IFN-γ Activation Research Articles

Primary Lung Dendritic Cell Cultures to Assess Efficacy of Spectinamide-1599 Against Intracellular Mycobacterium tuberculosis.

There is an urgent need to treat tuberculosis (TB) quickly, effectively and without side effects. Mycobacterium tuberculosis (Mtb), the causative organism of TB, can survive for long periods of time within macrophages and dendritic cells and these intracellular bacilli are difficult to eliminate with current drug regimens. It is well established that Mtb responds differentially to drug treatment depending on its extracellular and intracellular location and replicative state. In this study, we isolated and cultured lung derived dendritic cells to be used as a screening system for drug efficacy against intracellular mycobacteria. Using mono- or combination drug treatments, we studied the action of spectinamide-1599 and pyrazinamide (antibiotics targeting slow-growing bacilli) in killing bacilli located within lung derived dendritic cells. Furthermore, because IFN-γ is an essential cytokine produced in response to Mtb infection and present during TB chemotherapy, we also assessed the efficacy of these drugs in the presence and absence of IFN-γ. Our results demonstrated that monotherapy with either spectinamide-1599 or pyrazinamide can reduce the intracellular bacterial burden by more than 99.9%. Even more impressive is that when TB infected lung derived dendritic cells are treated with spectinamide-1599 and pyrazinamide in combination with IFN-γ a strong synergistic effect was observed, which reduced the intracellular burden below the limit of detection. We concluded that IFN-γ activation of lung derived dendritic cells is essential for synergy between spectinamide-1599 and pyrazinamide.

Abstract 2048: Interferon-γ triggers an anti-tumorigenic chain reaction in the tumor vessels of colorectal carcinoma

Abstract In colorectal carcinoma (CRC), a Th1-tumor microenvironment (TME) is associated with improved prognosis of the patients. The lead cytokine of the Th1-response is interferon (IFN)-γ. IFN-γ is predominantly regarded as an immunomodulatory cytokine. At present, its putative tumor blood vessel-directed anti-tumorigenic effects have not been investigated comprehensively. We demonstrate that the vascular effects of IFN-γ in CRC trigger a two-step anti-tumorigenic chain reaction. First, IFN-γ exerts direct angiostatic activity on endothelial cells. This was detected in vitro using primary endothelial cell cultures and in vivo in Th1-dominated inflammatory reactions of the colon using mouse models with specific deletion of the IFN-γ receptor-2 in endothelial cells and treatment with neutralizing anti-IFN-γ antibodies. Angiostatic IFN-γ effects could be validated in human CRC tissues using the cellular IFN-γ activation marker guanylate binding protein-1 which confirmed reduced angiogenic activity of vessels exposed to IFN-γ at that single cell level. IFN-γ-induced angiostasis was associated with a highly significantly improved cancer-related 5-year survival of the CRC patients (n=388). In a second step, angiostatic activity of IFN-γ resulted in the maintenance of mature tumor vessels in CRC tissues which expressed and secreted the anti-tumorigenic protein SPARCL1 in human CRC tissues (n = 42). This was significantly repressed in CRC tissues lacking a Th1 response. Functional analyses showed that SPARCL1 inhibited angiogenic activity of cultivated endothelial cells in different in vitro tests of angiogenesis (endothelial cell proliferation, migration, spreading, 3D-sprouting and capillary formation in matrigel), both after retrovirally triggered recombinant expression in endothelial cells or after adding the recombinantly purified human SPARCL1 protein. Interestingly, secreted SPARCL1 has also been shown to inhibit proliferation and migration of human CRC tumor cell lines. We could confirm the anti-tumor cell activity of soluble purified SPARCL1 using different tumor cell lines derived from mouse colon tumors (MC38) or mouse melanomas (B16F10), respectively. SPARCL1 inhibited proliferation and migration of both cell lines significantly. Using a mouse model system with a general knock out of the SPARCL1 gene, we obtained first evidence that the growth of metastatically spread MC38 cells in the lungs is repressed by SPARCL1 in vivo. Altogether our study demonstrates that the tumor vessel-directed effects of an IFN-γ-dominated tumor microenvironment in CRC trigger a two-step anti-tumorigenic chain reaction. This provides further insight into the potent anti-tumorigenic activity of a Th1-TME in CRC that may be of relevance for selection of patients' for anti-angiogenic therapy regiments. Moreover, our findings indicate novel pathways which may be prone to tumor immune evasion. Citation Format: Michael Stürzl, Victoria Langer, Daniela Regensburger, Clara Tenkerian, Annika Klingler, Maximilian Waldner, Christoph Becker, Valerie Meniel, Robert Grützmann, Carol-Immanuel Geppert, Nathalie Britzen-Laurent, Elisabeth Naschberger. Interferon-γ triggers an anti-tumorigenic chain reaction in the tumor vessels of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2048.

