Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Suppression of Ca2+ signals by EGR4 controls Th1 differentiation and anti-cancer immunity in vivo.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

Functional Conservation of a Developmental Switch in Mammals since the Jurassic Age.

ThPOK is a "master regulator" of T lymphocyte lineage choice, whose presence or absence is sufficient to dictate development to the CD4 or CD8 lineages, respectively. Induction of ThPOK is transcriptionally regulated, via a lineage-specific silencer element, SilThPOK. Here, we take advantage of the available genome sequence data as well as site-specific gene targeting technology, to evaluate the functional conservation of ThPOK regulation across mammalian evolution, and assess the importance of motif grammar (order and orientation of TF binding sites) on SilThPOK function in vivo. We make three important points: First, the SilThPOK is present in marsupial and placental mammals, but is not found in available genome assemblies of nonmammalian vertebrates, indicating that it arose after divergence of mammals from other vertebrates. Secondly, by replacing the murine SilThPOK in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial SilThPOK supports correct CD4 T lymphocyte lineage-specification in mice. To our knowledge, this is the first in vivo demonstration of functional equivalency for a silencer element between marsupial and placental mammals using a definitive knockin approach. Finally, we show that alteration of the position/orientation of a highly conserved region within the murine SilThPOK is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this "solid" synteny block is critical for silencer function. Dependence of SilThPOK function on motif grammar conserved since the mid-Jurassic age, 165 Ma, suggests that the SilThPOK operates as a silenceosome, by analogy with the previously proposed enhanceosome model.

Novel mutation in Teneurin 3 found to co-segregate in all affecteds in a multi-generation family with developmental dysplasia of the hip.

DDH is a debilitating condition characterized by incomplete formation of the acetabulum leading to dislocation of the hip, suboptimal joint function and accelerated wear of the articular cartilage resulting in early onset crippling arthritis of the hip in 20-40 year olds. Current diagnostic tests in newborns using physical manipulation of the femur or ultrasound either under or over-diagnose this condition. Developing an accurate, cost effective diagnostic test is a goal of this study. To better understand the biologic pathways involved in acetabular development, DNA from severely affected individuals in a four generation family that showed inter-generational transmission of the disorder was isolated and whole exome sequenced. A novel A to C transversion at position 183721398 on human chromosome four was found to co-segregate with the affected phenotype in this family. This mutation encodes a glutamine to proline change at position 2665 in the Teneurin 3 (TENM3) gene and was judged damaging by four prediction programs. Eight week old knock-in mutant mice show delayed development of the left acetabulum and the left glenoid fossa as shown by the presence of more Alcian blue staining on the socket rims of both the hip and the shoulder. We hypothesize that mutated TENM3 will slow chondrogenesis. MMP13 has been shown to impair extracellular matrix remodeling and suppress differentiation. Bone marrow cells from the knock-in mouse were found to overexpress MMP13 with or without BMP2 stimulation. This variant may elucidate pathways responsible for normal hip development and become part of an accurate test for DDH. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas.

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αßTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

F1-adenosine triphosphatase displays properties characteristic of an antigen presentation molecule for Vgamma9Vdelta2 T cells.

Human Vgamma9Vdelta2 T lymphocytes are activated by phosphoantigens provided exogenously or produced by tumors and infected cells. Activation requires a contact between Vgamma9Vdelta2 cells and neighboring cells. We previously reported a role for cell surface F1-adenosine triphosphatase (ATPase) in T cell activation by tumors and specific interactions between Vgamma9Vdelta2 TCRs and purified F1-ATPase. 721.221 cells do not express surface F1-ATPase and do not support phosphoantigen responses unless they are rendered apoptotic by high doses of zoledronate, a treatment that promotes F1-expression as well as endogenous phosphoantigen production. By monitoring calcium flux in single cells, we show in this study that contact of T cells with F1-ATPase on polystyrene beads can partially replace the cell-cell contact stimulus during phosphoantigen responses. Triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester, an adenylated derivative of isopentenyl pyrophosphate, can stably bind to F1-ATPase-coated beads and promotes TCR aggregation, lymphokine secretion, and activation of the cytolytic process provided that nucleotide pyrophosphatase activity is present. It also acts as an allosteric activator of F1-ATPase. In the absence of Vgamma9Vdelta2 cells, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester immobilized on F1-ATPase is protected from nucleotide pyrophosphatase activity, as is the antigenic activity of stimulatory target cells. Our experiments support the notion that Vgamma9Vdelta2 T cells are dedicated to the recognition of phosphoantigens on cell membranes in the form of nucleotide derivatives that can bind to F1-ATPase acting as a presentation molecule.

