ABSTRACT
Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Neoplasms, Experimental/immunology , Proto-Oncogene Proteins c-myb/immunology , Stem Cells/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , Cell Differentiation/immunology , Cell Line, Tumor , HEK293 Cells , Humans , Immunologic Memory/genetics , Lymphocytic Choriomeningitis/immunology , Lymphocytic Choriomeningitis/metabolism , Lymphocytic Choriomeningitis/virology , Lymphocytic choriomeningitis virus/immunology , Lymphocytic choriomeningitis virus/physiology , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/virology , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/metabolism , Stem Cells/metabolism , Stem Cells/virology , T Cell Transcription Factor 1/genetics , T Cell Transcription Factor 1/immunology , T Cell Transcription Factor 1/metabolismABSTRACT
OBJECTIVE: Hepatocellular carcinoma (HCC) represents a typical inflammation-associated cancer. Tissue resident innate lymphoid cells (ILCs) have been suggested to control tumour surveillance. Here, we studied how the local cytokine milieu controls ILCs in HCC. DESIGN: We performed bulk RNA sequencing of HCC tissue as well as flow cytometry and single-cell RNA sequencing of enriched ILCs from non-tumour liver, margin and tumour core derived from 48 patients with HCC. Simultaneous measurement of protein and RNA expression at the single-cell level (AbSeq) identified precise signatures of ILC subgroups. In vitro culturing of ILCs was used to validate findings from in silico analysis. Analysis of RNA-sequencing data from large HCC cohorts allowed stratification and survival analysis based on transcriptomic signatures. RESULTS: RNA sequencing of tumour, non-tumour and margin identified tumour-dependent gradients, which were associated with poor survival and control of ILC plasticity. Single-cell RNA sequencing and flow cytometry of ILCs from HCC livers identified natural killer (NK)-like cells in the non-tumour tissue, losing their cytotoxic profile as they transitioned into tumour ILC1 and NK-like-ILC3 cells. Tumour ILC composition was mediated by cytokine gradients that directed ILC plasticity towards activated tumour ILC2s. This was liver-specific and not seen in ILCs from peripheral blood mononuclear cells. Patients with high ILC2/ILC1 ratio expressed interleukin-33 in the tumour that promoted ILC2 generation, which was associated with better survival. CONCLUSION: Our results suggest that the tumour cytokine milieu controls ILC composition and HCC outcome. Specific changes of cytokines modify ILC composition in the tumour by inducing plasticity and alter ILC function.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Carcinoma, Hepatocellular/metabolism , Cytokines/metabolism , Humans , Immunity, Innate , Killer Cells, Natural/metabolism , Leukocytes, Mononuclear , Liver Neoplasms/metabolism , Lymphocytes , RNA/metabolism , Tumor MicroenvironmentABSTRACT
Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4(+) but not CD8(+) T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4(+) T lymphocytes have greater mitochondrial mass than CD8(+) T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4(+) T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4(+) T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.
Subject(s)
CD4-Positive T-Lymphocytes/pathology , Carcinogenesis , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Non-alcoholic Fatty Liver Disease/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Carcinogenesis/immunology , Carcinogenesis/pathology , Carcinoma, Hepatocellular/metabolism , Case-Control Studies , Choline/metabolism , Diet , Disease Models, Animal , Genes, myc , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Linoleic Acid/metabolism , Lipid Metabolism , Liver/immunology , Liver/pathology , Liver Neoplasms/metabolism , Male , Methionine/deficiency , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/pathology , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Oxidative Stress , Reactive Oxygen Species/metabolismABSTRACT
Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (nâ¯=â¯2) or with unknown underlying genetic defect (nâ¯=â¯3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.
