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1.
Sci Immunol ; 6(64): eabb6444, 2021 Oct 15.
Article in English | MEDLINE | ID: mdl-34623903

ABSTRACT

Interleukin-27 (IL-27) is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4+ T cells by IL-27, enhanced by transforming growth factor­ß (TGF-ß), and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient deprivation cell death without releasing high-mobility group box 1 (HMGB1). This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Ch25h-expressing CD4+ T cells that received IL-27 and TGF-ß signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27­treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worse outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Inflammation Mediators/metabolism , Inflammation/metabolism , Interleukin-27/metabolism , Skin/metabolism , Steroid Hydroxylases/metabolism , Animals , Cholesterol/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Steroid Hydroxylases/genetics
2.
Mucosal Immunol ; 1(4): 279-88, 2008 Jul.
Article in English | MEDLINE | ID: mdl-19079189

ABSTRACT

Loss of CD4(+) T cells in the gut is necessary but not sufficient to cause AIDS in animal models, raising the possibility that a differential loss of CD4(+) T-cell subtypes may be important. We found that CD4(+) T cells that produce interleukin (IL)-17, a recently identified lineage of effector CD4(+) T-helper cells, are infected by SIV(mac251)in vitro and in vivo, and are found at lower frequency at mucosal and systemic sites within a few weeks from infection. In highly viremic animals, Th1 cells predominates over Th17 T cells and the frequency of Th17 cells at mucosal sites is negatively correlated with plasma virus level. Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, our finding may explain the chronic enteropathy in human immunodeficiency virus (HIV) infection. Thus, therapeutic approaches that reconstitute an adequate balance between Th1 and Th17 may be beneficial in the treatment of HIV infection.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Interleukin-17/immunology , Mucous Membrane/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/physiology , Th1 Cells/immunology , Animals , Antigens, Viral/immunology , Humans , Lymphocytes/immunology , Macaca mulatta , Mucous Membrane/virology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/immunology , Virus Replication/physiology
5.
Proc Natl Acad Sci U S A ; 98(26): 15137-42, 2001 Dec 18.
Article in English | MEDLINE | ID: mdl-11752460

ABSTRACT

Differentiation of naive CD4(+) T cells into IFN-gamma-producing T helper 1 (T(H)1) cells is pivotal for protective immune responses against intracellular pathogens. T-bet, a recently discovered member of the T-box transcription factor family, has been reported to play a critical role in this process, promoting IFN-gamma production. Although terminal T(H)1 differentiation occurs over days, we now show that challenge of mice with a prototypical T(H)1-inducing stimulus, Toxoplasma gondii soluble extract, rapidly induced IFN-gamma and T-bet; T-bet induction was substantially lower in IFN-gamma-deficient mice. Naive T cells expressed little T-bet, but this transcription factor was induced markedly by the combination of IFN-gamma and cognate antigen. Human myeloid antigen-presenting cells showed T-bet induction after IFN-gamma stimulation alone, and this induction was antagonized by IL-4 and granulocyte/macrophage colony-stimulating factor. Although T-bet was induced rapidly and directly by IFN-gamma, it was not induced by IFN-alpha, lipopolysaccharide, or IL-1, indicating that this action of IFN-gamma was specific. Moreover, T-bet induction was dependent on Stat1 but not Stat4. These data argue for a model in which IFN-gamma gene regulation involves an autocrine loop, whereby the cytokine regulates a transcription factor that promotes its own production. These findings substantially alter the current view of T-bet in IFN-gamma regulation and promotion of cell-mediated immune responses.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Dendritic Cells/metabolism , Interferon-gamma/physiology , Macrophages/metabolism , Transcription Factors/biosynthesis , Animals , Antibodies, Monoclonal/immunology , Base Sequence , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/physiology , Cells, Cultured , DNA Primers , Dendritic Cells/immunology , Female , Humans , Macrophages/immunology , Mice , Mice, Inbred C57BL , T-Box Domain Proteins , Transcription Factors/physiology , Up-Regulation
6.
Mol Cell ; 8(5): 959-69, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11741532

ABSTRACT

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.


