Mouse Spleen Cells Research Articles

Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM.We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM.Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. DisclosuresLicht:Celgene: Research Funding.

Rapamycin Promote the Inhibition Rate of Proliferation on SHI-1 Cells and Enhance the Efficacy of Chemotherapy in Npg Leukemia Mice

Acute myeloid leukemia (AML) is a malignant hematologic disease. The remission rate of traditional chemotherapy is about 70-80%. In order to improve the efficacy of chemotherapy, some new drug which could enhance the efficacy of chemotherapy should be explored. Rapamycin is an immunosuppressive agent which can regulate cell proliferation, autophagy and other activities by inhibiting mTOR signal pathway. The studies of rapamycin as an anti-leukemia agent were limited. We had successfully establish a leukemia model in NPG mice with systemic leukemia infiltration using the human acute monocytic leukemia cell line SHI-1 cells. So in this study, the rapamycin and/or chemotherapy were used to treat the SHI-1 cells in vitro and NPG leukemia mice to investigate the efficacy of rapamycin in the treatment of AML. Rapamycin alone could effectively inhibit the proliferation of SHI-1 cells in vitro, and the inhibition rate were 35.4% which lower than the inhibition rate chemotherapy group (49.9%) using cytarabine and adriamycin. The combination with rapamycin and chemotherapy could significantly enhanced the inhibition rate to 60.3% (P<0.05) (Fig A). At day 19 after SHI-1 were inoculated to the NPG mice from tail vein, nearly 1% CD45 and CD33 positive cells were found in the peripheral blood. Then the NPG mice were divided to 4 groups to receive treatment. Control group without any treatment, rapamycin group were intraperitoneally injected with rapamycin (10mg/kg) every day for 5 days, chemotherapy group were intraperitoneally injected with cytarabine (100mg/kg for 5 days) and adriamycin (1mg/kg every other day for 3 times), combined group were treated with rapamycin, cytarabine and adriamycin. At the first day after treatment, one NPG mice were sacrificed randomly in four groups. The weight of the spleen in control group, rapamycin group, chemotherapy group and combination group were 78.6 mg, 48.9 mg, 16.1 mg, 11.7 mg respectively (Fig B). The ratio of human CD33 and CD45 positive cells in peripheral blood, bone marrow and spleen of NPG mice were 20.2%, 23.2% and 16.8% in control group, 2.02%, 2.68% and 3.31% in rapamycin group, 13.6%, 14.2% and 5.50% in chemotherapy group, 0.31%, 2.49% and 1.22% in combined group respectively (Fig C). Histopathological results showed that the degree of leukemia infiltration in the organs of NPG in the rapamycin group, chemotherapy group, and the combined group was significantly less than that of the control group. The median survival time of NPG mice in control group was 30.5 days. The treatment of rapamycin or chemotherapy could significantly prolong the media survival time of NPG mice to 35.0 and 34.0 days (P<0.05). When combined rapamycin with chemotherapy, the life time of NPG mice were significantly extended to 39 days (P<0.05) (Fig D). When NPG mice were died, more neoplasms were grow in the organs such as kidney, liver, stomach, mesenteric, mediastinum, bladder, spine, skin and neck in the NPG mice of control group, the treatment of rapamycin, chemotherapy and combined treatment would significantly decrease the counts of infiltrative organ with leukemia cells (Fig E). Altogether, our studies verified that rapamycin could inhibit the growth of SHI-1 cells and enhance the efficacy of chemotherapy in vitro. In NPG leukemia mice, the treatment of rapamycin alone could gain the efficacy as in chemotherapy group, when combined with chemotherapy, rapamycin had synergistic effect to significantly enhance the efficacy of chemotherapy to prolong the median survival time and decrease the degree of leukemia infiltration in NPG mice. DisclosuresNo relevant conflicts of interest to declare.

BST-236, a Novel Cytarabine Prodrug, Is Safer and As Effective As Cytarabine in In Vivo Leukemia Models

