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1.
Nat Immunol ; 17(4): 433-40, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26901150

ABSTRACT

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.


Subject(s)
Apoptosis/genetics , Autoimmunity/genetics , B-Lymphocytes/immunology , Immune Tolerance/genetics , MicroRNAs/genetics , Animals , Apoptosis/immunology , Apoptosis Regulatory Proteins/metabolism , Autoimmunity/immunology , Bcl-2-Like Protein 11 , Bone Marrow Transplantation , Cell Cycle Proteins/metabolism , Cell Proliferation/genetics , Disease Models, Animal , HEK293 Cells , Humans , Immune Tolerance/immunology , Immunoblotting , Lupus Erythematosus, Systemic/immunology , Membrane Proteins/metabolism , Mice , Mice, Inbred MRL lpr , MicroRNAs/immunology , Nuclear Proteins/metabolism , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA
2.
Immunity ; 44(6): 1284-98, 2016 06 21.
Article in English | MEDLINE | ID: mdl-27332731

ABSTRACT

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.


Subject(s)
DEAD-box RNA Helicases/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Forkhead Box Protein O1/metabolism , MicroRNAs/genetics , Multiple Sclerosis/immunology , Ribonuclease III/metabolism , Th17 Cells/physiology , Animals , Cells, Cultured , DEAD-box RNA Helicases/genetics , Forkhead Box Protein O1/genetics , Humans , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Interleukin-1 Type I/metabolism , Ribonuclease III/genetics , STAT3 Transcription Factor/metabolism
3.
Semin Cancer Biol ; 64: 102-107, 2020 08.
Article in English | MEDLINE | ID: mdl-32522353

ABSTRACT

Since the discovery of the first microRNA (miRNA) in 1993, thousands of miRNAs have been identified in humans and mice and many of them have been shown to control a large variety of cellular processes in different cell types including those composing the immune system. MicroRNAs regulate virtually all aspects of immune cell development, differentiation and function. Studies have shown that these molecules are involved in the maintenance of lymphocyte tolerance and, when dysregulated, promote the development of autoimmune diseases. In this review, we focus on the current knowledge about the roles of miRNAs in B cell tolerance and their contribution to autoimmunity, highlighting additional roles for some of these miRNAs in T cell tolerance. Finally, we will comment on miRNAs that promote both autoimmunity and lymphoma.


Subject(s)
Autoimmune Diseases/immunology , B-Lymphocytes/immunology , Immune Tolerance/immunology , MicroRNAs/genetics , Neoplasms/immunology , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/pathology , B-Lymphocytes/metabolism , Humans , Immune Tolerance/genetics , Neoplasms/genetics , Neoplasms/pathology
4.
PLoS Genet ; 13(2): e1006623, 2017 02.
Article in English | MEDLINE | ID: mdl-28241004

ABSTRACT

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.


Subject(s)
B-Lymphocytes/metabolism , Gene Expression Regulation , MicroRNAs/genetics , Protein Biosynthesis/genetics , Transcriptome/genetics , 5' Untranslated Regions/genetics , Animals , B-Lymphocytes/cytology , Base Sequence , Bcl-2-Like Protein 11/genetics , Bcl-2-Like Protein 11/metabolism , Cells, Cultured , Flow Cytometry , Gene Expression Profiling/methods , Immunoblotting , Mice, Knockout , Mice, Transgenic , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribosomes/genetics , Ribosomes/metabolism
5.
Front Immunol ; 13: 861655, 2022.
Article in English | MEDLINE | ID: mdl-35634349

ABSTRACT

A microRNA (miRNA) often regulates the expression of hundreds of target genes. A fundamental question in the field of miRNA research is whether a miRNA exerts its biological function through regulating a small number of key targets or through small changes in the expression of hundreds of target genes. We addressed this issue by performing functional analysis of target genes regulated by miR-148a. We previously identified miR-148a as a critical regulator of B cell central tolerance and found 119 target genes that may mediate its function. We selected 4 of them for validation and demonstrated a regulatory role for Bim, Pten, and Gadd45a in this process. In this study, we performed functional analysis of the other miR-148a target genes in in vitro and in vivo models of B cell central tolerance. Our results show that those additional target genes play a minimal role, if any, in miR-148a-mediated control of B cell central tolerance, suggesting that the function of miRNAs is mediated by a few key target genes. These findings have advanced our understanding of molecular mechanisms underlying miRNA regulation of gene expression and B cell central tolerance.