C-terminal truncation of IFN-\u03b3 inhibits proinflammatory macrophage responses and is deficient in autoimmune disease

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages. In acute peritonitis this signature was absent in Mmp12–/– mice and recapitulated in Mmp12+/+ mice treated with a MMP12-specific inhibitor. Similarly, loss-of-MMP12 increases IFN-γ–dependent proinflammatory markers and iNOS+/MHC class II+ macrophage accumulation with worse lymphadenopathy, arthritic synovitis and lupus glomerulonephritis. In active human systemic lupus erythematosus, MMP12 levels were lower and IFN-γ higher compared to treated patients or healthy individuals. Hence, macrophage proteolytic truncation of IFN-γ attenuates classical activation of macrophages as a prelude for resolving inflammation.

Mycobacterium smegmatis Induces Neurite Outgrowth and Differentiation in an Autophagy-Independent Manner in PC12 and C17.2 Cells.

Both pathogenic and non-pathogenic Mycobacteria can induce the differentiation of immune cells into dendritic cells (DC) or DC-like cells. In addition, pathogenic Mycobacteria is found to stimulate cell differentiation in the nerves system. Whether non-pathogenic Mycobacteria interacts with nerve cells remains unknown. In this study, we found that co-incubation with fast-growing Mycobacteria smegmatis induced neuron-like morphological changes of PC12 and C17.2 cells. Moreover, the M. smegmatis culture supernatant which was ultrafiltrated through a membrane with a 10 kDa cut-off, induced neurite outgrowth and differentiation in an autophagy-independent pathway in PC12 and C17.2 cells. Further analysis showed that IFN-γ production and activation of the PI3K-Akt signaling pathway were involved in the neural differentiation. In conclusion, our finding demonstrated that non-pathogenic M. smegmatis was able to promote neuronal differentiation by its extracellular proteins, which might provide a novel therapeutic strategy for the treatment of neurodegenerative disorders.

IL-2 enhances IFNγ signals in subpopulations of T and B lymphocytes from treatment naive relapsing remitting multiple sclerosis (RRMS) patients.

Abstract Multiple sclerosis (MS) is the most common demyelinating disorder where most patients exhibit an undulating relapsing remitting course of disease progression (RRMS). Dysregulated effector and regulatory arms of the adaptive immune system contribute to disease progression and severity. Low dose interleukin 2 (IL-2) therapy is being considered for the treatment of autoimmunity to control adaptive effector responses and promote regulatory T cell (Treg) activity. In RRMS, others have shown that IFNγ responsiveness leads to loss of Treg function. We tested the possibility that IL-2 signals will dampen IFNγ dependent STAT activation in subpopulations of effector and regulatory T and B lymphocytes in a disease specific context. We stimulated PBMC from treatment naive RRMS or as a disease control, RA patients, with IFNγ and/or IL-2. Using a multi-parameter phospho-flow approach we evaluated the activation of STAT1, STAT3, and STAT5 in each subpopulation of T and B cells. The data was analyzed using t-SNE and SPADE visualization tools to interrogate changes in the activation and proportion of cell populations. We found IFNγ stimulation broadly induced greater activation of STAT1 in MS than in RA within multiple lymphocyte populations. IL-2 co-stimulation further increased IFN-γ activation in MS but not in RA cell populations, including in Tregs. Our observations suggest that in MS (1) there are enhanced IFNγ induced effector responses and (2) IL-2 signals synergize with IFNγ induced STAT1 activation. Thus, low dose IL-2 as a therapy for MS is likely to further exacerbate RRMS pathogenesis.