Subject(s)
Bone Marrow Transplantation , Busulfan/administration & dosage , Cyclophosphamide/administration & dosage , Graft vs Host Disease , Pentostatin/administration & dosage , Transplantation Conditioning , Adolescent , Adult , Busulfan/adverse effects , Child , Child, Preschool , Cyclophosphamide/adverse effects , Disease-Free Survival , Female , Follow-Up Studies , Graft vs Host Disease/mortality , Graft vs Host Disease/prevention & control , Humans , Lymphocyte Transfusion , Male , Middle Aged , Pentostatin/adverse effects , Primary Immunodeficiency Diseases/mortality , Primary Immunodeficiency Diseases/therapy , Prospective Studies , Survival RateABSTRACT
Although chronic graft-versus-host disease (CGVHD) is the primary nonrelapse complication of allogeneic transplantation, understanding of its pathogenesis is limited. To identify the main operant pathways across the spectrum of CGVHD, we analyzed gene expression in circulating monocytes, chosen as in situ systemic reporter cells. Microarrays identified two interrelated pathways: 1) IFN-inducible genes, and 2) innate receptors for cellular damage. Corroborating these with multiplex RNA quantitation, we found that multiple IFN-inducible genes (affecting lymphocyte trafficking, differentiation, and Ag presentation) were concurrently upregulated in CGVHD monocytes compared with normal subjects and non-CGVHD control patients. IFN-inducible chemokines were elevated in both lichenoid and sclerotic CGHVD plasma and were linked to CXCR3+ lymphocyte trafficking. Furthermore, the levels of the IFN-inducible genes CXCL10 and TNFSF13B (BAFF) were correlated at both the gene and the plasma levels, implicating IFN induction as a factor in elevated BAFF levels in CGVHD. In the second pathway, damage-/pathogen-associated molecular pattern receptor genes capable of inducing type I IFN were upregulated. Type I IFN-inducible MxA was expressed in proportion to CGVHD activity in skin, mucosa, and glands, and expression of TLR7 and DDX58 receptor genes correlated with upregulation of type I IFN-inducible genes in monocytes. Finally, in serial analyses after transplant, IFN-inducible and damage-response genes were upregulated in monocytes at CGVHD onset and declined upon therapy and resolution in both lichenoid and sclerotic CGVHD patients. This interlocking analysis of IFN-inducible genes, plasma analytes, and tissue immunohistochemistry strongly supports a unifying hypothesis of induction of IFN by innate response to cellular damage as a mechanism for initiation and persistence of CGVHD.
Subject(s)
Graft vs Host Disease/immunology , Interferons/metabolism , Monocytes/physiology , Adult , Antigen Presentation , B-Cell Activating Factor/metabolism , Cell Differentiation , Cell Movement/genetics , Chemokine CXCL10/metabolism , Chronic Disease , DEAD Box Protein 58/metabolism , Female , Humans , Immunity, Innate , Male , Middle Aged , Receptors, CXCR3/metabolism , Receptors, Immunologic , Receptors, Pattern Recognition/metabolism , Signal Transduction , Toll-Like Receptor 7/metabolism , Transplantation, Homologous , Young AdultABSTRACT
IL-7 is essential for T-cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL-7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T-cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL-7. Moreover, RTEs express low levels of IL-7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL-7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL-7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL-7-induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL-7 signaling in RTEs preferentially upregulated expression of Bcl-2, which is anti-apoptotic but also anti-proliferative. In contrast, naïve T cells showed diminished Bcl-2 induction but greater proliferative response to IL-7. Collectively, these data indicate that IL-7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.
Subject(s)
Homeostasis , Interleukin-7/metabolism , Signal Transduction , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , Animals , Apoptosis/genetics , Apoptosis/immunology , Cell Movement/immunology , Cell Proliferation , Cell Survival/genetics , Cell Survival/immunology , DNA-Binding Proteins/deficiency , Immunophenotyping , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Interleukin-7/metabolismABSTRACT
Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Cell Line, Tumor , Cytokines/biosynthesis , Forkhead Transcription Factors/metabolism , Gene Deletion , Gene Targeting , Interferon-gamma/biosynthesis , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40(-/-) tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.
