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.

Randomized phase 2 trial of regadenoson for treatment of acute vaso-occlusive crises in sickle cell disease.

Adenosine A2A receptor (A2AR) agonists have been shown to decrease tissue inflammation induced by hypoxia/reoxygenation in mice with sickle cell disease (SCD). The key mediator of the A2AR agonist's anti-inflammatory effects is a minor lymphocyte subset, invariant natural killer T (iNKT) cells. We tested the hypothesis that administration of an A2AR agonist in patients with SCD would decrease iNKT cell activation and dampen the severity of vaso-occlusive (VO) crises. In a phase 2, randomized, placebo-controlled trial, we administered a 48-hour infusion of the A2AR agonist regadenoson (1.44 µg/kg per hour) to patients with SCD during VO crises to produce a plasma concentration of ∼5 nM, a concentration known from prior studies to suppress iNKT cell activation in SCD. The primary outcome measure was a >30% reduction in the percentage of activated iNKT cells. Ninety-two patients with SCD were randomized to receive a 48-hour infusion of regadenoson or placebo, in addition to standard-of-care treatment, during hospital admission for a VO crisis and had analyzable iNKT cell samples. The proportion of subjects who demonstrated a reduction of >30% in activated iNKT cells was not significantly different between the regadenoson and placebo arms (43% vs 23%; P = .07). There were also no differences between regadenoson and placebo groups in length of hospital stay, mean total opioid use, or pain scores. These data demonstrate that a low-dose infusion of regadenoson intended to reduce the activity of iNKT cells is not sufficient to produce a statistically significant reduction in such activation or in measures of clinical efficacy. This trial was registered at www.clinicaltrials.gov as #NCT01788631.

NF-κB is activated in CD4+ iNKT cells by sickle cell disease and mediates rapid induction of adenosine A2A receptors.

Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.

Sickle cell vaso-occlusion causes activation of iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson.

Adenosine A2A receptor (A2AR) agonists reduce invariant natural killer T (iNKT) cell activation and decrease inflammation in sickle cell disease (SCD) mice. We conducted a phase 1 trial of the A2AR agonist regadenoson in adults with SCD. The target dose was 1.44 µg/kg/h. iNKT cell activation was evaluated using antibodies targeting the p65 subunit of nuclear factor-κB (phospho-NF-κB p65), interferon-γ (IFN-γ), and A2AR. Regadenoson was administered to 27 adults with SCD. We examined 21 patients at steady state and 6 during painful vaso-occlusive crises (pVOC). iNKT cell activation was also measured in 14 African-American controls. During pVOC, the fraction of iNKT cells demonstrating increased phospho-NF-κB p65 and A2AR expression was significantly higher compared with controls (P < .01) and steady-state patients (P < .05). IFN-γ expression was also significantly higher compared with controls (P = .02). After a 24-hour infusion of regadenoson during pVOC, phospho-NF-κB p65 activation in iNKT cells decreased compared to baseline by a median of 48% (P = .03) to levels similar to controls and steady-state SCD. No toxicities were identified. Infusional regadenoson administered to adults with SCD at 1.44 µg/kg/h during pVOC decreases activation of iNKT cells without toxicity.

A model-based tool to predict the propagation of infectious disease via airports.

Epidemics of novel or re-emerging infectious diseases have quickly spread globally via air travel, as highlighted by pandemic H1N1 influenza in 2009 (pH1N1). Federal, state, and local public health responders must be able to plan for and respond to these events at aviation points of entry. The emergence of a novel influenza virus and its spread to the United States were simulated for February 2009 from 55 international metropolitan areas using three basic reproduction numbers (R(0)): 1.53, 1.70, and 1.90. Empirical data from the pH1N1 virus were used to validate our SEIR model. Time to entry to the U.S. during the early stages of a prototypical novel communicable disease was predicted based on the aviation network patterns and the epidemiology of the disease. For example, approximately 96% of origins (R(0) of 1.53) propagated a disease into the U.S. in under 75 days, 90% of these origins propagated a disease in under 50 days. An R(0) of 1.53 reproduced the pH1NI observations. The ability to anticipate the rate and location of disease introduction into the U.S. provides greater opportunity to plan responses based on the scenario as it is unfolding. This simulation tool can aid public health officials to assess risk and leverage resources efficiently.

Sickle cell disease (SCD), iNKT cells, and regadenoson infusion.

A humanized murine sickle cell-disease (SCD) model (NY1DD) has been used to study ischemia/reperfusion injury (IRI) in sickle cell anemia, and iNKT cells (a very small subset of murine and human T cells) have been found to instigate such injury in this model. Furthermore, levels of activated iNKT cells are generally elevated in the circulation of patients with SCD. Because activated iNKT cells are rich in adenosine A2A receptors which, when agonized, down-regulate the inflammatory cytokine expression that characterizes the cells, we have conducted a phase 1 trial of a constant infusion of low-dose regadenoson (an adenosine analogue with high A2A receptor specificity) to determine its safety and the capacity of a safe dose to down-regulate circulating iNKT cells in patients with SCD. We have found two dose rates that are both safe and effective and now plan a controlled Phase 2B clinical trial to determine whether our highest dose, administered as a 48-hour constant infusion, will induce faster remission in both painful vaso-occlusive crisis (pVOC) and acute chest syndrome (ACS).

An analysis on the detection of biological contaminants aboard aircraft.

The spread of infectious disease via commercial airliner travel is a significant and realistic threat. To shed some light on the feasibility of detecting airborne pathogens, a sensor integration study has been conducted and computational investigations of contaminant transport in an aircraft cabin have been performed. Our study took into consideration sensor sensitivity as well as the time-to-answer, size, weight and the power of best available commercial off-the-shelf (COTS) devices. We conducted computational fluid dynamics simulations to investigate three types of scenarios: (1) nominal breathing (up to 20 breaths per minute) and coughing (20 times per hour); (2) nominal breathing and sneezing (4 times per hour); and (3) nominal breathing only. Each scenario was implemented with one or seven infectious passengers expelling air and sneezes or coughs at the stated frequencies. Scenario 2 was implemented with two additional cases in which one infectious passenger expelled 20 and 50 sneezes per hour, respectively. All computations were based on 90 minutes of sampling using specifications from a COTS aerosol collector and biosensor. Only biosensors that could provide an answer in under 20 minutes without any manual preparation steps were included. The principal finding was that the steady-state bacteria concentrations in aircraft would be high enough to be detected in the case where seven infectious passengers are exhaling under scenarios 1 and 2 and where one infectious passenger is actively exhaling in scenario 2. Breathing alone failed to generate sufficient bacterial particles for detection, and none of the scenarios generated sufficient viral particles for detection to be feasible. These results suggest that more sensitive sensors than the COTS devices currently available and/or sampling of individual passengers would be needed for the detection of bacteria and viruses in aircraft.