Subject(s)
Protein-Tyrosine Kinases/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , COS Cells , Catalysis , Enzyme Inhibitors/pharmacology , Humans , Interleukin Receptor Common gamma Subunit , Janus Kinase 3 , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Pyrimidines/pharmacology , Receptors, Interleukin-7/genetics , Receptors, Interleukin-7/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Staurosporine/pharmacology , src-Family Kinases/antagonists & inhibitors
8.
Trends Immunol ; 22(10): 556-60, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11574279

ABSTRACT

The suggestion that antigen-presenting cells (APCs) produce interferon gamma (IFN-gamma) is controversial because it conflicts with the initial paradigm in which the production of IFN-gamma was restricted to lymphoid cells. However, some answers to this skepticism have been provided by recent findings of high-level production and intracellular expression of IFN-gamma by interleukin-12 (IL-12)-stimulated macrophages and dendritic cells. New data are now emerging to explain the mechanism of production of IFN-gamma vby APCs. As in lymphoid cells, IL-12-induced IFN-gamma production in APCs requires signal transducer and activator of transcription 4 (STAT4), although the precise molecular events that govern the transcription of the gene encoding IFN-gamma are enigmatic still. Understanding these processes in lymphoid, and now nonlymphoid, cells remains an important challenge.


Subject(s)
Antigen-Presenting Cells/immunology , Interferon-gamma/biosynthesis , Animals , DNA-Binding Proteins/immunology , Humans , Models, Immunological , STAT4 Transcription Factor , Trans-Activators/immunology
9.
Am J Hum Genet ; 69(2): 301-14, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11443543

ABSTRACT

Mutations in the extracellular domain of the 55-kD tumor-necrosis factor (TNF) receptor (TNFRSF1A), a key regulator of inflammation, define a periodic-fever syndrome, TRAPS (TNF receptor-associated periodic syndrome [MIM 142680]), which is characterized by attacks of fever, sterile peritonitis, arthralgia, myalgia, skin rash, and/or conjunctivitis; some patients also develop systemic amyloidosis. Elsewhere we have described six disease-associated TNFRSF1A mutations, five of which disrupt extracellular cysteines involved in disulfide bonds; four other mutations have subsequently been reported. Among 150 additional patients with unexplained periodic fevers, we have identified four novel TNFRSF1A mutations (H22Y, C33G, S86P, and c.193-14 G-->A), one mutation (C30S) described by another group, and two substitutions (P46L and R92Q) present in approximately 1% of control chromosomes. The increased frequency of P46L and R92Q among patients with periodic fever, as well as functional studies of TNFRSF1A, argue that these are low-penetrance mutations rather than benign polymorphisms. The c.193-14 G-->A mutation creates a splice-acceptor site upstream of exon 3, resulting in a transcript encoding four additional extracellular amino acids. T50M and c.193-14 G-->A occur at CpG hotspots, and haplotype analysis is consistent with recurrent mutations at these sites. In contrast, although R92Q also arises at a CpG motif, we identified a common founder chromosome in unrelated individuals with this substitution. Genotype-phenotype studies identified, as carriers of cysteine mutations, 13 of 14 patients with TRAPS and amyloidosis and indicated a lower penetrance of TRAPS symptoms in individuals with noncysteine mutations. In two families with dominantly inherited disease and in 90 sporadic cases that presented with a compatible clinical history, we have not identified any TNFRSF1A mutation, despite comprehensive genomic sequencing of all of the exons, therefore suggesting further genetic heterogeneity of the periodic-fever syndromes.


