Rag1 Mice Research Articles

MP83-04 SUPPRESSION OF HUMAN PROSTATE CANCER (CAP) BY ADOPTIVE TRANSFER OF PATIENT-DERIVED PSMA-SPECIFIC, TGF-ß-INSENSITIVE CD8+ T CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-04 SUPPRESSION OF HUMAN PROSTATE CANCER (CAP) BY ADOPTIVE TRANSFER OF PATIENT-DERIVED PSMA-SPECIFIC, TGF-ß-INSENSITIVE CD8+ T CELLS Qiang Zhang, Timothy Kuzel, Brian Helfand, Ximing J Yang, Weijun Qin, Chung Lee, Benebito Carneiro, and Francis J Giles Qiang ZhangQiang Zhang More articles by this author , Timothy KuzelTimothy Kuzel More articles by this author , Brian HelfandBrian Helfand More articles by this author , Ximing J YangXiming J Yang More articles by this author , Weijun QinWeijun Qin More articles by this author , Chung LeeChung Lee More articles by this author , Benebito CarneiroBenebito Carneiro More articles by this author , and Francis J GilesFrancis J Giles More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2572AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has previously been shown that tumor-derived TGF-β related potent immunosuppression and lack of tumor killing specificity promote CaP progression. Here we report a new immunotherapeutic approach using adoptive transfer of patient-derived PSMA-specific, TGF-β-insensitive human CD8+ T cells to inhibit solid xenograft CaP. METHODS Peripheral blood CD8+ T cells were collected from metastatic castration resistant CaP patient by leukapheresis and cultured in the FDA approved Cell Processing Work Station (CPWS, Panasonic) with CD-3 Biotin/CD28/Anti-Biotin Beads (Bead: Cell 1:2) and IL-2 (100units/ml). We developed a TβRIIDN-TK-IRES-PZ1 chimeric T cell receptor retroviral construct using an anti-PSMA IgTCR(ζ) gene (PZ1) and a dominant negative TGF-β type II receptor (TβRIIDN) that could induce CD8+ T cells to be PSMA reactive and insensitive to TGF-β. PC-3 cells (PSMA negative; ATCC) or PSMA positive PC-3-PSMA cells (Cleveland Clinic) were used for target cells. Subcutaneous injection of PC-3 and PC-3-PSMA cells (2x105 cells/each, infected with HSV1-tk-GFP-luciferase reporter) into the left and right flank region respectively in each of 48 immunodeficient RAG-1 mice was performed. One week later, the animals were randomly assigned to one of three adoptive transfer groups (16 mice /each group, 2x106 CD8+ T cells/each mice): Group 1: CD8+ T cells infected with TβRIIDN-TK-IRES-PZ1 (71.1% positive); Group 2: Naive CD8+ T cells; Group 3: No treatment group. Tumor growth was monitored by IVIS luciferase imaging. The animals were provided 2 weekly adoptive transfer treatments and sacrificed after 3 weeks. The size and the weight of the tumor were recorded, and the infiltration of CD8+ T cells and apoptosis was evaluated by immunofluorescence staining and TUNEL. RESULTS In Group 1, the average tumor weight and volume was significantly lower in the PC3-PSMA tumor (0.413g and 514.2mm3) compared to the PC3 tumor (2.75 g and 3165.2 mm3); (~3-6×, P<0.05). There was no difference between the PC3 tumor (2.36g and 2768.5mm3 ) compared to PC3-PSMA tumor (2.45g and 2411.99 mm3) in Group 2 or Group 3. H&E staining showed large amount of nuclear fusion, fragmentation and necrosis were found in PC3-PSMA tumors in Group 1 compared to Group 2 and 3. In Group 1, tumor apoptosis (72.5/1,000 µm2) and CD8+ T cell infiltration (45.5/1,000 µm2 ) in PC3-PSMA tumor parenchyma was significantly higher compared to PC3 tumor (6.7/1,000 µm2 and 3.1/1,000 µm2 respectively). There was no significant apoptosis or CD8+ T cells infiltration observed in either PC3 and PC3-PSMA tumor in Group 2 and Group 3. CONCLUSIONS Our approach combines TGF-β insensitive with PSMA selectivity that significantly enhance the specificity and tumor killing ability of patient CD8+ T cells, and simultaneously suppress the tumor derived TGF-βinduced immunosuppression. Therefore, this construct may offer a novel therapeutic intervention for both primary CaP treatment as well as for recurrence after prostatectomy. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1107 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Qiang Zhang More articles by this author Timothy Kuzel More articles by this author Brian Helfand More articles by this author Ximing J Yang More articles by this author Weijun Qin More articles by this author Chung Lee More articles by this author Benebito Carneiro More articles by this author Francis J Giles More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Platelet-activating factor (PAF) receptor as a promising target for cancer cell repopulation after radiotherapy

