Intrinsic endothelial hyperresponsiveness to inflammatory mediators drives acute episodes in models of Clarkson disease.

Clarkson disease, or monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS), is a rare, relapsing-remitting disorder featuring the abrupt extravasation of fluids and proteins into peripheral tissues, which in turn leads to hypotensive shock, severe hemoconcentration, and hypoalbuminemia. The specific leakage factor(s) and pathways in ISCLS are unknown, and there is no effective treatment for acute flares. Here, we characterize an autonomous vascular endothelial defect in ISCLS that was recapitulated in patient-derived endothelial cells (ECs) in culture and in a mouse model of disease. ISCLS-derived ECs were functionally hyperresponsive to permeability-inducing factors like VEGF and histamine, in part due to increased endothelial nitric oxide synthase (eNOS) activity. eNOS blockade by administration of N(γ)-nitro-l-arginine methyl ester (l-NAME) ameliorated vascular leakage in an SJL/J mouse model of ISCLS induced by histamine or VEGF challenge. eNOS mislocalization and decreased protein phosphatase 2A (PP2A) expression may contribute to eNOS hyperactivation in ISCLS-derived ECs. Our findings provide mechanistic insights into microvascular barrier dysfunction in ISCLS and highlight a potential therapeutic approach.

RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms.

Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

Immunogenetics associated with severe coccidioidomycosis.

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, less than 1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled patients with DCM and performed whole-exome sequencing. In an exploratory set of 67 patients with DCM, 2 had haploinsufficient STAT3 mutations, and defects in ß-glucan sensing and response were seen in 34 of 67 cases. Damaging CLEC7A and PLCG2 variants were associated with impaired production of ß-glucan-stimulated TNF-α from PBMCs compared with healthy controls. Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were overrepresented in DCM, including CLEC7A Y238* and PLCG2 R268W. A validation cohort of 111 patients with DCM confirmed the PLCG2 R268W, CLEC7A I223S, and CLEC7A Y238* variants. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived hydrogen peroxide [H2O2] in transfected cells. Heterozygous DUOX1 or DUOXA1 variants that impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired ß-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.

TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers.

Genetically engineered T cell therapy can induce remarkable tumor responses in hematologic malignancies. However, it is not known if this type of therapy can be applied effectively to epithelial cancers, which account for 80-90% of human malignancies. We have conducted a first-in-human, phase 1 clinical trial of T cells engineered with a T cell receptor targeting HPV-16 E7 for the treatment of metastatic human papilloma virus-associated epithelial cancers (NCT02858310). The primary endpoint was maximum tolerated dose. Cell dose was not limited by toxicity with a maximum dose of 1 × 1011 engineered T cells administered. Tumor responses following treatment were evaluated using RECIST (Response Evaluation Criteria in Solid Tumors) guidelines. Robust tumor regression was observed with objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease. Responses included extensive regression of bulky tumors and complete regression of most tumors in some patients. Genomic studies, which included intra-patient tumors with dichotomous treatment responses, revealed resistance mechanisms from defects in critical components of the antigen presentation and interferon response pathways. These findings demonstrate that engineered T cells can mediate regression of common carcinomas, and they reveal immune editing as a constraint on the curative potential of cellular therapy and possibly other immunotherapies in advanced epithelial cancer.

Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

Sequence conservation of mitochondrial (mt)DNA during expansion of clonal mammary epithelial populations suggests a common mtDNA template in CzechII mice.

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA "de novo" SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA "de novo" SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious "de novo" SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to "heteroplasmic-shifting". A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues.

Life history evolution and cellular mechanisms associated with increased size in high-altitude Drosophila.

Understanding the physiological and genetic basis of growth and body size variation has wide-ranging implications, from cancer and metabolic disease to the genetics of complex traits. We examined the evolution of body and wing size in high-altitude Drosophila melanogaster from Ethiopia, flies with larger size than any previously known population. Specifically, we sought to identify life history characteristics and cellular mechanisms that may have facilitated size evolution. We found that the large-bodied Ethiopian flies laid significantly fewer but larger eggs relative to lowland, smaller-bodied Zambian flies. The highland flies were found to achieve larger size in a similar developmental period, potentially aided by a reproductive strategy favoring greater provisioning of fewer offspring. At the cellular level, cell proliferation was a strong contributor to wing size evolution, but both thorax and wing size increases involved important changes in cell size. Nuclear size measurements were consistent with elevated somatic ploidy as an important mechanism of body size evolution. We discuss the significance of these results for the genetic basis of evolutionary changes in body and wing size in Ethiopian D. melanogaster.