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1.
J Immunol ; 166(9): 5540-9, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11313393

ABSTRACT

Ag recognition triggered at the interface between a T cell and an APC is conditioned by cell-cell adhesion and cytoskeletal remodeling. The role played in these phenomena by Lck and Itk, two protein tyrosine kinases essential for T cell signaling, was examined. Early T cell responses (membrane ruffling, Ca(2+) response, APC-T cell adhesion) were monitored in T cells overexpressing kinase-defective (KD) Lck and Itk mutants by combining fluorescence imaging and electron microscopy. Neither Lck nor Itk appears to be involved in the Ag-independent formation of a small and labile contact interface between T cells and APCS: By contrast, the Ag-induced Ca(2+) response in a cell population is similarly blunted in both KD transfectants. However, the underlying mechanisms are strikingly different for the two kinases. The major effect of Lck-KD is to reduce the probability of giving rise to quasi-normal Ca(2+) responses, whereas overexpression of Itk-KD results in a tuning down of all single-cell Ca(2+) responses. In addition, Lck, but not Itk, is required for the formation of a stable T/APC conjugate and for T cell polarization after Ag stimulation. Overall, our results lead to a clear distinction between Lck and ITK: Lck plays an ignition role, controlling all the downstream events tested here, whereas Itk amplifies the Ca(2+) response, but is dispensable for APC-induced adhesive and morphological responses.


Subject(s)
Antigen Presentation , Calcium/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Microscopy, Electron, Scanning , Microscopy, Video , Protein-Tyrosine Kinases/physiology , T-Lymphocytes/immunology , Animals , Antigen Presentation/genetics , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigen-Presenting Cells/ultrastructure , Calcium Signaling/genetics , Calcium Signaling/immunology , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Size/genetics , Cell Size/immunology , Gene Expression Regulation, Enzymologic , Humans , Hybridomas , L Cells , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/biosynthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/deficiency , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Mice , Microscopy, Video/methods , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , T-Lymphocytes/metabolism , T-Lymphocytes/ultrastructure , Transfection
2.
J Exp Med ; 192(11): 1611-24, 2000 Dec 04.
Article in English | MEDLINE | ID: mdl-11104803

ABSTRACT

The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220(+)CD43(+) stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)M(lo)IgD(hi) B cells. Although Tec/Btk(null) mice were able to form germinal centers, the response to T cell-dependent antigens was impaired. Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients.


Subject(s)
Antigens, CD , B-Lymphocytes/immunology , Protein-Tyrosine Kinases/physiology , Agammaglobulinaemia Tyrosine Kinase , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Cell Division , Cell Line , Female , Immunoglobulin A/blood , Immunoglobulin D/biosynthesis , Immunoglobulin G/blood , Immunoglobulin M/biosynthesis , Immunoglobulin M/blood , Leukocyte Common Antigens/biosynthesis , Leukosialin , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogens/pharmacology , Mutagenesis , Phenotype , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Recombination, Genetic , Sialoglycoproteins/biosynthesis , Spleen/cytology , T-Lymphocytes/cytology , T-Lymphocytes/immunology
3.
Nature ; 404(6776): 402-7, 2000 Mar 23.
Article in English | MEDLINE | ID: mdl-10746729

ABSTRACT

Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes.


Subject(s)
Isoenzymes/metabolism , Lymphocyte Activation , NF-kappa B/physiology , Protein Kinase C/metabolism , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/immunology , Animals , Antigens, CD/metabolism , Female , Hemocyanins/immunology , Humans , Isoenzymes/genetics , Leukopoiesis , Male , Mice , Mutagenesis , Protein Kinase C/genetics , Protein Kinase C-theta , Signal Transduction , T-Lymphocytes/enzymology , Thymus Gland/cytology
4.
Annu Rev Immunol ; 17: 523-54, 1999.
Article in English | MEDLINE | ID: mdl-10358767

ABSTRACT

The two major subsets of T lymphocytes in the peripheral immune system, the helper and cytotoxic T cells, are defined by their expression of either the CD4 or the CD8 glycoproteins, respectively. Expression of these molecules, which serve as coreceptors by interacting specifically with either MHC class II or class I molecules, also defines discrete stages of T cell development within the thymus. Thus, CD4+ and CD8+ single-positive (SP) thymocytes arise from common progenitor double positive (DP) cells that express both CD4 and CD8, during a process known as positive selection. The molecular mechanisms underlying the developmental choice toward the helper or cytotoxic lineage remain poorly understood. Because regulation of coreceptor gene expression appears to be coupled to the phenotypic choice of the differentiating T cell, it is likely that shared signaling pathways direct CD4 and CD8 transcription and the development of an uncommited DP thymocyte toward either the helper or cytotoxic lineage. Therefore, an understanding of how CD4 and CD8 expression is regulated will not only provide insights into transcriptional control mechanisms in T cells, but may also result in the identification of molecular factors that are involved in lineage choices during T cell development. In this review, we summarize recent progress that has been made toward an understanding of how CD4 and CD8 gene expression is regulated.


