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1.
Dev Comp Immunol ; 65: 31-40, 2016 12.
Article in English | MEDLINE | ID: mdl-27345169

ABSTRACT

Pig is a domestic species of major importance in the agro-economy and in biomedical research. Mononuclear phagocytes (MNP) are organized in subsets with specialized roles in the orchestration of the immune response and new tools are awaited to improve MNP subset identification in the pig. We cloned pig CD11c cDNA and generated a monoclonal antibody to pig CD11c which showed a pattern of expression by blood and skin MNP subsets similar to humans. We also developed a porcine XCL1-mCherry dimer which specifically reacted with the XCR1-expressing dendritic cell subset of the type 1 lineage in blood and skin. These original reagents will allow the efficient identification of pig MNP subsets to study their role in physiological and pathological processes and also to target these cells in novel intervention and vaccine strategies for veterinary applications and preclinical evaluations.


Subject(s)
CD11c Antigen/metabolism , Dendritic Cells/physiology , Mononuclear Phagocyte System , Phagocytes/immunology , Receptors, G-Protein-Coupled/metabolism , Skin/metabolism , Swine/immunology , Animals , Antibodies, Monoclonal/metabolism , CD11c Antigen/immunology , Cloning, Molecular , Disease Models, Animal , Humans , Immunologic Tests/methods , Receptors, G-Protein-Coupled/immunology , Veterinary Medicine
2.
PLoS Biol ; 14(3): e1002401, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26942442

ABSTRACT

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive "Fas apoptosis" to "Fas multisignals" paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.


Subject(s)
Evolution, Molecular , Neoplasms/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , fas Receptor/metabolism , src-Family Kinases/metabolism , Amino Acid Sequence , Apoptosis , Endocytosis , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Protein Structure, Tertiary
3.
PLoS One ; 7(9): e46213, 2012.
Article in English | MEDLINE | ID: mdl-23029439

ABSTRACT

BACKGROUND: Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results. METHODOLOGY AND MAIN FINDINGS: A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2(Lp) protein in mutant mice compared to the wild type mice. CONCLUSION: Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Carrier Proteins/chemistry , Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Neural Tube Defects/genetics , Point Mutation , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Polarity/genetics , Gene Expression , Heterozygote , Homozygote , Humans , Immunoprecipitation , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Tube Defects/diagnosis , Protein Binding , Protein Multimerization , Proteomics , Surface Plasmon Resonance
4.
J Biol Chem ; 287(15): 11942-50, 2012 Apr 06.
Article in English | MEDLINE | ID: mdl-22362765

ABSTRACT

The identification of a neutralizing mAb against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication but also as an extracellular toxin able to disrupt the immune system. We showed previously that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi and selected a mAb named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat as an unfolded protein. mAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Epitopes/immunology , HIV-1/immunology , tat Gene Products, Human Immunodeficiency Virus/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibody Affinity , Antibody Specificity , Apoptosis , Cell Proliferation/drug effects , Epitope Mapping , HIV-1/genetics , HeLa Cells , Humans , Jurkat Cells/drug effects , Jurkat Cells/physiology , Lymphocytes/drug effects , Lymphocytes/metabolism , Mice , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino Acid , Transcriptional Activation , tat Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , tat Gene Products, Human Immunodeficiency Virus/chemistry
5.
Cancer Res ; 71(5): 1607-14, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21343393

ABSTRACT

The discovery of a targeted therapeutic compound along with its companion predictive biomarker is a major goal of clinical development for a personalized anticancer therapy to date. Here we present evidence of the predictive value of TLR3 expression by tumor cells for the efficacy of Poly (A:U) dsRNA in 194 breast cancer patients enrolled in a randomized clinical trial. Adjuvant treatment with double-stranded RNA (dsRNA) was associated with a significant decrease in the risk of metastatic relapse in TLR3 positive but not in TLR3-negative breast cancers. Moreover, we show the functional relevance of TLR3 expression by human tumor cells for the antitumor effects mediated by dsRNA in several preclinical mouse models carried out in immunocompromised animals. These 2 independent lines of evidence relied upon the generation of a novel tool, an anti-TLR3 antibody (40F9.6) validated for routine detection of TLR3 expression on paraffin-embedded tissues. Altogether, these data suggest that dsRNA mediates its therapeutic effect through TLR3 expressed on tumor cells, and could therefore represent an effective targeted treatment in patients with TLR3-positive cancers.


