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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. Mouse models of severe congenital neutropenia using patient-derived mutations in the GFI1 locus are used to determine the mechanisms by which the disease progresses.

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 Ca(2+) 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 Ca(2+) 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.

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, Sil(ThPOK). 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 Sil(ThPOK) function in vivo. We make three important points: First, the Sil(ThPOK) 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 Sil(ThPOK) in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial Sil(ThPOK) 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 Sil(ThPOK) is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this “solid” synteny block is critical for silencer function. Dependence of Sil(ThPOK) function on motif grammar conserved since the mid-Jurassic age, 165 Ma, suggests that the Sil(ThPOK) operates as a silenceosome, by analogy with the previously proposed enhanceosome model.

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.

Altered signaling via the T cell antigen receptor (TCR) promotes an adipose-tissue-like phenotype in invariant natural killer cells (iNKT cells) during thymic development and causes selective enrichment for iNKT cells in adipose tissues.

The molecular basis of differentiation of immature thymocyte precursors to alternate CD4 or CD8 lineage cells remains poorly understood, but appears to be controlled by differences in TCR signaling. Importantly, we have shown that the transcription factor ThPOK acts as a master regulator of this process, such that its presence or absence dictates development to the CD4 or CD8 lineages, respectively. We have also shown that ThPOK expression in thymocytes and mature T cells is regulated primarily at the transcriptional level via several stage- and lineage-specific cis elements. Of particular importance is the ultraconserved ThPOK silencer, whose deletion is sufficient to cause promiscuous expression of ThPOK in thymocytes and diversion of all developing thymocytes to the CD4 lineage. In this review, we provide an overview of the role of ThPOK in cd4 versus cd8 commitment, as well as its transcriptional regulation and additional important roles of ThPOK in development and function of other T cell subsets.

Efforts to understand the genomic impact of human-disease-relevant genetic lesions and how they disrupt the normal sequence of cell-state transitions is hampered by a lack of defined hierarchical cellular states and corresponding networks of regulatory genes (transcription factors). Severe congenital neutropenia (SCN) patients display inherited and de novo mutations in Growth factor independent-1 (GFI1), which encodes a zinc-finger transcription factor. We identified known (e.g. N382S in zinc finger 5) and novel GFI1 sequence changes in SCN patients, then used lentiviral mediated expression to functionally evaluate them. GFI1-N382S, GFI1-K403R and GFI1-R412X mutations (in zinc finger 6) significantly elevated the expression of the Gfi1 target gene, Irf8. We generated mice with these patient-derived SCN-associated mutations in the murine Gfi1 locus. Neonatal and adult Gfi1N382S/- and Gfi1R412X/-mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. The resulting steady-state dysgranulopoiesis in adult mice was further pronounced in neonates. We noted that Gfi1R412X/-mice accumulate less Gfi1 protein than Gfi1+/+, while Gfi1R412X/R412Xhomozygous alleles genetically rescued both the hypomorphic protein defect and substantially restored neutrophil numbers (though not to normal). In contrast, functional challenge with neutrophil-dependent pathogens in vivo revealed a broad susceptibility for all Gfi1-mutant mice. To determine the underlying mechanism of neutropenia and immune defects, we first used novel flow cytometry analyses and Fluidigm C1 single cell RNA-Seq to establish the successive genomic states encompassing normal granulocyte specification and commitment. Independent CITE-Seq/10x sequencing analysis provided direct correlation between flow cytometry populations and genomic information, while also establishing the trajectory through genomic states traversed during terminal granulopoiesis. Next, using a novel bioinformatics algorithm (cellHarmony) we assigned Gfi1-mutant cells to their respective wild-type cell states and then determined differential gene expression. We find few genes deregulated across granulopoiesis, and that the bulk of transcriptional impact on Gfi1 target genes is specific to successive granulopoietic cell states. These insights facilitated Gfi1R412X/- Irf8+/-genetic rescue of granulocytic specification, but not post-commitment defects. We noted that a portion of Gfi1R412X/-gene deregulation unrepaired by genetic rescue was enriched for chromosome organization, proteolysis, and innate immune effectors. Electron microscopy revealed uncondensed chromatin in mature Gfi1R412X/-neutrophils while SWATH proteomics identified a loss of neutrophil granule proteins and members of the NADPH oxidase complex (potentially linking SCN with chronic granulomatous disease genes). To this end, we functionally validated impaired NADPH oxidase complex function in neutrophils from Gfi1-mutant mice. We noted Gfi1 mutant mice have consistently elevated levels of granulocyte colony stimulating factor (but not other cytokines), and so we extended our analysis of oxidative burst to GCSF-rescued human SCN patients to find profound defects; underscoring the inability of genetic or cytokine rescued specification to resolve post-commitment defects. We illustrate a work flow that can be broadly applied to molecularly dissect translationally relevant mouse models of disease, and underscores the necessity of evaluating mutations within the context of relevant cell states. Disclosures Dwivedi: Abbvie: Employment. Myers:Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Nazor:BioLegend: Employment.

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 (Sbr)L. donovaniinduces 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 SbrL. donovaniis 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.

