G protein ß4 as a structural determinant of enhanced nucleotide exchange in the A2AAR-Gs complex.

Adenosine A2A receptors (A2AAR) evoke pleiotropic intracellular signaling events via activation of the stimulatory heterotrimeric G protein, Gs. Here, we used cryoEM to solve the agonist-bound structure of A2AAR in a complex with full-length Gs α and Gß4γ2 (A2AAR-Gs α:ß4γ2). The orthosteric binding site of A2AAR-Gs α:ß4γ2 was similar to other structures of agonist-bound A2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gß4 versus the closest analog, A2AAR-miniGs α:ß1γ2. Consequently, there are fewer interactions between the switch II in Gs α and the Gß4 torus. In reconstitution experiments Gß4γ2 displayed a ten-fold higher efficiency over Gß1γ2 in catalyzing A2AAR dependent GTPγS binding to Gs α. We propose that the less constrained switch II in A2AAR-Gs α:ß4γ2 accounts for this increased efficiency. These results suggest that Gß4 functions as a positive allosteric enhancer versus Gß1.

Improved survival of SARS COV-2-infected K18-hACE2 mice treated with adenosine A2AR agonist.

A life-threatening manifestation of Covid-19 infection is a cytokine storm that requires hospitalization and supplemental oxygen. Various strategies to reduce inflammatory cytokines have had some success in limiting cytokine storm and improving survival. Agonists of adenosine A2A receptors (A2AR) reduce cytokine release from most immune cells. Apadenoson is a potent and selective anti-inflammatory adenosine analog that reduces inflammation. When administered by subcutaneous osmotic pumps to mice infected with SARS CoV-2, Apadenoson was found to improve the outcomes of infection as measured by a decrease in weight loss, improved clinical symptoms, reduced levels of proinflammatory cytokines and chemokines in bronchial lavage (BAL) fluid, and enhanced survival of K18-hACE2 transgenic mice. These results support further examination of A2AR agonists as therapies for treating cytokine storm due to COVID-19.

Regadenoson for the treatment of COVID-19: A five case clinical series and mouse studies.

BACKGROUND: Adenosine inhibits the activation of most immune cells and platelets. Selective adenosine A2A receptor (A2AR) agonists such as regadenoson (RA) reduce inflammation in most tissues, including lungs injured by hypoxia, ischemia, transplantation, or sickle cell anemia, principally by suppressing the activation of invariant natural killer T (iNKT) cells. The anti-inflammatory effects of RA are magnified in injured tissues due to induction in immune cells of A2ARs and ecto-enzymes CD39 and CD73 that convert ATP to adenosine in the extracellular space. Here we describe the results of a five patient study designed to evaluate RA safety and to seek evidence of reduced cytokine storm in hospitalized COVID-19 patients. METHODS AND FINDINGS: Five COVID-19 patients requiring supplemental oxygen but not intubation (WHO stages 4-5) were infused IV with a loading RA dose of 5 µg/kg/h for 0.5 h followed by a maintenance dose of 1.44 µg/kg/h for 6 hours, Vital signs and arterial oxygen saturation were recorded, and blood samples were collected before, during and after RA infusion for analysis of CRP, D-dimer, circulating iNKT cell activation state and plasma levels of 13 proinflammatory cytokines. RA was devoid of serious side effects, and within 24 hours from the start of infusion was associated with increased oxygen saturation (93.8 ± 0.58 vs 96.6 ± 1.08%, P<0.05), decreased D-dimer (754 ± 17 vs 518 ± 98 ng/ml, P<0.05), and a trend toward decreased CRP (3.80 ± 1.40 vs 1.98 ± 0.74 mg/dL, P = 0.075). Circulating iNKT cells, but not conventional T cells, were highly activated in COVID-19 patients (65% vs 5% CD69+). RA infusion for 30 minutes reduced iNKT cell activation by 50% (P<0.01). RA infusion for 30 minutes did not influence plasma cytokines, but infusion for 4.5 or 24 hours reduced levels of 11 of 13 proinflammatory cytokines. In separate mouse studies, subcutaneous RA infusion from Alzet minipumps at 1.44 µg/kg/h increased 10-day survival of SARS-CoV-2-infected K18-hACE2 mice from 10 to 40% (P<0.001). CONCLUSIONS: Infused RA is safe and produces rapid anti-inflammatory effects mediated by A2A adenosine receptors on iNKT cells and possibly in part by A2ARs on other immune cells and platelets. We speculate that iNKT cells are activated by release of injury-induced glycolipid antigens and/or alarmins such as IL-33 derived from virally infected type II epithelial cells which in turn activate iNKT cells and secondarily other immune cells. Adenosine released from hypoxic tissues, or RA infused as an anti-inflammatory agent decrease proinflammatory cytokines and may be useful for treating cytokine storm in patients with Covid-19 or other inflammatory lung diseases or trauma.

