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Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.

Glycoprotein 2 (GP2) is a widely distributed protein in the digestive tract, contributing to mucosal barrier maintenance, immune homeostasis, and antigen-specific immune response, while also being linked to inflammatory bowel disease (IBD) pathogenesis. This review sheds light on the extensive distribution of GP2 within the gastrointestinal tract and its intricate interplay with the immune system. Furthermore, the significance of GP2 autoantibodies in diagnosing and categorizing IBD is underscored, alongside the promising therapeutic avenues for modulating GP2 to regulate immunity and maintain mucosal balance.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

HIV-infected individuals have increased risk for cardiovascular disease (CVD) despite suppressive antiretroviral therapy (ART). This is likely a result of persistent immune activation and systemic inflammation. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and may drive inflammation contributing to CVD. EVs were characterized in plasma from 74 HIV-infected individuals on combination antiretroviral therapy (cART) and 64 HIV-uninfected controls with paired carotid intima-media thickness (cIMT) assessment. EVs were profiled with markers reflecting lymphoid, myeloid, and endothelial origin. Seventeen plasma inflammatory biomarkers were also assessed. Human umbilical vein endothelial cell (HUVEC) apoptosis was quantified after EV exposure. A significant correlation was observed in HIV-infected participants between cIMT and EVs expressing CD16, and the monocyte-related markers CD4, CD14, and CX3CR1 showed a similar but nonsignificant association with cIMT. No significant correlation between cIMT measurements from HIV-uninfected individuals and EVs was observed. Levels of serum amyloid A, C-reactive protein, and myeloperoxidase significantly correlated with CD14+, CD16+, and CX3CR1+ EVs. No correlation was noted between cIMT and soluble inflammatory markers. HUVECs showed increased necrosis after exposure to the EV-containing fraction of plasma derived from HIV-infected individuals compared to uninfected controls. Our study reveals that EVs expressing monocyte markers correlated with cIMT in HIV-infected individuals on cART. Moreover, EV fractions derived from HIV-infected individuals lead to greater endothelial cell death via necrotic pathways. Collectively, EVs have potential as biomarkers of and therapeutic targets in the pathogenesis of CVD in the setting of treated HIV disease. IMPORTANCE HIV-infected individuals have a 2-fold-increased risk of cardiovascular disease compared with the general population, yet the mechanisms underlying this comorbidity are unclear. Extracellular vesicles have emerged as important mediators in cell-cell communication and, given what we know of their biology, may drive inflammation contributing to cardiovascular disease in this vulnerable population.

Natural killer (NK) cells can detect antibody-coated cells through recognition by the CD16 Fc receptor. The importance of CD16 in human NK cell biology has long been appreciated, but how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function. We reveal that the mouse CD3ζ transmembrane domain adopts a tightly packed confirmation, preventing association with CD16, whereas human CD3ζ adopts a versatile configuration that accommodates receptor assembly.

Intelectins are carbohydrate-binding proteins implicated in innate immunity and highly conserved across chordate evolution, including both ascidians and humans. Human intelectin-1 (ITLN1) is highly abundant within the intestinal mucosa and binds microbial but not host glycans. Genome-wide association studies identified SNPs in ITLN1 that are linked to susceptibility for Crohn's disease. Moreover, ITLN1 has been implicated in the pathophysiology of obesity and associated metabolic disease. To gain insight on biological activities of human ITLN1 in vivo, we developed a C57BL/6 mouse model genetically targeting the gene encoding the functional mouse ortholog. In wild-type C57BL/6 mice, both mRNA and protein analysis showed high expression of Itln1 in the small intestine, but manifold lower levels in colon and other extraintestinal tissues. Whereas intestinal expression of human ITLN1 localizes to goblet cells, our data confirm that mouse Itln1 is expressed in Paneth cells. Compared to wild-type littermate controls, mice homozygous for the Itln1 hypomorphic trapping allele had reduced expression levels of Itln1 expression (~10,000-fold). The knockout mice exhibited increased susceptibility in an acute model of experimentally induced colitis with 2% w/v dextran sulfate sodium (DSS). In a model of chronic colitis using a lower dose of DSS (1.5% w/v), which enabled a detailed view of disease activity across a protracted period, no differences were observed in body weight, fecal texture, hemoccult scores, food/water intake, or colon length at necropsy, but there was a statistically significant genotype over time effect for the combined fecal scores of disease activity. In model of diet-induced obesity, using two western-style diets, which varied in amounts of sugar (as sucrose) and saturated fat (as lard), mice with Itln1 expression ablated showed no increased susceptibility, in terms of weight gain, food intake, plasma markers of obesity compared to wildtype littermates. While the mouse genetic knockout model for Itln1 holds promise for elucidating physiological function(s) for mammalian intelectins, results reported here suggest that Itln1, a Paneth cell product in C57BL/6 mice, likely plays a minor role in the pathophysiology of chemically induced colitis or diet-induced obesity.