Subject(s)
Antigens, CD/genetics , Familial Mediterranean Fever/genetics , Genetic Heterogeneity , Mutation/genetics , Receptors, Tumor Necrosis Factor/genetics , Alternative Splicing/genetics , Amino Acid Sequence , Amyloidosis/genetics , Antigens, CD/chemistry , Base Sequence , DNA Mutational Analysis , Ethnicity/genetics , Exons/genetics , Female , Haplotypes/genetics , Humans , Introns/genetics , Male , Microsatellite Repeats/genetics , Models, Molecular , Molecular Sequence Data , Pedigree , Penetrance , Polymorphism, Single Nucleotide/genetics , Protein Structure, Tertiary , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor, Type I
10.
Curr Opin Immunol ; 13(3): 363-73, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11406370

ABSTRACT

Discovered during the past ten years, Janus kinases and signal transducers and activators of transcription have emerged as critical elements in cytokine signaling and immunoregulation. Recently, knockout mice for all the members of these families have been generated, with remarkably specific outcomes. Equally exciting is the discovery of a new class of inhibitors, the suppressor of cytokine signaling family. The phenotypes of mice deficient in these molecules are also striking, underscoring the importance of negative regulation in cytokine signaling.


Subject(s)
Receptors, Cytokine/classification , Receptors, Cytokine/physiology , Signal Transduction/immunology , Animals , Humans
11.
J Biol Chem ; 276(27): 25378-85, 2001 Jul 06.
Article in English | MEDLINE | ID: mdl-11349123

ABSTRACT

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.


Subject(s)
Cytokines/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, T-Cell/metabolism , Animals , CD3 Complex/metabolism , Calcium/metabolism , Enzyme Activation , Humans , Janus Kinase 3 , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mice , Phosphorylation , Rabbits , Receptors, Interleukin-2/metabolism , Spleen/cytology , T-Lymphocytes/enzymology , Transfection , Tumor Cells, Cultured , ZAP-70 Protein-Tyrosine Kinase
12.
J Immunol ; 166(7): 4446-55, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11254700

ABSTRACT

Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.


Subject(s)
DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Dendritic Cells/metabolism , Macrophages/metabolism , Trans-Activators/biosynthesis , Trans-Activators/physiology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Autocrine Communication/genetics , Autocrine Communication/immunology , CD8 Antigens/biosynthesis , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Cytokines/physiology , DNA-Binding Proteins/genetics , Dendritic Cells/cytology , Dendritic Cells/immunology , Immunity, Cellular/genetics , Immunity, Innate/genetics , Injections, Intravenous , Interferon-gamma/biosynthesis , Interleukin-12/physiology , Lipopolysaccharides/administration & dosage , Macrophage Activation/genetics , Macrophages/immunology , Macrophages/parasitology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , STAT4 Transcription Factor , Signal Transduction/genetics , Signal Transduction/immunology , Th2 Cells/immunology , Th2 Cells/metabolism , Toxoplasma/immunology , Toxoplasma/pathogenicity , Trans-Activators/genetics , Up-Regulation/genetics , Up-Regulation/immunology
13.
Curr Protoc Immunol ; Chapter 11: Unit 11.9A, 2001 Nov.
Article in English | MEDLINE | ID: mdl-18432708

ABSTRACT

This unit, along with Unit 11.9B, provides a summary of our current knowledge about various signaling pathways critical to the function of immune cells. Here, our understanding of T cell receptor (TCR)- and B cell receptor (BCR)-mediated signaling is summarized. A schematic representation of immunologically relevant cytokine receptors and the Janus Family Kinases (JAKs) that is activated through these receptors is provided, along with details about molecules involved in interleukin 2 mediated signal transduction.


Subject(s)
Cytokines/metabolism , Lymphocyte Activation , Protein Kinases/metabolism , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, T-Cell/immunology , Animals , B-Lymphocytes/immunology , Cytokines/immunology , Humans , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , T-Lymphocytes/immunology
14.
Genes Immun ; 2(8): 422-32, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11781709

