Systemic hypertension, preeclampsia, and pulmonary arterial hypertension (PAH) are diseases of high blood pressure in the systemic or pulmonary circulation. Beyond the well-defined contribution of more traditional pathophysiological mechanisms, such as changes in the renin-angiotensin-aldosterone system, to the development of these hypertensive disorders, there is substantial clinical evidence supporting an important role for inflammation and immunity in the pathogenesis of each of these three conditions. Over the last decade, work in small animal models, bearing targeted deficiencies in specific cytokines or immune cell subsets, has begun to clarify the immune-mediated mechanisms that drive changes in vascular structure and tone in hypertensive disease. By summarizing the clinical and experimental evidence supporting a contribution of the immune system to systemic hypertension, preeclampsia, and PAH, the current review highlights the cellular and molecular pathways that are common to all three hypertensive disorders. These mechanisms are centered on an imbalance in CD4 helper T cell populations, defined by excessive Th17 responses and impaired T activity, as well as the excessive activation or impairment of additional immune cell types, including macrophages, dendritic cells, CD8 T cells, B cells, and natural killer cells. The identification of common immune mechanisms in systemic hypertension, preeclampsia, and PAH raises the possibility of new therapeutic strategies that target the immune component of hypertension across multiple disorders.

Natural killer (NK) cells are cytotoxic innate lymphoid cells with an established role in the regulation of vascular structure in pregnancy and cancer. Impaired NK cell function has been identified in patients with pulmonary arterial hypertension (PAH), a disease of obstructive vascular remodeling in the lungs, as well as in multiple rodent models of disease. However, the precise contribution of NK cell impairment to the initiation and progression of PAH remains unknown. Here, we report the development of spontaneous pulmonary hypertension in two independent genetic models of NK cell dysfunction, including Nfil3−/− mice, which are deficient in NK cells due to the absence of the NFIL3 transcription factor, and Ncr1-Gfp mice, which lack the NK activating receptor NKp46. Mouse models of NK insufficiency exhibited increased right ventricular systolic pressure and muscularization of the pulmonary arteries in the absence of elevated left ventricular end-diastolic pressure, indicating that the development of pulmonary hypertension was not secondary to left heart dysfunction. In cases of severe NK cell impairment or loss, a subset of mice failed to develop pulmonary hypertension and instead exhibited reduced systemic blood pressure, demonstrating an extension of vascular abnormalities beyond the pulmonary circulation into the systemic vasculature. In both mouse models, the development of PAH was linked to elevated interleukin-23 production, whereas systemic hypotension in Ncr1-Gfp mice was accompanied by a loss of angiopoietin-2. Together, these results support an important role for NK cells in the regulation of pulmonary and systemic vascular function and the pathogenesis of PAH.

Allelic variants of the pan-haematopoietic cell marker CD45, identified as CD45.1 and CD45.2, have been established as a marker system to track haematopoietic cells following congenic mouse bone marrow transplants. Despite the frequent use of this model for studying the impact of genetic modifications on relative differentiation potential, it is now evident that a bias exists in CD45.1 versus CD45.2 cell reconstitution. While this bias has been demonstrated by reduced reconstitution potential in B cells of CD45.1 origin, differences in the development of other lymphocytes, as well as the impact of sex on this bias, remain uncertain. We performed bone marrow transplants with wild-type CD45.1 and CD45.2 donor cells, and characterised haematopoietic cell reconstitution in dual-expressing CD45.1/2 host mice. We report an increase in CD45.2 reconstitution in the bone marrow that persists in the spleen, thymus and blood. Through the use of CD45.1/2 hosts, we demonstrate the intrinsic bias towards CD45.2 reconstitution is independent of an immunogenic response to the CD45.1 epitope. Furthermore, we identify a sex-specific difference in reconstitution efficiencies, with female mice exhibiting a greater bias towards CD45.2 reconstitution than males. This work sheds new light on the limitations of the CD45.1/CD45.2 congenic system for tracking lymphocyte development.