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell-mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell-mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell-mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell-mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

BackgroundIt is difficult to distinguish between a tumor and its liver segment with traditional use of indocyanine green (ICG) alone. In the present study, a method was used to limit ICG to the liver segment adjacent to a tumor. A spectrally-distinct fluorescently-labeled tumor-specific antibody against human carcinoembryonic antigen-related cell-adhesion molecules was used to label the metastatic tumor in a patient-derived orthotopic xenograft mouse model to enable color-coded visualization and distinction of a colon-cancer liver metastases and its adjacent liver segment.Materials and methodsNude mice received surgical orthotopic implantation in the liver of colon-cancer liver metastases derived from two patients. An anti- carcinoembryonic antigen-related cell-adhesion molecules monoclonal antibody (mAb 6G5j) was conjugated to a near-infrared dye IR700DX (6G5j-IR700DX). After three weeks, mice received 6G5j-IR700DX via tail-vein injection 48 hours before surgery. ICG was intravenously injected after ligation of the left or left lateral Glissonean pedicle resulting in labeling of the segment with preserved blood-flow in the liver. Imaging was performed with the Pearl Trilogy and FLARE Imaging Systems.ResultsThe metastatic liver tumor had a clear fluorescence signal due to selective tumor targeting by 6G5j-IR700DX, which was imaged on the 700 nm channel. The adjacent liver segment, with preserved blood-flow in the liver, had a clear fluorescence ICG 800 nm signal, while the left or left lateral segment had no fluorescence signal. Overlay of the images showed clear color-coded differentiation between the tumor fluorescing at 700 nm and the adjacent liver segment fluorescing at 800 nm.ConclusionsColor-coding of a liver tumor and uninvolved liver segment has the potential for improved liver resection.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Intelectins are a family of multimeric secreted proteins that bind microbe-specific glycans. Both genetic and functional studies have suggested that intelectins have an important role in innate immunity and are involved in the etiology of various human diseases, including inflammatory bowel disease. Experiments investigating the role of intelectins in human disease using mouse models are limited by the fact that there is not a clear one-to-one relationship between intelectin genes in humans and mice, and that the number of intelectin genes varies between different mouse strains. In this study we show by gene sequence and gene expression analysis that human intelectin-1 (ITLN1) has multiple orthologues in mice, including a functional homologue Itln1; however, human intelectin-2 has no such orthologue or homologue. We confirm that all sub-strains of the C57 mouse strain have a large deletion resulting in retention of only one intelectin gene, Itln1. The majority of laboratory strains have a full complement of six intelectin genes, except CAST, SPRET, SKIVE, MOLF and PANCEVO strains, which are derived from different mouse species/subspecies and encode different complements of intelectin genes. In wild mice, intelectin deletions are polymorphic in Mus musculus castaneus and Mus musculus domesticus. Further sequence analysis shows that Itln3 and Itln5 are polymorphic pseudogenes due to premature truncating mutations, and that mouse Itln1 has undergone recent adaptive evolution. Taken together, our study shows extensive diversity in intelectin genes in both laboratory and wild-mice, suggesting a pattern of birth-and-death evolution. In addition, our data provide a foundation for further experimental investigation of the role of intelectins in disease.

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan β-D-galactofuranose or protein-protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

B cells infiltrate pancreatic ductal adenocarcinoma (PDAC) and in preclinical cancer models, can suppress T cell immunosurveillance in cancer. Here, we conducted a pilot study to assess the safety and feasibility of administering lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin to target tumor cells along with CART cells redirected against CD19 to deplete B cells. Both CARs contained 4-1BB and CD3ζ signaling domains. Three patients with chemotherapy-refractory PDAC received 1.5 g/m2 cyclophosphamide prior to separate infusions of lentiviral-transduced T cells engineered to express chimeric anti-mesothelin immunoreceptor SS1 (CART-Meso, 3 × 107/m2) and chimeric anti-CD19 immunoreceptor (CART-19, 3 × 107/m2). Treatment was well tolerated without dose-limiting toxicities. Best response was stable disease (1 of 3 patients). CART-19 (compared to CART-Meso) cells showed the greatest expansion in the blood, although persistence was transient. B cells were successfully depleted in all subjects, became undetectable by 7-10 days post-infusion, and remained undetectable for at least 28 days. Together, concomitant delivery of CART-Meso and CART-19 cells in patients with PDAC is safe. CART-19 cells deplete normal B cells but at the dose tested in these 3 subjects did not improve CART-Meso cell persistence.

