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1.  Physiological control of NKT cell-dependent hepatitis induction by extracellular adenosine 
European journal of immunology  2014;44(4):1119-1129.
Extracellular adenosine regulates inflammatory responses via A2A adenosine receptor (A2AR). A2AR-deficiency results in much exaggerated acute hepatitis, indicating non-redundancy of adenosine-A2AR pathway in inhibitory mechanisms of immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. A2AR agonist abolished NKT cell-dependent induction of acute hepatitis by Con A or α-galactosylceramide (α-GalCer), corresponding to down-regulation of activation markers and cytokines in NKT cells and of NK cell co-activation. These results show that A2AR signaling can down-regulate NKT cell activation and suppress NKT cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-GalCer induced more severe hepatitis in A2AR−/− mice than in wild-type controls. Transfer of A2AR−/− NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway.
PMCID: PMC4482763  PMID: 24448964
NKT cell; adenosine; A2A adenosine receptor; hepatitis; immunoregulation
2.  The development and immunosuppressive functions of CD4+ CD25+ FoxP3+ regulatory T cells are under influence of the adenosine-A2A adenosine receptor pathway 
The A2A adenosine receptor (A2AR)-mediated immunosuppression is firmly implicated in the life-saving down-regulation of collateral tissue damage during the anti-pathogen immune response and in highly undesirable protection of cancerous tissues during anti-tumor immune response. Therefore, depending on specific clinical situation there is a need to either weaken or strengthen the intensity of A2AR signal. While the A2AR-mediated immunosuppression was shown to be T cell autonomous in studies of effector T cells, it was not clear how A2AR stimulation affects regulatory T cells (Treg). Here we show in parallel assays that while A2AR stimulation on T cells directly inhibits their activation, there is also indirect and longer-lasting T cell inhibitory effect through modulation of Treg. A2AR stimulation expanded CD4+ CD25hi FoxP3+ cells, which also express CD39, CD73, and CTLA-4. Treg cultured with A2AR agonist showed increased expression of CTLA-4 and stronger immunosuppressive activity. There was a significant increase of Treg cell number after A2AR stimulation. The CD4+ FoxP3+ population contained those induced from CD4+ CD25− cells, but CD4+ FoxP3+ cells predominantly derived from CD4+ CD25+ natural Treg. Thus, A2AR stimulation numerically and functionally enhanced Treg-mediated immunosuppressive mechanism. These data suggest that the A2AR-mediated stimulation of lymphocytes using A2AR agonists should be considered in protocols for ex vivo expansion of Treg before the transfer to patients in different medical applications.
PMCID: PMC3389649  PMID: 22783261
regulatory T cells; adenosine; immunosuppression; A2A adenosine receptor; cancer; autoimmune; transplantation
3.  In vivo T Cell Activation in Lymphoid Tissues is Inhibited in the Oxygen-Poor Microenvironment 
Activation of immune cells is under control of immunological and physiological regulatory mechanisms to ensure adequate destruction of pathogens with the minimum collateral damage to “innocent” bystander cells. The concept of physiological negative regulation of immune response has been advocated based on the finding of the critical immunoregulatory role of extracellular adenosine. Local tissue oxygen tension was proposed to function as one of such physiological regulatory mechanisms of immune responses. In the current study, we utilized in vivo marker of local tissue hypoxia pimonidazole hydrochloride (Hypoxyprobe-1) in the flowcytometric analysis of oxygen levels to which lymphocytes are exposed in vivo. The level of exposure to hypoxia in vivo was low in B cells and the levels increased in the following order: T cells < NKT cells < NK cells. The thymus was more hypoxic than the spleen and lymph nodes, suggesting the variation in the degree of oxygenation among lymphoid organs and cell types in normal mice. Based on in vitro studies, tissue hypoxia has been assumed to be suppressive to T cell activation in vivo, but there was no direct evidence demonstrating that T cells exposed to hypoxic environment in vivo are less activated. We tested whether the state of activation of T cells in vivo changes due to their exposure to hypoxic tissue microenvironments. The parallel analysis of more hypoxic and less hypoxic T cells in the same mouse revealed that the degree of T cell activation was significantly stronger in better-oxygenated T cells. These observations suggest that the extent of T cell activation in vivo is dependent on their localization and is decreased in environment with low oxygen tension.
PMCID: PMC3342240  PMID: 22566817
T cell; oxygen; hypoxia; hyperoxia; Hypoxyprobe-1; cytometry; tumor
4.  In vitro induction of T cells that are resistant to A2 adenosine receptor-mediated immunosuppression 
British Journal of Pharmacology  2008;156(2):297-306.
Background and purpose:
The increased levels of extracellular adenosine in inflamed tissues down-regulate activated immune cells via the A2A adenosine receptor. This A2A adenosine receptor-mediated immunosuppression is a disqualifying obstacle in cancer immunotherapy as it protects cancerous tissues from adoptively transferred anti-tumour T cells. The aim of this study was to test whether the negative selection of T cells will produce T cells that are resistant to inhibition by extracellular adenosine.
Experimental approach:
Cytotoxic T lymphocytes (CTL) were developed by mixed lymphocyte culture in the presence or absence of the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA). The sensitivity of CTL to adenosine analogues was characterized by cAMP induction, interferon-γ production and cytotoxicity.
Key results:
CTL that could proliferate even in the presence of NECA were less susceptible to inhibition by A2A adenosine receptor agonists, as shown by a much smaller accumulation of cAMP and less inhibition of interferon-γ production compared with control CTL. The successful protocol to produce CTL that are both resistant to adenosine-mediated immunosuppression and maintain strong cytotoxicity and interferon-γ secretion required NECA to be added only during the expansion stage after the establishment of CTL. In contrast, the priming of resting T cells in the presence of NECA resulted in T cells with impaired effector functions.
Conclusions and implications:
Adenosine-resistant effector T cells were successfully obtained by exposure of activated T cells to NECA. These in vitro studies form the basis for future attempts to produce anti-tumour T cells that are more effective in adoptive immunotherapy.
PMCID: PMC2697837  PMID: 19076726
Cytotoxic T lymphocytes; cancer; adenosine; A2A adenosine receptor; adoptive immunotherapy; mouse

Results 1-4 (4)