Inhibition of Cytokine Production and Cytotoxic Activity of Human Antimelanoma Specific CD8+ and CD4+ T Lymphocytes by Adenosine-Protein Kinase A Type I Signaling (original) (raw)
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Cancer Research, 2006
Adenosine is an important signaling molecule that regulates multiple physiologic processes and exerts major anti-inflammatory actions. Tumors have high concentrations of adenosine, which could inhibit the function of tumor-infiltrating lymphoid cells. We investigated the ability of adenosine and its stable analogue 2-chloroadenosine (CADO) to inhibit cytokine production and cytotoxic activity of lymphokine-activated killer (LAK) cells and determined whether both these effects are initiated via a common pathway. CADO strongly inhibited cytotoxic activity of LAK cells and attenuated the production of IFN-;, granulocyte macrophage colony-stimulating factor, tumor necrosis factor A, and macrophage inflammatory protein-1A by LAK cells stimulated by cross-linking of the Ly49D receptor. These inhibitory effects were associated with the ability of CADO to stimulate cyclic AMP (cAMP) production and activate protein kinase A (PKA). Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKA types I and II, we found that activation of PKA I, but not PKA II, mimicked the inhibitory effects of CADO on LAK cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKI 14-22 peptide) failed to abrogate the inhibitory effects of CADO whereas Rp-8-Br-cAMPS, an antagonist of the RI subunit, blocked the inhibitory effects of CADO. We conclude that the inhibitory effects of adenosine are probably mediated via cAMP-dependent activation of the RI subunits of PKA I but are independent of the catalytic activity of PKA. Tumor-produced adenosine could be a potent tumor microenvironmental factor inhibiting the functional activity of tumor-infiltrating immune cells. (Cancer Res 2006; 66(15): 7758-65) Requests for reprints:
Adenosine and Adenosine Receptors in the Immunopathogenesis and Treatment of Cancer
Journal of cellular physiology, 2017
Tumor cells overcome anti-tumor responses in part through immunosuppressive mechanisms. There are several immune modulatory mechanisms. Among them, adenosine is an important factor which is generated by both cancer and immune cells in tumor microenvironment to suppress anti-tumor responses. Two cell surface expressed molecules including CD73 and CD39 catalyze the generation of adenosine from adenosine triphosphate (ATP). The generation of adenosine can be enhanced under metabolic stress like tumor hypoxic conditions. Adenosine exerts its immune regulatory functions through four different adenosine receptors including A1, A2A, A2B, and A3 which are expressed on various immune cells. Several studies have indicated the overexpression of adenosine generating enzymes and adenosine receptors in various cancers which was correlated with tumor progression. Since the signaling of adenosine receptors enhances tumor progression, their manipulation can be promising therapeutic approach in cance...
2019
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.
Extracellular adenosine metabolism in immune cells in melanoma
Cancer Immunology, Immunotherapy, 2014
subpopulation that co-expressed also PD-1. ectonucleotidase expression was also up-regulated in CD4 + and CD8 + T cells upon activation. In addition, these ectoenzymes were largely found to be expressed on memory T cell compartment (in particular, on effector memory cells). Our data suggest that extracellular adenosine produced by regulatory T cells (Tregs) and MDsCs can suppress T cell effector functions through paracrine signaling. another mechanism involves its production also by effector T cells and an inhibition of their anti-tumor reactivity via autocrine signaling as a part of the negative feedback loop. This mode of adenosine signaling could be also used by Tregs and MDsCs to enhance their immunosuppressive activity.
Adenosine A2A Receptors Intrinsically Regulate CD8+ T Cells in the Tumor Microenvironment
Cancer Research, 2014
Adenosine A 2A receptor (A 2A R) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A 2A R deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a À/À mice within 2 weeks of tumor inoculation. A 2A R deletion in the host reduced numbers of CD8 þ T cells and effector-memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell-specific deletion of A 2A R. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector-memory differentiation, and reduced antiapoptotic IL7Ra (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8 þ T-cell density within tumors in wild-type hosts. We found that A 2A R-proficient CD8 þ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A 2A R signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A 2A R blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector-memory differentiation. Thus, our findings imply that the efficacious application of A 2A R inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. Cancer Res; 74(24); 7239-49. Ó2014 AACR.
Blood, 1997
(2) the A2a, but not the A1 or A3, receptors are the major absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined im-expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-munodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a recep-T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of tor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhi-the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor a chain (CD25) mole-bition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of cules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic re-of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular ceptors on T cells as a potentially attractive pharmacologic target. toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP ᭧ tively low concentrations of adenosine rather then the direct,
Purinergic Signalling, 2013
The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutininstimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A 2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A 1 , A 2A , and A 2B ARs. Cell proliferation was measured by [ 3 H]thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A 1), BAY60-6583 (A 2B), and IB-MECA (A 3), and the antagonists PSB-36 (A 1), MSX-2 (A 2A), and PSB-10 (A 3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A 2A), and the antagonists preladenant (SCH-420814, A 2A), PSB-1115 (A 2B), and PSB-603 (A 2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC 50 value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms. Keywords Adenosine receptors. Real-time PCR. Human lymphocytes. Jurkat T cells. Proliferation. [ 3 H]Thymidine incorporation. Receptor-independent mechanisms Abbreviations ADA Adenosine deaminase AR Adenosine receptor BAY60-6583 2-[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2ylsulfanyl]acetamide cAMP Cyclic AMP CGS-21680 (2-p-[2-Carboxyethyl]phenethylamino)-5′-N-ethylcarboxamidoadenosine CI-IB-MECA 2-Chloro-N 6-(3-iodobenzyl)-9-[5-(methyl-carbamoyl)-β-d-ribofuranosyl] adenine CPA N 6-Cyclopentyladenosine DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid FCS Fetal calf serum GPCR(s) G protein-coupled receptor(s) IB-MECA N 6-(3-Iodobenzyl)-5′-Nmethylcarboxamidoadenosine Anke C. Schiedel and Svenja K. Lacher contributed equally.