Identification of tumor-specific cell surface antigens has proven challenging, as the vast majority of tumor-associated antigens are also expressed in normal tissues. In mesothelioma, we identified a highly specific tumor cell surface antigen that can be targeted for therapy development. Mesothelioma is caused by malignant transformation of the mesothelium, is incurable, and can be categorized into three histologic subtypes: epithelioid, biphasic, and sarcomatoid. To identity novel mesothelioma cell surface antigens with broad subtype coverage and high tissue specificity, we have previously selected phage antibody display libraries on live mesothelioma cells and tissues following counterselection on normal cells and identified a panel of human antibodies that bind all subtypes of mesothelioma, but not normal mesothelium. One of the antibodies, M25, showed high specificity against an antigen we identify here as ALPPL2. IHC on normal human tissues found that ALPPL2 is expressed only on placental trophoblasts, but not on any other normal tissues. This significant tissue specificity and broad tumor type coverage suggest that ALPPL2 could be an excellent cell surface target for therapeutic development against mesothelioma. To evaluate therapeutic potential of ALPPL2 targeting, an ALPPL2-targeted antibody-drug conjugate was developed and demonstrated potent and specific tumor killing in vitro and in vivo against both epithelioid and sarcomatoid mesothelioma. Thus, ALPPL2 belongs to a rare class of cell surface antigens classified as truly tumor specific and is well suited for therapy development against ALPPL2-expressing tumors. SIGNIFICANCE: These findings identify ALPP2 as a true tumor-specific cell surface antigen whose tissue specificity enables the development of novel therapies.

To identify candidate biomarkers associated with neuromyelitis optica spectrum disorder (NMOSD) using high-throughput technologies that broadly assay the concentrations of serum analytes and frequencies of immune cell subsets. Sera, peripheral blood mononuclear cells (PBMCs), and matched clinical data from participants with NMOSD and healthy controls (HCs) were obtained from the Collaborative International Research in Clinical and Longitudinal Experience Study NMOSD biorepository. Flow cytometry panels were used to measure the frequencies of 39 T-cell, B-cell, regulatory T-cell, monocyte, natural killer (NK) cell, and dendritic cell subsets in unstimulated PBMCs. In parallel, multiplex proteomics assays were used to measure 46 serum cytokines and chemokines in 2 independent NMOSD and HC cohorts. Multivariable regression models were used to assess molecular and cellular profiles in NMOSD compared with HC. NMOSD samples had a lower frequency of CD16+CD56+ NK cells. Both serum cohorts and multivariable logistic regression revealed increased levels of B-cell activating factor associated with NMOSD. Interleukin 6, CCL22, and CCL3 were also elevated in 1 NMOSD cohort of the 2 analyzed. Multivariable linear regression of serum analyte levels revealed a correlation between CX3CL1 (fractalkine) levels and the number of days since most recent disease relapse. Integrative analyses of cytokines, chemokines, and immune cells in participants with NMOSD and HCs provide congruence with previously identified biomarkers of NMOSD and highlight CD16+CD56+ NK cells and CX3CL1 as potential novel biomarker candidates.

ObjectiveThe recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries.ConclusionsThese data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P

Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a co-regulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1,3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host's attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.

The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

ObjectivesIn a genome-wide association study, we discovered chromosome 12q15 (defined as rs73329476) as a silica-related pneumoconiosis susceptibility region. However, the causal variants in this region have not yet been reported.MethodsWe systematically screened eight potentially functional single-neucleotide polymorphism (SNPs) in the genes near rs73329476 (carboxypeptidase M (CPM) and cleavage and polyadenylation specific factor 6 (CPSF6)) in a case-control study including 177 cases with silicosis and 204 healthy controls, matched to cases with years of silica dust exposure. We evaluated the associations between these eight SNPs and the development of silicosis. Luciferase reporter gene assays were performed to test the effects of selected SNP on the activity of CPM in the promoter. In addition, a two-stage case-control study was performed to investigate the expression differences of the two genes in peripheral blood leucocytes from a total of 64 cases with silicosis and 64 healthy controls with similar years of silica dust exposure as the cases.ResultsWe found a strong association between the mutant rs12812500 G allele and the susceptibility of silicosis (OR=1.45, 95% CI 1.03 to 2.04, p=0.034), while luciferase reporter gene assays indicated that the mutant G allele of rs12812500 is strongly associated with increased luciferase levels compared with the wild-type C allele (p