Subject(s)
Central Tolerance , MicroRNAs , B-Lymphocytes/metabolism , Cell Line, Tumor , MicroRNAs/genetics , MicroRNAs/metabolism
6.
Cancers (Basel) ; 13(22)2021 Nov 12.
Article in English | MEDLINE | ID: mdl-34830805

ABSTRACT

Cancer immunology research has mainly focused on the role of protein-coding genes in regulating immune responses to tumors. However, despite more than 70% of the human genome is transcribed, less than 2% encodes proteins. Many non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), have been identified as critical regulators of immune cell development and function, suggesting that they might play important roles in orchestrating immune responses against tumors. In this review, we summarize the scientific advances on the role of ncRNAs in regulating adaptive tumor immunity, and discuss their potential therapeutic value in the context of cancer immunotherapy.

7.
Front Immunol ; 12: 722320, 2021.
Article in English | MEDLINE | ID: mdl-34950130

ABSTRACT

The inhibition of anabolic pathways, such as aerobic glycolysis, is a metabolic cornerstone of memory T cell differentiation and function. However, the signals that hamper these anabolic pathways are not completely known. Recent evidence pinpoints the chemokine receptor CCR5 as an important player in CD4+ T cell memory responses by regulating T cell antigen receptor (TCR) nanoclustering in an antigen-independent manner. This paper reports that CCR5 specifically restrains aerobic glycolysis in memory-like CD4+ T cells, but not in effector CD4+ T cells. CCR5-deficient memory CD4+ T cells thus show an abnormally high glycolytic/oxidative metabolism ratio. No CCR5-dependent change in glucose uptake nor in the expression of the main glucose transporters was detected in any of the examined cell types, although CCR5-deficient memory cells did show increased expression of the hexokinase 2 and pyruvate kinase M2 isoforms, plus the concomitant downregulation of Bcl-6, a transcriptional repressor of these key glycolytic enzymes. Further, the TCR nanoclustering defects observed in CCR5-deficient antigen-experienced CD4+ T cells were partially reversed by incubation with 2-deoxyglucose (2-DG), suggesting a link between inhibition of the glycolytic pathway and TCR nanoscopic organization. Indeed, the treatment of CCR5-deficient lymphoblasts with 2-DG enhanced IL-2 production after antigen re-stimulation. These results identify CCR5 as an important regulator of the metabolic fitness of memory CD4+ T cells, and reveal an unexpected link between T cell metabolism and TCR organization with potential influence on the response of memory T cells upon antigen re-encounter.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Immunologic Memory , Receptors, Antigen, T-Cell/metabolism , Receptors, CCR5/physiology , Animals , Antigens/immunology , Cells, Cultured , Gene Expression Regulation , Glucose/metabolism , Glycolysis/genetics , Ligands , Mice , Mice, Transgenic , Mitochondria/metabolism , Nanostructures , Ovalbumin/immunology , Oxygen Consumption , Peptide Fragments/immunology , Proto-Oncogene Proteins c-bcl-6/biosynthesis , Proto-Oncogene Proteins c-bcl-6/genetics , Receptors, CCR5/deficiency , Specific Pathogen-Free Organisms
8.
J Immunother Cancer ; 8(1)2020 06.
Article in English | MEDLINE | ID: mdl-32591431