Subject(s)
CD40 Antigens/metabolism , Hepatitis/immunology , Myeloid Cells/immunology , Adoptive Transfer , Alanine Transaminase/blood , Animals , Antigens, CD1d/biosynthesis , Arginase/antagonists & inhibitors , Arginase/biosynthesis , Arginase/metabolism , Aspartate Aminotransferases/blood , B7-1 Antigen/biosynthesis , B7-2 Antigen/biosynthesis , CD11b Antigen/metabolism , CD40 Antigens/biosynthesis , CD40 Antigens/genetics , Cell Line , Concanavalin A/pharmacology , Female , Galactosylceramides/pharmacology , Hepatitis/genetics , Hepatocytes/immunology , Hepatocytes/pathology , Liver/cytology , Liver/injuries , Liver Neoplasms/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mitogens/pharmacology , Myeloid Cells/transplantation , Reactive Oxygen Species/metabolism , Receptors, Chemokine/metabolismABSTRACT
Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b(+)F4/80(hi)CD206(hi) and CD11b(+)F4/80(hi)CD124(hi)) macrophages and CD4(+)Foxp3(+) regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.
Subject(s)
Cancer Vaccines/immunology , Neoplasm Recurrence, Local/immunology , Neoplasms/immunology , Tumor Microenvironment/immunology , Animals , Antigens, Differentiation/immunology , Antigens, Differentiation/metabolism , CD11b Antigen/immunology , CD11b Antigen/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Disease Models, Animal , Female , Flow Cytometry , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Humans , Interleukin-4 Receptor alpha Subunit/immunology , Interleukin-4 Receptor alpha Subunit/metabolism , Kaplan-Meier Estimate , Lectins, C-Type/immunology , Lectins, C-Type/metabolism , Macrophages/immunology , Macrophages/metabolism , Male , Mannose Receptor , Mannose-Binding Lectins/immunology , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred C57BL , Neoplasms/pathology , Neoplasms/surgery , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Treatment Failure , Vaccination/methodsABSTRACT
Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b(+) Gr-1(high) granulocytic MDSCs. Coculture of CD11b(+) Gr-1(high) granulocytic MDSCs with antigen-stimulated T cells and simultaneous blockade of IFN-γ by the use of anti-IFN-γ blocking antibody, IFN-γ(-/-) effector T cells, IFN-γR(-/-) MDSCs or STAT1(-/-) MDSCs led to upregulation of Bcl2a1 in CD11b(+) Gr-1(high) cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM-CSF released by antigen-stimulated CD8(+) T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN-γ by a direct interaction of phosphorylated STAT-1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN-γ/ STAT1-dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.
Subject(s)
Apoptosis/immunology , CD11b Antigen/immunology , Interferon-gamma/immunology , Myeloid Cells/immunology , Proto-Oncogene Proteins c-bcl-2/immunology , Receptors, Chemokine/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line , Female , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Minor Histocompatibility Antigens , STAT1 Transcription Factor/immunology , Up-Regulation/immunologyABSTRACT
BACKGROUND: Gene therapy is currently under investigation as a means of managing a variety of pulmonary diseases. Unfortunately, gene transfer to bronchial epithelium has been hampered by the lack of stable and efficient transduction. Recent studies have shown that gene vectors could be tethered to the metallic surfaces of intra-arterial stents. This approach enables efficacious and site-specific adenoviral gene delivery to the vascular endothelium. OBJECTIVES: We hypothesized that airway mesh stents impregnated with viral gene vectors could be used for local gene delivery to benign and malignant bronchial epithelium. METHODS: Serotype 5 adenoviral vectors (Ad5, E1-/E3-) containing the reporter genes green fluorescent protein (Ad.GFP) or ß-galactoside/LacZ (Ad.LacZ), or a therapeutic gene, Ad.INF-ß, were coupled to either metallic mesh disks or stents via anti-Ad knob antibodies. These platforms were assessed for their ability to transfect bronchial epithelial cells from both rats and humans, as well as murine (L1C2) and human (A549) lung cancer cell lines. Gene transfer was quantified by fluorescent microscopy, scanning fluorimetry for Ad.GFP, and light microscopy studies assessing ß-galactosidase staining for Ad.LacZ. Metallic mesh and stent-mediated gene transfer was also performed in a murine flank tumor model and in a rat endotracheal tumor model in order to evaluate the therapeutic potential. RESULTS: In these studies, murine and human non-small cell lung cancer (NSCLC) cells were successfully transfected with reporter genes in vitro. Ad.LacZ-complexed mesh successfully transfected reporter genes into established murine flank NSCLC tumors. In addition, Ad.LacZ-tethered stents could effectively transfect both tracheobronchial epithelium and submucosal glands in rats. Similar epithelial transfection was achieved in ex vivo human bronchial epithelium. Pilot in vivo experimentation provided data supporting the concept that therapeutic genes could also be delivered with this technology. In additional pilot in vivo experiments, the growth of murine flank tumors was inhibited by placement of mesh disks coupled with Ad.muINF-ß, and rats bearing endotracheal tumors demonstrated a trend towards prolonged survival with insertion of Ad.ratINF-ß-tethered stents. CONCLUSIONS: Stent-mediated gene delivery successfully enabled site-specific vector administration to target rat and human airway cells in cell culture, organ culture and in vivo. Local tracheobronchial gene delivery via stents could provide a viable clinical solution for overcoming the difficulties encountered with vector delivery within the lungs, in particular by lowering requisite vector titers and by directing desired vectors to areas of interest. This strategy may prove valuable for treating tumors involving the tracheobronchial tree, as well as other nonmalignant tracheobronchial disorders.