Cell migration is essential for proper development and the defense against pathogens. Our previous work detailed a pathway of REversion-inducing-Cysteine-rich protein with Kazal motifs (RECK) isoform-mediated invasion in which a shorter RECK protein competes with MMP9 for interaction with the canonical RECK protein on the cell surface. Here we demonstrate that the mechanism through which RECK isoforms affect cell migration is mediated through changes in the levels of post-translational modifications (PTM) of α-tubulin. We show that both the canonical and short RECK isoforms modulate levels of tubulin acetylation and detyrosination. We demonstrate that these changes are sufficient to modulate the rate of fibroblast migration. If these tubulin PTMs are not altered, the effects of the canonical RECK isoform on cell migration are reversed. In defining the molecular pathway linking RECK and tubulin PTMs, we found that MMP9 and integrin activity both act as upstream regulators of tubulin acetylation and detyrosination. Overall, we propose a mechanism in which RECK isoforms on the cell surface have opposing effects on cell migration through MMP9-modulated changes to integrin-extracellular matrix (ECM) interactions that, in turn, affect microtubule PTMs.

UNLABELLED: Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editors assessment is that all the issues have been addressed (see decision letter).

PURPOSE OF REVIEW:Luminal chemosensing is a term used to describe how small molecules in the gut lumen interact with the host through surface receptors or via transport into the submucosa. In this review, we have summarized recent advances of understanding luminal chemosensing in the gastroduodenal mucosa, with a particular emphasis on how chemosensing affects mucosal protective responses and the metabolic syndrome. RECENT FINDINGS:In the past decade, data have supported the hypothesis that gut luminal chemosensing not only is important for the local or remote regulation of gut function but also contributes to the systemic regulation of metabolism, energy balance and food intake. We have provided examples of how luminal nutrients such as long-chain fatty acids (LCFAs), endogenous compounds such as bile acids, bacterial metabolites such as short-chain fatty acids (SCFAs) and bacterial components such as lipopolysaccharide (LPS) activate cognate receptors expressed on key effector cells such as enteroendocrine cells and inflammatory cells in order to profoundly affect organ function through the initiation or suppression of inflammatory pathways, altering gut barrier function and nutrient uptake, altering gut motility and visceral pain pathways, and preventing mucosal injury. SUMMARY:These recent discoveries in this area have provided new possibilities for identifying novel molecular targets for the treatment of mucosal injury, metabolic disorders and abnormal visceral sensation. Understanding luminal chemosensory mechanisms may help to identify novel molecular targets for the treatment and prevention of mucosal injury, metabolic disorders and abnormal visceral sensation.

Intestinal production of endocannabinoid and oleoylethanolamide (OEA) is impaired in high-fat diet/obese rodents, leading to reduced satiety. Such diets also alter the intestinal microbiome in association with enhanced intestinal permeability and inflammation; however, little is known of these effects in humans. This study aimed to 1) evaluate effects of lipid on plasma anandamide (AEA), 2-arachidonyl- sn-glycerol (2-AG), and OEA in humans; and 2) examine relationships to intestinal permeability, inflammation markers, and incretin hormone secretion. Twenty lean, 18 overweight, and 19 obese participants underwent intraduodenal Intralipid infusion (2 kcal/min) with collection of endoscopic duodenal biopsies and blood. Plasma AEA, 2-AG, and OEA (HPLC/tandem mass spectrometry), tumor necrosis factor-α (TNFα), glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) (multiplex), and duodenal expression of occludin, zona-occludin-1 (ZO-1), intestinal-alkaline-phosphatase (IAP), and Toll-like receptor 4 (TLR4) (by RT-PCR) were assessed. Fasting plasma AEA was increased in obese compared with lean and overweight patients ( P < 0.05), with no effect of BMI group or ID lipid infusion on plasma 2-AG or OEA. Duodenal expression of IAP and ZO-1 was reduced in obese compared with lean ( P < 0.05), and these levels related negatively to plasma AEA ( P < 0.05). The iAUC for AEA was positively related to iAUC GIP ( r = 0.384, P = 0.005). Obese individuals have increased plasma AEA and decreased duodenal expression of ZO-1 and IAP compared with lean and overweight subjects. The relationships between plasma AEA with duodenal ZO-1, IAP, and GIP suggest that altered endocannabinoid signaling may contribute to changes in intestinal permeability, inflammation, and incretin release in human obesity.