Subject(s)
CD4 Antigens/genetics , CD8 Antigens/genetics , T-Lymphocytes/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Humans , Mice , Models, Biological , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , T-Lymphocytes/cytology
5.
Immunity ; 9(4): 485-96, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9806635

ABSTRACT

We and others have recently identified a CD8 locus enhancer (E8) that directs expression in mature CD8 single-positive thymocytes and peripheral CD8+ T cells and in extrathymically derived intestinal intraepithelial lymphocytes (IEL). In this study, we show that deletion of E8, by homologous recombination results in reduced CD8alphaalpha homodimer expression on IEL. Since CD8 expression on thymus-derived T cells was normal, other enhancers regulate CD8 expression in these cells. By exploiting a transgenic reporter expression assay, we identified three additional enhancers that directed expression in diverse thymocyte subsets and mature T cells but not in CD8alphaalpha+ IEL. The results suggest that CD8alpha expression is primarily regulated by E8, in IEL and by the novel enhancers in the thymus-dependent lineages.


Subject(s)
CD8 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , Enhancer Elements, Genetic , Animals , CD8 Antigens/chemistry , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Digestive System/cytology , Digestive System/immunology , Dimerization , Epithelial Cells/immunology , Gene Deletion , Gene Expression Regulation, Developmental , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Conformation , Recombination, Genetic , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology , Thymus Gland/immunology
6.
Immunity ; 7(4): 537-47, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9354474

ABSTRACT

Positive selection of CD4+CD8+ T cells to the CD4+CD8- helper and CD4- CD8+ cytotoxic lineages is a multistep process that involves complex regulation of coreceptor gene expression. By analyzing expression of a reporter gene in transgenic mice, we have identified a DNA segment, located between the murine CD8beta and CD8alpha genes, that has enhancer activity restricted to CD8 lineage cells. Remarkably, this enhancer functions in thymocytes undergoing positive selection to the CD4-CD8+ phenotype but not in immature double-positive thymocytes. The enhancer also functions in gut intraepithelial lymphocytes that express CD8alpha but not CD8beta, suggesting that it is specific for CD8alpha expression. The tight correlation between activation of this enhancer and the final step in positive selection has important implications for understanding the mechanism of lineage commitment in thymocytes.


Subject(s)
CD8 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , Enhancer Elements, Genetic , T-Lymphocyte Subsets/immunology , Animals , CD2 Antigens/genetics , DNA Footprinting , Deoxyribonuclease I , Gene Expression Regulation, Developmental , Genes, Reporter , Humans , Immunity, Mucosal , Leukopoiesis , Mice , Mice, Transgenic , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , Thymus Gland/cytology , Transcription, Genetic
7.
Nature ; 381(6584): 661-6, 1996 Jun 20.
Article in English | MEDLINE | ID: mdl-8649511

ABSTRACT

Entry of HIV-1 into target cells requires cell-surface CD4 and additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T-cell lines was recently identified and named fusin. However, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains in vivo. The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the beta-chemokines RANTES, MIP-1alpha and MIP-1beta.


Subject(s)
CD4 Antigens/metabolism , HIV-1/physiology , Macrophages/virology , Receptors, Cytokine/metabolism , Receptors, HIV/metabolism , 3T3 Cells , Animals , Cell Line , Chemokines/metabolism , Chemokines/physiology , DNA , Gene Products, env/metabolism , HeLa Cells , Humans , Macrophages/immunology , Membrane Fusion , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Receptors, CCR5 , Receptors, CXCR4 , Receptors, Cytokine/genetics , Receptors, HIV/genetics , Recombinant Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/virology , Virus Replication
8.
Genomics ; 32(1): 155-8, 1996 Feb 15.
Article in English | MEDLINE | ID: mdl-8786109

