Abstract
BackgroundSeveral 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.MethodsCD8+ 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.ResultsHere, 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.ConclusionsOur 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.
Highlights
Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth
The 25th to 75th percentiles, the median and min-max of the values are represented. ***P < 0.001, ****P < 0.0001, one-way ANOVA test. (d) Cumulative data showing the frequency of cytokine production (IL-2 and TNF-α) and CD107 expression by CD8+ T cells stimulated overnight with anti-CD3/anti-CD28 coated beads in unconditioned media or in presence of Ado
Effects of Ado on CD8+ T cell functional avidity and evaluation of Adenosine receptor (AdoR) expression. (a) Cumulative data of the functional sensitivity (IC50 of IL-2 and TNF- α production) to Ado measured in distinct CD8+ T cell memory subsets after overnight stimulation with anti-CD3/ CD28 beads in presence of decreasing concentrations of Ado
Summary
Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. In the tumor microenvironment (TME), multiple mechanisms such as overexpression of immune checkpoint molecules [6] and loss of nutrients [7, 8] were recently shown to impair T cell metabolic fitness, impairing immune control of tumor growth [9, 10]. It remains largely unexplored whether and how immunosuppressive molecules, such as adenosine (Ado), impair T cell functions by subverting their metabolic activity. The relative sensitivity of T cell subsets to Ado and the relevant mediators downstream of Ado receptors (AdoRs) have not yet been determined
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