, pharmacologic P2RX7 blockade after establishment of chronic infection leads to a decrease in WT LCMV-specific CD8+ T-cell numbers, indicating P2RX7-mediated eATP sensing is constantly required for CD8+ T-cells to survive during chronic infection. Finally, we used adoptive transfer of in vitro activated WT vs P2RX7KO OT-I cells (in which we observed similar initial activation) and observed that CD8+ T-cells require cell-intrinsic P2RX7 to infiltrate B16-OVA melanoma tumors. Additionally, P2RX7KO mice have impaired B16 melanoma growth control. These results show that the danger signal eATP can aid in CD8+ T-cell long-term viability and function in the presence of chronic antigen. This will open the possibility of using this signaling pathway to design new alternatives to boost cancer immunotherapies, for example by harnessing the P2RX7/eATP pathway in tumor-specific CD8+ T-cells in adoptive transfer-based immunotherapies, which can be combined with existing checkpoint blockade therapies. Citation Format: Henrique Borges da Silva, Stephen C. Jameson. The extracellular ATP receptor P2RX7 is required for CD8+ T-cells to maintain and respond to chronic virus and melanoma tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A173.