Adenosine 2A Receptor Inhibition Research Articles

Increased spinal adenosine impairs phrenic long-term facilitation in aging rats.

Moderate acute intermittent hypoxia (mAIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long-term facilitation (pLTF). In middle-aged male and geriatric female rats, mAIH-induced pLTF is attenuated through unknown mechanisms. In young adults, mAIH activates competing intracellular signaling cascades, initiated by serotonin 2 and adenosine 2A (A2A) receptors, respectively. Spinal A2A receptor inhibition enhances mAIH-induced pLTF, meaning, serotonin dominates, and adenosine constrains mAIH-induced plasticity in the daily rest phase. Thus, we hypothesized elevated basal adenosine levels in the ventral cervical spinal cord of aged rats shifts this balance, undermining mAIH-induced pLTF. A selective A2A receptor antagonist (MSX-3) or vehicle was delivered intrathecally at C4 in anesthetized young (3-6 mo) and aged (20-22 mo) Sprague-Dawley rats before mAIH (3,5-min episodes; arterial Po2 = 45-55 mmHg). In young males, spinal A2A receptor inhibition enhanced pLTF (119 ± 5%) vs. vehicle (55 ± 9%), consistent with prior reports. In old males, pLTF was reduced to 25 ± 11%, but A2A receptor inhibition increased pLTF to levels greater than in young males (186 ± 19%). Basal adenosine levels in ventral C3-C5 homogenates are elevated two- to threefold in old vs. young males. These findings advance our understanding of age as a biological variable in phrenic motor plasticity and will help guide translation of mAIH as a therapeutic modality to restore respiratory and nonrespiratory movements in older populations afflicted with clinical disorders that compromise movement.NEW & NOTEWORTHY Advanced age undermines respiratory motor plasticity, specifically phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH). We report that spinal adenosine increases in aged male rats, undermining mAIH-induced pLTF via adenosine 2A (A2A) receptor activation, an effect reversed by selective spinal adenosine 2A receptor inhibition. These findings advance our understanding of mechanisms that impair neuroplasticity, and the ability to compensate for the onset of lung or neural injury with age, and may guide efforts to harness mAIH as a treatment for clinical disorders that compromise breathing and other movements.

Upregulated adenosine 2A receptor accelerates post-infectious irritable bowel syndrome by promoting CD4+ T cells' T helper 17 polarization.

BACKGROUNDPost-infectious irritable bowel syndrome (PI-IBS) is generally regarded as a functional disease. Several recent studies have reported the involvement of low-grade inflammation and immunological dysfunction in PI-IBS. T helper 17 (Th17) polarization occurs in IBS. Adenosine and its receptors participate in intestinal inflammation and immune regulation.AIMTo investigate the role of Th17 polarization of CD4+ T cells regulated by adenosine 2A receptor (A2AR) in PI-IBS.METHODSA PI-IBS model was established by infecting mice with Trichinella spiralis. The intestinal A2AR and CD4+ T lymphocytes were detected by immunohistochemistry, and the inflammatory cytokines were detected by enzyme-linked immunoassay. CD4+ T lymphocytes present in the animal’s spleen were separated and cultured with or without A2AR agonist and antagonist. Western blotting and real-time quantitative polymerase chain reaction were performed to determine the effect of A2AR on the cells and intestinal tissue. Cytokine production was determined. The protein and mRNA levels of A2AR associated signaling pathway molecules were also evaluated. Furthermore, A2AR agonist and antagonist were injected into the mouse model and the clinical features were observed.RESULTSThe PI-IBS mouse model showed increased expression of ATP and A2AR (P < 0.05), and inhibition of A2AR improved the clinical features in PI-IBS, including the abdominal withdrawal reflex and colon transportation test (P < 0.05). The number of intestinal CD4+ T cells and interleukin-17 (IL-17) protein levels increased during PI-IBS, which was reversed by administration of the A2AR antagonist (P < 0.05). CD4+ T cells expressed A2AR and produced IL-17 in vitro, which was regulated by the A2AR agonist and antagonist. The A2AR antagonist increased the production of IL-17 by CD4+ T cells via the Janus kinase-signal transducer and activator of transcription-receptor-related orphan receptor γ signaling pathway.CONCLUSIONThe results of the present study suggested that the upregulation of A2AR increases PI-IBS by promoting the Th17 polarization of CD4+ T cells.