Introduction: Acute myeloid leukemia (AML) is associated with poor outcomes in older and medically unfit patients, largely due to the severe toxicity associated with cytarabine treatment, which precludes the administration of effective cytarabine doses. BST-236 is a prodrug of cytarabine, inactive in its prodrug form and designed to deliver cytarabine to leukemia cells with reduced systemic toxicity, thus to enable delivery of high cytarabine doses to leukemia cells with relative sparing of normal tissues.Results: BST-236 is a conjugate of cytarabine and asparagine (Figure 1). It was demonstrated that BST-236 is inactive as an intact prodrug and that its activity is exerted by release of cytarabine via non-enzymatic hydrolysis. Unlike free cytarabine, the bound cytarabine in BST-236 is not phosphorylated into its active metabolite Ara-CTP and it is protected by the asparagine residue from deamination into its inactive form Ara-U. In vitro studies demonstrate that BST-236 enters into leukemia cells, accompanied by cellular accumulation of free cytarabine, which is released from BST-236 (Figure 2). Like cytarabine, treatment with BST-236 result in induction of cell death of various leukemia cell lines via apoptosis, an activity which is dependent on the human equilibrative nucleoside transporter 1 (hENT1). The in vitro kinetics of BST-236-induced toxicity were found to be delayed compared to administration of free cytarabine, correlating with an observed delayed cellular availability of cytarabine.In vivo studies in mice and dogs demonstrate that BST-236 concentrations in the plasma are dose-proportional, with a prodrug-typical profile and only ~5% of free cytarabine present in the plasma. The maximal tolerated dose of BST-236 was found to be several-fold higher than reported for cytarabine, with mainly hematological effects and no unexpected toxicities.In vivo head-to-head studies in human leukemia mouse models with equimolar doses of cytarabine and BST-236 demonstrate similar efficacy of complete elimination of the leukemia cells in the bone marrow, spleen, and peripheral blood by both molecules (Figure 3A). However, while cytarabine treatment was associated with significant toxicity including weight loss, dramatic reduction in spleen size and number of mouse spleen cells, and delayed normal murine white blood cell recovery, equimolar BST-236 doses enabled spleen and BM recovery with minimal weight loss and no observed clinical signs (Figure 3B, 3C).Summary: in vitro and in vivo studies demonstrate that BST-236 is a prodrug of cytarabine, which enables the delivery of cytarabine to target cells, resulting in elimination of the leukemia with reduced systemic toxicity compared to free cytarabine. The data also suggest that while the mechanism of cell death induced by BST-236 and cytarabine is similar, the observed differential kinetics of the delivery of cytarabine by BST-236 and its metabolism may explain its reduced systemic toxicity. Our nonclinical findings are in line with the clinical results of the BST-236 Phase 1/2 study (ASH 2017 abstract no 893, manuscript in preparation) and suggest that BST-236 may enable delivery of high cytarabine doses to older and medically-unfit patients who currently cannot benefit from an effective cytarabine therapy. This suggestion is to be confirmed by an ongoing Phase 2b study. [Display omitted] DisclosuresTessler:Biosight: Employment. Gengrinovitch:Biosight: Employment. Ben Yakar:Biosight: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Flaishon:Biosight: Employment.

Hydrolysable tannins, gallic acid, and ellagic acid in walnut reduced 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) reduction in T-Cells cultured from the spleen of mice

Walnut is an allergenic food in some populations. Although some allergenic proteins have been identified in walnuts, few small molecule components in the food have been evaluated for their immunomodulatory functions. To examine the immunomodulatory functions, we cultured T-cells from the spleen of mice with small molecule compounds in walnut, measured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction in the cells and assessed the T-cell activation and growth. Of the nine compounds of walnut, three compounds decreased the T-cell activation and growth in anti-CD3/CD28 antibody-stimulated spleen cells of mice, whereas five compounds increased that. Because the three compounds, pedunculagin, casuarictin, and pentagalloylglucose that decreased the T-cell activation and growth belong to the hydrolysable tannin class, we inferred that the tannins would be hydrolyzed in a culture containing fetal bovine serum to yield gallic acid and/or ellagic acid. Therefore, we assessed the effect of these two compounds on the T-cell activation and growth and cytokine secretion in the culture. Both compounds decreased the T-cell activation/growth and the secretion of interleukin-4, interleukin-10, and interferon-γ. These results suggested that walnuts contained compounds that either suppressed or promoted T-cell growth and that supplementation of hydrolysable tannins to walnut-containing foods might ameliorate allergic reaction by walnut consumption.

Biochemical characterisation of lectin from wild chickpea (Cicer reticulatum L.) with potential inhibitory action against human cancer cells.