Cell migration is a highly conserved process involving cytoskeletal reorganization and restructuring of the surrounding extracellular matrix. Although there are many studies describing mechanisms underlying cell motility, little has been reported about the contribution of alternative isoform use toward cell migration. Here, we investigated whether alternative isoform use can affect cell migration focusing on reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), an established inhibitor of cell migration. We found that a shorter isoform of RECK is more highly expressed in proliferating fibroblasts, in TGF-β-treated fibroblasts, and in tumors compared with differentiated tissue. Knockdown of this short RECK isoform reduces fibroblast migration through Matrigel. Thus, this short isoform of RECK generated by a combination of alternative splicing and alternative polyadenylation plays an opposing role to the canonical RECK isoform, as knockdown of canonical RECK results in faster cell migration through Matrigel. We show that the short RECK protein competes with matrix metalloprotease 9 (MMP9) for binding to the Kazal motifs of canonical RECK, thus liberating MMP9 from an inactivating interaction with canonical RECK. Our studies provide a new paradigm and a detailed mechanism for how alternative isoform use can regulate cell migration by producing two proteins with opposing effects from the same genetic locus.

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

The epithelial and epidermal innate cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) have pivotal roles in the initiation of allergic inflammation in asthma and atopic dermatitis (AD). However, the mechanism by which the expression of these innate cytokines is regulated remains unclear. Intelectin (ITLN) is expressed in airway epithelial cells and promotes allergic airway inflammation. We hypothesized that ITLN is required for allergen-induced IL-25, IL-33, and TSLP expression. In two asthma models, Itln knockdown reduced allergen-induced increases in Il-25, Il-33, and Tslp and development of type 2 response, eosinophilic inflammation, mucus overproduction, and airway hyperresponsiveness. Itln knockdown also inhibited house dust mite (HDM)-induced early upregulation of Il-25, Il-33, and Tslp in a model solely inducing airway sensitization. Using human airway epithelial cells, we demonstrated that HDM-induced increases in ITLN led to phosphorylation of epidermal growth factor receptor and extracellular-signal regulated kinase, which were required for induction of IL-25, IL-33, and TSLP expression. In two AD models, Itln knockdown suppressed expression of Il-33, Tslp, and Th2 cytokines and eosinophilic inflammation. In humans, ITLN1 expression was significantly increased in asthmatic airways and in lesional skin of AD. We conclude that ITLN contributes to allergen-induced Il-25, Il-33, and Tslp expression in asthma and AD.

The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as 'Trojan horses' and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14+CD16+ monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14+ monocytes was significantly decreased, while the CD14+CD16+ monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14+CD16+ monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14+CD16+ monocytes in particular.

Interaction between the activating NKG2D receptor on lymphocytes and its ligands MICA, MICB, and ULBP1-6 modulate T and NK cell activity and may contribute to the pathogenesis of Crohn's disease (CD). NKG2D ligands are generally not expressed on the cell surface of normal, non-stressed cells, but expression of MICA and MICB in CD intestine has been reported. In this exploratory study, we further characterize the expression of NKG2D and its ligands, including the less well-described ULBP4-6, in CD, and test if NKG2D ligand interactions are involved in the migration of activated T cells into the affected mucosal compartments. Intestinal tissue from CD patients and healthy controls were analyzed by flow cytometry, mass cytometry, and immunohistochemistry for expression of NKG2D and ligands, and for cytokine release. Furthermore, NKG2D-dependent chemotaxis of activated CD8+ T cells across a monolayer of ligand-expressing human intestinal endothelial cells was examined. Activated lymphocytes down-regulated NKG2D expression upon accumulation in inflamed CD intestine. NKG2D expression on CD56+ T and γδ T cells from inflamed tissue seemed inversely correlated with CRP levels and cytokine release. B cells, monocytes, mucosal epithelium, and vascular endothelium expressed NKG2D ligands in inflamed CD intestine. The expression of NKG2D ligands was correlated with cytokine release, but was highly variable between patients. Stimulation of vascular intestinal endothelial cells in vitro induced expression of NKG2D ligands, including MICA/B and ULBP2/6. Blockade of NKG2D on CD8+ T cells inhibited the migration over ligand-expressing endothelial cells. Intestinal induction of NKG2D ligands and ligand-induced down-regulation of NKG2D in CD suggest that the NKG2D-ligand interaction may be involved in both the activation and recruitment of NKG2D+ lymphocytes into the inflamed CD intestine.