ABSTRACT

BACKGROUND: Tumor-infiltrating lymphocytes (TILs), mainly CD8+ cytotoxic T lymphocytes (CTL), are linked to immune-mediated control of human cancers and response to immunotherapy. Tumors have nonetheless developed specific mechanisms that selectively restrict T cell entry into the tumor microenvironment. The extracellular superoxide dismutase (SOD3) is an anti-oxidant enzyme usually downregulated in tumors. We hypothesize that upregulation of SOD3 in the tumor microenvironment might be a mechanism to boost T cell infiltration by normalizing the tumor-associated endothelium. RESULTS: Here we show that SOD3 overexpression in endothelial cells increased in vitro transmigration of naïve and activated CD4+ and CD8+ T cells, but not of myeloid cells. Perivascular expression of SOD3 also specifically increased CD4+ and CD8+ effector T cell infiltration into tumors and improved the effectiveness of adoptively transferred tumor-specific CD8+ T cells. SOD3-induced enhanced transmigration in vitro and tumor infiltration in vivo were not associated to upregulation of T cell chemokines such as CXCL9 or CXCL10, nor to changes in the levels of endothelial adhesion receptors such as intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). Instead, SOD3 enhanced T cell infiltration via HIF-2α-dependent induction of specific WNT ligands in endothelial cells; this led to WNT signaling pathway activation in the endothelium, FOXM1 stabilization, and transcriptional induction of laminin-α4 (LAMA4), an endothelial basement membrane component permissive for T cell infiltration. In patients with stage II colorectal cancer, SOD3 was associated with increased CD8+ TIL density and disease-free survival. SOD3 expression was also linked to a T cell-inflamed gene signature using the COAD cohort from The Cancer Genome Atlas program. CONCLUSION: Our findings suggest that SOD3-induced upregulation of LAMA4 in endothelial cells boosts selective tumor infiltration by T lymphocytes, thus transforming immunologically "cold" into "hot" tumors. High SOD3 levels are associated with human colon cancer infiltration by CD8+ T cells, with potential consequences for the clinical outcome of these patients. Our results also uncover a cell type-specific, distinct activity of the WNT pathway for the regulation of T cell infiltration into tumors.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , CD8-Positive T-Lymphocytes/immunology , Endothelial Cells/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms/immunology , Superoxide Dismutase/metabolism , Superoxide Dismutase/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Female , Humans , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/metabolism , Neoplasms/pathology , Signal Transduction , Superoxide Dismutase/genetics , Tumor Cells, Cultured , Tumor Microenvironment
9.
Nat Commun ; 11(1): 5850, 2020 11 17.
Article in English | MEDLINE | ID: mdl-33203876

ABSTRACT

HIV broadly neutralizing antibodies (bnAbs) can suppress viremia and protect against HIV infection. However, their elicitation is made difficult by low frequencies of appropriate precursor B cell receptors and the complex maturation pathways required to generate bnAbs from these precursors. Antibody genes can be engineered into B cells for expression as both a functional antigen receptor on cell surfaces and as secreted antibody. Here, we show that HIV bnAb-engineered primary mouse B cells can be adoptively transferred and vaccinated in immunocompetent mice resulting in the expansion of durable bnAb memory and long-lived plasma cells. Somatic hypermutation after immunization indicates that engineered cells have the capacity to respond to an evolving pathogen. These results encourage further exploration of engineered B cell vaccines as a strategy for durable elicitation of HIV bnAbs to protect against infection and as a contributor to a functional HIV cure.


Subject(s)
AIDS Vaccines/immunology , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , B-Lymphocytes/physiology , B-Lymphocytes/transplantation , Broadly Neutralizing Antibodies/blood , Broadly Neutralizing Antibodies/genetics , Female , Genetic Engineering/methods , HEK293 Cells , HIV Antibodies/blood , HIV Antibodies/genetics , HIV Antibodies/immunology , HIV Infections , Humans , Immunization , Immunologic Memory/genetics , Lymphocyte Activation , Mice, Inbred C57BL , Somatic Hypermutation, Immunoglobulin
11.
Elife ; 82019 01 17.
Article in English | MEDLINE | ID: mdl-30648968

ABSTRACT

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.