Subject(s)
Bronchial Neoplasms/therapy , Carcinoma, Non-Small-Cell Lung/therapy , Gene Transfer Techniques/instrumentation , Respiratory Mucosa/pathology , Stents , Transgenes , Animals , Bronchial Neoplasms/genetics , Bronchial Neoplasms/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Galactosides/genetics , Genetic Vectors , Green Fluorescent Proteins/genetics , Humans , Interferon-beta/genetics , RatsABSTRACT
BACKGROUND & AIMS: Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease. METHODS: The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages. RESULTS: An accumulation of MDSC was found in mice with HCC irrespective of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL6, IL1ß) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF overexpression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib. CONCLUSIONS: Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.
Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Movement , Cell Proliferation , Disease Models, Animal , Liver Neoplasms/pathology , Myeloid Cells/pathology , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Diethylnitrosamine/adverse effects , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Heterografts , Humans , Liver Neoplasms/chemically induced , Liver Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Myeloid Cells/drug effects , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Phenylurea Compounds/pharmacology , Proto-Oncogene Proteins c-myc/metabolism , SorafenibABSTRACT
A rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g., dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multiparameter fluorescence activated cell sorting. Cell lysis, DNA extraction, and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid online hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25-20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25-20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20 000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and nonrelapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g., T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple sclerosis.
Subject(s)
DNA/chemistry , Deoxyadenosines/chemistry , Deuterium/chemistry , Animals , Cell Proliferation , Chromatography, Gas , Flow Cytometry , Isotope Labeling , Mice , Software , Tandem Mass SpectrometryABSTRACT
IL-7 signaling is required for thymocyte development and its loss has a severe deleterious effect on thymus function. Thymocyte-stromal cell interactions and other mechanisms tightly regulate IL-7 expression. We show that disruption of that regulation by over-expression of IL-7 inhibits T-cell development and promotes extensive B-cell lymphopoiesis in the thymus. Our data reveal that high levels of IL-7 negate Notch-1 function in thymocytes found in IL-7 transgenic mice and in co-culture with OP9-DL1 cells. While high levels of IL-7R are present on thymocytes, increased suppressor of cytokine signaling-1 expression blunts IL-7 downstream signaling, resulting in hypo-phosphorylation of proteins in the PI3K-Akt pathway. Consequently, GSK3ß remains active and inhibits Notch-1 signaling as observed by decreased Hes-1 and Deltex expression in thymic progenitors. This is the first demonstration that high levels of IL-7 antagonize Notch-1 signaling and suggest that IL-7 may affect T- versus B-lineage choice in the thymus.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation , Interleukin-7/immunology , Lymphopoiesis , Receptor, Notch1/metabolism , T-Lymphocytes/cytology , Thymocytes/cytology , Animals , Coculture Techniques , Interleukin-7/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction , Stromal Cells/cytology , Stromal Cells/immunology , Suppressor of Cytokine Signaling Proteins/immunology , Thymus Gland/cytology , Thymus Gland/growth & development , Thymus Gland/immunology , Thymus Gland/metabolismABSTRACT
Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity.