Subject(s)
Antibodies, Monoclonal , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/analysis , Breast Neoplasms/drug therapy , RNA, Double-Stranded/therapeutic use , Toll-Like Receptor 3/biosynthesis , Animals , Antibody Specificity , Breast Neoplasms/metabolism , Female , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Multicenter Studies as Topic , Randomized Controlled Trials as Topic , Retrospective Studies , Toll-Like Receptor 3/analysis
6.
J Cell Sci ; 123(Pt 14): 2391-401, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20551181

ABSTRACT

Cilia and flagella are evolutionary conserved organelles that generate fluid movement and locomotion, and play roles in chemosensation, mechanosensation and intracellular signalling. In complex organisms, cilia are highly diversified, which allows them to perform various functions; however, they retain a 9+0 or 9+2 microtubules structure connected to a basal body. Here, we describe FOR20 (FOP-related protein of 20 kDa), a previously uncharacterized and highly conserved protein that is required for normal formation of a primary cilium. FOR20 is found in PCM1-enriched pericentriolar satellites and centrosomes. FOR20 contains a Lis1-homology domain that promotes self-interaction and is required for its satellite localization. Inhibition of FOR20 expression in RPE1 cells decreases the percentage of ciliated cells and the length of the cilium on ciliated cells. It also modifies satellite distribution, as judged by PCM1 staining, and displaces PCM1 from a detergent-insoluble to a detergent-soluble fraction. The subcellular distribution of satellites is dependent on both microtubule integrity and molecular motor activities. Our results suggest that FOR20 could be involved in regulating the interaction of PCM1 satellites with microtubules and motors. The role of FOR20 in primary cilium formation could therefore be linked to its function in regulating pericentriolar satellites. A role for FOR20 at the basal body itself is also discussed.


Subject(s)
Centrosome/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cilia/metabolism , Proteins/metabolism , Retinal Pigment Epithelium/metabolism , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Autoantigens/genetics , Autoantigens/immunology , Autoantigens/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/immunology , Cell Cycle Proteins/metabolism , Cell Differentiation/genetics , Cell Line, Transformed , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/immunology , Cilia/pathology , Gene Expression Regulation, Developmental/genetics , Humans , Hybridomas , Microtubules/metabolism , Microtubules/pathology , Phylogeny , Protein Engineering , Proteins/genetics , RNA, Small Interfering/genetics , Rats , Retinal Pigment Epithelium/immunology , Retinal Pigment Epithelium/pathology
7.
J Immunol ; 183(11): 7212-22, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19915064

ABSTRACT

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.


Subject(s)
Antibodies, Monoclonal/immunology , Lymphocyte Activation/immunology , Receptor, Notch1/biosynthesis , Receptor, Notch2/biosynthesis , Animals , Antibody Specificity , Cell Differentiation , Cell Membrane/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Fluorescent Antibody Technique , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Receptor, Notch1/immunology , Receptor, Notch2/immunology , Signal Transduction/immunology , Stem Cells/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism
8.
J Immunol ; 181(12): 8199-203, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-19050235

ABSTRACT

Interactions between Notch1 receptors on lymphoid progenitors and Delta-like 4 (DL4) ligands on cortical thymic epithelial cells (cTEC) are essential for T cell lineage commitment, expansion, and maturation in the thymus. Using a novel mAb against DL4, we show that DL4 levels on cTEC are very high in the fetal and neonatal thymus when thymocyte expansion is maximal but decrease dramatically in the adult when steady-state homeostasis is attained. Analysis of mutant mouse strains where thymocyte development is blocked at different stages indicates that lymphostromal interactions ("thymus crosstalk") are required for DL4 down-regulation on cTEC. Reconstitution of thymocyte development in these mutant mice further suggests that maturation of thymocytes to the CD4(+)CD8(+) stage and concomitant expansion are needed to promote DL4 down-regulation on cTEC. Collectively, our data support a model where thymic crosstalk quantitatively regulates the rate of Notch1-dependent thymopoiesis by controlling DL4 expression levels on cTEC.