BackgroundPancreatic cancer is the fourth most deadly cancer in the United States, and is expected to be the second most deadly by 2030. The major difficulty in treating pancreatic cancer is the late onset of symptoms. Generally, patients show metastatic disease by the time of diagnosis, with a survival rate of 5% beyond 5 years. In patients without metastatic disease, surgical resection increases 5 year survival rate to 25%. The remaining 75% succumb to undetected metastases. Clearly, improvements to both detection, surgical intervention, and therapeutic strategies will be needed to improve patient outcome in pancreatic cancer.MethodsRecent literature has been surveyed and atomic models of new therapeutic approaches were generated.Results and conclusionsHere, we focus on the recent progress employing monoclonal antibodies (mAbs) to target pancreatic cancer associated markers, and more specifically on recent chemical and protein engineering efforts to improve the homogeneity, stability, and administration of mAbs to precisely deliver imaging agents and cytotoxins to sites of disease.

Cholestatic liver fibrosis is caused by obstruction of the biliary tract and is associated with early activation of portal fibroblasts (PFs) that express Thy-1, fibulin 2, and the recently identified marker mesothelin (MSLN). Here, we have demonstrated that activated PFs (aPFs) and myofibroblasts play a critical role in the pathogenesis of liver fibrosis induced by bile duct ligation (BDL). Conditional ablation of MSLN+ aPFs in BDL-injured mice attenuated liver fibrosis by approximately 50%. Similar results were observed in MSLN-deficient mice (Msln-/- mice) or mice deficient in the MSLN ligand mucin 16 (Muc16-/- mice). In vitro analysis revealed that MSLN regulates TGF-β1-inducible activation of WT PFs by disrupting the formation of an inhibitory Thy-1-TGFβRI complex. MSLN also facilitated the FGF-mediated proliferation of WT aPFs. Therapeutic administration of anti-MSLN-blocking Abs attenuated BDL-induced fibrosis in WT mice. Liver specimens from patients with cholestatic liver fibrosis had increased numbers of MSLN+ aPFs/myofibroblasts, suggesting that MSLN may be a potential target for antifibrotic therapy.

Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.

BACKGROUND & AIMS: The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohns disease (CD) cause significant morbidity and are increasing in prevalence among all populations, including African Americans. More than 200 susceptibility loci have been identified in populations of predominantly European ancestry, but few loci have been associated with IBD in other ethnicities. METHODS: We performed 2 high-density, genome-wide scans comprising 2345 cases of African Americans with IBD (1646 with CD, 583 with UC, and 116 inflammatory bowel disease unclassified) and 5002 individuals without IBD (controls, identified from the Health Retirement Study and Kaiser Permanente database). Single-nucleotide polymorphisms (SNPs) associated at P

Evidence suggests European American (EA) women have two- to five-fold increased odds of having pelvic organ prolapse (POP) when compared with African American (AA) women. However, the role of genetic ancestry in relation to POP risk is not clear. Here we evaluate the association between genetic ancestry and POP in AA women from the Women's Health Initiative Hormone Therapy trial. Women with grade 1 or higher classification, and grade 2 or higher classification for uterine prolapse, cystocele or rectocele at baseline or during follow-up were considered to have any POP (N = 805) and moderate/severe POP (N = 156), respectively. Women with at least two pelvic exams with no indication for POP served as controls (N = 344). We performed case-only, and case-control admixture-mapping analyses using multiple logistic regression while adjusting for age, BMI, parity and global ancestry. We evaluated the association between global ancestry and POP using multiple logistic regression. European ancestry at the individual level was not associated with POP risk. Case-only and case-control local ancestry analyses identified two ancestry-specific loci that may be associated with POP. One locus (Chromosome 15q26.2) achieved empirically-estimated statistical significance and was associated with decreased POP odds (considering grade ≥2 POP) with each unit increase in European ancestry (OR: 0.35; 95% CI: 0.30, 0.57; p-value = 1.48x10-5). This region includes RGMA, a potent regulator of the BMP family of genes. The second locus (Chromosome 1q42.1-q42.3) was associated with increased POP odds with each unit increase in European ancestry (Odds ratio [OR]: 1.69; 95% confidence interval [CI]: 1.28, 2.22; p-value = 1.93x10-4). Although this region did not reach statistical significance after considering multiple comparisons, it includes potentially relevant genes including TBCE, and ACTA1. Unique non-overlapping European and African ancestry-specific susceptibility loci may be associated with increased POP risk.