Background: Natural killer (NK) cell impairment is a feature of pulmonary arterial hypertension (PAH) and contributes to vascular remodeling in animal models of disease. Although mutations in BMPR2 , the gene encoding the BMP (bone morphogenetic protein) type-II receptor, are strongly associated with PAH, the contribution of BMPR2 loss to NK cell impairment remains unknown. We explored the impairment of IL (interleukin)-15 signaling, a central mediator of NK cell homeostasis, as both a downstream target of BMPR2 loss and a contributor to the pathogenesis of PAH. Methods: The expression, trafficking, and secretion of IL-15 and IL-15Rα (interleukin 15 α-type receptor) were assessed in human pulmonary artery endothelial cells, with or without BMPR2 silencing. NK cell development and IL-15/IL-15Rα levels were quantified in mice bearing a heterozygous knock-in of the R899X-BMPR2 mutation ( bmpr2 +/R899X ). NK-deficient Il15 −/− rats were exposed to the Sugen/hypoxia and monocrotaline models of PAH to assess the impact of impaired IL-15 signaling on disease severity. Results: BMPR2 loss reduced IL-15Rα surface presentation and secretion in human pulmonary artery endothelial cells via impaired trafficking through the trans-Golgi network. bmpr2 +/R899X mice exhibited a decrease in NK cells, which was not attributable to impaired hematopoietic development but was instead associated with reduced IL-15/IL-15Rα levels in these animals. Il15 −/− rats of both sexes exhibited enhanced disease severity in the Sugen/hypoxia model, with only male Il15 −/− rats developing more severe PAH in response to monocrotaline. Conclusions: This work identifies the loss of IL-15 signaling as a novel BMPR2 -dependent contributor to NK cell impairment and pulmonary vascular disease.

Background: AXL receptor tyrosine kinase (AXL) is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer, stromal, and select immune cells, and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL can be mediated by its ligand, growth arrest specific protein 6 (GAS6), or via ligand-independent homo/hetero-dimerization, both of which facilitate AXL phosphorylation, initiation of signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective AXL inhibitor, AB801. Materials and Methods: The potency and selectivity of AB801 against AXL and other kinases were determined using a panel of HTRF KinEASE-TK assays and via a competition binding assay utilizing DNA-tagged kinases. The effects of AB801 were further assessed by a cell-based phospho-AXL ELISA. The pharmacokinetic (PK) profile of the molecule was evaluated in preclinical species. AB801 was characterized in routine in vitro safety assays, including hERG inhibition. Downstream signaling of AXL was evaluated by phospho-array, Western blot, and qPCR. Pharmacodynamics (PD) and anti-tumor efficacy in combination with standard of care (SOC) therapies were assessed in murine cancer models. Results: The novel AXL inhibitor AB801 is potent, reversible, and selective. AB801 exhibits cellular activity at low nanomolar concentrations and retains significant activity in 100% human serum. Excellent selectivity was observed against MERTK (860x), TYRO3 (1,400x), and the overall kinome. Importantly, AB801 does not show significant CYP450 or hERG inhibition. Favorable preclinical PK is consistent with projected once-a-day oral administration in humans. AB801 increases sensitivity to SOC therapeutics such as chemotherapy, and results in increased DNA damage. Moreover, AB801 treatment sensitizes tumors to checkpoint blockade by increasing immune cell activation. Significant anti-tumor efficacy is observed in combination SOC therapies in multiple in vivo models. Conclusions: AXL inhibition is a promising therapeutic mechanism for impairing the growth of tumors resistant to SOC therapeutics. AB801 exhibits improved potency, selectivity, and safety profiles compared to other AXL inhibitors currently advancing into clinical development. Citation Format: Susan Lee Paprcka, Armon Goshayeshi, Suan Liu, Ruben Flores, Lauren Rocha, Jhansi L. Leslie, Dillon H. Miles, Corinne N. Foley, Shiwei Qu, Manjunath Lamani, Srinivas Paladugu, Hsin-Ting Huang, Nidhi Tribewal, Ada Chen, Joseph Kulusich, Stefan Garrido-Shaqfeh, Patricia Fabila, Salema Jafri, Anuja Devarajan, Ester Fernandez-Salas. AB801 is a highly potent and selective AXL kinase inhibitor that demonstrates significant anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 518.