ABSTRACT

CpG islands were identified and localized to chromosome 1p36 by means of pulsed-field gel blot hybridization with 1p36-specific microclone probes. Five CpG islands, designated CpG17, CpG28, CpG60, CpG112a, and CpG112b, were molecularly cloned from corresponding cosmids. All five islands are associated with transcribed sequences, as shown by RNA blot hybridizations. Screening of cDNA libraries with the island-specific genomic probes led to the isolation of two cDNA clones to date. These encode the human transcription factor E2F-2 and the dominant-negative helix-loop-helix gene ID3, respectively. Pulsed-field gel electrophoresis analysis also revealed that these two genes are located next to each other at a distance of about 25 kb.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , Chromosomes, Human, Pair 1/genetics , CpG Islands , DNA-Binding Proteins , Neoplasm Proteins , Chromosome Mapping , Cloning, Molecular , Cosmids , DNA Probes , DNA, Complementary/genetics , E2F Transcription Factors , E2F2 Transcription Factor , Electrophoresis, Gel, Pulsed-Field , Helix-Loop-Helix Motifs/genetics , Humans , Inhibitor of Differentiation Proteins , RNA/genetics , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1 , Transcription Factors/genetics
9.
Genomics ; 29(3): 607-15, 1995 Oct 10.
Article in English | MEDLINE | ID: mdl-8575752

ABSTRACT

In an approach to mapping physically the most distal 30 Mb of human chromosome 1p, region-specific clone libraries were generated by microdissection and microcloning. PFGE blot hybridization of single or low-copy microclones against rare-cutter digests of genomic DNA revealed physical linkage for groups of markers. Supplementary PFGE analysis of 31 1p36-p35-specific probes for genetically mapped loci established a total of 15 grouped sets, consisting of altogether 69 markers. Twelve of the grouped sets were located in 1pter-p36.12, as revealed by microcell hybrid mapping; the remaining three were localized proximal to 1p36.12. Regional assignment and ordering of most grouped sets was achieved either by evaluating the included genetic markers or by fluorescence in situ hybridization of representative probes. The genomic extent of individual grouped sets encompassed between 1100 and 2100 kb, covering a total of approximately 22 Mb of the distal chromosome 1p region. One particular grouped set was shown to contain seven polymorphic marker loci that were previously suggested to be distributed across the entire 1pter-p35 region. The increase in the number of hybridization marker probes in 1p36 and their physical mapping is expected to facilitate positional cloning experiments in this region; in particular, the construction of clone contigs may be greatly facilitated.


Subject(s)
Chromosomes, Human, Pair 1 , Blotting, Southern , Cell Line , Chromosome Mapping , Cloning, Molecular , DNA/chemistry , DNA/isolation & purification , DNA Probes , Gene Library , Genetic Linkage , Genetic Markers , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Restriction Mapping
10.
Dev Dyn ; 203(2): 163-73, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7655079

ABSTRACT

The family of dominant-negative helix-loop-helix (dnHLH) transcriptional modulators consists of four mammalian genes known to date: Id1, Id2, Id3 (Id3 was formerly designated HLH462 or HEIR1 or HLH1R21), and Id4. We have performed a detailed study of Id3 expression in mouse embryos in order to gain insight into the possible developmental control functions of this gene and to compare its expression to that of other mammalian dnHLH genes. Employing RNA in situ hybridization on sectioned mouse embryos of gestational stages E9.5-17.5 and neonatal head, we generally detected a high abundance of transcripts at early stages that gradually declined at most sites toward E15.5. Expression of Id3 in embryos was detected in brain, spinal cord, olfactory system, branchial arches, limbs, sclerotome, endocardiac cushions, the outer lining of the gut, lung, retina, the collecting system of the kidney, and in tooth anlagen. Although the abundance of mRNA decreased toward later stages in most tissues, it remained high in teeth and kidney. This expression pattern suggests that Id3 functions both in undifferentiated tissues and in organs which are in the process of differentiation. When compared to the expression of other dnHLH genes, it becomes obvious that the pattern of Id3 mainly coincides with that of Id1. This may reflect a partial redundancy in gene function. Furthermore, previous experiments suggested a mutually exclusive expression of the proto-oncogene N-myc an Id3. The results of the present study confirm these data.