Endogenous Plastic Somatic (ePS) cells isolated from adult human tissues exhibit extensive lineage plasticity in vitro and in vivo. Here we visualize these rare ePS cells in a latent state, i.e. lacking SOX2, OCT3/4 and NANOG (SON) expression, in non-diseased breast specimens through immunohistochemical analysis of previously identified ePS-specific biomarkers (CD73+, EpCAM+ and CD90-). We also report a novel mechanism by which these latent ePS cells acquire SON expression and plasticity in vitro. Four extracellular factors are necessary for the acquisition of SON expression and lineage plasticity in ePS cells: adenosine (which is produced by the 5' ecto-nucleotidase CD73 and activates in turn the PKA-dependent IL6/STAT3 pathway through the adenosine receptor ADORA2b), IL6, FGF2 and ACTIVIN A. Blocking any pathway component renders ePS cells incapable of SON expression and lineage plasticity. Notably, hESCs do not use adenosine or IL6 nor they express CD73 or ADORA2b and inhibition of adenosine signaling does not ablate their plasticity. Therefore, the data presented here delineate novel circuitry and physiological signals for accessing SON expression in rare, undifferentiated human cells.

While immunotherapy may offer promising new approaches for high grade meningiomas, little is currently known of the immune landscape in meningiomas. We sought to characterize the immune microenvironment and a potentially targetable antigen mesothelin across WHO grade I-III cases of meningiomas, and how infiltrating immune populations relate to patient outcomes. Immunohistochemistry was performed on tissue microarrays constructed from 96 meningioma cases. The cohort included 16 WHO grade I, 62 WHO grade II, and 18 WHO grade III tumors. Immunohistochemistry was performed using antibodies against CD3, CD8, CD20, CD68, PD-L1, and mesothelin. Dual staining using anti-PD-L1 and anti-CD68 antibodies was performed, and automated cell detection and positive staining detection algorithms were utilized. Greater degree of PD-L1 expression was found in higher grade tumors. More specifically, higher grade tumors contained increased numbers of intratumoral CD68-, PD-L1+ cells (p = 0.022), but did not contain higher numbers of infiltrating CD68+, PD-L1+ cells (p = 0.30). Higher PD-L1+/CD68- expression was independently predictive of worse overall survival in our cohort when accounting for grade, performance status, extent of resection, and recurrence history (p = 0.014). Higher expression of PD-L1+/CD68- was also present in tumors that had undergone prior radiotherapy (p = 0.024). Approximately quarter of meningiomas overexpressed mesothelin to levels equivalent to those found in pancreatic carcinomas and malignant mesotheliomas. The association with poor survival outcomes in our study suggests that PD-L1 may play a significant biologic role in the aggressive phenotype of higher grade meningiomas. Thus, immunotherapeutic strategies such as checkpoint inhibition may have clinical utility in PD-L1 overexpressing meningiomas.

BackgroundDiffuse gliomas are poorly immunogenic, fatal brain tumors. The basis for insufficient antitumor immunity in diffuse gliomas is unknown. Gain-of-function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma.MethodsWe analyzed the database of The Cancer Genome Atlas for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligand expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza-2'deoxycytodine (decitabine) as a potential NK cell sensitizing agent in IDH mutant cells.ResultsIDH mutant glioma stemlike cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes are resistant to NK cell-mediated lysis. Decitabine increases NKG2D ligand expression and restores NK-mediated lysis of IDH mutant cells in an NKG2D-dependent manner.ConclusionsIDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULBP3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.

γδ T cells expressing Vδ2 may be instrumental in the control of malaria, because they inhibit the replication of blood-stage parasites in vitro and expand during acute malaria infection. However, Vδ2 T-cell frequencies and function are lower among children with heavy prior malaria exposure. It remains unclear whether malaria itself is driving this loss. Here we measure Vδ2 T-cell frequency, cytokine production, and degranulation longitudinally in Ugandan children enrolled in a malaria chemoprevention trial from 6 to 36 months of age. We observed a progressive attenuation of the Vδ2 response only among children incurring high rates of malaria. Unresponsive Vδ2 T cells were marked by expression of CD16, which was elevated in the setting of high malaria transmission. Moreover, chemoprevention during early childhood prevented the development of dysfunctional Vδ2 T cells. These observations provide insight into the role of Vδ2 T cells in the immune response to chronic malaria.

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.

To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.