Aging Impairs Phrenic Long‐Term Facilitation in Rats by an Adenosine‐Dependent Mechanism

Phrenic long‐term facilitation (pLTF) is a form of respiratory plasticity elicited by acute intermittent hypoxia (AIH). AIH activates competing intracellular signaling cascades, initiated by phrenic motor neuron serotonin 2 and adenosine 2A (A2A) receptors, respectively. With moderate AIH (mAIH), the serotonin‐dependent Q pathway to phrenic motor facilitation dominates, with an adenosine constraint. Consequently, spinal A2Areceptor inhibition enhances mAIH‐induced pLTF. In middle‐aged male, and geriatric acyclic female rats, pLTF is attenuated through unknown mechanisms. We hypothesize that basal spinal adenosine levels are elevated in aged rats, thereby undermining mAIH‐induced pLTF. Thus, we predict that spinal A2A receptor inhibition in aged male rats will restore pLTF to levels equal to young male rats with A2A receptor inhibition. A selective A2A receptor antagonist (MSX‐3; 10 uM, 12 uL) or vehicle (0.9% sterile saline) were delivered intrathecally (C4) in anesthetized, vagotomized, paralyzed and ventilated young (3‐6 months) and aged (20‐22 months) male Sprague‐Dawley rats prior to mAIH (3, 5 min hypoxic episodes; arterial Po2= 45‐55 mmHg). In young males, mAIH elicits ~ 60% pLTF at 60 min post‐AIH. Spinal A2A receptor inhibition significantly enhanced mAIH‐induced pLTF in young males (119 + 5%; n = 6) vs vehicle controls (55 + 9%; n = 6; p &lt; 0.001) and time controls (12 + 4%; n = 6; p &lt; 0.001). In aged males, A2A receptor inhibition enhanced mAIH‐induced pLTF to a greater extent (186 + 19%; n = 7) vs vehicle controls (25 + 11%; n = 7; p &lt; 0.001) and time controls (3 + 5%; n = 4; p &lt; 0.001). Measured basal adenosine levels in ventral C3‐C5 homogenates were significantly elevated in aged vs young males (p = 0.008). Preliminary data suggest aged female rats (16‐18 months) also have enhanced mAIH‐induced pLTF with A2A receptor inhibition (173+ 24%; n = 3) vs vehicle controls (53 + 7%; n = 3) and time controls (5 + 17%; n = 2) in persistent estrus. These findings advance our understanding of mechanisms whereby age undermines mAIH‐induced phrenic motor plasticity. Understanding age and sex effects on AIH‐induced respiratory motor plasticity is crucial as we harness therapeutic AIH to treat severe neuromuscular disorders that compromise breathing.

High glutamate concentration reverses the inhibitory effect of microglial adenosine 2A receptor on NLRP3 inflammasome assembly and activation

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A2AR) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A2AR on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A2AR on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A2AR activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A2AR activation or the glutamate concentration. We further demonstrated that A2AR activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A2AR on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A2AR on neuroinflammation under different pathological conditions.

Abstract 1568: A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer

Abstract Extracellular adenosine impairs immune function in tumors and limits the efficacy of anti-CTAL4 immune checkpoint blockade. We previously showed that adenosine 2A receptor (A2aR) blockade with AZD4635 moderately inhibited tumor growth, in part by improving antigen presentation and cytotoxic T cell (CTL) activity in mouse Pten-deficient prostate cancer. Here, we continue to investigate the antitumor activity of AZD4635 and its effects when combined with CTLA4 blockade (aCTLA4) in preclinical models of Pten-deficient prostate cancer. Prostate tumors from Pten conditional knockout (KO) mice treated with aCTLA4 for five days had increased expression levels of adenosinergic genes. Furthermore, gene expression, flow cytometric and IHC analyses showed increased T regulatory cell differentiation and accumulation that was offset with the addition of AZD4635. Moreover, expression levels of genes associated with Th 1 cell differentiation and T cell-mediated cytotoxicity were increased in AZD4635/aCTLA4 treated tumors. Adding AZD4635 to a four-week regimen of aCTLA4 therapy improved the antitumor response by two-fold in an early-stage intervention model of Pten-deficient prostate cancer. Androgen deprivation (AD) has the potential to promote T cell infiltration in prostate tumors and using the AZD4635/aCTLA4 treatment combination as neoadjuvant therapy to AD via surgical castration led to a greater antitumor response. The antitumor activity of this treatment combination was further examined and confirmed in a subcutaneous syngeneic genome-derived allograft model using tumor-bearing conditional Pten-deficient knockout mice grafted with Pten-deficient CRPC tumor fragments. In a Pten/Trp53 conditional double knockout (DKO) mouse model of advanced prostate cancer, the AZD4635/aCTLA4 treatment combination did not improve overall survival rates. However, when AZD4635/aCTLA4 was used as neoadjuvant therapy to AR inhibition with the anti-androgen apalutamide, it improved median survival time from 21 days in monotherapy treated mice to 34 days in AZD4635/aCTLA4 treated mice. These results provide preclinical evidence to support the rational combination of A2AR blockade with AZD4635 and aCTLA4 immune checkpoint inhibition for PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Alwin Schuller, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1568.

Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated.MethodsWe report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition.ResultsWe provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies.ConclusionWe provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.

A phase I, open-label, multicenter study to assess the safety, pharmacokinetics, and preliminary antitumor activity of AZD4635 both as monotherapy and in combination in patients with advanced solid malignancies: Results from prostate cancer patients (NCT02740985).

5518 Background: AZD4635 inhibits adenosine 2a receptor (A2aR) signaling and improves immune activation and anti-tumor activity in preclinical models. This phase I study assessed the safety, pharmacokinetics, pharmacodynamics and efficacy of AZD4635 monotherapy (mono) and in combination (combo) with durvalumab (durva) in patients (pts) with refractory solid tumors. Here we present data for immune checkpoint-naïve pts with metastatic castrate-resistant prostate cancer (mCRPC). Methods: Pts with refractory mCRPC received AZD4635 mono (75 mg or 100 mg QD oral nanosuspension [(RP2D]) or in combination (75 mg or 100mg QD) with durva 1.5g IV q4wk. Results: Between 30Aug16 and 20Jun19 (data cutoff [DCO]) 94 mCRPC pts were treated with mono (n = 49) or combo (n = 45): median age 70.5 yrs; ECOG 0-1 = 99%. The median number of prior treatment regimens was 5 (range = 1-10); 61% of pts (57/94) had prior chemotherapy, 90% had prior new hormonal therapy. PK data suggest AZD4635 concentrations at 75-100 mg QD are above the in vitro IC50 for A2aR inhibition throughout the dosing interval. Modeling predicts 80-90% A2aR occupancy at steady state for doses at ≥75 mg QD. Most common treatment-related AEs ( &gt; 10%) were nausea, vomiting, fatigue, decreased appetite, dizziness, and diarrhea. At the DCO in this ongoing study, 70 pts were evaluable for response by RECIST v1.1 (mono = 33, combo = 37). Confirmed response occurred in 8 pts: mono = ORR 6.1% (2PRs) and combo = 16.2% (2CRs, 4PRs). The duration of response across cohorts ranged from 1-18.5 mo (5 pts ongoing). PSA response (defined as ≥50% decrease from baseline of ≥1ng/ml) was observed in 6.4% (3/47 pts; 95% CI, 1.3-17.5) of mono-treated patients and 20% (9/45 pts; 95% CI, 9.6-34.6) of combo-treated patients. Patients with high adenosine (ADO) gene expression signature (N = 46) in peripheral blood, showed a median PFS of 21 weeks v. 8.7 weeks in ADO signature low patients (N = 46) (HR 0.5, CI 0.3-0.9) (DCO 20Jun19). In addition, baseline TCR clonality and diversity were linked with response. Conclusions: In mCRPC pts, AZD4635 alone or in combination with durva was tolerable and associated with clinical benefit. A mCRPC phase II trial is ongoing with continued exploration of predictors of response to treatment. Clinical trial information: NCT02740985 .