A major drawback of radiotherapy is the accelerated growth of the surviving tumor cells. Radiotherapy generates a variety of lipids that bind to the receptor for platelet-activating factor, expressed by cells in the tumor microenvironment. In the present study, using the TC-1 tumor cell line, we found that irradiation induced a twofold increase in receptor expression and generated agonists of receptor. Irradiated cells induced a 20-fold increase in live TC-1 proliferation in vitro. Furthermore, subcutaneous co-injection of irradiated TC-1 cells with TC-1 expressing luciferase (TC-1 fluc+) markedly increased TC-1 fluc+ proliferation in a receptor-dependent way. Moreover we used a human carcinoma cell line not expressing the PAF receptor (KBM) and the same cell transfected with the receptor gene (KBP). Following co-injection of live KBP cells with irradiated KBM in RAG mice, the tumor growth was significantly increased compared with tumor formed following co-injection of live KBM with irradiated KBM. This tumor cell repopulation correlated with increased infiltration of tumor-promoting macrophages (CD206+). We propose that receptor represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation.

Varying proliferative and clonogenic potential in NRAS-mutated congenital melanocytic nevi according to size.

Congenital melanocytic nevi (CMN) are benign proliferations that may be associated with various consequences depending on their size. They are characterized by a specific molecular signature, namely a postzygotic somatic NRAS or BRAF mutation. We have recently reported that large CMN (lCMN), which are classically associated with an increased melanoma risk, harbour cell subpopulations with specific clonogenic and tumorigenic potential. We wished to ascertain whether cells displaying similar properties persisted postnatally in medium CMN (mCMN). Eighteen medium M1, nine large and one giant NRAS-mutated CMN were prospectively included in the study. Subpopulations of mCMN cells expressed stem cell/progenitor lineage markers such as Sox10, nestin and Oct4, as was the case in lCMN. Nevertheless, conversely to lCMN, mCMN cells with clonogenic properties were rarer. In vitro, approximatively one in 1500 cells isolated from fresh mCMN formed colonies that could be passaged. In vivo, mCMN seemed to harbour cells with less proliferative potential than the larger lesions as lCMN biopsies displayed a threefold expansion compared to mCMN when xenografted in Rag2(-/-) mice. Thus, our data revealed variations in clonogenicity and tumorigenic properties in NRAS-mutated CMN according to size.

EMT reversal in human cancer cells after IR knockdown in hyperinsulinemic mice.

Type 2 diabetes (T2D) is associated with increased cancer risk and cancer-related mortality. Data herein show that we generated an immunodeficient hyperinsulinemic mouse by crossing the Rag1(-/-) mice, which have no mature B or T lymphocytes, with the MKR mouse model of T2D to generate the Rag1(-/-) (Rag/WT) and Rag1(-/-)/MKR(+/+) (Rag/MKR) mice. The female Rag/MKR mice are insulin resistant and have significantly higher nonfasting plasma insulin levels compared with the Rag/WT controls. Therefore, we used these Rag/MKR mice to investigate the role of endogenous hyperinsulinemia on human cancer progression. In this study, we show that hyperinsulinemia in the Rag/MKR mice increases the expression of mesenchymal transcription factors, TWIST1 and ZEB1, and increases the expression of the angiogenesis marker, vascular endothelial growth factor A (VEGFA). We also show that silencing the insulin receptor (IR) in the human LCC6 cancer cells leads to decreased tumor growth and metastases, suppression of mesenchymal markers vimentin, SLUG, TWIST1 and ZEB1, suppression of angiogenesis markers, VEGFA and VEGFD, and re-expression of the epithelial marker, E-cadherin. The data in this paper demonstrate that IR knockdown in primary tumors partially reverses the growth-promoting effects of hyperinsulinemia as well as highlighting the importance of the insulin receptor signaling pathway in cancer progression, and more specifically in epithelial-mesenchymal transition.