Expression of complement receptor 3 (CR3) and regulatory protein CD46 on dendritic cells of antiretroviral naïve and treated HIV-1 infected individuals: Correlation with immune activation status

During infection and budding, human immunodeficiency virus-1 (HIV-1) acquires regulators of Complement Activation (RCAs) along with the host cell membrane on the viral envelope. Activation of host complement system results in opsonization of virus by complement fragments, however the virus evades complement mediated lysis (CoML) by virtue of the RCAs on the viral envelope. The RCAs on HIV-1 envelope process complement protein C3 into various fragments that promote viral entry and infection of cells through different complement receptors. Complement opsonized HIV-1 has been shown in vitro to infect dendritic cells (DCs) in a CR3 dependent manner, although the role of CR3 and CD46 in natural HIV-1 infection is not clear. Surface expression of CR3 and CD46 on DC subsets of 30 antiretroviral naïve, 31 treated (cART) HIV-1 infected individuals and 30 seronegative controls was measured by flow cytometry and plasma levels of cytokines and complement activity (C3c levels) were quantitated by sandwich ELISA. Significantly lower surface expression of CR3 and CD46 was observed on DC subsets in naïve and treated HIV-1 infected individuals compared to controls. Significantly higher complement activation and plasma levels of IL-4, IL-8, IL-10 and IFN-γ were observed in treatment naïve HIV-1 infected individuals than controls. Significantly lower plasma levels of IL-4, IL-6, IL-8 and IL-10 were observed in treated vs. naïve HIV-1 infected individuals. Our findings suggest that alterations in expression of CR3 and CD46 on DCs along with complement activity could be factors that influence viral persistence and HIV-1 disease progression and need to be further evaluated.

Development of a M cell-targeted microparticulate platform, BSK02™, for oral immunization against the ovarian cancer antigen, sperm protein 17.

Although it only accounts for approximately 5% of all female cancer cases, ovarian cancer (OC) ranks as the fifth leading cause of death due to cancer in women. We have evaluated the potential of an orally administered microparticulate vaccine incorporating an immunodominant epitope peptide derived from the cancer/testis antigen sperm protein 17 (SP17) aberrantly expressed in OC, to retard the progression of the disease. The peptide antigen and the immune-stimulatory toll-like receptor 9 ligand CpG oligonucleotide were incorporated into spray dried microparticles composed of enteric and sustained release polymers together with the Aleuria aurantia lectin targeting microfold cells present in the gut-associated lymphoid tissue. These particles were administered via oral route to mice challenged week prior with SP17-expressing ID8 OC cells. Analysis of splenocytes harvested from vaccinated mice revealed strong activation of IFN-γ+/CD8+ lymphocytes in response to re-stimulation with the SP17 antigen. Moreover, vaccinated animals showed significant retardation of ascites/tumor volume in comparison to placebo-treated animals four weeks after the tumor challenge (p = 0.005). Taken together, our results suggest that vaccination against SP17 using orally administered microparticles could potentially be used as an effective consolidation strategy for OC patients with residual tumor or high probability for relapse following first-line treatments. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 29-36, 2019.

Magnesium enhances the chondrogenic differentiation of mesenchymal stem cells by inhibiting activated macrophage-induced inflammation

Magnesium deficiency increases the generation of pro-inflammatory cytokines, which is consistently accompanied by the sensitization of cells such as neutrophils, macrophages and endothelial cells. We investigated the potential of magnesium to regulate macrophage polarization and macrophage-induced inflammation with or without lipopolysaccharide (LPS) and interferon-γ (IFN-γ) activation and further elucidated whether these effects impact the inhibitory functions of activated macrophage-induced inflammation on cartilage regeneration. The results showed that magnesium inhibited the activation of macrophages, as indicated by a significant reduction in the percentage of CCR7-positive cells, while the percentage of CD206-positive cells decreased to a lesser degree. After activation, both pro-inflammatory and anti-inflammatory cytokines were down-regulated at the mRNA level and certain cytokines (IL-1β, IL-6 and IL-10) were decreased in the cell supernatant with the addition of magnesium. Moreover, magnesium decreased the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB) to impede its activation. A modified micromass culture system was applied to assess the effects of activated macrophage-conditioned medium with or without magnesium treatment on the chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Magnesium enhanced the chondrogenic differentiation of hBMSCs by reversing the adverse effects of activated macrophage-induced inflammation.

Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein.

Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects.

Indoleamine 2,3-Dioxygenase Activity Increases NAD+ Production in IFN-γ-Stimulated Human Primary Mononuclear Cells.

IFN-γ activation of mononuclear phagocytes significantly increases indoleamine 2,3-dioxygenase (IDO) and flux through the kynurenine pathway (KP). However, the effect of IDO on NAD+ synthesis, the end product of KP metabolism, is unknown. To investigate this, primary human peripheral blood mononuclear cells were cultured up to 10 days and activated with IFN-γ in the presence or absence of a poly(ADP-ribose) polymerase (PARP) inhibitor. Day 10 macrophages had significantly higher NAD+ levels compared with monocytes. IFN-γ activation of macrophages resulted in the highest induction of IDO but decreased intracellular NAD+ concentrations at both 24 and 48 hours. However, IFN-γ activation of both day 6 and day 10 macrophages in the presence of a PARP inhibitor resulted in significantly higher intracellular NAD+ levels at 24 hours. This study provides evidence for the first time that an immune-mediated increase in IDO activity increases NAD+ biosynthesis concomitantly with an increase in NAD+ catabolism in primary human macrophages.

Identification and functional characterization of interferon regulatory factor 7 involved in activation JAK/STAT pathway in miiuy croaker

Interferon regulatory factor (IRF) family is a transcription factor family which plays an important role in the regulation of natural immunity and immune cell differentiation. IRF7 is important to regulate the response of type I interferon (IFN) to viral infection. Thus, more researches of the characteristic and functions of IRF7 should be done to get better understanding of the mechanisms underlying immune reactions. Here, the characterization of full-length cDNA of IRF7 was reported from miiuy croaker. Gene characterization analysis of mmiIRF7 showed conservative with other fish and inferred that the difference of tryptophan residues in IRF7 may occurred in the period of fish-specific genome duplication (FSGD) or earlier. Syntenic analysis of IRF7 showed that fish IRF7 had more highly conserved synteny than the higher vertebrates IRF7. Luciferase reporter assays result showed the ability of mmiIRF7 for activation of IFNα, IFNβ, IFNγ and ISRE luciferase reporter. In this study, we systematically and comprehensively analyzed evolution and function of mmiIRF7, which will provide the basis for future research on fish IRF family.

Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection.

Klebsiella pneumoniae is a significant cause of nosocomial pneumonia and an alarming pathogen owing to the recent isolation of multidrug resistant strains. Understanding of immune responses orchestrating K. pneumoniae clearance by the host is of utmost importance. Here we show that type I interferon (IFN) signaling protects against lung infection with K. pneumoniae by launching bacterial growth-controlling interactions between alveolar macrophages and natural killer (NK) cells. Type I IFNs are important but disparate and incompletely understood regulators of defense against bacterial infections. Type I IFN receptor 1 (Ifnar1)-deficient mice infected with K. pneumoniae failed to activate NK cell-derived IFN-γ production. IFN-γ was required for bactericidal action and the production of the NK cell response-amplifying IL-12 and CXCL10 by alveolar macrophages. Bacterial clearance and NK cell IFN-γ were rescued in Ifnar1-deficient hosts by Ifnar1-proficient NK cells. Consistently, type I IFN signaling in myeloid cells including alveolar macrophages, monocytes and neutrophils was dispensable for host defense and IFN-γ activation. The failure of Ifnar1-deficient hosts to initiate a defense-promoting crosstalk between alveolar macrophages and NK cell was circumvented by administration of exogenous IFN-γ which restored endogenous IFN-γ production and restricted bacterial growth. These data identify NK cell-intrinsic type I IFN signaling as essential driver of K. pneumoniae clearance, and reveal specific targets for future therapeutic exploitations.