Regadenoson Reduces Soluble Receptor for Advanced Glycation End-Products in Lung Recipients.

BACKGROUND: The selective adenosine A2A receptor (A2AR) agonist regadenoson reduces inflammation due to lung ischemia-reperfusion injury (IRI). The objective of this study was to investigate molecular and cellular mechanisms by which regadenoson reduces IRI in lung transplant recipients. METHODS: Fourteen human lung transplant recipients were infused for 12 hours with regadenoson and 7 more served as untreated controls. Plasma levels of high mobility group box 1 and its soluble receptor for advanced glycation end-products (sRAGE) were measured by Luminex. Matrix metalloproteinase (MMP) 2 and 9 were measured by gelatin zymography. Tissue inhibitor of metalloproteinase 1 was measured by mass spectroscopy. A2AR expression on leukocytes was analyzed by flow cytometry. MMP-9-mediated cleavage of RAGE was evaluated using cultured macrophages in vitro. RESULTS: Regadenoson treatment during lung transplantation significantly reduced levels of MMP-9 (P < .05), but not MMP-2, and elevated levels of tissue inhibitor of metalloproteinase 1 (P < .05), an endogenous selective inhibitor of MMP-9. Regadenoson infusion significantly reduced plasma levels of sRAGE (P < .05) during lung reperfusion compared with control subjects. A2AR expression was highest on invariant natural killer T cells and higher on monocytes than other circulating immune cells (P < .05). The shedding of RAGE from cultured monocytes/macrophages was increased by MMP-9 stimulation and reduced by an MMP inhibitor or by A2AR agonists, regadenoson or ATL146e. CONCLUSIONS: In vivo and in vitro studies suggest that A2AR activation reduces sRAGE in part by inhibiting MMP-9 production by monocytes/macrophages. These results suggest a novel molecular mechanism by which A2AR agonists reduce primary graft dysfunction.

Unlocking antitumor immunity with adenosine receptor blockers.

Tumors survive by creating a tumor microenvironment (TME) that suppresses antitumor immunity. The TME suppresses the immune system by limiting antigen presentation, inhibiting lymphocyte and natural killer (NK) cell activation, and facilitating T cell exhaustion. Checkpoint inhibitors like anti-PD-1 and anti-CTLA4 are immunostimulatory antibodies, and their blockade extends the survival of some but not all cancer patients. Extracellular adenosine triphosphate (ATP) is abundant in inflamed tumors, and its metabolite, adenosine (ADO), is a driver of immunosuppression mediated by adenosine A2A receptors (A2AR) and adenosine A2B receptors (A2BR) found on tumor-associated lymphoid and myeloid cells. This review will focus on adenosine as a key checkpoint inhibitor-like immunosuppressive player in the TME and how reducing adenosine production or blocking A2AR and A2BR enhances antitumor immunity.

Feeding desensitizes A1 adenosine receptors in adipose through FOXO1-mediated transcriptional regulation.

OBJECTIVE: Adipose tissue is a critical regulator of energy balance that must rapidly shift its metabolism between fasting and feeding to maintain homeostasis. Adenosine has been characterized as an important regulator of adipocyte metabolism primarily through its actions on A1 adenosine receptors (A1R). We sought to understand the role A1R plays specifically in adipocytes during fasting and feeding to regulate glucose and lipid metabolism. METHODS: We used Adora1 floxed mice with an inducible, adiponectin-Cre to generate FAdora1-/- mice, where F designates a fat-specific deletion of A1R. We used these FAdora1-/- mice along with specific agonists and antagonists of A1R to investigate changes in adenosine signaling within adipocytes between the fasted and fed state. RESULTS: We found that the adipose tissue response to adenosine is not static, but changes dynamically according to nutrient conditions through the insulin-Akt-FOXO1 axis. We show that under fasted conditions, FAdora1-/- mice had impairments in the suppression of lipolysis by insulin on normal chow and impaired glucose tolerance on high-fat diet. FAdora1-/- mice also exhibited a higher lipolytic response to isoproterenol than WT controls when fasted, however this difference was lost after a 4-hour refeeding period. We demonstrate that FOXO1 binds to the A1R promoter, and refeeding leads to a rapid downregulation of A1R transcript and desensitization of adipocytes to A1R agonism. Obesity also desensitizes adipocyte A1R, and this is accompanied by a disruption of cyclical changes in A1R transcription between fasting and refeeding. CONCLUSIONS: We propose that FOXO1 drives high A1R expression under fasted conditions to limit excess lipolysis during stress and augment insulin action upon feeding. Subsequent downregulation of A1R under fed conditions leads to desensitization of these receptors in adipose tissue. This regulation of A1R may facilitate reentrance into the catabolic state upon fasting.