A wild chickpea lectin (WCL) from the seeds of Cicer reticulatum L. was chromato-purified using DEAE-Cellulose and SP-Sephadex ion exchange chromatography. WCL was thermostable upto 60°C with broad pH optima (pH 5-9) and various divalent metal ions did not influence its activity. WCL demonstrated DNA protection in a dose-dependent manner. The lectin exerted antifungal activity against diverse fungal pathogens. WCL augmented the mitogenic response of mouse spleen cells at 10µg/ml concentration and showed an inhibition of HIV-1 reverse transcriptase at IC50 of 200µM. Against human cancer cell lines, lectin demonstrated anticancer potential. The cell viability assay in HepG2, Ishikawa, MCF-7 and MDA-MB-231 cell line demonstrated IC50 values of 61.8, 54.4, 37.5 and 44.2µg/ml respectively. PRACTICAL APPLICATIONS: WCL exhibited distinct medicinal properties vis-à-vis antiproliferative, mitogenic, antifungal/bacterial and HIV-1 reverse transcriptase inhibiting activities. The potential of WCL can be a subject of exploration from a pharmacological standpoint.

Application of a novel nanoemulsion adjuvant for rabies vaccine which stabilizes a Krebs cycle intermediate (SDH) in an animal model

ABSTRACTRabies is the most lethal zoonotic, vaccine-preventable viral disease in the world. Its treatment is complicated by insufficient vaccine supply and the requirement for four to five repeated injections, as commercially available inactivated rabies lack adjuvant and have low immunogenicity. In this study, we focused on the role of a Krebs cycle intermediate, succinate dehydrogenase (SDH), in the innate immune response to cytokine production. We formulated a novel nanoemulsion adjuvant, Golden03, which stabilizes mouse SDH activity and contains more coenzyme Q10 and succinic acid than the classic MF59 adjuvant. Mice were immunized on days 1, 3, and 7, with seroconversion rate results suggesting that Golden03 significantly enhanced vaccine-stimulated antibody production against the rabies virus. Neutralizing antibody concentration testing by RFFIT indicated that treatment with Golden03 could result in antibody levels of up to 0.74 IU/mL 5 days post infection (DPI). ELISPOT for IFN-γ in mouse spleen cells showed that Golden03 enhanced immune responses at 14 DPI, inducing a rapid and powerful cellular response compared to the control group. Furthermore, the Vaccine-Golden03 group displayed no obvious weight loss or death after intracranial injection with CVS-11. An additional advantage is that Golden03 allowed for a three-quarter reduction in dose, while maintaining its efficacy and rapid stimulation effect. We suggest that Golden03 could be developed as a potential adjuvant for use in human rabies vaccine.

Newly designed hemagglutinin-Der p 2 chimera is a potential candidate for allergen specific immunotherapy

AimTo investigate the immunomodulatory potential of a chimera composed of the receptor-binding domain of hemagglutinin 1 (H1s) from Influenza virus and Der p 2 (D2) allergen for allergen-specific immunotherapy of house-dust mite allergy (HDM). Main methodsH1sD2 chimera and D2 allergen were produced by genetic engineering in E. coli. Recombinant antigens were extracted from inclusion bodies by urea, then refolded and purified by immobilized- metal affinity chromatography (IMAC). Purity was verified by 2D-PAGE and secondary structures were assessed by CD spectroscopy. IgE reactivity of H1sD2 and D2 was tested in western blot with sera from 8 persons with clinical history of HDM allergy. Immunogenicity of H1sD2 and D2 were analyzed in Balb/c mice. Cytokine profile was analyzed by ELISA after stimulation of mouse spleen cells with H1sD2 and D2. Leukocyte population abundance of cells isolated from spleen and lymph node was assessed by flow cytometry. Key findingsPurified recombinant proteins H1sD2 (42 kDa) and D2 (15 kDa) revealed well defined secondary structures, and preserved IgE reactive epitopes. Analysis of supernatants of mouse spleen cells after stimulation with H1sD2 and D2, revealed a qualitatively different cytokine profile from H1sD2 immunized mouse cells (increase in IL10). CD8+ cells were decreased in the lymph node of D2 immunized mice, whereas H1sD2 immunization led to an increase of CD8+ cells in both the lymph node and the spleen. SignificanceH1sD2 chimera attenuates Der p 2-inherent Th2 response and directs the immune response toward Th1 and Treg phenotype.

The immune influence of a parabiosis model on tumour-bearing mice.