Since the discovery that CD4 and CD8 T cells exhibit selective TCR specificity for MHC class II or I antigen-presenting molecules, respectively, the basis of CD4 and CD8 lineage commitment in the thymus has remained the focus of intense study. Studying the mechanism by which bipotential double-positive (DP) progenitors diverge into single-positive (SP) CD4 or CD8 thymocytes is important both for understanding formation of the adaptive immune system, as well as for elucidating the bipotential fate decisions in general. Current information strongly suggests that duration and intensity of TCR signaling controls lineage choice, as proposed by the kinetic signaling and quantitative instructive models, respectively. However, the molecular pathways by which alternate TCR signals trigger alternate lineage fates remain to be precisely elucidated. Recently, identification of the transcription factors, ThPOK (T helper–inducing POZ Kruppel) and Runx, as key regulators of lineage choice and lineage-specific gene expression has provided an important breakthrough in this regard. ThPOK is selectively expressed in class II–restricted cells at the CD4+8lo stage and is necessary and sufficient to dictate development toward the CD4 lineage, while Runx3 is selectively required for CD8 T cell differentiation. Given the central role of ThPOK and Runx in lineage commitment and differentiation, understanding their upstream regulation and downstream modes of action is expected to reveal further important aspects of the molecular circuitry underlying CD4/CD8 lineage commitment. Accordingly, this article focuses mainly on the molecular basis of CD4/CD8 lineage commitment, describing the TCR signaling requirements and transcriptional regulatory circuits that control this process with a special emphasis on ThPOK and Runx.

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-αβTCR 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.

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-γ and tumor necrosis factor-α 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-γ 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-γ 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-γ and/or tumor necrosis factor-α, 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-γ-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.

The transcription factors TCF-1 and LEF-1 have diverse roles in differentiation into the CD4+ lineage through means both dependent on and independent of the transcription factor Th-POK.

Human Vγ9Vδ2 T lymphocytes are activated by phosphoantigens provided exogenously or produced by tumors and infected cells. Activation requires a contact between Vγ9Vδ2 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 Vγ9Vδ2 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 Vγ9Vδ2 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 Vγ9Vδ2 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.

1 pages; International audience; 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.

An aqueous extract of human placenta (HPE) was found to offer protection against established experimental visceral leishmaniasis in BALB/c mice and hamsters, whether the Leishmania donovani strain involved was one that was sensitive or resistant to pentavalent antimony. Intraperitoneal administration of the extract, into mice or hamsters that had been infected 2 months previously, led to antileishmanial T-cell proliferation among splenic mononuclear cells, the generation of host-protective cytokines (interferon-gamma, tumour necrosis factor and interleukin-12) and the upregulation of the expression of inducible nitric oxide synthase (and subsequent NO generation) in splenocytes. Furthermore, splenic macrophages from the HPE-treated mice showed increased generation of reactive oxygen species and enhanced surface expression of antigens of major histocompatibility complex class II (MHCII), and the extract restored the otherwise-defective antigen-presenting ability of the macrophages. Thus, in mice and hamsters infected with L. donovani, HPE therapy can stimulate both arms of the host's immune system and favour the complete resolution of the leishmanial infection. Among five human cases of visceral leishmaniasis, 30 daily intramuscular injections of HPE, at doses much lower than those used in the experimental infections, also gave very promising results. Based on the results of this pilot study, a further evaluation of the efficacy of HPE therapy, which may offer a cost-effective way of improving the treatment of antimony-resistant cases of visceral leishmaniasis, is being undertaken.

Background In search of a suitable GSH-depleting agent, a novel copper complex viz., copper N-(2-hydroxyacetophenone) glycinate (CuNG) has been synthesized, which was initially found to be a potential resistance modifying agent and later found to be an immunomodulator in mice model in different doses. The objective of the present work was to decipher the effect of CuNG on reactive oxygen species (ROS) generation and antioxidant enzymes in normal and doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing Swiss albino mice. Methods The effect of CuNG has been studied on ROS generation, multidrug resistance-associated protein1 (MRP1) expression and on activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Results CuNG increased ROS generation and reduced MRP1 expression in EAC/Dox cells while only temporarily depleted glutathione (GSH) within 2 h in heart, kidney, liver and lung of EAC/Dox bearing mice, which were restored within 24 h. The level of liver Cu was observed to be inversely proportional to the level of GSH. Moreover, CuNG modulated SOD, CAT and GPx in different organs and thereby reduced oxidative stress. Thus nontoxic dose of CuNG may be utilized to reduce MRP1 expression and thus sensitize EAC/Dox cells to standard chemotherapy. Moreover, CuNG modulated SOD, CAT and and GPx activities to reduce oxidative stress in some vital organs of EAC/Dox bearing mice. CuNG treatment also helped to recover liver and renal function in EAC/Dox bearing mice. Conclusion Based on our studies, we conclude that CuNG may be a promising candidate to sensitize drug resistant cancers in the clinic.

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.

The emergence of an increasing number of Leishmania donovani strains resistant to pentavalent antimonials (SbV), the first line of treatment for visceral leishmaniasis worldwide, accounts for decreasing efficacy of chemotherapeutic interventions. A kinetoplastid membrane protein-11 (KMP-11)-encoding construct protected extremely susceptible golden hamsters from both pentavalent antimony responsive (AG83) and antimony resistant (GE1F8R) virulent L. donovani challenge. All the KMP-11 DNA vaccinated hamsters continued to survive beyond 8 mo postinfection, with the majority showing sterile protection. Vaccinated hamsters showed reversal of T cell anergy with functional IL-2 generation along with vigorous specific anti-KMP-11 CTL-like response. Cytokines known to influence Th1- and Th2-like immune responses hinted toward a complex immune modulation in the presence of a mixed Th1/Th2 response in conferring protection against visceral leishmaniasis. KMP-11 DNA vaccinated hamsters were protected by a surge in IFN-γ, TNF-α, and IL-12 levels along with extreme down-regulation of IL-10. Surprisingly the prototype candidature of IL-4, known as a disease exacerbating cytokine, was found to have a positive correlation to protection. Contrary to some previous reports, inducible NO synthase was actively synthesized by macrophages of the protected hamsters with concomitant high levels of NO production. This is the first report of a vaccine conferring protection to both antimony responsive and resistant Leishmania strains reflecting several aspects of clinical visceral leishmaniasis.