Subject(s)
Antigen-Antibody Reactions/genetics , B-Lymphocytes/immunology , Gene Editing/methods , Antibodies, Neutralizing/immunology , Antibody Specificity , CRISPR-Cas Systems , Cell Line , Cytidine Deaminase/metabolism , HIV Antibodies/immunology , Humans , Immunoglobulin Heavy Chains/genetics , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology
12.
Oncogene ; 24(43): 6533-44, 2005 Sep 29.
Article in English | MEDLINE | ID: mdl-16007183

ABSTRACT

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3) has antitumor activity in addition to its classical action on calcium metabolism and bone tissue biology. It is thought to regulate the expression of multiple target genes and thus modulate processes critical for tumor growth and metastases. Here we show that 1alpha,25(OH)2D3 differentially regulates the expression of Id1 and Id2 genes, members of a family of transcriptional regulators of cell proliferation and differentiation. 1alpha,25(OH)2D3 induced epithelial differentiation in SW480-ADH human colon carcinoma cell line by promoting expression of the proteins implicated in adherent junction formation, including E-cadherin, and by inhibiting beta-catenin transcriptional activity. 1alpha,25(OH)2D3 activated the human Id1 gene promoter and rapidly induced Id1 RNA and protein. Ectopic overexpression of Id1 was not sufficient to induce E-cadherin, which was critical for the morphological changes induced by 1alpha,25(OH)2D3 in SW480-ADH cells. Conversely, Id2 transcription rate, RNA and protein levels were decreased by 1alpha,25(OH)2D3. Id2 downregulation by 1alpha,25(OH)2D3 mediated the antiproliferative effect of 1alpha,25(OH)2D3 on SW480-ADH cells. In addition, we showed that 1alpha,25(OH)2D3 changed the levels of the inducer of angiogenesis, vascular endothelial growth factor and the potent antiangiogenic factor thrombospondin-1, leading to a balanced change in the angiogenic potential of SW480-ADH human colon carcinoma cells.


Subject(s)
Carcinoma/genetics , Colonic Neoplasms/genetics , DNA-Binding Proteins/genetics , Neovascularization, Pathologic/drug therapy , Repressor Proteins/genetics , Transcription Factors/genetics , Vitamin D/analogs & derivatives , Animals , Cadherins/drug effects , Cadherins/metabolism , Carcinoma/drug therapy , Carcinoma/pathology , Cell Proliferation/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Cytoskeletal Proteins/drug effects , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitor of Differentiation Protein 1 , Inhibitor of Differentiation Protein 2 , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Repressor Proteins/drug effects , Trans-Activators/drug effects , Trans-Activators/metabolism , Transcription Factors/drug effects , Tumor Cells, Cultured , Vitamin D/pharmacology , beta Catenin
13.
Cancer Res ; 64(16): 5632-42, 2004 Aug 15.
Article in English | MEDLINE | ID: mdl-15313901

ABSTRACT

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.


Subject(s)
Eye Proteins , Melanoma/blood supply , Melanoma/therapy , Neovascularization, Pathologic/therapy , Nerve Growth Factors , Proteins/physiology , Serpins/physiology , Animals , Cell Division/physiology , Cell Line, Tumor , Cell Movement/physiology , Humans , Liver Neoplasms/prevention & control , Liver Neoplasms/secondary , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Melanoma/genetics , Melanoma/secondary , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Neovascularization, Pathologic/genetics , Protein Biosynthesis , Proteins/genetics , Proteins/metabolism , Serpins/biosynthesis , Serpins/genetics , Serpins/metabolism , Transduction, Genetic , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays
14.
Nat Commun ; 7: 12207, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27481093

ABSTRACT

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.


Subject(s)
B-Lymphocytes/physiology , Immune Tolerance/genetics , Lymphocyte Activation/genetics , MicroRNAs/physiology , PTEN Phosphohydrolase/genetics , Animals , Cell Differentiation/genetics , Cells, Cultured , Female , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal
15.
J Exp Med ; 213(9): 1901-19, 2016 08 22.
Article in English | MEDLINE | ID: mdl-27481129

ABSTRACT

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4(+) T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and function.


