Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8 + T cells

J Immunother Cancer. 2019 Oct 10;7(1):257. doi: 10.1186/s40425-019-0719-5.

Abstract

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.

Keywords: Adenosine; CD8 T cells; Metabolism; TILs; mTOR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / immunology
  • Adenosine / metabolism*
  • Adult
  • Aged
  • Aged, 80 and over
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism
  • Cell Line, Tumor
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Disease Progression
  • Female
  • Humans
  • Lymphocytes, Tumor-Infiltrating / immunology
  • Lymphocytes, Tumor-Infiltrating / metabolism
  • Male
  • Mechanistic Target of Rapamycin Complex 1 / metabolism
  • Middle Aged
  • Neoplasms / immunology*
  • Neoplasms / metabolism
  • Primary Cell Culture
  • Receptor, Adenosine A2A / metabolism
  • Signal Transduction / immunology*
  • Tumor Escape
  • Tumor Microenvironment / immunology*

Substances

  • ADORA2A protein, human
  • Receptor, Adenosine A2A
  • Mechanistic Target of Rapamycin Complex 1
  • Cyclic AMP-Dependent Protein Kinases
  • Adenosine