Kinetic profiling and functional characterization of 8-phenylxanthine derivatives as A2B adenosine receptor antagonists.

A2B adenosine receptor (A2BAR) antagonists have therapeutic potential in inflammation-related diseases such as asthma, chronic obstructive pulmonary disease and cancer. However, no drug is currently clinically approved, creating a demand for research on novel antagonists. Over the last decade, the study of target binding kinetics, along with affinity and potency, has been proven valuable in early drug discovery stages, as it is associated with improved in vivo drug efficacy and safety. In this study, we report the synthesis and biological evaluation of a series of xanthine derivatives as A2BAR antagonists, including an isothiocyanate derivative designed to bind covalently to the receptor. All 28 final compounds were assessed in radioligand binding experiments, to evaluate their affinity and for those qualifying, kinetic binding parameters. Both structure-affinity and structure-kinetic relationships were derived, providing a clear relationship between affinity and dissociation rate constants. Two structurally similar compounds, 17 and 18, were further evaluated in a label-free assay due to their divergent kinetic profiles. An extended cellular response was associated with long A2BAR residence times. This link between a ligand's A2BAR residence time and its functional effect highlights the importance of binding kinetics as a selection parameter in the early stages of drug discovery.

Developmentally distinct CD4+ Treg lineages shape the CD8+ T cell response to acute Listeria infection.

SignificanceThe CD4+ Treg response following acute Listeria infection is heterogeneous and deploys two distinct modes of suppression coinciding with initial pathogen exposure and resolution of infection. This bimodal suppression of CD8+ T cells during priming and contraction is mediated by separate Treg lineages. These findings make a significant contribution to our understanding of the functional plasticity inherent within Tregs, which allows these cells to serve as a sensitive and dynamic cellular rheostat for the immune system to prevent autoimmune pathology in the face of inflammation attendant to acute infection, enable expansion of the pathogen-specific response needed to control the infection, and reestablish immune homeostasis after the threat has been contained.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Targeting Adenosine with Adenosine Deaminase 2 to Inhibit Growth of Solid Tumors.

Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity-dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer. SIGNIFICANCE: This study identifies ADA2 as a prognostic factor associated with prolonged cancer patient survival and introduces the potential of enzymatic removal of adenosine with engineered ADA2 for cancer immunotherapy.

Clearance of apoptotic cells by lung alveolar macrophages prevents development of house dust mite-induced asthmatic lung inflammation.

BACKGROUND: Poor clearance of apoptotic cells has been suggested to contribute to severe asthma, but whether uptake of apoptotic cells by lung phagocytes might dampen house dust mite (HDM)-induced lung inflammation has not been shown. OBJECTIVES: This study investigated whether apoptotic cell engulfment in the murine lung impacts the development of allergen-induced asthmatic airway inflammation and which immune modulating mechanisms were activated. METHODS: Apoptotic cells were infused into the lungs of mice challenged with HDM allergen and lung inflammation, expression of suppressive molecules, and induction of regulatory T cells were monitored. Additionally, an adenosine receptor agonist was tested to study the mechanism of suppression elicited by apoptotic cells. RESULTS: Apoptotic cell uptake by lung alveolar macrophages suppressed HDM-driven allergic asthma. This was associated with promoting the regulatory T cell-inducing molecule retinoic acid, inhibiting inflammatory cytokine production, and making macrophages more susceptible to receiving suppressive signals from adenosine. Correspondingly, adenosine receptor agonist treatment also limited HDM-driven allergic airway inflammation through an action on alveolar macrophages. CONCLUSIONS: These data provide insight into the mechanisms by which lung macrophages dampen allergen-induced airway inflammation. They suggest that targeting lung macrophages to increase their phagocytic capacity, enhance their ability to make retinoic acid, dampen their capacity to make inflammatory cytokines, and increase their responsiveness to adenosine, could be useful to suppress allergic responses.