Exosomes Derived from HIV-1 Infected DCs Mediate Viral trans-Infection via Fibronectin and Galectin-3

Exosomes are membrane enclosed nano-sized vesicles actively released into the extracellular milieu that can harbor genomic, proteomic and lipid cargos. Functionally, they are shown to regulate cell-cell communication and transmission of pathogens. Though studies have implicated a role for exosomes in HIV-1 pathogenesis, their mechanisms are not well defined. Here, we characterized exosomes derived from uninfected or HIV-1 infected T-cells and DCs. We demonstrate substantial differences in morphological, molecular and biogenesis machinery between exosomes derived from these two immune cell types. In addition, exosomes derived from HIV-1 infected DCs were 4 fold more infective than either cell free HIV-1 or exosomes derived from T-cells. Molecular analysis of exosomes detected the presence of fibronectin and galectin-3 in those derived from DCs, whereas T-cell exosomes lacked these molecules. Addition of anti-fibronectin antibody and β-lactose, a galectin-3 antagonist, significantly blocked DC exosome-mediated HIV-1 infection of T-cells. We also observed increased gene expression of the pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β and RANTES and activation of p38/Stat pathways in T-cells exposed to exosomes derived from HIV-1 infected DCs. Our study provides insight into the role of exosomes in HIV pathogenesis and suggests they can be a target in development of novel therapeutic strategies against viral infection.

Highlights of This Issue

Low-dose DNA demethylating agent decitabine has been used and has shown clinical effectiveness for the treatment of solid tumors, while the previous researches mainly focused on its effects on cancer cells. Li and colleagues examined whether low-dose decitabine treatment directly influences T-cell activation and cytolysis-mediated antitumor responses. Based on in vitro, in vivo and clinical trial studies, low-dose decitabine therapy could enhance the activation, proliferation and cytolytic activity of human IFN-γ+ T cells, which was correlated with improved therapeutic responses and survival of cancer patients, thus highlighting the important role of antitumor immune regulation during decitabine-based therapy.Current treatment strategies including single agent endocrine agents or combinations with CDK4/6 inhibitors increased capabilities of effective treatment for patients with hormone-receptor positive (HR+) metastatic breast cancer. Liquid biopsies are emerging as an important area of biomarker development for assessment and monitoring of disease heterogeneity. Paollillo and colleagues performed a prospective study to evaluate estrogen-receptor expression and ESR1 mutations in patients with HR+ disease with measurable CTC by standard method. They demonstrated the feasibility of single cells molecular analysis and discovered both phenotypic and genotypic heterogeneity a phenomenon that can only be described by using CTC.ER+/FGFR1-amplified breast cancer cells and tumors retain proliferation and ERα genomic activity despite estrogen deprivation therapy. Formisano and colleagues show that combined inhibition of ERα and FGFR1 induced regression of ER+/FGFR1-amplified breast cancer cells and patient-derived xenografts, supporting further development of combinations of ER and FGFR antagonists for the treatment of patients with this subtype of breast cancer.Abundant nontumor cells are enriched in glioma tissues, which might dilute the purity of glioma cells and play important roles in glioma biology. To characterize the biological and clinical features of glioma purity, Zhang and colleagues performed a comprehensive analysis on more than 2,000 glioma tissues. Glioma purity conferred critical implications in multiple dimensions. Low purity cases were characterized by malignant phenotypes, poor prognosis, distinct genomic alterations and infiltration of specific immune cells. This study highlights the importance of glioma purity and the infiltrated nontumor cells, which may provide insights into precise classification, clinical prediction, and effective therapeutic strategy.

The IDO inhibitor 1-methyl tryptophan activates the aryl hydrocarbon receptor response in mesenchymal stromal cells.