Abstract 3297: Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition

Abstract Background. Intestinal mucus layer disruption and gut microflora modification in conjunction with tight junction (TJ) changes can increase colonic permeability that allows bacterial dissemination and intestinal and systemic disease. Employing Citrobacter rodentium (CR) as a model system to mimic the pathogenic mechanisms of Enteropathogenic and Enterohaemorrhagic E. coli, we showed previously that a functional cross-talk between the Notch and Wnt/β-catenin pathways regulates hyperplasia and/or colitis following CR infection. Aim: To test the hypothesis that CR infection combined with chronic Notch inhibition will alter the composition of the colonic mucus to promote dysbiosis, tight junction disruption, loss of multipotent intestinal stem cells (ISCs) and colitis. Material/methods: Immune-competent NIH:Swiss outbred mice were infected with CR (108 CFUs) and treated with Notch blocker Dibenzazepine [DBZ, i.p. @ 10μmol/Kg body weight]. Mucus composition, fecal 16S rDNA analysis and components of TJ integrity were measured and correlated with changes in components of the Notch pathway. 6% Pectin diet served as a SCFA-delivery system to the colon. Results: In the colons of CR-infected/DBZ-treated mice, mucus analysis revealed dramatic alterations in the composition of trace O-glycans and complex type and hybrid N-glycans, compared to CR-infected alone that preceded alterations in 16S rDNA microbial community structure. Indeed, mucin degrading and colitogenic bacterium, Akkermansia muciniphila exhibited dramatic increases in the feces of CR-infected and DBZ-treated mice. At the same time, colons from CR-infected/DBZ-treated mice had decreased expression of antimicrobial peptides Angiogenin-4, Retnlβ, Intelectin-1 and Intelectin-2, respectively. Expression levels of both TJ and adherens junction proteins (e.g., Claudin-5, ZO-1, ZO-2, E-Cadherin, β-catenin) also decreased significantly in the colonic crypts of CR-infected/DBZ-treated mice that paralleled loss of Notch signaling. Both C3H/HeN mice that exhibit exaggerated response to CR infection and T- and B-cell deficient Rag-1 mice following CR-infection and DBZ-treatment, exhibited dramatic increases in paracellular permeability, goblet cell metaplasia and exacerbation of colitis. Chronic blockade of the Notch pathway depleted colonic epithelium of ISC markers Dclk1 and Lgr5 concomitant with exacerbation of colitis. 6% Pectin diet ameliorated colitis by restoring barrier integrity via recruitment of Bacteroides vulgatus, Streptococcus gordonii and Lactococcus lactis, bacteria with potential anti-inflammatory functions. Conclusions: 1. These studies help us delineate the mechanistic basis of colitis development in the aftermath of chronic Notch inhibition. 2. Our findings also caution against the use of Notch inhibitors for patients suffering from colitis-associated colon cancer. Citation Format: Ishfaq Ahmed, Badal Roy, Rita-marie McFadden, Shrikant Anant, Seth Septer, Shahid Umar. Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3297.

Role of Axl in T-Lymphocyte Survival in Salt-Dependent Hypertension.

Survival of immune and nonimmune cells relies on Axl, a receptor tyrosine kinase, which is implicated in hypertension. Activated T lymphocytes are involved in regulation of high blood pressure. The goal of the study was to investigate the role of Axl in T-lymphocyte functions and its contribution to salt-dependent hypertension. We report increased apoptosis in peripheral blood from Axl(-/-) mice because of lower numbers of white blood cells mostly lymphocytes. In vitro studies showed modest reduction in interferon gamma production in Axl(-/-) type 1 T helper cells. Axl did not affect basic proliferation capacity or production of interleukin 4 in Axl(-/-) type 2 T helper cells. However, competitive repopulation of Axl(-/-) bone marrow or adoptive transfer of Axl(-/-) CD4(+) T cells to Rag1(-/-) mice showed robust effect of Axl on T lymphocyte expansion in vivo. Adoptive transfer of Axl(-/-) CD4(+) T cells was protective in a later phase of deoxycorticosterone-acetate and salt hypertension. Reduced numbers of CD4(+) T cells in circulation and in perivascular adventitia decreased vascular remodeling and increased vascular apoptosis in the late phase of hypertension. These findings suggest that Axl is critical for survival of T lymphocytes, especially during vascular remodeling in hypertension.

