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1.
Autoimmunity ; 48(1): 29-39, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25483245

ABSTRACT

Subunit vaccines are typically poorly immunogenic when administered alone, and require adjuvants for robust responses. Triggering TLRs to boost antigen-specific adaptive immunity is an attractive approach to increase the potency and quality of vaccines. However, recent reports suggest that alterations in TLR expression are associated with the pathogenesis of inflammatory and autoimmune diseases. To compare genetic studies with adjuvant studies, we examined whether stimulation through a TLR agonist induces or increases the autoimmune phenotype of healthy or autoimmune mice. C57BL/6, MRL/lpr, and Fcγr2b-deficient mice were dosed i.p. with Poly I:C every other day for 3 weeks, and monitored for signs of autoimmunity over 3 months. A separate group of mice was vaccinated three times i.m. with rPA anthrax antigen with or without Poly I:C with 2 weeks between doses. Immunized groups exhibited robust responses to vaccine and C57BL/6 and MRL/lpr mice showed a statistically significant increase in anti-rPA IgG responses in the presence of Poly I:C. Interestingly, Fcγr2b-/- mice showed increases with the base rPA vaccine, which was not significantly increased when Poly I:C was used as an adjuvant. In the chronically dosed groups, we also observed subtle alterations in levels of total antibody and some autoantibodies. However, there were no statistically significant differences in autoimmune syndrome, as measured by proteinurea, kidney pathology, weight loss, and mortality, with Poly I:C administration in chronic or vaccination mode. Taken together, these results suggest that administration of TLR3 agonists in chronic or vaccination mode does not induce or exacerbate models of systemic lupus erythematosus.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Anthrax/prevention & control , Antibodies, Bacterial/biosynthesis , Lupus Erythematosus, Systemic/immunology , Poly I-C/administration & dosage , Toll-Like Receptor 3/immunology , Animals , Anthrax/immunology , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/immunology , Autoantibodies/biosynthesis , Autoimmunity , Bacterial Toxins/administration & dosage , Bacterial Toxins/immunology , Disease Models, Animal , Female , Gene Expression , Immunization , Injections, Intramuscular , Injections, Intraperitoneal , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Transgenic , Receptors, IgG/deficiency , Receptors, IgG/genetics , Receptors, IgG/immunology , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/genetics
2.
PLoS One ; 7(9): e45276, 2012.
Article in English | MEDLINE | ID: mdl-23028902

ABSTRACT

BACKGROUND: The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1 germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur. CONCLUSIONS/SIGNIFICANCE: These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.


Subject(s)
Nerve Tissue Proteins/genetics , Receptors, Antigen, T-Cell/genetics , Th1 Cells/metabolism , Th1-Th2 Balance , Th2 Cells/metabolism , Actins/genetics , Actins/immunology , Animals , Cell Communication/genetics , Cell Differentiation , Cytokines/biosynthesis , Cytokines/immunology , Discs Large Homolog 1 Protein , Gene Expression Regulation, Developmental , Genetic Engineering , Germ-Line Mutation , Lymphocyte Activation , Mice , Mice, Knockout , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/immunology , Receptors, Antigen, T-Cell/immunology , SAP90-PSD95 Associated Proteins , Signal Transduction , Th1 Cells/cytology , Th1 Cells/immunology , Th2 Cells/cytology , Th2 Cells/immunology
3.
J Immunol ; 187(6): 2993-3002, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21849673

ABSTRACT

TCR engagement triggers the polarized recruitment of membrane, actin, and transducer assemblies within the T cell-APC contact that amplify and specify signaling cascades and T effector activity. We report that caveolin-1, a scaffold that regulates polarity and signaling in nonlymphoid cells, is required for optimal TCR-induced actin polymerization, synaptic membrane raft polarity, and function in CD8, but not CD4, T cells. In CD8(+) T cells, caveolin-1 ablation selectively impaired TCR-induced NFAT-dependent NFATc1 and cytokine gene expression, whereas caveolin-1 re-expression promoted NFATc1 gene expression. Alternatively, caveolin-1 ablation did not affect TCR-induced NF-κB-dependent Iκbα expression. Cav-1(-/-) mice did not efficiently promote CD8 immunity to lymphocytic choriomeningitis virus, nor did cav-1(-/-) OT-1(+) CD8(+) T cells efficiently respond to Listeria monocytogenes-OVA after transfer into wild-type hosts. Therefore, caveolin-1 is a T cell-intrinsic orchestrator of TCR-mediated membrane polarity and signal specificity selectively employed by CD8 T cells to customize TCR responsiveness.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Caveolin 1/immunology , Immunological Synapses/immunology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , Actins/metabolism , Animals , CD8-Positive T-Lymphocytes/metabolism , Caveolin 1/metabolism , Cell Polarity , Cell Separation , Flow Cytometry , Gene Expression , Gene Expression Regulation/immunology , Immunoblotting , Immunological Synapses/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Receptors, Antigen, T-Cell/metabolism , Reverse Transcriptase Polymerase Chain Reaction
4.
J Immunol ; 182(9): 5283-95, 2009 May 01.
Article in English | MEDLINE | ID: mdl-19380775