Subject(s)
Cell Communication/immunology , Epithelial Cells/immunology , Epithelial Cells/metabolism , Membrane Proteins/biosynthesis , Thymus Gland/cytology , Thymus Gland/immunology , Adaptor Proteins, Signal Transducing , Animals , Animals, Newborn , Calcium-Binding Proteins , Cell Communication/genetics , Down-Regulation/immunology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Lymphopoiesis/genetics , Lymphopoiesis/immunology , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Immunological , Rats , Receptor, Notch1/physiology , Stromal Cells/cytology , Stromal Cells/immunology , Stromal Cells/metabolism , Thymus Gland/embryology , Thymus Gland/metabolism
9.
J Exp Med ; 205(11): 2515-23, 2008 Oct 27.
Article in English | MEDLINE | ID: mdl-18824585

ABSTRACT

Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.


Subject(s)
Cell Differentiation/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Receptor, Notch1/metabolism , Signal Transduction/immunology , T-Lymphocytes/cytology , Thymus Gland/cytology , Adaptor Proteins, Signal Transducing , Animals , Calcium-Binding Proteins , Flow Cytometry , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Mice , Mice, Transgenic , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , beta-Galactosidase
10.
Proc Natl Acad Sci U S A ; 104(9): 3384-9, 2007 Feb 27.
Article in English | MEDLINE | ID: mdl-17360655

ABSTRACT

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ, and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ. NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122(+)NK1.1(+)DX5(-) stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo. Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.


Subject(s)
Gene Expression Regulation/immunology , Killer Cells, Natural/cytology , Membrane Glycoproteins/metabolism , Receptors, Immunologic/metabolism , Animals , Antigens, Ly , Cell Differentiation/immunology , Diphtheria Toxin/toxicity , Flow Cytometry , Green Fluorescent Proteins/metabolism , Haplorhini , Immunophenotyping , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Membrane Glycoproteins/genetics , Mice , Mice, Transgenic , Microscopy, Fluorescence , Natural Cytotoxicity Triggering Receptor 1 , Promoter Regions, Genetic/genetics , Receptors, Immunologic/genetics
11.
J Biol Chem ; 281(29): 20283-90, 2006 Jul 21.
Article in English | MEDLINE | ID: mdl-16709568

ABSTRACT

Fluorescence resonance energy transfer and native PAGE analytical techniques were employed to assess the quaternary structure of ABCA1, an ATP binding cassette transporter playing a crucial role in cellular lipid handling. These experimental approaches support the conclusion that ABCA1 is associated in dimeric structures that undergo transition into higher order structures, i.e. tetramers, during the ATP catalytic cycle. Our data hence underline molecular assembly as a crucial parameter in ABCA1 function and the advantage of native PAGE as analytical tool for intractable membrane proteins.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/metabolism , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Catalysis , Dimerization , Electrophoresis, Polyacrylamide Gel , Fluorescence Resonance Energy Transfer , HeLa Cells , Humans , Kinetics , Macromolecular Substances , Plasmids , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
12.
J Neurochem ; 97(2): 345-55, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16539677

ABSTRACT

The notion that the ATP-binding cassette transporter-A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimer's disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome-related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin(+) cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimer's lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Brain/cytology , Neurons/metabolism , Stem Cells/physiology , Animals , Biomarkers/metabolism , Blotting, Western/methods , CD24 Antigen/metabolism , Cell Differentiation/physiology , Cells, Cultured , Embryo, Mammalian , Fluorescent Antibody Technique/methods , Gene Expression/physiology , Gene Expression Regulation, Developmental/physiology , Glial Fibrillary Acidic Protein/metabolism , Glutamic Acid/metabolism , Humans , Luminescent Proteins/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomal-Associated Membrane Protein 2/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Neurons/classification , Rats , Subcellular Fractions/metabolism , Time Factors , Transfection/methods , gamma-Aminobutyric Acid/metabolism
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