Subject(s)
Antigens, CD/immunology , Dendritic Cells/immunology , Immune Tolerance/immunology , T-Lymphocytes, Regulatory/immunology , Animals , B7-H1 Antigen , Cells, Cultured , Female , Flow Cytometry , Graft vs Host Disease/immunology , Humans , Lymphocyte Activation/immunology , Mice , Signal Transduction/immunologyABSTRACT
Since previous work using a nonreplicating adenovirus-expressing mouse interferon-ß (Ad.mIFNß) showed promising preclinical activity, we postulated that a vector-expressing IFNß at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNß (VV.mIFNß) was tested. VV.mIFNß-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNß had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNß vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.
Subject(s)
Immunotherapy/methods , Interferon-beta/metabolism , Vaccinia virus/genetics , Animals , Cell Line, Tumor , Female , Interferon-beta/genetics , Lung Neoplasms/therapy , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Vaccinia virus/immunology , Virus Replication/genetics , Virus Replication/physiologyABSTRACT
CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Interleukin-2/therapeutic use , Lymphopenia/drug therapy , Receptors, Interleukin-2/immunology , T-Lymphocytes, Regulatory/drug effects , Adolescent , Adult , Animals , CD4-Positive T-Lymphocytes/immunology , Child , Female , Forkhead Transcription Factors/analysis , Homeostasis/immunology , Humans , Interleukin-2/administration & dosage , Interleukin-2/immunology , Lymphocyte Transfusion , Lymphopenia/chemically induced , Lymphopenia/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Receptors, Interleukin-2/metabolism , Recombinant Proteins/therapeutic use , Sarcoma/complications , Sarcoma/drug therapy , T-Lymphocytes, Regulatory/immunologyABSTRACT
The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non-small-cell lung cancer (NSCLC). Anti-murine-CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8(+) T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.
Subject(s)
Chemokine CCL2/antagonists & inhibitors , Lung Neoplasms/therapy , Macrophages/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antibodies, Monoclonal/pharmacology , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/secondary , Carcinoma, Non-Small-Cell Lung/therapy , Cell Line, Tumor , Chemokine CCL2/immunology , Female , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Lymphocyte Activation , Lymphocyte Depletion , Mesothelioma/immunology , Mesothelioma/pathology , Mesothelioma/therapy , Mice , Mice, 129 Strain , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , PhenotypeABSTRACT
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-ß and subsequent high-level induction of IFN-ß-dependent proteins, such as myxovirus resistance 1 (Mx1) and 2',5'-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-ß system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and "first responders" in the early stages of viral pandemics or bioterror attacks.
Subject(s)
Respiratory Tract Infections/prevention & control , Respiratory Tract Infections/virology , Virus Diseases/prevention & control , Animals , Antineoplastic Agents/pharmacology , Bronchi/virology , Epithelial Cells/virology , Female , Humans , Immune System , Immunity, Innate , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Respiratory Tract Infections/immunology , Virus Diseases/immunology , Xanthones/pharmacologyABSTRACT
The most widely used approach to cancer immunotherapy is vaccines. Unfortunately, the need for multiple administrations of antigens often limits the use of one of the most effective vaccine approaches, immunogene therapy using viral vectors, because neutralizing antibodies are rapidly produced. We hypothesized that after viral immunogene therapy "primed" an initial strong antitumor immune response, subsequent "boosts" could be provided by sequential courses of chemotherapy. Three adenoviral (Ad)-based immunogene therapy regimens were administered to animals with large malignant mesothelioma and lung cancer tumors followed by three weekly administrations of a drug regimen commonly used to treat these tumors (Cisplatin/Gemcitabine). Immunogene therapy followed by chemotherapy resulted in markedly increased antitumor efficacy associated with increased numbers of antigen-specific, activated CD8(+) T-cells systemically and within the tumors. Possible mechanisms included: (i) decreases in immunosuppressive cells such as myeloid-derived suppressor cells (MDSC), T-regulatory cells (T-regs), and B-cells, (ii) stimulation of memory cells by intratumoral antigen release leading to efficient cross-priming, (iii) alteration of the tumor microenvironment with production of "danger signals" and immunostimulatory cytokines, and (iv) augmented trafficking of T-cells into the tumors. This approach is currently being tested in a clinical trial and could be applied to other trials of viral immunogene therapy.