The aim of this study was to analyse the immune influence of a parabiosis model on tumour-bearing mice. Parabiosis was established between C57BL/6 wild-type mice expressing green fluorescent protein (GFP+) and C57BL/6 wild-type mice without green fluorescent protein (GFP) to ensure blood cross-circulation between animals, and then the expression of CD4+ T cells, CD8+ T cells and interleukins 2, 4 and 10, and interferon-gamma (INF-γ) in spleen cells of parabiosis model mice were examined with flow cytometry. At day 8 and day 14 after conjoined surgery, we were aiming to sample tumour tissue in the parabiosis mice and observe changes of CD3, CD4, CD8, CD31, IFN-γ and vascular endothelial growth factor (VEGF) through immunohistochemical analysis. The interaction of blood was established on the third day with modelling rate of 85.7% after blood interaction. The healthy cells of GFP+ C57 mice entered the blood circulation of tumour-bearing mice via a connecting capillary network, playing a role in stimulating CD4+ and CD8+ cells in the tumour-bearing mice so that CD4+ cells increased more in tumour-bearing mice than in the positive control group (p <0.05). The number of GFP+ cells that were detected in a tumour-bearing mouse was small, but GFP+ cells can stimulate the mouse itself to generate more CD4+/interleukin (IL)-4, CD4+/IL-10 (p <0.05).The numbers of CD4+/IL-2, CD4+/IL-4 and CD4+/IL-10 among the GFP+ mice were higher than those in the negative control group(p <0.05).The levels of IFN-γ in both mice in the parabiosis model were decreased (p <0.05). The rate of CD4+/CD8+ in parabiosis GFP+ mice was higher than in the negative control group (p <0.05). In immunohistochemical tests, the rates of CD3, CD4, CD8 and IFN-γ positive cells was higher than in the positive control group, with their optical densities of 0.32 ± 0.63, 0.33 ± 0.00, 0.31 ± 0.91 and 0.28 ± 0.14 respectively (p <0.05). The expression of CD31 (0.19 ± 0.50) and VEGF (0.19 ± 0.21) were lower when compared with the positive control group, with no significant difference. CD31 and VEFG cell expression was low, at 0.19 ± 0.50 and 0.19 ± 0.21, respectively, compared with the positive control group (p >0.05). Values for CD31 and VEGF cells in the positive control group were higher, at 0.32 ± 0.35 and 0.29 ± 0.35, respectively, but when compared with the parabiosis tumour-bearing group, there was no significant difference. The expression of CD3, CD4, CD8 and IFN-γ cells at day 8 was low: 0.22, 0.17, 0.15 and 0.16, respectively. When compared with the parabiosis tumour-bearing group, there was no significant difference. The established allogeneic parabiosis mice model can be well adapted to the conjoined state of mice and be applied in wide medical experiments. The parabiosis model has played an important role in studying immune regulation, which provides a basis for the future tumour immunotherapy. Parabiosis models can stimulate tumour-bearing mice to generate CD3, CD4, CD8 and IFN-γ, and play a notable role in immune regulation and tumour destruction. The positive expression rates of CD31 and VEFG cells in the parabiosis tumour-bearing group were lower; however, when compared with the positive control group, there was no significant difference.

Exosomes as adjuvants for the recombinant hepatitis B antigen: First report

Over the past few years, exosomes, a class of extracellular vesicles (EVs), have emerged as key players for inter-cellular communication ultimately modulating the behavior of target cells with countless outcomes. Nevertheless, the potential role of exosomes as vaccine adjuvants remains largely unexplored. Herein, we hypothesized that exosomes derived from immune cells may have an immunostimulatory effect and could constitute a good target towards the development of new fine-tuned vaccine adjuvants. To accomplish this goal, exosomes isolated from lipopolysaccharide endotoxin (LPS)-stimulated human monocytic cell line (THP-1) were characterized and tested for their non-specific immunostimulatory activity when administered subcutaneously to healthy mice; additionally, exosomes’ vaccine adjuvant ability was also disclosed after their inclusion in vaccine formulations.The results obtained suggested that the isolated exosomes evoked a pro-inflammatory profile in spleen cells of healthy mice through the induction of cytokines such as tumor necrosis factor alpha (TNF-α), chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES) and interleukin 1 beta (IL-1β).Moreover, subcutaneous vaccination of mice with exosomes combined with a solution of hepatitis B recombinant antigen (HBsAg) or combined with a suspension containing HBsAg loaded poly-ε-caprolactone (PCL)/chitosan nanoparticles (NPs), induced a humoral immune response quite similar to the one achieved with the experimental control group (HBsAg solution without exosomes). However, exosomes triggered an immunomodulator effect on the cellular immune response, highlighted by the enhancement of IFN-γ secretion.To the best of authors knowledge, this is the first report describing extensively the role of unmodified exosomes as adjuvants and co-adjuvants for hepatitis B vaccination strategies.