Subject(s)
MicroRNAs/physiology , Nuclear Proteins/physiology , Proto-Oncogene Proteins c-rel/physiology , Signal Transduction/physiology , T-Lymphocytes, Helper-Inducer/immunology , Ubiquitin-Protein Ligases/physiology , Animals , CD40 Ligand/analysis , Cell Differentiation , Mice , Mice, Inbred C57BL , NF-kappa B/physiology , T-Lymphocytes, Cytotoxic/immunology
16.
Front Genet ; 6: 340, 2015.
Article in English | MEDLINE | ID: mdl-26697058

ABSTRACT

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5'- and 3'-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

17.
Oncotarget ; 4(12): 2288-301, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24317954

ABSTRACT

Beyond their ability to inhibit cholesterol biosynthesis, the statins have pleiotropic effects that include anti-inflammatory and immunomodulatory activities. Statins could have clinical utility, alone or in combination with other chemotherapeutics, in the treatment of cancer. The mechanisms that underlie the anti-tumor activity of the statins are nonetheless poorly defined. No studies have analyzed how they alter the tumor-associated leukocyte infiltrate, a central factor that influences tumor stroma and cancer evolution. Here we used HER2/neu transgenic (Tg-neu) mice to analyze the effect of lovastatin (Lov) on the inflammatory reaction of spontaneous mammary tumors. Lov treatment of tumor-bearing Tg-neu mice did not alter growth of established tumors, but significantly reduced the number of new oncogenic lesions in these mice. Moreover, Lov inhibited the growth of newly implanted Tg-neu tumors in immunocompetent but not in immunodeficient mice. We found that Lov enhanced tumor infiltration by effector T cells, and reduced the number of immunosuppressive and pro-angiogenic M2-like tumor-associated macrophages (TAM). Concomitantly, the drug improved the structure and function of the tumor vasculature, measured as enhanced tumor oxygenation and penetration of cytotoxic drugs. Microarray analysis identified a Lov-elicited genetic program in Tg-neu tumors that might explain these effects; we observed Lov-induced downregulation of placental growth factor, which triggers aberrant angiogenesis and M2-like TAM polarization. Our results identify a role for lovastatin in the shaping and re-education of the inflammatory infiltrate in tumors, with functional consequences in angiogenesis and antitumor immunity.


Subject(s)
Anticholesteremic Agents/pharmacology , Lovastatin/pharmacology , Macrophages/drug effects , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/immunology , T-Lymphocytes/drug effects , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Polarity/drug effects , Doxorubicin/pharmacology , Female , Lovastatin/administration & dosage , Macrophages/immunology , Macrophages/pathology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
18.
Oncoimmunology ; 1(1): 106-108, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22720226

ABSTRACT

Despite intensive study, the role of CCR5 in cancer remains elusive. We showed that CCR5 expression by both CD4+ and CD8+ T cells is necessary to boost anti-tumor responses by optimizing helper-dependent CD8+ T cell priming. Our findings could have implications for cancer treatment in patients with defective CCR5 expression.

19.
Anticancer Agents Med Chem ; 12(9): 1045-57, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22583417

ABSTRACT

Extensive evidence implicates CCR5 and its ligands in the biology of tumors, although there is considerable controversy regarding the role of this chemokine receptor in cancer progression. The discrepancies between the pro- and anti-tumor effects of CCR5 might derive from its expression by cell types with opposing functions in tumor progression and the context in which tumors originate. We propose that CCR5 is necessary for optimal activation of the adaptive immune response to tumors, and for the success of certain immunotherapeutic strategies. Since efficient activation of T cell responses has broad implications in the success of some chemoand radiotherapy protocols, activation of CCR5, rather than its inhibition, might provide new therapeutic opportunities for cancer treatment.


Subject(s)
Immunotherapy/methods , Neoplasms/immunology , Neoplasms/therapy , Receptors, CCR5/immunology , Adaptive Immunity/drug effects , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Chemokines/immunology , Humans , Immunologic Factors/chemistry , Immunologic Factors/pharmacology
20.
Cancer Res ; 71(16): 5455-66, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21715565

ABSTRACT

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.


Subject(s)
Adoptive Transfer , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Neoplasms, Experimental/immunology , Receptors, CCR5/immunology , Animals , Base Sequence , Cell Division/immunology , DNA Primers , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms, Experimental/pathology
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