Engineered red blood cells (activating antigen carriers) drive potent T cell responses and tumor regression in mice.

Activation of T cell responses is essential for effective tumor clearance; however, inducing targeted, potent antigen presentation to stimulate T cell responses remains challenging. We generated Activating Antigen Carriers (AACs) by engineering red blood cells (RBCs) to encapsulate relevant tumor antigens and the adjuvant polyinosinic-polycytidylic acid (poly I:C), for use as a tumor-specific cancer vaccine. The processing method and conditions used to create the AACs promote phosphatidylserine exposure on RBCs and thus harness the natural process of aged RBC clearance to enable targeting of the AACs to endogenous professional antigen presenting cells (APCs) without the use of chemicals or viral vectors. AAC uptake, antigen processing, and presentation by APCs drive antigen-specific activation of T cells, both in mouse in vivo and human in vitro systems, promoting polyfunctionality of CD8+ T cells and, in a tumor model, driving high levels of antigen-specific CD8+ T cell infiltration and tumor killing. The efficacy of AAC therapy was further enhanced by combination with the chemotherapeutic agent Cisplatin. In summary, these findings support AACs as a potential vector-free immunotherapy strategy to enable potent antigen presentation and T cell stimulation by endogenous APCs with broad therapeutic potential.

OGR1-dependent regulation of the allergen-induced asthma phenotype.

The proton-sensing receptor, ovarian cancer G protein-coupled receptor (OGR1), has been shown to be expressed in airway smooth muscle (ASM) cells and is capable of promoting ASM contraction in response to decreased extracellular pH. OGR1 knockout (OGR1KO) mice are reported to be resistant to the asthma features induced by inhaled allergen. We recently described certain benzodiazepines as OGR1 activators capable of mediating both procontractile and prorelaxant signaling in ASM cells. Here we assess the effect of treatment with the benzodiazepines lorazepam or sulazepam on the asthma phenotype in wild-type (WT) and OGR1KO mice subjected to inhaled house dust mite (HDM; Dermatophagoides pteronyssius) challenge for 3 wk. In contrast to previously published reports, both WT and OGR1KO mice developed significant allergen-induced lung inflammation and airway hyperresponsiveness (AHR). In WT mice, treatment with sulazepam (a Gs-biased OGR1 agonist), but not lorazepam (a balanced OGR1 agonist), prevented allergen-induced AHR, although neither drug inhibited lung inflammation. The protection from development of AHR conferred by sulazepam was absent in OGR1KO mice. Treatment of WT mice with sulazepam also resulted in significant inhibition of HDM-induced collagen accumulation in the lung tissue. These findings suggest that OGR1 expression is not a requirement for development of the allergen-induced asthma phenotype, but OGR1 can be targeted by the Gs-biased OGR1 agonist sulazepam (but not the balanced agonist lorazepam) to protect from allergen-induced AHR, possibly mediated via suppression of chronic bronchoconstriction and airway remodeling in the absence of effects on airway inflammation.

Regulation of ovarian cancer G protein-coupled receptor-1 expression and signaling.

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pHo) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pHo or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pHo and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pHo or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins.

α-Catenin-dependent cytoskeletal tension controls Yap activity in the heart.

Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. At the same time, the N-cadherin/catenin cell adhesion complex accumulates at the cell termini, creating a specialized type of cell-cell contact called the intercalated disc (ICD). To investigate the relationship between ICD maturation and proliferation, αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes were deleted to generate cardiac-specific α-catenin double knockout (DKO) mice. DKO mice exhibited aberrant N-cadherin expression, mislocalized actomyosin activity and increased cardiomyocyte proliferation that was dependent on Yap activity. To assess effects on tension, cardiomyocytes were cultured on deformable polyacrylamide hydrogels of varying stiffness. When grown on a stiff substrate, DKO cardiomyocytes exhibited increased cell spreading, nuclear Yap and proliferation. A low dose of either a myosin or RhoA inhibitor was sufficient to block Yap accumulation in the nucleus. Finally, activation of RhoA was sufficient to increase nuclear Yap in wild-type cardiomyocytes. These data demonstrate that α-catenins regulate ICD maturation and actomyosin contractility, which, in turn, control Yap subcellular localization, thus providing an explanation for the loss of proliferative capacity in the newborn mammalian heart.

Bitter Taste Receptor Agonists Mitigate Features of Allergic Asthma in Mice.

Asthma is characterized by airway inflammation, mucus secretion, remodeling and hyperresponsiveness (AHR). Recent research has established the bronchodilatory effect of bitter taste receptor (TAS2R) agonists in various models. Comprehensive pre-clinical studies aimed at establishing effectiveness of TAS2R agonists in disease models are lacking. Here we aimed to determine the effect of TAS2R agonists on features of asthma. Further, we elucidated a mechanism by which TAS2R agonists mitigate features of asthma. Asthma was induced in mice using intranasal house dust mite or aerosol ova-albumin challenge, and chloroquine or quinine were tested in both prophylactic and treatment models. Allergen challenge resulted in airway inflammation as evidenced by increased immune cells infiltration and release of cytokines and chemokines in the lungs, which were significantly attenuated in TAS2R agonists treated mice. TAS2R agonists attenuated features of airway remodeling including smooth muscle mass, extracellular matrix deposition and pro-fibrotic signaling, and also prevented mucus accumulation and development of AHR in mice. Mechanistic studies using human neutrophils demonstrated that inhibition of immune cell chemotaxis is a key mechanism by which TAS2R agonists blocked allergic airway inflammation and exerted anti-asthma effects. Our comprehensive studies establish the effectiveness of TAS2R agonists in mitigating multiple features of allergic asthma.

Alpha-catenins control cardiomyocyte proliferation by regulating Yap activity.

RATIONALE: Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood. OBJECTIVE: To investigate the role of α-catenins in the heart using cardiac-specific αE- and αT-catenin double knockout mice. METHODS AND RESULTS: We used 2 cardiac-specific Cre transgenes to delete both αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of α-catenins increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the α-catenin double knockout hearts. Fetal genes were increased in the α-catenin double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of α-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of α-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction. CONCLUSIONS: These studies demonstrate that α-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting α-catenins might be a useful strategy to promote myocardial regeneration after injury.