Cellular sensing of extracellular purine nucleosides triggers an innate IFN-ß response.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-ß (IFN-ß) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-ß. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-ß. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-ß production.

Adenosine A2A receptor agonist (regadenoson) in human lung transplantation.

BACKGROUND: Currently, there are no clinically approved treatments for ischemia-reperfusion injury after lung transplantation. Pre-clinical animal models have demonstrated a promising efficacy of adenosine 2A receptor (A2AR) agonists as a treatment option for reducing ischemia-reperfusion injury. The purpose of this human study, is to conduct a Phase I clinical trial for evaluating the safety of continuous infusion of an A2AR agonist in lung transplant recipients. METHODS: An adaptive, two-stage continual reassessment trial was designed to evaluate the safety of regadenoson (A2AR agonist) in the setting of lung transplantation. Continuous infusion of regadenoson was administered to lung transplant recipients that was started at the time of skin incision. Adverse events and dose-limiting toxicities, as pre-determined by a study team and assessed by a clinical team and an independent safety monitor, were the primary end-points for safety in this trial. RESULTS: Between January 2018 and March 2019, 14 recipients were enrolled in the trial. Of these, 10 received the maximum infused dose of 1.44 µg/kg/min for 12 hours. No dose-limiting toxicities were observed. The steady-state plasma regadenoson levels sampled before the reperfusion of the first lung were 0.98 ± 0.46 ng/ml. There were no mortalities within 30 days. CONCLUSIONS: Regadenoson, an A2AR agonist, can be safely infused in the setting of lung transplantation with no dose-limiting toxicities or drug-related mortality. Although not powered for the evaluation of secondary end-points, the results of this trial and the outcome of pre-clinical studies warrant further investigation with a Phase II randomized controlled trial.

The Expression of Adenosine A2B Receptor on Antigen-Presenting Cells Suppresses CD8+ T-cell Responses and Promotes Tumor Growth.

Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immunosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103+ dendritic cells, which was associated with more effective cross-priming of adoptively transferred tumor antigen-specific CD8+ T cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdim antigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific conditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metastasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8+ T-cell responses and promoted metastasis. These data provide a strong rationale to combine A2BR inhibition with T-cell-based immunotherapy for the treatment of tumor growth and metastasis.

Contrast-enhanced ultrasound detects changes in microvascular blood flow in adults with sickle cell disease.

In patients with sickle cell disease (SCD), poor outcome measures compromise the potential success of clinical trials. Contrast-enhanced ultrasound (CEUS) is a technique that can non-invasively quantify deep tissue microvascular blood flow. We tested the hypothesis that CEUS of forearm skeletal muscle could be used to: 1) assess microvascular abnormalities that occur during vaso-occlusive crisis; and 2) test new therapies for SCD that are targeted to improving the status of the microcirculation. We performed a prospective study, CEUS perfusion imaging of resting forearm muscle was performed in adults with SCD: 1) during and after a pain episode, and 2) before, during, and after a 24-hour infusion of the investigative agent, regadenoson, an adenosine A2A agonist. CEUS destruction-replenishment time-intensity data were analyzed to measure microvascular blood flow, as well as its components, microvascular blood volume and flux rate. Serial CEUS measurements were obtained in 32 adults with SCD. For the studies during crisis, there was a 30% reduction in microvascular blood flow compared to steady-state (p = 0.031), a reduction that was largely due to microvascular flux rate. For the regadenoson group, a non-significant 25% increase in flux rate and 9% increase in microvascular blood flow compared to baseline were detected during infusion. In a study of adults with SCD, CEUS detected changes in microvascular blood flow associated with vaso-occlusive crises. No changes were found during an infusion of the adenosine A2A agonist, regadenoson. This study provides preliminary evidence that CEUS could detect blood flow changes consistent with SCD physiology.

Purine Release, Metabolism, and Signaling in the Inflammatory Response.