Fingolimod promotes peripheral nerve regeneration via modulation of lysophospholipid signaling.

BackgroundThe lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are pleiotropic signaling molecules with a broad range of physiological functions. Targeting the S1P1 receptor on lymphocytes with the immunomodulatory drug fingolimod has proven effective in the treatment of multiple sclerosis. An emerging body of experimental evidence points to additional direct effects on cells of the central and peripheral nervous system. Furthermore, fingolimod has been reported to reduce LPA synthesis via inhibition of the lysophospholipase autotaxin. Here we investigated whether modulation of particular signaling aspects of S1P as well as LPA by fingolimod might propagate peripheral nerve regeneration in vivo and independent of its anti-inflammatory potency.MethodsSciatic nerve crush was performed in wildtype C57BL/6, in immunodeficient Rag1−/− and Foxn1−/− mice. Analyses were based on walking track analysis and electrophysiology, histology, and cAMP formation. Quantification of different LPA species was performed by liquid chromatography coupled to tandem mass spectrometry. Furthermore, functional consequences of autotaxin inhibition by the specific inhibitor PF-8380 and the impact of fingolimod on early cytokine release in the injured sciatic nerve were investigated.ResultsClinical and electrophysiological measures indicated an improvement of nerve regeneration under fingolimod treatment that is partly independent of its anti-inflammatory properties. Fingolimod treatment correlated with a significant elevation of axonal cAMP, a crucial factor for axonal outgrowth. Additionally, fingolimod significantly reduced LPA levels in the injured nerve. PF-8380 treatment correlated with improved myelin thickness. Sciatic nerve cytokine levels were not found to be significantly altered by fingolimod treatment.ConclusionsOur findings provide in vivo evidence for direct effects of fingolimod on cells of the peripheral nervous system that may propagate nerve regeneration via a dual mode of action, differentially affecting axonal outgrowth and myelination by modulating relevant aspects of S1P and LPA signaling.

Ozone-Induced Nasal Type 2 Immunity in Mice Is Dependent on Innate Lymphoid Cells.

Epidemiological studies suggest that elevated ambient concentrations of ozone are associated with activation of eosinophils in the nasal airways of atopic and nonatopic children. Mice repeatedly exposed to ozone develop eosinophilic rhinitis and type 2 immune responses. In this study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced eosinophilic rhinitis by using lymphoid-sufficient C57BL/6 mice, Rag2(-/-) mice that are devoid of T cells and B cells, and Rag2(-/-)Il2rg(-/-) mice that are depleted of all lymphoid cells including ILCs. The animals were exposed to 0 or 0.8 ppm ozone for 9 consecutive weekdays (4 h/d). Mice were killed 24 hours after exposure, and nasal tissues were selected for histopathology and gene expression analysis. ILC-sufficient C57BL/6 and Rag2(-/-) mice exposed to ozone developed marked eosinophilic rhinitis and epithelial remodeling (e.g., epithelial hyperplasia and mucous cell metaplasia). Chitinase-like proteins and alarmins (IL-33, IL-25, and thymic stromal lymphopoietin) were also increased morphometrically in the nasal epithelium of ozone-exposed C57BL/6 and Rag2(-/-) mice. Ozone exposure elicited increased expression of Il4, Il5, Il13, St2, eotaxin, MCP-2, Gob5, Arg1, Fizz1, and Ym2 mRNA in C57BL/6 and Rag2(-/-) mice. In contrast, ozone-exposed ILC-deficient Rag2(-/-)Il2rg(-/-) mice had no nasal lesions or overexpression of Th2- or ILC2-related transcripts. These results indicate that ozone-induced eosinophilic rhinitis, nasal epithelial remodeling, and type 2 immune activation are dependent on ILCs. To the best of our knowledge, this is the first study to demonstrate that ILCs play an important role in the nasal pathology induced by repeated ozone exposure.

Selective Sphingosine-1-Phosphate Receptor 1 Modulation Attenuates Experimental Intracerebral Hemorrhage.

