Sergey Apasov

A2A adenosine receptor protects tumors from antitumor T cells

The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in ≈60% of A2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A2 receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. The data suggest that effects of A2AR are T cell autonomous. The inhibition of antitumor T cells via their A2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the “Hellstrom paradox”). We propose to target the hypoxia→adenosine→A2AR pathway as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A2AR-deficient mice suggest that A2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia→adenosine→A2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage.

Differential Effects of Physiologically Relevant Hypoxic Conditions on T Lymphocyte Development and Effector Functions

Direct measurements revealed low oxygen tensions (0.5–4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with 20% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., IL-2 and IFN-γ) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during 20% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient 20% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.

Memory of Extracellular Adenosine A2A Purinergic Receptor-mediated Signaling in Murine T Cells

Accumulation of extracellular and intracellular adenosine (Ado) under hypoxic conditions or in the absence of adenosine deaminase results in lymphocyte depletion and in severe combined immunodeficiency, which are currently explained by direct intracellular lymphotoxicity of Ado metabolites. In support of the alternative, “signaling” mechanism, we show that extracellular Ado (extAdo) suppresses all tested T cell receptor (TCR)-triggered effector functions of T lymphocytes including the TCR-triggered FasL mRNA up-regulation in cytotoxic T lymphocytes. Strong evidence against the intracellular lymphotoxicity of Ado (and in support of the signaling model) is provided by abrogation of TCR-triggered growth inhibition in Ado-exposed T cells. The brief exposure to Ado was sufficient to observe inhibition of TCR-triggered effector functions. The “memory” of T cells to exposure to extAdo is best explained by sustained increases in cAMP. Selective agonist (CGS21680) and antagonist (ZM241385) of A2A adenosine receptor were used in functional assays and cDNA probes for different sybtypes of adenosine receptors were used in Northern blot studies. A2Areceptors are identified as the predominantly expressed subtype of Gs-coupled Ado receptors in T cells. The demonstration of cross-talk between the A2A receptors and TCR in both directions support the possible role of A2A receptors in mechanisms of extAdo-mediated immunosuppression in vivounder adenosine deaminase deficiency and hypoxic conditions in,e.g., solid tumors.

Cutting edge: Physiologic attenuation of proinflammatory transcription by the Gs protein-coupled A2A adenosine receptor in vivo.

The A2A adenosine receptor plays a critical role in the physiologic immunosuppressive pathway that protects normal tissues from excessive collateral damage by overactive immune cells and their proinflammatory cytokines. In this study, we examine and clarify the mechanism of tissue protection by extracellular adenosine using A2AR-deficient mice and show that the A2AR inhibits TLR-induced transcription of proinflammatory cytokines in vivo. The observed increase in proinflammatory cytokines mRNA in A2AR-deficient mice was associated with enhanced activity of the NF-κB transcription factor. These observations provide the genetic in vivo evidence for attenuation of proinflammatory transcriptional activity of NF-κB by a “metabokine” adenosine and point to the need to re-evaluate the regulation of other transcription factors in hypoxic and adenosine-rich microenvironments of inflamed normal tissues and solid tumors.

Role of Extracellular ATP and P1 and P2 Classes of Purinergic Receptors in T‐cell Development and Cytotoxic T Lymphocyte Effector Functions

The extracellular ATP (ATPQ) model of cell-mediated cytotoxicity was originally suggested as an aiternalive cytotoxic pathway (Trenn et al. 1987) that does not require extracellular Ca-^ and complements the perforin-mediated pathway (FilIppini et al. 1990a,b, diVirgilio et al. 1990). The straightforward consideration of ATPo as a CTL-produced. lethal hit-delivering lytic intermediate has evolved into the more general model of ATPo involvement (as the source of extracellular adenosine. as a phosphate donor, and as a transmembrane signaling ligand) in both T-cell development and elTector functions. Extracellular ATP (ATP,)) is strongly implicated as a fast excitatory transmitter at synapses between neurons (Edwards et al. 1992) and into the ATP-reccptor mediated cell-to-cell spread of calcium signals between mast cells (Osipchuk & Cahalan 1992). Extracellular ATP and e.xtraccllular phosphorylation were suggested to be involved in the cell-cell contact interactions leading to the lymphocyte activation and effector functions of T lymphocytes {Filippini et al. 1990a.b. Redegeld et al. !991. 1993). The presence of highly active ecto-ATPase (Filippini etal. 1990a) and the Ag-receptor-triggered accumulation of extracellular ATP (Filippi-

Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, A1, and A3 adenosine receptors in lymphoid organs

Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice demonstrated that the cyclic AMP-elevating A2a receptor plays a non-redundant role in protection from excessive acute inflammatory tissue damage and in the down-regulation of proinflammatory cytokine production, it remained to be established whether genetic deficiency of the A2a receptor is accompanied by a compensatory up-regulation of the cAMP-elevating A2b receptor and/or other adenosine receptors. Here, we show that most of the cAMP response to adenosine is abolished in lymphoid tissues of A2a receptor-deficient mice, although some response remains in splenocytes. No significant changes were observed in A2b, A1, and A3 mRNA levels in the thymus or lymph nodes of A2a receptor-deficient mice, but small increases in mRNA expression of these receptors were detected in the spleen. These data suggest that regulation of the expression of A2b, A1, and A3 receptors is not affected significantly by the absence of A2a receptors and may provide further explanation of earlier in vivo observations of increased tissue damage and of longer persistence of proinflammatory cytokines in animals with inactivated A2a receptors.

Phosphorylation of T-lymphocyte plasma membrane-associated proteins by ectoprotein kinases: implications for a possible role for ectophosphorylation in T-cell effector functions

Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.

Phosphorylation of Extracellular Domains of T-Lymphocyte Surface Proteins CONSTITUTIVE SERINE AND THREONINE PHOSPHORYLATION OF THE T CELL ANTIGEN RECEPTOR ECTODOMAINS

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and 32Pi metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that αβTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [γ-32P]ATP or [γ-32P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both α and β TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that αβTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of αβTCR-mediated T-lymphocytes response.

Study of A2A adenosine receptor gene deficient mice reveals that adenosine analogue CGS 21680 possesses no A2A receptor-unrelated lymphotoxicity

Cell surface A2A adenosine receptor (A2AR) mediated signalling affects a variety of important processes and adenosine analogues possess promising pharmacological properties. Demonstrating the receptor specificity of potentially lymphotoxic adenosine‐based drugs facilitates their development for clinical applications. To distinguish between the receptor‐dependent and ‐independent lymphotoxicity and apoptotic activity of adenosine and its analogues we used lymphocytes from A2AR‐deficient mice. Comparison of A2AR‐expressing (+/+) and A2AR‐deficient (−/−) cells in cyclic AMP accumulation assays confirmed that the A2AR agonist CGS 21680 is indeed selective for A2A receptors in T‐lymphocytes. Incubation of A2AR‐expressing thymocytes with extracellular adenosine or CGS 21680 in vitro results in the death of about 7–15% of thymocytes. In contrast, no death was induced in parallel assays in cells from A2AR‐deficient mice, providing genetic evidence that CGS 21680 does not display adenosine receptor‐independent intracellular cytotoxicity. The A2A receptor‐specific lymphotoxicity of CGS 21680 is also demonstrated in a long‐term (6‐day) in vitro model of thymocyte positive selection where addition of A2AR antagonist ZM 241,385 did block the effects of CGS 21680, allowing the survival of T cells. The use of cells from adenosine receptor‐deficient animals is proposed as a part of the screening process for potential adenosine‐based drugs for their receptor‐independent cytotoxicity and lymphotoxicity.