Specific requirements for Vgamma9Vdelta2 T cell stimulation by a natural adenylated phosphoantigen.

Human Vgamma9Vdelta2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vgamma9Vdelta2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vgamma9Vdelta2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vgamma9Vdelta2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vgamma9Vdelta2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vgamma9Vdelta2 cells.

Inhibition of ABC transporters abolishes antimony resistance in Leishmania Infection.

The emergence of antimony (Sb) resistance has jeopardized the treatment of visceral leishmaniasis in various countries. Previous studies have considered the part played by leishmanial parasites in antimony resistance, but the involvement of host factors in the clinical scenario remained to be investigated. Here we show that unlike infection with Sb-sensitive (Sbs) Leishmania donovani, infection with Sb-resistant (Sb r) L. donovani induces the upregulation of multidrug resistance-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) in host cells, resulting in a nonaccumulation of intracellular Sb following treatment with sodium antimony gluconate (SAG) favoring parasite replication. The inhibition of MRP1 and P-gp with resistance-modifying agents such as lovastatin allows Sb accumulation and parasite killing within macrophages and offers protection in an animal model in which infection with Sb r L. donovani is otherwise lethal. The occurrence of a similar scenario in clinical cases is supported by the findings that unlike monocytes from SAG-sensitive kala-azar (KA) patients, monocytes from SAG-unresponsive KA patients overexpress P-gp and MRP1 and fail to accumulate Sb following in vitro SAG treatment unless pretreated with inhibitors of ABC transporters. Thus, the expression status of MRP1 and P-gp in blood monocytes may be used as a diagnostic marker for Sb resistance and the treatment strategy can be designed accordingly. Our results also indicate that lovastatin, which can inhibit both P-gp and MRP1, might be beneficial for reverting Sb resistance in leishmaniasis as well as drug resistance in other clinical situations, including cancer.

Overcoming drug-resistant cancer by a newly developed copper chelate through host-protective cytokine-mediated apoptosis.

PURPOSE: Previously, we have synthesized and characterized a novel Cu(II) complex, copper N-(2-hydroxy acetophenone) glycinate (CuNG). Herein, we have determined the efficacy of CuNG in overcoming multidrug-resistant cancer using drug-resistant murine and human cancer cell lines. EXPERIMENTAL DESIGN: Action of CuNG following single i.m. administration (5 mg/kg body weight) was tested in vivo on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing mice and doxorubicin-resistant sarcoma 180-bearing mice. Tumor size, ascitic load, and survival rates were monitored at regular intervals. Apoptosis of cancer cells was determined by cell cycle analysis, confocal microscopy, Annexin V binding, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay ex vivo. IFN-gamma and tumor necrosis factor-alpha were assayed in the culture supernatants of in vivo and in vitro CuNG-treated splenic mononuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Source of IFN-gamma and changes in number of T regulatory marker-bearing cells in the tumor site following CuNG treatment were investigated by flow cytometry. Supernatants of in vitro CuNG-treated cultures of peripheral blood mononuclear cells from different drug-insensitive cancer patients were tested for presence of the apoptogenic cytokine IFN-gamma and its involvement in induction of apoptosis of doxorubicin-resistant CEM/ADR5000 cells. RESULTS: CuNG treatment could resolve drug-resistant cancers through induction of apoptogenic cytokines, such as IFN-gamma and/or tumor necrosis factor-alpha, from splenic mononuclear cells or patient peripheral blood mononuclear cells and reduce the number of T regulatory marker-bearing cells while increase infiltration of IFN-gamma-producing T cells in the ascetic tumor site. CONCLUSION: Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers irrespective of multidrug resistance phenotype.

Sodium antimony gluconate induces generation of reactive oxygen species and nitric oxide via phosphoinositide 3-kinase and mitogen-activated protein kinase activation in Leishmania donovani-infected macrophages.

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.