Preclinical studies and a proof-of-concept clinical study have shown that sphingosine-1-phosphate receptor (S1PR) modulator, fingolimod, improves the clinical outcome of intracerebral hemorrhage (ICH). However, the specific subtype of the S1PRs through which immune modulation provides protection in ICH remains unclear. In addition, fingolimod-induced adverse effects could limit its use in patients with stroke because of interactions with other S1PR subtypes, particularly with S1PR3. RP101075 is a selective S1PR1 agonist with superior cardiovascular safety profile. In this study, we investigated the impact of RP101075 treatment in a mouse model of ICH. ICH was induced by injection of autologous blood in 294 male C57BL/6J and Rag2(-/-) mice. ICH mice randomly received vehicle, RP101075, or RP101075 plus S1PR1 antagonist W146 by daily oral gavage for three consecutive days, starting from 30 minutes after surgery. Neurodeficits, brain edema, brain infiltration of immune cells, blood-brain barrier integrity, and cell death were assessed after ICH. RP101075 significantly attenuated neurological deficits and reduced brain edema in ICH mice. W146 blocked the effects of RP101075 on neurodeficits and brain edema. RP101075 reduced the counts of brain-infiltrating lymphocytes, neutrophils, and microglia, as well as cytokine expression after ICH. Enhanced blood-brain barrier integrity and alleviated neuronal death were also seen in ICH mice after RP101075 treatment. S1PR1 modulation via RP101075 provides protection in experimental ICH. Together with the advantageous pharmacological features of RP101075, these results warrant further investigations of its mechanisms of action and translational values in ICH patients.

HTLV-1 infection and neuropathogenesis in the context of Rag1−/−γc−/− (RAG1) and BLT mice

Abstract HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a disabling chronic inflammatory disease of the central nervous system (CNS) with similarities to multiple sclerosis (MS). To date, the lack of a suitable small animal model has hindered our quest to understand the immuno- and neuropathogenesis of HTLV-1 in an in vivo system. Previous work from others have established that host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34+ hematopoietic stem cells and followed HTLV-1 infection. Flow cytometry and histological analyses revealed reconstitution of Rag1 and BLT mice with human immune cells, including macrophages, dendritic cells, T cells and B cells. Proviral load (PVL) was determined in the peripheral blood, spleen, and other organs of neonatal and adult Rag1 and BLT hu-mice by droplet digital PCR. Tax showed peak expression at 14 wpi in Rag1 mice with continued expression until 16 weeks. Within blood, PVL and viral protein Tax was detected as early as 2 wks post-infection (wpi) in Rag-1 and BLT hu-mice. Successful infection was followed by immune activation and Tax expression within lymphoid organs. Moreover, lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption were observed in the spinal cord of the infected mice. These data represents the first attempt to establish HTLV-1 neuropathogenesis in the context of RAG1 and BLT mice suggesting possibility of developing a small animal model of HAM/TSP in humanized mice.

Influence of phthiocerol dimycocerosate on CD4+ T cell priming and persistence during Mycobacterium tuberculosis infection

The characterisation of mycobacterial factors that influence or modulate the host immune response may aid the development of more efficacious TB vaccines. We have previously reported that Mycobacterium tuberculosis deficient in export of Phthiocerol Dimycocerosates (DIM) (MT103(ΔdrrC)) is more attenuated than wild type M.tuberculosis and provides sustained protective immunity compared to the existing BCG vaccine. Here we sought to define the correlates of immunity associated with DIM deficiency by assessing the impact of MT103(ΔdrrC) delivery on antigen presenting cell (APC) function and the generation of CD4(+) T cell antigen-specific immunity. MT103(ΔdrrC) was a potent activator of bone marrow derived dendritic cells, inducing significantly greater expression of CD86 and IL-12p40 compared to BCG or the MT103 parental strain. This translated to an increased ability to initiate early invivo priming of antigen-specific CD4(+) T cells compared to BCG with enhanced release of IFN-γ and TNF upon antigen-restimulation. The heightened immunity induced by MT103(ΔdrrC) correlated with greater persistence within the spleen compared to BCG, however both MT103(ΔdrrC) and BCG were undetectable in the lung at 70 days post-vaccination. In immunodeficient RAG (-/-) mice, MT103(ΔdrrC) was less virulent than the parental MT103 strain, yet MT103(ΔdrrC) infected mice succumbed more rapidly compared to BCG-infected animals. These results suggest that DIM translocation plays a role in APC stimulation and CD4(+) T cell activation during M.tuberculosis infection and highlights the potential of DIM-deficient strains as novel TB vaccine candidates.

Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects.

Omenn syndrome (OS) is caused by hypomorphic Rag mutations and characterized by a profound immunodeficiency associated with autoimmune-like manifestations. Both in humans and mice, OS is mediated by oligoclonal activated T and B cells. The role of microbial signals in disease pathogenesis is debated. Here, we show that Rag2(R229Q) knock-in mice developed an inflammatory bowel disease affecting both the small bowel and colon. Lymphocytes were sufficient for disease induction, as intestinal CD4 T cells with a Th1/Th17 phenotype reproduced the pathological picture when transplanted into immunocompromised hosts. Moreover, oral tolerance was impaired in Rag2(R229Q) mice, and transfer of wild-type (WT) regulatory T cells ameliorated bowel inflammation. Mucosal immunoglobulin A (IgA) deficiency in the gut resulted in enhanced absorption of microbial products and altered composition of commensal communities. The Rag2(R229Q) microbiota further contributed to the immunopathology because its transplant into WT recipients promoted Th1/Th17 immune response. Consistently, long-term dosing of broad-spectrum antibiotics (ABXs) in Rag2(R229Q) mice ameliorated intestinal and systemic autoimmunity by diminishing the frequency of mucosal and circulating gut-tropic CCR9(+) Th1 and Th17 T cells. Remarkably, serum hyper-IgE, a hallmark of the disease, was also normalized by ABX treatment. These results indicate that intestinal microbes may play a critical role in the distinctive immune dysregulation of OS.

Commensal A4 bacteria inhibit intestinal Th2-cell responses through induction of dendritic cell TGF-β production.

It has been shown that while commensal bacteria promote Th1, Th17 and Treg cells in lamina propria (LP) in steady-state conditions, they suppress mucosal Th2 cells. However, it is still unclear whether there are specific commensal organisms down-regulating Th2 responses, and the mechanism involved. Here we demonstrate that commensal A4 bacteria, a member of the Lachnospiraceae family, which produce an immunodominant microbiota CBir1 antigen, inhibits LP Th2-cell development. When transferred into the intestines of RAG(-/-) mice, CBir1-specific T cells developed predominately towards Th1 cells and Th17 cells, but to a lesser extent into Th2 cells. The addition of A4 bacterial lysates to CD4(+) T-cell cultures inhibited production of IL-4. A4 bacteria stimulated dendritic cell production of TGF-β, and blockade of TGF-β abrogated A4 bacteria inhibition of Th2-cell development in vitro and in vivo. Collectively, our data show that A4 bacteria inhibit Th2-cell differentiation by inducing dendritic cell production of TGF-β.

T-bet as a key regulator of mucosal immunity.

Initially understood to be a key regulator of interferon-γ-producing helper T cells, our knowledge of T-bet's functional roles has expanded to encompass a growing range of cellular lineages. In addition to regulating other interferon-γ-producing adaptive immune cells, it is now clear that T-bet plays a fundamental role in the regulation of innate immune responses across mucosal surfaces. This homeostatic role is demonstrated by the spontaneous colitis that occurs when T-bet is deleted from innate immune cells in RAG(-/-) mice. Using this model as a focal point, we review our understanding of T-bet's regulation of adaptive and innate immune systems, focusing particularly on mucosal populations including innate lymphoid cells, dendritic cells and intraepithelial lymphocytes. With the increasingly diverse effects of T-bet on different lineages, the classical binding-centric paradigm of T-bet's molecular functionality has increasingly struggled to account for the versatility of T-bet's biological effects. Recent recognition of the synergistic interactions between T-bet and other canonical transcription factors has led to a co-operative paradigm that has provided greater explanatory power. Synthesizing insights from ChIP-seq and comparative biology, we expand the co-operative paradigm further and suggest a network approach as a powerful way to understand and model T-bet's diverse functionality.

Smad7 Knockdown Restores Aryl Hydrocarbon Receptor-mediated Protective Signals in the Gut.