Subject(s)
Embryonic and Fetal Development , Gene Expression , Helix-Loop-Helix Motifs , Animals , Base Sequence , Genes, myc , In Situ Hybridization , Kidney/embryology , Mice , Mice, Inbred Strains , Molecular Probes/genetics , Molecular Sequence Data , Spine/embryology , Tooth/embryology
11.
EMBO J ; 11(7): 2563-71, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1628620

ABSTRACT

We have isolated a novel human gene encoding a helix-loop-helix (HLH) protein by molecularly cloning chromosome 1p36-specific CpG islands. The gene termed heir-1 was localized to the neuroblastoma consensus deletion at 1p36.2-p36.12. Its predicted protein is 95.8% identical to the mouse HLH462 protein and has clear homology to the mouse Id and Drosophila emc proteins. Heir-1 does not encode a basic DNA binding domain as found in basic HLH proteins. The gene is expressed specifically at high abundance in adult lung, kidney and adrenal medulla, but not in adult brain. Despite prominent heir-1 expression in adrenal medulla, which is a prime target for neuroblastomas, 10 out of 12 neuroblastoma-derived cell lines revealed very low levels of heir-1 mRNA. Low heir-1 expression was generally found in tumor cell lines with N-myc overexpression, whereas the two cell lines displaying high heir-1 levels did not overexpress N-myc. Mutually exclusive expression of both genes was also found by in situ hybridization in developing mouse tissues, particularly in the forebrain neuroectoderm. We conclude that heir-1 expression is reduced specifically in the majority of neuroblastomas and suggest an inverse correlation between heir-1 and N-myc expression in neuroblastoma tumors and in embryonic development.


Subject(s)
Gene Expression , Genes, myc , Neoplasm Proteins/genetics , Neuroblastoma/genetics , Adrenal Medulla/embryology , Adrenal Medulla/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Brain/embryology , Brain/metabolism , Chromosomes, Human, Pair 1 , Cloning, Molecular , DNA , Dinucleoside Phosphates/genetics , Electrophoresis, Gel, Pulsed-Field , Humans , Inhibitor of Differentiation Proteins , Kidney/embryology , Kidney/metabolism , Lung/embryology , Lung/metabolism , Mice , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Nucleic Acid Hybridization , Proteins/genetics , Restriction Mapping , Tumor Cells, Cultured
12.
Brain Pathol ; 2(3): 195-208, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1343834

ABSTRACT

Of all human tumors, neuroblastomas bear the most prominent genetic changes. Amplifications and deletions of chromosomal DNA can be identified by light microscopy on chromosomal spreads of neuroblastoma cells with remarkable frequency and consistency. Consequently, extensive studies have been undertaken to elucidate the molecular basis of these cytogenetic changes. A rich body of information has accumulated on the role played by dominant oncogenes and recessive tumor suppressor genes in the pathogenesis of this disease. Most notably, it was found that amplification of N-myc is responsible for the presence of double minutes and homogeneously staining regions in neuroblastoma chromosomes. It has also been discovered that N-myc amplification is a prognostic sign of malignancy. More recently, recessive genetic alterations in neuroblastoma, such as deletion of putative tumor-suppressing genes have received increasing attention, and considerable efforts are being made to identify such genetic elements. Finally, the susceptibility of neuroblastoma cells to differentiating stimuli has made them a popular in vitro system for neurobiological and pharmacological research. The need for suitable in vivo systems has spurred the development of several animal models employing tumor viruses and transgenic technologies.


Subject(s)
Brain Neoplasms/genetics , Neuroblastoma/genetics , Amino Acid Sequence , Animals , Brain Neoplasms/pathology , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Neuroblastoma/pathology , Sequence Deletion
13.
EMBO J ; 10(11): 3513-22, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1833188

ABSTRACT

We have analysed nucleocytoplasmic export of mRNAs in primate cells with the aim of identifying signals promoting RNA transport. Our results demonstrate that sequences directing either histone mRNA 3' processing or cleavage/polyadenylation of mRNA stimulate nucleocytoplasmic RNA transport. To elucidate the nature of this stimulation, we engineered test gene transcripts which could obtain a mature histone 3' end by the RNA cleaving activity of a cis-acting ribozyme, thus circumventing the cellular 3' end processing machinery. However, such transcripts were found to be transport deficient and accumulated in the nuclear compartment. Our experiments provide genetic evidence that there is a linkage between 3' end formation and the export of RNA transcripts from the nucleus. Analysis of a similar series of histone mRNAs in which the mature 3' end was generated by means of ribozyme cleavage led to the discovery of a second export mechanism which relies on features specific for mature histone RNA and which can be uncoupled from the cellular processing machinery. The presence of histone mRNA sequences and of the highly conserved histone hairpin structure, positioned close to the 3' terminus, are critical determinants for this export mechanism.


Subject(s)
Cell Nucleus/metabolism , RNA, Messenger/metabolism , Animals , Base Sequence , Biological Transport , Drug Resistance/genetics , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Molecular Sequence Data , Neomycin/pharmacology , Nucleic Acid Conformation , Plasmids , Primates , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Ribonucleoproteins, Small Nuclear , Transcription, Genetic
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