A polysaccharide from Antrodia cinnamomea mycelia exerts antitumor activity through blocking of TOP1/TDP1-mediated DNA repair pathway

A polysaccharide (termed ACPS-1) from mycelia of Antrodia cinnamomea under submerged culture was purified by hot water extraction and successive DEAE-52 cellulose and Sephadex G-100 column chromatography, and structurally characterized by FTIR, NMR, periodate oxidation, Smith degradation, and GC–MS. ACPS-1 (MW 2.296 × 104 Da) was composed primarily of Man, Xyl, Ara, Fuc and Rha with a molar ratio of 31.27:1.77:1.44:1.34:1.00, and its backbone consisted of repeating α-(1 → 3), α-(1 → 6), α-(1 → 2), and α-(1 → 4) glycosidic linkages. ACPS-1 displayed strong in vitro growth-inhibitory effects on several human and mouse cancer cell lines (HeLa, A431, H22 and S180), and were not cytotoxic to normal mouse spleen cells. Studies of the inhibitory mechanism revealed that ACPS-1 induced apoptosis and cell cycle arrest (cells remained in G2/M phase) through blocking of topoisomerase I/tyrosyl-DNA phosphodiesterase I (TOP1/TDP1)-mediated DNA repair pathway. Our findings suggest that ACPS-1 has strong potential applications in pharmaceutical and food industries, and as a novel anticancer agent based on its dual TOP1/TDP1 inhibitory effect.

Effect of Immune Responses Against Hydatid Cyst Antigens on Growth of Melanoma Tumor

Background: Hydatid cyst is the larval stage of the tape worm parasite, Echinococcus granulosus. Human is infected by ingestion of parasite ova excreted in dog feces. The anticancer activity of different antigens of hydatid cyst has been reported in the previous works. In this research, the role of immune system in induction of this anticancer activity has been investigated.Materials and Methods: Spleen cells of mice immunized with hydatid cyst antigens were transferred to the recipient mice that had already been challenged with melanoma cells. Also, mice with melanoma cancer were injected with antisera raised against hydatid cyst antigens. In all above mice, tumor growth was measured using a caliper. Results: Passive transfer of spleen cells resulted in significant inhibition of melanoma cancer growth. However passive transfer of antisera did not affect cancer growth in the recipient mice.Conclusion: According to the results of this work, cellular immunity but not humoral immunity may be involved in induction of anticancer activities of hydatid cyst.

Abstract A68: Targeting metabolic vulnerabilities of MDSCs to enhance the antitumor activity of PD-1 blockade

Abstract Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that differentiate from bone marrow precursors and expand in cancer-bearing hosts. MDSCs significantly contribute to tumor-induced immune suppression and represent a promising target for cancer immune therapies. Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. However, their potential effects on the tumor microenvironment are largely unknown. Here we report that phenformin, a mitochondrial complex I inhibitor, selectively inhibits granulocytic myeloid-derived suppressor cells (G-MDSCs) in spleens of tumor bearing mice and ex vivo. Metabolomics analysis reveal that, compared to CD11b+Ly6G- cells, CD11b+Ly6G+ MDSCs have lower levels of glutathione and NADPH/NADP+ ratio, suggesting these cells have higher level of endogenous ROS. Interestingly, phenformin further induces production of ROS in G-MDSC, whereas co-treatment of the antioxidant N-acetylcysteine dampens ROS levels in these cells and rescues the effect of phenformin on reducing the numbers of G-MDSCs. Importantly, co-treatment of phenformin enhances the effect of anti-PD-1 antibody therapy on inhibiting tumor growth in the BRAF V600E/PTEN null melanoma mouse model. Combination of phenformin and anti PD-1 cooperatively induces CD8+ T cell infiltration and decreases levels of proteins that are critical for immune suppressive activities of MDSCs. Our findings demonstrate a selective, inhibitory effect of phenformin on G-MDSCs-driven immune suppression and support that phenformin improves the anti-tumor activity of PD-1 blockade immunotherapy in melanoma. Citation Format: Sun Hye Kim, Man Li, Sebastian Trousil, bin zheng. Targeting metabolic vulnerabilities of MDSCs to enhance the antitumor activity of PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A68.

