TY - JOUR T1 - Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8<sup>+</sup> T cells JF - Journal for ImmunoTherapy of Cancer JO - J Immunother Cancer DO - 10.1186/s40425-019-0719-5 VL - 7 IS - 1 SP - 257 AU - Beatris Mastelic-Gavillet AU - Blanca Navarro Rodrigo AU - Laure Décombaz AU - Haiping Wang AU - Giuseppe Ercolano AU - Rita Ahmed AU - Leyder Elena Lozano AU - Angela Ianaro AU - Laurent Derré AU - Massimo Valerio AU - Thomas Tawadros AU - Patrice Jichlinski AU - Tu Nguyen-Ngoc AU - Daniel E. Speiser AU - Grégory Verdeil AU - Nicolas Gestermann AU - Olivier Dormond AU - Lana Kandalaft AU - George Coukos AU - Camilla Jandus AU - Christine Ménétrier-Caux AU - Christophe Caux AU - Ping-Chih Ho AU - Pedro Romero AU - Alexandre Harari AU - Selena Vigano Y1 - 2019/12/01 UR - http://jitc.bmj.com/content/7/1/257.abstract N2 - Background Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. Among these, the accumulation of adenosine (Ado) contributes to tumor progression and represents a promising immunotherapeutic target. Ado has been shown to impair T cell effector function, but the role and mechanisms employed by Ado/Ado receptors (AdoRs) in modulating human peripheral and tumor-infiltrating lymphocyte (TIL) function are still puzzling.Methods CD8+ T cell cytokine production following stimulation was quantified by intracellular staining and flow cytometry. The cytotoxic capacity of tumor infiltrating lymphocytes (TILs) was quantified by the chromium release assay following co-culture with autologous or anti-CD3-loaded tumor cell lines. The CD8+ T cell metabolic fitness was evaluated by the seahorse assay and by the quantification of 2-NBDG uptake and CD71/CD98 upregulation upon stimulation. The expression of AdoRs was assessed by RNA flow cytometry, a recently developed technology that we validated by semiquantitative RT-PCR (qRT-PCR), while the impact on T cell function was evaluated by the use of selective antagonists and agonists. The influence of Ado/AdoR on the PKA and mTOR pathways was evaluated by phosphoflow staining of p-CREB and p-S6, respectively, and validated by western blot.Results Here, we demonstrate that Ado signaling through the A2A receptor (A2AR) in human peripheral CD8+ T cells and TILs is responsible for the higher sensitivity to Ado-mediated suppression of T central memory cells. We confirmed that Ado is able to impair peripheral and tumor-expanded T cell effector functions, and we show for the first time its impact on metabolic fitness. The Ado-mediated immunosuppressive effects are mediated by increased PKA activation that results in impairment of the mTORC1 pathway.Conclusions Our findings unveil A2AR/PKA/mTORC1 as the main Ado signaling pathway impairing the immune competence of peripheral T cells and TILs. Thus, p-CREB and p-S6 may represent useful pharmacodynamic and efficacy biomarkers of immunotherapies targeting Ado. The effect of Ado on T cell metabolic fitness reinforces the importance of the adenosinergic pathway as a target for next-generation immunotherapy.Beatris Mastelic-Gavillet and Blanca Navarro Rodrigo contributed equally to this work.Abbreviations:AdoAdenosineAdoRAdenosine receptorE:TEffect:target (ratio)PBMCsPeripheral blood mononuclear cellsPKAProtein kinase APMAPhorbol 12-myristate 13-acetateqRT-PCRSemiquantitative real-time PCRREPRapid expansion protocolTCMT central memory cellsTCRT cell receptorTILsTumor-infiltrating lymphocytesTMETumor microenvironment ER -