BackgroundThe REversion-inducing Cysteine-rich protein with Kazal motif (RECK) is a well-known inhibitor of matrix metalloproteinases (MMPs) and cellular invasion. Although high expression levels of RECK have already been correlated with a better clinical outcome for several tumor types, its main function, as well as its potential prognostic value for breast cancer patients, remain unclear.MethodsThe RECK expression profile was investigated in a panel of human breast cell lines with distinct aggressiveness potential. RECK functional analysis was undertaken using RNA interference methodology. RECK protein levels were also analyzed in 1040 cases of breast cancer using immunohistochemistry and tissue microarrays (TMAs). The association between RECK expression and different clinico-pathological parameters, as well as the overall (OS) and disease-free (DFS) survival rates, were evaluated.ResultsHigher RECK protein expression levels were detected in more aggressive breast cancer cell lines (T4-2, MDA-MB-231 and Hs578T) than in non-invasive (MCF-7 and T47D) and non-tumorigenic (S1) cell lines. Indeed, silencing RECK in MDA-MB-231 cells resulted in elevated levels of pro-MMP-9 and increased invasion compared with scrambled (control) cells, without any effect on cell proliferation. Surprisingly, by RECK immunoreactivity analysis on TMAs, we found no association between RECK positivity and survival (OS and DFS) in breast cancer patients. Even considering the different tumor subtypes (luminal A, luminal B, Her2 type and basal-like) or lymph node status, RECK remained ineffective for predicting the disease outcome. Moreover, by multivariate Cox regression analysis, we found that RECK has no prognostic impact for OS and DFS, relative to standard clinical variables.ConclusionsAlthough it continues to serve as an invasion and MMP inhibitor in breast cancer, RECK expression analysis is not useful for prognosis of these patients.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.

BackgroundThere is considerable heterogeneity in asthma treatment response.ObjectiveWe sought to identify biomarkers of corticosteroid treatment response in children with asthma and evaluate the utility and mechanistic basis of these biomarkers.MethodsChildren (5-18 years) presenting to the emergency department with an acute asthma exacerbation were recruited and followed during hospitalization. Nasal epithelial cells were collected on presentation to the emergency department (T0) and 18 to 24 hours later (T1), and T1/T0 gene expression ratios were analyzed to identify genes associated with good and poor corticosteroid treatment response phenotypes. The utility of these genes in discriminating between systemic corticosteroid treatment response groups was then tested prospectively in a new cohort of patients. A gene candidate (vanin-1 [VNN1]) that consistently distinguished good versus poor response phenotypes was further studied in an experimental asthma model, and VNN1 promoter methylation was measured by means of bisulfite pyrosequencing in patients.ResultsVNN1 mRNA expression changes were associated with systemic corticosteroid treatment response in children with acute asthma, and VNN1 was required for optimal response to corticosteroid treatment in an experimental asthma model. A CpG site within the VNN1 promoter was differentially methylated between good versus poor treatment response groups, and methylation at this site correlated with VNN1 mRNA expression.ConclusionsWe have identified a biological basis for poor corticosteroid treatment response that can be used to distinguish a subgroup of asthmatic children who respond poorly to systemic corticosteroid treatment. VNN1 contributes to corticosteroid responsiveness, and changes in VNN1 nasal epithelial mRNA expression and VNN1 promoter methylation might be clinically useful biomarkers of treatment response in asthmatic children.

BackgroundThe interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity.ResultsBy examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin.ConclusionsThese results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

The restriction factor BST2 (tetherin) prevents the release of enveloped viruses from the host cell and is counteracted by HIV-1 Vpu. Vpu and BST2 interact directly via their transmembrane domains. This interaction enables Vpu to induce the surface down-regulation and the degradation of BST2, but neither of these activities fully accounts for the ability of Vpu to enhance virion release. During a study of naturally occurring Vpu proteins, we found that a tryptophan residue near the Vpu C terminus is particularly important for enhancing virion release. Vpu proteins with a W76G polymorphism degraded and down-regulated BST2 from the cell surface, yet they inefficiently stimulated virion release. Here we explore the mechanism of this anomaly. We find that Trp-76 is critical for the ability of Vpu to displace BST2 from sites of viral assembly in the plane of the plasma membrane. This effect does not appear to involve a general reorganization of the membrane microdomains associated with virion assembly, but rather is a specific effect of Vpu on BST2. Using NMR spectroscopy, we find that the cytoplasmic domain of Vpu and Trp-76 specifically interact with lipids. Moreover, paramagnetic relaxation enhancement studies show that Trp-76 inserts into the lipid. These data are consistent with a model whereby Trp-76 anchors the C terminus of the cytoplasmic tail of Vpu to the plasma membrane, enabling the movement of Vpu-bound BST2 away from viral assembly sites.

Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrP(Sc).