ATP, NAD+, and nucleic acids are abundant purines that, in addition to having critical intracellular functions, have evolved extracellular roles as danger signals released in response to cell lysis, apoptosis, degranulation, or membrane pore formation. In general ATP and NAD+ have excitatory and adenosine has anti-inflammatory effects on immune cells. This review focuses on recent advances in our understanding of purine release mechanisms, ectoenzymes that metabolize purines (CD38, CD39, CD73, ENPP1, and ENPP2/autotaxin), and signaling by key P2 purinergic receptors (P2X7, P2Y2, and P2Y12). In addition to metabolizing ATP or NAD+, some purinergic ectoenzymes metabolize other inflammatory modulators, notably lysophosphatidic acid and cyclic GMP-AMP (cGAMP). Also discussed are extracellular signaling effects of NAD+ mediated by ADP-ribosylation, and epigenetic effects of intracellular adenosine mediated by modification of S-adenosylmethionine-dependent DNA methylation.

Induction of antiinflammatory purinergic signaling in activated human iNKT cells.

Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define "purinergic Th2-type cytokine bias" as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.

A2AR Adenosine Signaling Suppresses Natural Killer Cell Maturation in the Tumor Microenvironment.

Extracellular adenosine is a key immunosuppressive metabolite that restricts activation of cytotoxic lymphocytes and impairs antitumor immune responses. Here, we show that engagement of A2A adenosine receptor (A2AR) acts as a checkpoint that limits the maturation of natural killer (NK) cells. Both global and NK-cell-specific conditional deletion of A2AR enhanced proportions of terminally mature NK cells at homeostasis, following reconstitution, and in the tumor microenvironment. Notably, A2AR-deficient, terminally mature NK cells retained proliferative capacity and exhibited heightened reconstitution in competitive transfer assays. Moreover, targeting A2AR specifically on NK cells also improved tumor control and delayed tumor initiation. Taken together, our results establish A2AR-mediated adenosine signaling as an intrinsic negative regulator of NK-cell maturation and antitumor immune responses. On the basis of these findings, we propose that administering A2AR antagonists concurrently with NK cell-based therapies may heighten therapeutic benefits by augmenting NK cell-mediated antitumor immunity.Significance: Ablating adenosine signaling is found to promote natural killer cell maturation and antitumor immunity and reduce tumor growth. Cancer Res; 78(4); 1003-16. ©2017 AACR.

Pediatric tolerogenic DCs expressing CD4 and immunoglobulin-like transcript receptor (ILT)-4 secrete IL-10 in response to Fc and adenosine.

We characterized a novel population of tolerogenic myeloid dendritic cells (tmDCs) defined as CD11c+ CD11b+ CD14+ CD4+ and immunoglobulin-like transcript receptor (ILT)-4+ that are significantly more abundant in the circulation of infants and young children than in adults. TmDCs secrete the immunosuppressive lymphokine interleukin (IL)-10 when stimulated with the heavy constant region of immunoglobulins (Fc) and express high levels of the adenosine A2A receptor (A2A R), which, when activated by adenosine, inhibits the release of pro-inflammatory cytokines from most immune cells. Here we show that stimulation of the A2A R on tmDCs by regadenoson or N-ethylcarboxamidoadenosine (NECA) rapidly increases cyclic AMP accumulation and enhances IL-10 production under Fc stimulatory conditions. In co-culture experiments, tmDCs inhibit the differentiation of naïve T cells to a pro-inflammatory phenotype. In conclusion, although DCs are classically viewed as antigen presenting cells that activate T cells, we show an independent role of tmDCs in pediatric immune regulation that may be important for suppressing T cell responses to neoantigens in infants and young children.

Exercise versus vasodilator stress limb perfusion imaging for the assessment of peripheral artery disease.

PURPOSE: Our aim was to determine whether pharmacologic vasodilation is an alternative to exercise stress during limb perfusion imaging for peripheral artery disease (PAD). METHODS: Quantitative contrast-enhanced ultrasound (CEU) perfusion imaging of the bilateral anterior thigh and calf was performed in nine control subjects and nine patients with moderate to severe PAD at rest and during vasodilator stress with dipyridamole. For those who were able, CEU of the calf was then performed during modest plantar flexion exercise (20 watts). CEU time-intensity data were analyzed to quantify microvascular blood flow (MBF) and its parametric components of microvascular blood volume and flux rate. RESULTS: Thigh and calf skeletal muscle MBF at rest was similar between control and PAD patients. During dipyridamole, MBF increased minimally (