The catabolism of tryptophan (Trp) by indoleamine 2,3-dioxygenase (IDO) is a key step in tolerance effected by a variety of cell types, including mesenchymal stromal cells (MSCs). Trp catabolism generates molecules known as kynurenines, whose tolerance mechanisms involve activation of the Aryl Hydrocarbon Receptor (AHR). A synthetic analog of Trp, 1-methyl tryptophan (1MT), is a selective inhibitor of IDO enzymatic activity being utilized in cancer immunotherapy trials. We hypothesized 1MT might activate AHR independently of its effects on IDO. We demonstrate MSCs express AHR protein, and that in vitro treatment with 1MT causes AHR nucleotranslocation. Upon analyzing mRNA, we observed transcriptional upregulation of cytochrome p450 1a1 and 1b1 by 1MT racemic mixture (R-MT), consistent with AHR-activation. RNA-sequencing identified Nrf2, MAPK12 and IL-1a as downstream targets of 1MT. We demonstrate 1a1 and 1b1 activation by 1MT in IDO+ MSC following interferon-γ (IFN-γ) activation, suggesting AHR signaling is uncoupled from IDO catalytic function. Such a mechanism of action for 1MT may extend its usage to a wider range of patients, irrespective of tumor IDO expression. These observations support a novel paradigm by which AHR-activating compounds like 1MT can be used in cancer immunotherapy to stimulate a pro-inflammatory response.

Markedly different immune responses and virus kinetics in littermates infected with porcine circovirus type 2 or porcine parvovirus type 1

Porcine parvovirus type 1 (PPV1) and porcine circovirus type 2 (PCV2) are small single-stranded DNA viruses with high prevalence in the global pig population. The aim of this study was to compare and contrast PCV2 and PPV1 infections in high-health status pigs and to describe PCV2 long-term infection dynamics. Six caesarian-derived colostrum-deprived pigs were randomly divided into two groups and were experimentally infected with PCV2 or PPV1 at 5 weeks of age. All pigs had detectable viremia by day (D) 3 post-infection. Pigs infected with PPV1 had a detectable INF-α response by D3 followed by a high IFN-γ response by D6. The PPV1 pigs developed antibodies against PPV1 by D6 resulting in decreasing virus titers until PPV1 DNA became undetectable from D28 until D42. In contrast, PCV2-infected pigs had no detectable INF-α or IFN-γ response after PCV2 infection. PCV2-infected pigs had no detectable anti-PCV2 humoral response until D49 and had a sustained high level of PCV2 DNA for the duration of the study. While PPV1-infected pigs were clinically normal, PCV2-infected pigs developed severe clinical illness including fatal systemic porcine circovirus associated disease (PCVAD) by D28, fatal enteric PCVAD by D56 and chronic PCVAD manifested as decreased weight gain and periods of diarrhea. Microscopically, all three PCV2-infected pigs had lymphoid lesions consistent with PCVAD and associated with low (chronic disease) to high (acute disease) levels of PCV2 antigen. Under the study conditions, there was a lack of early IFN-γ and INF-α activation followed by a delayed and low humoral immune response and persisting viremia with PCV2 infection. In contrast, PPV1-infected pigs had IFN-γ and INF-α activation and an effective immune response to the PPV1 infection.

Selective activation of anti-CD73 mechanisms in control of primary tumors and metastases

ABSTRACTThe emerging role for CD73 in driving cancer growth and metastasis has presented opportunities to develop anti-CD73 monoclonal antibodies (mAbs) in the treatment of human cancers. Blockade of CD73 by antagonistic CD73 mAbs ameliorates tumor growth and metastasis via the inhibition of enzymatic and non-enzymatic CD73 pathways. In this study, we investigated whether Fc-receptor cross-linking represented a non-redundant mechanism by which anti-CD73 mAbs exert potent suppression of solid tumors and metastases. We engineered four anti-CD73 mAbs, each different in their ability to modulate CD73 enzymatic function and bind Fc receptors. mAbs recognizing a similar epitope of CD73 (CD73–04, TY/23 and 2C5) displayed the greatest antitumor activity. Importantly, we observed that the optimal control of metastasis by anti-CD73 mAbs involved primarily Fc receptor engagement, while suppression of solid tumors required both, enzyme inhibition and activation of Fc receptors. Engagement of Fc-receptors was also essential for optimal anti-metastatic effect in combination with either A2AR inhibitor or anti-PD-1 mAb treatment. The control of experimental metastases relied on the activation of host NK cells and IFNγ, while NK cells, CD8+ T cells and IFNγ were needed for effective antitumor effect in the spontaneous metastases model. These observations advance our understanding of the enzymatic and non-enzymatic functions of anti-CD73 mAbs in solid tumors and metastases. Altogether, these findings will greatly assist in the design of anti-CD73 mAbs to be used as either single agents or in combination with other immunotherapeutic molecules or targeted therapies.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Immune Function (II).