Expression of a short antibody heavy chain peptide effectively antagonizes adenosine 2A receptor in vitro and in vivo

ABSTRACT Background Adenosine 2A receptor (A2AR) is involved in many physiological and pathological functions and serves as an important drug target. Inhibition of A2AR may alleviate symptoms associated with a variety of neuropsychiatric disorders. However, the currently used A2AR antagonists have specificity limitations. Research design and methods A Fab fragment (Fab2838) of an A2AR mouse monoclonal antibody can specifically bind to A2AR to form a complex and inhibit the activity of its receptor. We constructed the vector AntiA2AR, a small-molecule peptide that binds to and inhibits A2AR based on Fab2838. Results Experiments in HEK293T cells showed that peptide AntiA2AR of 29 peptides was the most effective among the synthesized peptides in inhibiting the A2AR downstream signal cAMP/PKA/CREB. In neurons, the AntiA2AR reversed the calcium flow change induced by the A2AR agonist CGS21680 (1 μM). Furthermore, AntiA2AR expression in the mice striatum weakened the p-PKA/p-CREB signal, enhanced locomotor abilities and increased time spent in the center area in the home-cage observation experiment and increased anxiolytic behavior in the elevated-plus maze test. Conclusions Antagonistic peptide AntiA2AR can effectively block the A2AR signaling pathway. This provides a new strategy for the specific inhibition of A2AR and provides information needed for drug development.

Pharmacological targeting of immune checkpoint A2aR improves function of anti-CD19 CAR T cells in vitro

In spite of impressive results in the treatment of acute lymphoblastic B cell leukemia (B-ALL) with chimeric antigen receptor (CAR) T cells, the clinical outcome of some hematological cancers like follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL) has not been very promising likely due to immunosuppressive networks within tumor microenvironment. Hypoxia in the microenvironment of hematological malignancies and consequently generation of adenosine molecule is appeared to be correlated with immunosuppression, tumor progression, and relapse. Herein, we hypothesized that whether pharmacological targeting of adenosine 2a receptor (A2aR) can enhance antitumor activity of anti-CD19 CAR T cells in vitro. Prior to functional assays, A2aR expression was assessed in CAR-expressing T cells. Our results showed that A2aR was not only up-regulated in the fully human anti-CD19 CAR T cells (hereafter referred to as huCAR19 T cells) but also was further overexpressed following re-stimulation with target cells. Although pharmacological inhibition of A2aR could significantly increase proliferation capacity and cytokine production of huCAR19 T cells following treatment with an adenosine analog, cytotoxic activity of huCAR19 T cells was not significantly improved. Considering A2aR overexpression in huCAR19 T cells in the tumor microenvironment, our results indicated that pharmacological targeting of A2aR could not only improve huCAR19 T cells functionality in a hostile tumor microenvironment but also could have a therapeutic advantage, and sought to assess the possibility in a pre-clinical setting.

Targeting an adenosine-mediated "don't eat me signal" augments anti-lymphoma immunity by anti-CD20 monoclonal antibody.

A growing body of evidence suggests that macrophage immune checkpoint molecules are potential targets in the era of cancer immunotherapy. Here we showed that extracellular adenosine, an abundant metabolite in the tumor microenvironment, critically impedes the therapeutic efficacy of anti-CD20 monoclonal antibodies (mAbs) against B-cell lymphoma. Using a syngeneic B-cell lymphoma model, we showed that host deficiency of adenosine 2A receptor (A2AR), but not A2BR, remarkably improved lymphoma control by anti-CD20 mAb therapy. Conditional deletion of A2AR in myeloid cells, and to a lesser extent in NK cells, augmented therapeutic efficacy of anti-CD20 mAb. Indeed, adenosine signaling impaired antibody-mediated cellular phagocytosis (ADCP) by macrophages and limited the generation of anti-lymphoma CD8+ T cells. Pharmacological inhibition of A2AR overcame the adenosine-mediated negative regulation of ADCP by rituximab in a xeno-transplanted lymphoma model. Moreover, aberrant overexpression of CD39, an apical ecto-enzyme for adenosine generation, showed a negative impact on prognosis in patients with diffuse large B-cell lymphoma, as well as on preclinical efficacy of rituximab. Together, adenosine acts as a "don't eat me signal", and may be a potential target to harness anti-lymphoma immunity.

Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells

BackgroundCAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR).MethodsIn this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software.ResultsOur experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells.ConclusionAltogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

Abstract A87: The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression in tumor microenvironment and enhances antitumor immunity partly by enhancing CD103+ dendritic cells

Abstract Adenosine signaling via the adenosine 2A receptor (A2AR) is emerging as an important regulatory mechanism of immune responses. Adenosine levels are increased in the tumor microenvironment by the CD39/CD73 axis and adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects that promote tumor growth and limit the efficacy of immune checkpoint inhibitors. We provide evidence that AZD4635, an oral A2AR antagonist, inhibits downstream signaling causing enhanced antigen presentation by CD103+ DCs and increased T-cell activation. In our in vitro assays, NECA (a stable analog of adenosine) caused potent suppression of CD3/CD28 stimulated IFNγ secretion in T cells with 1 μM NECA leading to 80% suppression of IFNγ secretion. Incubation of CD8+ T cells with AZD4635 restored IFNγ secretion back to baseline levels with an observed EC50 of 0.053μM and 0.598 μM at 0.1 and 1 μM NECA, respectively. Treatment of mice bearing CT26 colorectal carcinoma and MC38 colorectal carcinoma with AZD4635 monotherapy led to tumor growth inhibition with (TGI) of 44% and 73%, respectively, and in combination with anti-PD-1 with a TGI of 73% and 91%, respectively. Ex vivo analysis of MC38 OVA tumor-bearing mice showed increase in intratumoral CD8 T cells and CD103+DCs in treated groups compared to control. In particular, combination treatment increased the frequency of OVA antigen specific CD8+ T cells as measured by % of cells expressing the activation marker CD25 (2-fold increase) or the proliferation marker Ki67 (2-fold increase). Aligned with increased tumor antigen-specific T-cell response was a 2-fold increase of intratumoral CD103+ DCs. CD103+DCs play a critical role in tumor antigen trafficking and cross-priming to T cells, which are crucial for successful tumor rejection. In vitro, treatment of CD103+ DCs with NECA suppressed about 50% of pinocytosis (uptake of fluorescent beads) and antigen presentation (OVA peptides), which was almost completely rescued by AZD4635 treatment. We extend these studies into human DCs, and show adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen presentation and T-cell costimulation by DCs, leading to 50% increase in priming and expansion of antigen-specific T cells compared to adenosine-treated group. Our results support the role of adenosine signaling as an intrinsic negative regulator of CD103+ DC maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. Our preclinical data support the evaluation of AZD4635 as a single agent and in combination with durvalumab (αPD-L1 antibody) in patients with solid malignancies (www.clinicaltrials.gov- NCT02740985, NCT03980821). Citation Format: Dinesh Chandra, Christine M. Barbon, Alexandra Borodovsky, Yanjun Wang, Minwei Ye, Laura Prickett, Kris Sachsenmeier, Wenlin Shao, Carl Barrett, Stephen Fawell, Deanna A. Mele, Alwin Schuller. The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression in tumor microenvironment and enhances antitumor immunity partly by enhancing CD103+ dendritic cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A87.

Severe sustained hypoxia elicits long‐lasting phrenic motor facilitation (pMF) by an adenosinergic mechanism

Moderate acute intermittent hypoxia (AIH; 3, 5‐min episodes) elicits serotonin‐dependent phrenic long‐term facilitation (pLTF); this form of pLTF is enhanced by spinal adenosine 2A (A2A) receptor inhibition. Severe AIH also elicits pLTF, but via an adenosine A2A receptor dependent (serotonin independent) mechanism. This shift may reflect greater spinal adenosine release/formation during severe AIH. Moderate sustained hypoxia (SH; 9–20‐min) fails to elicit pMF, demonstrating pMF pattern‐sensitivity. However, since severe SH may increase extracellular adenosine levels, we hypothesized that severe SH would elicit pMF via an A2A receptor dependent mechanism. Severe SH (PaO2=25–35; 25 min) was studied in anesthetized, paralyzed and ventilated rats +/− spinal A2A receptor inhibition (MSX‐3, i.t., C4). Severe SH elicits pMF (53%±12% at 60 min post‐SH) vs. time controls (10%±3%; p&lt;0.05, both n=8). MSX‐3 pre‐treatment attenuated severe SH induced pMF (10%±5%; p&lt;0.05, n=7). Thus, severe SH and AIH both elicit spinal A2A‐dependent pMF, unlike moderate SH and AIH (Devinney et al., ibid), indicating that adenosine‐dependent pMF is not pattern sensitive. Understanding mechanisms of pattern sensitivity in respiratory plasticity may enable us to “harness” plasticity as a treatment for respiratory insufficiency.[Supported by NIH HL080209 and T32 007654 and the Francis Families Foundation]