P2RY2 is a purinergic immune checkpoint linking extracellular ATP to immune evasion and adaptive resistance to immunotherapy Hu, Zhaoqing Matsuo, Hitoshi Du, Shangce Berzain Battion, Cecilia Jassowicz, Lena Carretero, Rafael Sator-Schmit, Melanie Zhao, Xiyue Miao, Beiping Eris, Cansu Engel, Helena Mahmoud, Mohamed A.A. Lapor, Elke Xiao, Yanling Hofmann, Ilse Herold-Mende, Christel Sun, Chong P2RY2 Immunotherapy ATP We thank the core facilities at the German Cancer Research Centre (DKFZ) in Heidelberg, including Flow Cytometry, the Centre for Preclinical Research, Next-Generation Sequencing, Proteomics, and Light Microscopy facilities, for their support. This work is supported by the European Research Council: ERC starting grant, DRILL, 101078722 (to CS) (Funded by the European Union. The views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them); DKFZ/Bayer Innovation Alliance grant P2RY2 (to CS); DKFZ-MOST (Israel Ministry of Science and Technology) cooperation program grant, Ca 208 (to CS). Extracellular ATP (eATP) accumulates substantially in the tumor microenvironment (TME) and rises further during immunotherapy. While canonically an immune-activating “danger” signal, eATP also promotes immunosuppression in tumors, thus far largely attributed to its metabolite, adenosine. Here, we identify direct eATP signaling through P2RY2 as a dominant, adenosine- independent mechanism of immune resistance. Specifically, eATP–P2RY2 signaling serves as the primary upstream driver of COX-1/2 upregulation and consequent accumulation of immunosuppressive PGE₂ in the TME, uncovering the long-sought TME-specific trigger of pathological COX–PGE₂ hyperactivation in solid tumors. Genetic deletion or pharmacologic inhibition of P2RY2 eliminates both baseline and therapy-induced intratumoral PGE₂, restores antitumor T cell responses, and reverses resistance to CAR-T, TCR-T, checkpoint blockade, and TIL therapies. Given that persistently elevated eATP is a hallmark of solid tumors, our work reveals a fundamental mechanism by which tumors hijack innate “danger” signaling to establish immune suppression and develop adaptive resistance to immunotherapy. These findings establish P2RY2 as a purinergic immune checkpoint with translational potential for combinatorial cancer immunotherapies. 2025-12-04T11:07:46Z 2025-12-04T11:07:46Z 2025-10-10 preprint Hu, Z. et al. (2025). P2RY2 is a purinergic immune checkpoint linking extracellular ATP to immune evasion and adaptive resistance to immunotherapy. BioRxiv. Preprint doi: https://doi.org/10.1101/2025.10.09.681049 https://hdl.handle.net/10481/108582 10.1101/2025.10.09.681049 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional bioRxiv