In Crohn's disease [CD], the pathological process is driven by an excessive immune response that is poorly counterbalanced by regulatory mechanisms. One such a mechanism involves aryl hydrocarbon receptor [AhR], a transcription factor that delivers protective signals in the gut. Expression of AhR is reduced in CD lamina propria mononuclear cells [LPMC] even though factors accounting for such a defect remain unknown. Since CD LPMC express elevated levels of Smad7, an inhibitor of transforming growth factor beta 1 [TGF-β1] activity, and TGF-β1 regulates AhR in other systems, we examined the link between AhR and Smad7 in the gut. AhR and interleukin [IL]-22 were evaluated in normal LPMC stimulated with TGF-β1 and 6-formylindolo[3,2-b]carbazole [Ficz], an activator of AhR, and in CD LPMC incubated with a Smad7 antisense oligonucleotide and then stimulated with Ficz and TGF-β1. AhR and IL-22 expression was evaluated in LPMC of Smad7-transgenic mice. Finally, we evaluated the protective effect of Ficz on colitis in RAG1 mice injected with naïve or Smad7-overexpressing T cells. In normal LPMC, TGF-β1 induced AhR and this event was associated with increased production of IL-22 following stimulation with Ficz. Treatment of CD LPMC with Smad7 antisense oligonucleotide enabled TGF-β1 to enhance AhR expression. Consistently, AhR expression and Ficz-induced IL-22 production were markedly reduced in T cells of Smad7-transgenic mice. In RAG1 mice, Ficz ameliorated colitis induced by wild type T cells but did not affect colitis induced by transfer of Smad7-overexpressing T cells. The inverse correlation between Smad7 and AhR expression helps to propagate inflammatory signals in the gut.

Targeting Ornithine Decarboxylase by α-Difluoromethylornithine Inhibits Tumor Growth by Impairing Myeloid-Derived Suppressor Cells.

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.

Defective CD8 Signaling Pathways Delay Rejection in Older Recipients.

CD8+ T cells play a cardinal feature in response to alloantigens and are able to generate effector/memory T cells independently from CD4+ T cells. To investigate the impact of aging on CD8 T cells, we used a fully mismatched mouse skin transplant model. Our findings showed a prolonged allograft survival in older recipients associated with a significant increase of CD4+ and CD8+ CD44high CD62Llow effector/memory T cells and a reduced systemic IFNγ production. When reconstituting young CBA Rag-1 mice that lack mature T and B cells with old CD8+ T cells expressing clonal anti-H2K T cell receptor (TCR) alloreactive for MHC I, graft survival was significantly prolonged and comparable to those receiving young CD8+ T cells. Moreover, our data showed that reduced systemic IFNγ levels observed in old recipients had been linked to a compromised expression of the IL-2R β subunit (CD122) by old CD8+ T cells. In addition, we observed an impaired IFNγ production on IL-2 receptor activation. At the same time, gene profiling analysis of old CD8 T cells demonstrated reduced chemokine ligand-3 and CD40L expression that resulted in compromised CD8+ T cell/dendritic cell communication, leading to impaired migratory and phagocytic activity of CD11c cells.Collectively, our study demonstrated that aging delays allograft rejection. CD8 T cells play a critical role in this process linked to a compromised production of IFNγ, in addition to a defective IL-2 receptor signaling machinery and a defective communication between CD8 T cells and dendritic cells.

IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo

Biogenesis of the immune synapse at the interface between antigen-presenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4(+) T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells. We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4(+) T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag(-/-) mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo.

IRAK1 Drives Intestinal Inflammation by Promoting the Generation of Effector Th Cells with Optimal Gut-Homing Capacity.

IL-1R-associated kinase (IRAK) 1 is an important component of the IL-1R and TLR signaling pathways, which influence Th cell differentiation. In this study, we show that IRAK1 promotes Th17 development by mediating IL-1β-induced upregulation of IL-23R and subsequent STAT3 phosphorylation, thus enabling sustained IL-17 production. Moreover, we show that IRAK1 signaling fosters Th1 differentiation by mediating T-bet induction and counteracts regulatory T cell generation. Cotransfer experiments revealed that Irak1-deficient CD4(+) T cells have a cell-intrinsic defect in generating Th1 and Th17 cells under inflammatory conditions in spleen, mesenteric lymph nodes, and colon tissue. Furthermore, IRAK1 expression in T cells was shown to be essential for T cell accumulation in the inflamed intestine and mesenteric lymph nodes. Transcriptome analysis ex vivo revealed that IRAK1 promotes T cell activation and induction of gut-homing molecules in a cell-intrinsic manner. Accordingly, Irak1-deficient T cells failed to upregulate surface expression of α4β7 integrin after transfer into Rag1(-/-) mice, and their ability to induce colitis was greatly impaired. Lack of IRAK1 in recipient mice provided additional protection from colitis. Therefore, IRAK1 plays an important role in intestinal inflammation by mediating T cell activation, differentiation, and accumulation in the gut. Thus, IRAK1 is a promising novel target for therapy of inflammatory bowel diseases.

