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

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.

Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells

“…Adenosine signaling also attenuates T cell cytotoxicity and cytokine production [8]. Enhanced adenosine signaling on T cells could be a consequence of two independent phenomena; increased levels of extracellular adenosine, caused by hypoxic conditions and altered cellular metabolic activity, and increased expression of the receptor as an immune checkpoint molecule on T cells due to activation of the cells following antigen recognition [13,20,28]. It is less clear that ex vivo manipulation of T cells used in cancer immunotherapy can influence the expression of A2aR and other inhibitory receptors prior to their administration.…”

Standardization of sampling and sample preparation for analysis of human monocyte subsets in peripheral blood

“…High throughput metabolic profiling by Seahorse extracellular flux assays made it possible to observe differences between quiescent, activated, and memory immune cells [425][426][427], evaluate the effects of changes in the cellular microenvironment [428][429][430],…”

The New Era of Cancer Immunotherapy: Targeting Myeloid-Derived Suppressor Cells to Overcome Immune Evasion

“…The activity of NADPH oxidase enzyme translates into an increase in ROS production which mediates tumor immunosuppression and might thus represent a potential target for therapeutic intervention. Other important mediators involved in cancer immunosuppression are IDO and the adenosine (ADO)-producing enzymes CD39 and CD73 (249)(250)(251)(252)(253). It has been reported that ADO-producing enzymes are expressed in MDSCs isolated from the peripheral blood of NSCLC patients and favor their immunosuppressive function.…”