MBD2 regulates differentiation and function of Th17 cells in neutrophils- dominant asthma via HIF-1\u03b1

BackgroundT helper 17 (Th17) cells have proven to be crucial in the pathogenesis of neutrophils-dominant asthma. Hypoxia inducible factor-1α (HIF-1α) is involved in allergic responses in asthma. Our previous studies indicated that Methtyl-CpG binding domain protein 2 (MBD2) expression was increased in asthma patients. The aim of the present study is to understand how MBD2 interacts with HIF-1α to regulate Th17 cell differentiation and IL-17 expression in neutrophils-dominant asthma.MethodsA neutrophils-dominant asthma mouse model was established using female C57BL/6 mice to investigate Th17 cell differentiation and MBD2 and HIF-1α expression regulation using flow cytometry, western blot or qRT-PCR. MBD2 and HIF-1α genes were silenced or overexpressed through lentiviral transduction to explore the roles of MBD2 in Th17 cell differentiation and IL-17 release in neutrophils-dominant asthma.ResultsA neutrophilic inflammatory asthma phenotype model was established successfully. This was characterized by airway hyperresponsiveness (AHR), increased BALF neutrophil granulocytes, activated Th17 cell differentiation, and high IL-17 levels. MBD2 and HIF-1α expression were significantly increased in the lung and spleen cells of mice with neutrophils-dominant asthma. Through overexpression or silencing of MBD2 and HIF-1α genes, we have concluded that MBD2 and HIF-1α regulate Th17 cell differentiation and IL-17 secretion. Moreover, MBD2 was also found to regulate HIF-1α expression.ConclusionsOur findings have uncovered new roles for MBD2 and HIF-1α, and provide novel insights into the epigenetic regulation of neutrophils-dominant asthma.

Abstract 1877: Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia

Abstract SINE compounds (KPT-330/selinexor and KPT-8602/eltanexor) are a novel class of small molecules with therapeutic potential across a broad range of cancers. SINE compounds target exportin 1 (XPO1), which is an essential nucleo-cytoplasmic transport protein, responsible for the nuclear export of &amp;gt;200 cargo proteins including many tumor-suppressor proteins (TSPs) and oncogenes, such as p53, IκB, BCL-2, eIF4E and MYC. XPO1 is overexpressed in numerous solid and hematologic malignancies, often correlating with poor prognosis. An important family of oncoproteins regulated by SINE compounds are the pro-survival BCL-2 family proteins, BCL-2 and MCL-1, as both are transported from the nucleus by forming a complex with the XPO1 cargo protein, elF4E. BCL-2 and MCL-1 are dysregulated and linked to maintenance and survival in numerous cancers. Early studies with venetoclax (VEN/ABT-199), a potent, selective inhibitor of BCL-2, have revealed response rates from 50-80% across a variety of hematologic malignancies. However, resistance often arises via upregulation of other BCL-2 family antiapoptotic proteins, most commonly MCL-1. Thus, we hypothesized that co-treatment of VEN and SINE compounds would show enhanced cell killing in AML due to the ability of SINE to further block the prosurvival signals of both BCL-2 and MCL-1. Co-treatment with eltanexor and VEN demonstrated synergistic loss of viability in multiple hematologic malignancy cell lines, particularly MV-4-11 cells. Further in vitro analysis showed this enhanced cell death was due to an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays. We used in vivo studies to test the effect of this combination in a systemic AML xenograft model. For this, MV-4-11 cells were transplanted into NSGS mice and allowed to engraft before treating the mice with vehicle, eltanexor, VEN, or a combination of the two compounds. When the vehicle mice became moribund at 4 weeks after transplant, all treatment groups were sacrificed to compare leukemic burden in the hematopoietic tissues. Flow cytometric analysis revealed a drastic elimination of leukemic cells in the bone marrow, spleen, and peripheral blood of the combination-treated mice compared to vehicle and single-treatment groups. Immunohistochemical analysis of the spleen and bone marrow confirmed this finding, and also revealed the reduction in leukemic cells was due, in part, to an induction of apoptosis (an increase in cleaved caspase 3 and decrease in survivin) in splenic tissue sections, in agreement with the in vitro results. Finally, the dramatic clearance of leukemia cells in the mouse caused by eltanexor and VEN treatment led to prolonged survival. Our results demonstrate the potential of this combination regimen in the treatment of AML. Citation Format: Melissa A. Fischer, Pia M. Arrate, Hua Chang, Agnieszka E. Gorska, Londa S. Fuller, Haley E. Ramsey, Trinayan Kashyap, Christian Argueta, William Senapedis, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Michael R. Savona. Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia [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 1877.