ABSTRACT

Mutations of the Janus kinase 3 (JAK3) have been previously described to cause an autosomal recessive variant of severe combined immunodeficiency (SCID) usually characterized by the near absence of T and NK cells, but preserved numbers of B lymphocytes (T-B+SCID). We now report a family whose JAK3 mutations are associated with the persistence of circulating T cells, resulting in previously undescribed clinical presentations, ranging from a nearly unaffected 18-year-old subject to an 8-year-old sibling with a severe lymphoproliferative disorder. Both siblings were found to be compound heterozygotes for the same deleterious JAK3 mutations: an A96G initiation start site mutation, resulting in a dysfunctional, truncated protein product and a G2775(+3)C mutation in the splice donor site sequence of intron 18, resulting in a splicing defect and a predicted premature stop. These mutations were compatible with minimal amounts of functional JAK3 expression, leading to defective cytokine-dependent signaling. Activated T cells in these patients failed to express Fas ligand (FasL) in response to IL-2, which may explain the accumulation of T cells with an activated phenotype and a skewed T cell receptor (TcR) Vbeta family distribution. We speculate that residual JAK3 activity accounted for the maturation of thymocytes, but was insufficient to sustain IL-2-mediated homeostasis of peripheral T cells via Fas/FasL interactions. These data demonstrate that the clinical spectrum of JAK3 deficiency is quite broad and includes immunodeficient patients with accumulation of activated T cells, and indicate an essential role for JAK3 in the homeostasis of peripheral T cells in humans.


Subject(s)
Protein-Tyrosine Kinases/deficiency , Adolescent , Amino Acid Sequence , B-Lymphocytes/metabolism , Cell Line, Transformed , Child , DNA, Complementary , Fas Ligand Protein , Female , Gene Expression , Humans , Immunophenotyping , Interleukin-2/metabolism , Janus Kinase 3 , Male , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Mutation , Pedigree , Protein-Tyrosine Kinases/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Interleukin-2/metabolism , Sequence Analysis, DNA , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/immunology , Severe Combined Immunodeficiency/pathology , Signal Transduction , T-Lymphocytes , Up-Regulation
15.
Arthritis Res ; 2(1): 16-32, 2000.
Article in English | MEDLINE | ID: mdl-11094415

ABSTRACT

Cytokines play a critical role in the normal development and function of the immune system. On the other hand, many rheumatologic diseases are characterized by poorly controlled responses to or dysregulated production of these mediators. Over the past decade tremendous strides have been made in clarifying how cytokines transmit signals via pathways using the Janus kinase (Jak) protein tyrosine kinases and the Signal transducer and activator of transcription (Stat) proteins. More recently, research has focused on several distinct proteins responsible for inhibiting these pathways. It is hoped that further elucidation of cytokine signaling through these pathways will not only allow for a better comprehension of the etiopathogenesis of rheumatologic illnesses, but may also direct future treatment options.


Subject(s)
Adjuvants, Immunologic/physiology , Cytokines/metabolism , Protein-Tyrosine Kinases/physiology , Signal Transduction/physiology , Transcription, Genetic/physiology , Animals , Humans
16.
J Immunol ; 165(11): 6221-8, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11086056

ABSTRACT

Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.


Subject(s)
Interleukin-12/metabolism , Receptors, Interleukin/deficiency , Receptors, Interleukin/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Animals , Binding Sites/genetics , Binding Sites/immunology , Concanavalin A/pharmacology , DNA-Binding Proteins/metabolism , Female , Gene Targeting , Immune Tolerance/genetics , Interferon-gamma/biosynthesis , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Lymphocyte Activation/genetics , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Phosphorylation , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/physiology , Receptors, Interleukin/physiology , Receptors, Interleukin-12 , STAT4 Transcription Factor , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Trans-Activators/metabolism
17.
Ann Rheum Dis ; 59 Suppl 1: i115-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11053101

ABSTRACT

Cytokines have critical functions in regulating immune responses. A large number of these factors bind related receptors termed the Type I and Type II families of cytokine receptors. These receptors activate Janus kinases (Jaks) and Stat family of transcription factors. The essential and specific function of Jaks and Stats is particularly well illustrated by human and mouse mutations. The possibility that these molecules could be targeted to produce novel immunosuppressive compounds is considered in this review.


Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Immunosuppressive Agents/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Trans-Activators/antagonists & inhibitors , Animals , DNA-Binding Proteins/immunology , Humans , Mice , Protein-Tyrosine Kinases/immunology , Trans-Activators/immunology
18.
Hum Genet ; 106(1): 73-9, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10982185

ABSTRACT

JAK3 deficiency in humans results in autosomal recessive T-B+ severe combined immunodeficiency disease (SCID), a severe immunodeficiency that can only be cured by bone marrow transplantation. We unraveled the complete organization of the human JAK3 gene, which includes 23 exons. This information allowed us to set up a molecular screening test that enabled us to diagnose JAK3 deficiency in 14 patients from 12 unrelated families with T-B+ SCID. In order to define the mutations, we used a nonradioactive single-strand conformation polymorphism (SSCP)/heteroduplex (HD) assay based on exon-specific polymerase chain reaction (PCR). In this cohort of patients, 15 independent JAK3 gene mutations have been identified, including 7 that have not been described previously. Mutation analysis information was used for genetic counseling and prenatal diagnosis.


Subject(s)
DNA Mutational Analysis/methods , Protein-Tyrosine Kinases/genetics , Severe Combined Immunodeficiency/genetics , Consanguinity , Exons , Female , Heterozygote , Homozygote , Humans , Introns , Janus Kinase 3 , Male , Models, Genetic , Molecular Sequence Data , Polymorphism, Single-Stranded Conformational , Protein-Tyrosine Kinases/deficiency
19.
Blood ; 96(5): 1844-52, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10961885

ABSTRACT

Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4(-/-) mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12-induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12-induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38alpha and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38alpha/STAT4 pathway as an important mediator of IL-12 actions. (Blood. 2000;96:1844-1852)


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/metabolism , Interleukin-12/pharmacology , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , Serine/metabolism , Trans-Activators/metabolism , 3T3 Cells , Animals , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Cell Line , DNA-Binding Proteins/genetics , Humans , Isoenzymes/metabolism , Jurkat Cells , MAP Kinase Kinase 4 , MAP Kinase Kinase 6 , Mice , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/genetics , Mutation , Phosphorylation/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , STAT4 Transcription Factor , Serine/genetics , Signal Transduction/drug effects , Trans-Activators/genetics , Transcriptional Activation/drug effects , Transfection , Tyrosine/metabolism , p38 Mitogen-Activated Protein Kinases
20.
Mol Ther ; 1(2): 145-53, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10933924

ABSTRACT

Mutations of the common gamma chain (gammac) of cytokine receptors cause X-linked severe combined immunodeficiency (XSCID), a candidate disease for gene therapy. Using an XSCID murine model, we have tested the feasibility of stem cell gene correction. XSCID bone marrow (BM) cells were transduced with a retroviral vector expressing the murine gammac (mgammac) and engrafted in irradiated XSCID animals. Transplanted mice developed mature B cells, naive T cells, and mature natural killer (NK) cells, all of which were virtually absent in untreated mice. The mgammac transgene was detected in all treated mice, and we could demonstrate mgammac expression in newly developed lymphocytes at both the RNA and protein level. In addition, treated mice showed T cell proliferation responses to mitogens and production of antigen-specific antibodies upon immunization. Four of seven treated animals showed a clear increase of the transgene positive cells, suggesting in vivo selective advantage for gene-corrected cells. Altogether, these results show that retroviral-mediated gene transfer can improve murine XSCID and suggest that similar strategies may prove beneficial in human clinical trials.


Subject(s)
Bone Marrow Cells/immunology , Genetic Linkage , Genetic Therapy , Lymphatic System/growth & development , Lymphatic System/physiology , X Chromosome/genetics , X Chromosome/immunology , Animals , B-Lymphocytes/immunology , Bone Marrow Transplantation/immunology , Cell Division , Cell Line , Flow Cytometry , Genetic Vectors , Immunoglobulin G/metabolism , Immunophenotyping , Killer Cells, Natural/immunology , Lymphatic System/immunology , Lymphocyte Count , Mice , Mice, Knockout , Mice, SCID , Receptors, Cytokine/genetics , Receptors, Cytokine/immunology , Receptors, Interleukin-2/genetics , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/immunology , Transduction, Genetic , Transgenes/genetics
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