Abstract B28: Dianhydrogalactitol inhibits the growth of glioblastoma multiforme stem and non-stem cultures, in vitro and in vivo

Abstract Glioblastoma multiforme (GBM) is the most common malignant brain cancer in adults and it presents some of the greatest challenges in the management of cancer patients worldwide. The standard of care for GBM patients is surgical resection followed by temozolomide (TMZ) and irradiation (XRT), but the prognosis for GBM patients remains poor, quality of life is low, and median survival for patients with recurrent GBM is &amp;lt;6 months. TMZ-resistance has emerged as a significant unmet medical need, as DNA repair enzyme 0-6-methylguanine DNA methyltransferase (MGMT) removes the TMZ-mediated methyl-group adducts at the O6 position of guanine. Dianhydrogalactitol (VAL-083) is a structurally unique bi-functional DNA alkylating agent that causes DNA crosslinks at the N7 position of guanine. VAL-083 crosses the blood brain barrier and accumulates in brain tumor tissue, and previous clinical trials suggest that VAL-083 has activity against a range of tumors, including GBM. Because VAL-083's N7 adducts are not subject to MGMT-mediated repair, it may be an effective chemotherapeutic in the treatment of TMZ-resistant GBM. We have recently shown that TMZ activity is similar in cancer stem cells (CSC) and their paired non-CSC from primary GBM tissues, and that the activity is MGMT-dependent. We thus sought to investigate how our CSC and non-CSC panel would respond to VAL-083 alone or in combination with XRT. We further investigated the activity of VAL-083 in in vivo models of drug-resistant GBM in comparison to TMZ. Rag2 mice bearing intracranial human GBM xenograft tumors of either MGMT-positive and TMZ-resistant origin (BT74), or MGMT-negative and TMZ-sensitive origin (U251) were treated. VAL-083 was given i.p. 3 times/week x 3 weeks, and the efficacy of VAL-083 in controlling tumor growth compared to TMZ (30 mg/kg). Disease progression was evaluated by overall survival, clinical observations and body weight measurements. Our in vitro results show that VAL-083 is a potent inhibitor of all tested primary GBM cultures, irrespective of MGMT status. VAL-083 causes cell cycle arrest and loss of cell viability in TMZ-resistant cells, and at lower concentrations than TMZ in TMZ-sensitive cells. Furthermore, VAL-083 is not affected by cell culture condition (Stem vs. Non-Stem). Low dose VAL-083 combined with XRT exhibited an additive effect in all cultures tested, suggesting that VAL-083 might act as a radiosensitizer. In the in vivo U251 model, the median survival time for mice treated with 4 mg/kg VAL-083 was significantly increased to 72 days compared to 48 days for controls (p&amp;lt;0.0001). Median survival time for 3 mg/kg VAL-083 was 54 days. Body weight loss was observed in mice treated with 5 mg/kg and treatment was stopped after 4 doses after which the animals recovered and their median survival was 57 days. Animals treated with TMZ were terminated at day 102 at the end of the study. BT74: study is ongoing, data will be presented at the meeting. In conclusion, VAL-083 is highly efficacious against both stem and non-stem GBM cell cultures in vitro, the activity is independent of MGMT and VAL-083 appears to act as a radiosensitizer in GBM. In vivo xenograft GBM models further validate the benefits of VAL-083 in the treatment of GBM and support ongoing clinical research with VAL-083, which is currently in a clinical trial for GBM patients with recurrent disease. Citation Format: Shaun D. Fouse, Anne Steino, Nicholas Butowski, Jeffrey A. Bacha, Sarath Kanekal, Nancy Dos Santos, Joseph F. Costello, Dennis M. Brown. Dianhydrogalactitol inhibits the growth of glioblastoma multiforme stem and non-stem cultures, in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B28.