Abstract 126: MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells

Abstract The MYC oncogene is commonly overexpressed in hematopoietic cancers. In a transgenic mouse model of MYC-induced T-cell lymphoma, we found that there was a systemic reduction in NK cell (CD3- NKp46High) numbers and activation in lymphoid organs such as spleen and bone marrow. MYC inactivation in lymphoma-bearing MYCON mice partially restored NK cell numbers and activation. Hence, the MYC oncogene appears to suppress NK cell-mediated immune surveillance of lymphomas. We evaluated whether reduction in mature and activated NK cells (CD3- NKp46High) in MYCON mice was due to increased death of NK cells in these mice when compared to normal and MYCOFF mice. Using flow cytometry, NK cells were measured in age matched spleens and bone marrows of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. Surprisingly, we observed a significant reduction in proportions of dead NK cells (NKp46+ PI+) in spleen and bone marrow of MYCON mice, in comparison to normal and MYCOFF cohorts. Hence, the reduction in activated NK cell numbers during MYC-driven lymphomagenesis does not occur because of increased death of NK cells. Next, we investigated whether MYC arrests early NK cell development in the bone marrow of lymphoma mice, leading to systemic NK suppression. NK cell lineage specification begins with CD122 expression, that is maintained throughout NK development starting at the precursor stage. NK precursors (NKP) transition to immature NK (iNK) cells by expressing the cytotoxicity marker NKp46. iNK cells leave the bone marrow to populate peripheral lymphoid organs. We measured NKP (CD122+ NKP46-), and iNK (CD122+ NKp46+) cells in bone marrow of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. We observed no significant changes in the percentages of NKP. However, the proportions of iNK cells were significantly reduced in MYCON mice, in comparison to MYCOFF and normal mice. Our results suggest that MYC may block transition from NKP to iNK stage during early NK cell development. The reduction of iNK cells in spleens of MYCON mice was concordant with the reduction of iNK cells in bone marrow from the same mice. We conclude that MYC-induced lymphomagenesis blocks early NK cell development, thereby suppressing NK-mediated immune surveillance. Citation Format: Line D. Heftdal, Srividya Swaminathan, Dean W. Felsher. MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells [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 126.

NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells.

NK-4 is the main component of the antiallergic drug Lumin, which has been in popular usage since the early 1950s. In this study, we examined whether NK-4 exerts a regulatory effect on the activation and effector function of Th2 cells. NK-4 inhibited IL-4 production by anti-CD3ε mAb-stimulated BALB/c mouse spleen cells, whereas NK-4 had little effect on IFN-γ production. IL-4 and IL-5 secretion by anti-CD3ε mAb- or antigen-stimulated Th2 cells (D10.G4.1) was abrogated by NK-4 without affecting cell numbers, whereas IFN-γ secretion by activated Th1 cells was unchanged. Mechanistic analysis revealed that NK-4 inhibited mRNA expression of the Th2-associated transcription factors GATA-3 and NFATc1 in anti-CD3ε mAb-stimulated D10.G4.1 cells. Regarding the regulation of Th2 cell effector functions, NK-4 inhibited the secretion of eotaxin and thymus and activation-regulated chemokine (TARC) by normal human dermal fibroblasts in response to IL-4 and/or TNF-α. NK-4 achieved TARC attenuation comparable to what is observed with suplatast tosilate, an antiallergic drug that selectively inhibits Th2 cytokine production, at 14-fold lower concentrations of suplatast tosilate. Dexamethasone increased TARC production by 2.2- to 2.6-fold of control cultures. NK-4 successfully inhibited the STAT6 signaling pathway, suggesting a potential mechanism for down-regulating chemokines expression. In addition, NK-4 abrogated IL-4-driven modulation of cytokine production profile in human monocytic THP-1 cells from proinflammatory to anti-inflammatory response, as seen in the inverted ratio of TNF-α to IL-10 produced in response to LPS. These results suggest that NK-4 could prevent IL-4-driven polarization to alternatively activated macrophages, which are proposed to have pathogenic roles in allergic asthma. The importance of Th2 cytokines and chemokines in the development and progression of type 2 inflammatory disorders has been highlighted by recent advance in our understanding the immunological mechanism underlying allergic disease. Our results support the use of NK-4 as a reasonable therapeutic option to alleviate Th2-mediated allergic inflammation.

Transcriptomics of the Vaccine Immune Response: Priming With Adjuvant Modulates Recall Innate Responses After Boosting.

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In this study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. Mice were primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. mRNA sequencing was performed on blood samples collected 1, 2, and 7 days after priming and after boosting. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared with the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared with mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen-presenting cells, and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody-secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly reprograms the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response.

In Vitro Anti-inflammatory Effects of the Phenylbutyric Acid Metabolite Phenylacetyl Glutamine.