Taurine deficient mice lacking cysteine sulfinic acid decarboxylase (CSAD KO) were developed for investigating the various physiological roles of taurine including the development of the brain and eye as well as immune function. Due to severe abnormalities of immune function in a taurine deficient cat, the immune function including adoptive and innate immunity in taurine-deficient mice have been studied. Previously we demonstrated that B cell function in CSAD KO was reduced in both females and males. However, T cell function was significantly reduced only in females. In this study, we have examined innate immunity using macrophage activation with LPS or/and IFN-γ and polymorphonuclear leukocytes (PMN) activation with phorbol myristate acetate (PMA). Pro- and anti-inflammatory cytokines including IL-6, TNF-α and IL-10 as well as nitric oxide (NO) were determined using ELISA and Griess reagent, respectively. Peritoneal macrophages were activated with 1 μg/mL of lipopolysaccharide (LPS) and/or 50 U/mL of IFN-γ. In addition, superoxide anion was measured using peritoneal PMN activated with PMA in the presence and absence of superoxide dismutase. Superoxide anion production in activated PMN from CSAD KO homozygotes (HO) was not significantly different from wild-type (WT) with and without 25 mM taurine. IL-10 and TNF-α production in both female and male CSAD KO were not significantly different. IL-6 and NO were significantly lower only in females as previously observed in Con A-activated cellular proliferation of splenocytes. Cytokine production with 10 mM of taurine was not different, indicating the reduction of NO and IL-6 in females may be due to the absence of the CSAD gene, not due to low taurine concentrations.These data indicate that some measures of innate immunity were altered in female CSAD mice.

Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium

A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.

ChAT-positive neurons participate in subventricular zone neurogenesis after middle cerebral artery occlusion in mice

The mechanisms of post-stroke neurogenesis in the subventricular zone (SVZ) are unclear. However, neural stem cell-intrinsic and neurogenic niche mechanisms, as well as neurotransmitters, have been shown to play important roles in SVZ neurogenesis. Recently, a previously unknown population of choline acetyltransferase (ChAT)+ neurons residing in rodent SVZ were identified to have direct control over neural stem cell proliferation by indirectly activating fibroblast growth factor receptor (FGFR). This finding revealed possible neuronal control over SVZ neurogenesis. In this study, we assessed whether these ChAT+ neurons also participate in stroke-induced neurogenesis. We used a permanent middle cerebral artery occlusion (MCAO) model produced by transcranial electrocoagulation in mice, atropine (muscarinic cholinergic receptor [mAchR] antagonist), and donepezil (acetylcholinesterase inhibitor) to investigate the role of ChAT+ neurons in stroke-induced neurogenesis. We found that mAchRs, phosphorylated protein kinase C (p-PKC), and p-38 levels in the SVZ were upregulated in mice on day 7 after MCAO. MCAO also significantly increased the number of BrdU/doublecortin-positive cells and protein levels of phosphorylated–neural cell adhesion molecule and mammalian achaete scute homolog-1. FGFR was activated in the SVZ, and doublecortin-positive cells increased in the peri-infarction region. These post-stroke neurogenic effects were enhanced by donepezil and partially decreased by atropine. Neither atropine nor donepezil affected peri-infarct microglial activation or serum concentrations of TNF-α, IFN-γ, or TGF-β on day 7 after MCAO. We conclude that ChAT+ neurons in the SVZ may participate in stroke-induced neurogenesis, suggesting a new mechanism for neurogenesis after stroke.