Sodium 4-phenylbutyrate (PBA), which exerts a wide range of anti-inflammatory effects, is rapidly cleared from the body (approximately 98%) by urinary excretion by 24 h after oral treatment in humans. PBA was almost entirely excreted to urine as phenylacetyl glutamine (PAGln). However, no data describe the potential anti-inflammatory effects of PAGln. The purpose of this study was to evaluate the anti-inflammatory effects of PAGln on mouse spleen cells and peritoneal cavity cells, and explore the potential mechanism underlying this effect. PAGln was added to mouse spleen cell cultures stimulated by concanavalin A, or mouse peritoneal cavity cell cultures stimulated by lipopolysaccharide. After 72 h of culture, levels of inflammatory cytokines in culture supernatants were measured using a sandwich enzyme-linked immunosorbent assay system, and levels of inflammatory proteins were assessed by Western blotting. PAGln significantly inhibited inflammatory cytokine (interferon-γ, interleukin-6, and tumor necrosis factor-α) production, decrease of cell number in the spleen cell, and suppressed the expression of inflammatory proteins (nuclear factor κB, and inducible nitric oxide synthase). These results suggest that PAGln possesses anti-inflammatory activity via inhibition of T cell activation and Toll-like receptor 4 signaling. This study of the anti-inflammatory mechanism of PAGln provides useful information about its potential for therapeutic applications.

Production of monoclonal antibodies against recombinant nucleoprotein of avian influenza virus, serotype H9N2

Background: Avian influenza viruses (AIVs) including the subtype H9N2 cause considerable financial losses to poultry industries. Rapid and accurate diagnosis of avian influenza (AI) infection is important in control and eradication programs. OBJECTIVES: The aim of this study was to produce monoclonal antibodies (MAbs) specific for the nucleocapsid protein )NP (of AIV H9N2 subtype to improve diagnostic assays. METHODS: Recombinant NP protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen for mice immunization. Spleen cells of the immunized mice were fused with SP2/0 myeloma cells. Next, culture supernatants of primary hybridoma clones were screened by indirect ELISA. After three rounds of sub cloning, the reactivity of the MAbs with recombinant and natural antigens was assessed by Western blotting. RESULTS: Six MAbs showed specific binding to recombinant and natural NP from AIV H9N2 in Western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay. Cross-reactivity with genetically non-related including Newcastle viruse (Paramyxoviridae family) was not detected. CONCLUSIONS: Based on the results, the MAbs generated in this study could be used for the development of rapid diagnostic assays for recognition of AIV.

The 180 splice variant of NCAM-containing exon 18-is specifically expressed in small cell lung cancer cells.

The Neural Cell Adhesion Molecule (NCAM) is a glycoprotein expressed as 120, 140 and/or 180 kDa isoforms, all derived through alternative splicing of a single gene. NCAM 120 contains no intracellular domain, whereas NCAM 140 and 180 have different intracellular domains determined by alternative splicing of exon 18. NCAM has been described as a biomarker to discriminate small cell lung cancer (SCLC) from non-SCLC (NSCLC). However, peripheral blood mononuclear cells (PBMC) also express NCAM. We studied the expression of NCAM splice variants in cell lines, tumor tissues and control cells. Using reverse transcriptase-PCR we evaluated the expression of NCAM exon 18 splice variants in lung cancers cell lines, control cell lines, PBMC of healthy controls and SCLC tissue. In addition we studied the expression of the NCAM exon 18 encoded protein (E18) in SCLC by immunocytochemistry and flow cytometry using an E18-specific monoclonal antibody obtained by hybridoma fusion of E18-immunized mouse spleen cells. Finally we looked at immune responses to E18 in mice. We found expression of RNA encoding the NCAM 180 variant in all SCLC cell lines. NCAM exon 18 was not expressed in 23/28 (82%) of the other tumor and leukemia cell lines tested and PBMC. Next, we also evaluated the expression of NCAM exon 18 in human SCLC tissue. Expression of NCAM exon 18 in 8 of the 10 (80%) SCLC biopsy samples was found. The newly raised E18-specific antibodies stained NCAM at the adherent junctions between adjacent cells in SCLC cell lines. The data demonstrate the intracellular location of E18 in SCLC. Furthermore, a specific cytotoxic T cell (CTL) response and significant antibody titers were found in mice upon immunization with recombinant E18 and its encoding DNA. The results of this study can be applied in the diagnosis and immunotherapy of SCLC. A larger study investigating E18 as a marker for SCLC is indicated.