ABSTRACT

T cell burst size is regulated by the duration of TCR engagement and balanced control of Ag-induced activation, expansion, and apoptosis. We found that galectin-1-deficient CD8 T cells undergo greater cell division in response to TCR stimulation, with fewer dividing cells undergoing apoptosis. TCR-induced ERK signaling was sustained in activated galectin-1-deficient CD8 T cells and antagonized by recombinant galectin-1, indicating galectin-1 modulates TCR feed-forward/feedback loops involved in signal discrimination and procession. Furthermore, recombinant galectin-1 antagonized binding of agonist tetramers to the TCR on activated OT-1 T cells. Finally, galectin-1 produced by activated Ag-specific CD8 T cells negatively regulated burst size and TCR avidity in vivo. Therefore, galectin-1, inducibly expressed by activated CD8 T cells, functions as an autocrine negative regulator of peripheral CD8 T cell TCR binding, signal transduction, and burst size. Together with recent findings demonstrating that gal-1 promotes binding of agonist tetramers to the TCR of OT-1 thymocytes, these studies identify galectin-1 as a tuner of TCR binding, signaling, and functional fate determination that can differentially specify outcome, depending on the developmental and activation stage of the T cell.


Subject(s)
CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Galectin 1/physiology , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Autocrine Communication/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/transplantation , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Division/genetics , Cell Division/immunology , Cell Proliferation , Down-Regulation/genetics , Down-Regulation/immunology , Female , Galectin 1/biosynthesis , Galectin 1/deficiency , Galectin 1/genetics , Lymphocyte Activation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Protein Binding/genetics , Protein Binding/immunology , Receptors, Antigen, T-Cell/antagonists & inhibitors , Receptors, Antigen, T-Cell/deficiency , Receptors, Antigen, T-Cell/physiology , Signal Transduction/genetics
5.
Blood ; 112(1): 120-30, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18323414

ABSTRACT

During thymocyte development, the T-cell receptor (TCR) can discriminate major histocompatibility complex (MHC)/peptide ligands over a narrow range of affinities and translate subtle differences into functional fate decisions. How small differences in TCR input are translated into absolute differences in functional output is unclear. We examined the effects of galectin-1 ablation in the context of class-I-restricted thymocyte development. Galectin-1 expression opposed TCR partial agonist-driven positive selection, but promoted TCR agonist-driven negative selection of conventional CD8(+) T cells. Galectin-1 expression also promoted TCR agonist-driven CD8alphaalpha intestinal intraepithelial lymphocytes (IEL) development. Recombinant galectin-1 enhanced TCR binding to agonist/MHC complexes and promoted a negative-selection-signaling signature, reflected in intensified rapid and transient extracellular signal-regulated kinase (ERK) activation. In contrast, galectin-1 expression antagonized ERK activity in thymocytes undergoing positive selection. We propose that galectin-1 aids in discriminating TCR-directed fate decisions by promoting TCR binding to agonist/MHC complexes and enforcing agonist-driven signals, while opposing partial-agonist signals. In this way, galectin-1 widens the distinction between TCR-directed functional fate cues.


Subject(s)
Galectin 1/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Base Sequence , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , DNA Primers/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Galectin 1/genetics , Galectin 1/metabolism , Galectin 1/pharmacology , Gene Expression , H-Y Antigen/genetics , H-Y Antigen/metabolism , Histocompatibility Antigens Class I/metabolism , MAP Kinase Signaling System , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Recombinant Proteins/pharmacology , T-Lymphocytes/cytology
6.
Nat Immunol ; 8(2): 154-61, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17187070

ABSTRACT

Tyrosine kinases couple the T cell receptor (TCR) to discrete signaling cascades, each of which is capable of inducing a distinct functional outcome. Precisely how TCR signals are channeled toward specific targets remains unclear. TCR stimulation triggers 'alternative' activation of the mitogen-activated protein kinase p38, whereby the Lck and Zap70 tyrosine kinases directly activate p38. Here we report that alternatively activated p38 associated with the Dlgh1 MAGUK scaffold protein. 'Knockdown' of Dlgh1 expression blocked TCR-induced activation of p38 and the transcription factor NFAT but not of the mitogen-activated protein kinase Jnk or transcription factor NF-kappaB. A Dlgh1 mutant incapable of binding p38 failed to activate NFAT. Along with reports that the CARMA1 MAGUK scaffold protein coordinates activation of Jnk and NF-kappaB but not of p38 or NFAT, our findings identify MAGUK scaffold proteins as 'orchestrators' of TCR signal specificity.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Membrane Proteins/metabolism , NFATC Transcription Factors/metabolism , Receptors, Antigen, T-Cell/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cells, Cultured , Discs Large Homolog 1 Protein , Enzyme Activation , Guanylate Kinases , Membrane Proteins/genetics , Mice , NF-kappa B/metabolism , Phosphorylation , Protein Binding , RNA, Small Interfering/genetics , Signal Transduction , p38 Mitogen-Activated Protein Kinases/genetics
7.
J Exp Med ; 201(3): 419-30, 2005 Feb 07.
Article in English | MEDLINE | ID: mdl-15699074

ABSTRACT

Lipid raft membrane compartmentalization and membrane-associated guanylate kinase (MAGUK) family molecular scaffolds function in establishing cell polarity and organizing signal transducers within epithelial cell junctions and neuronal synapses. Here, we elucidate a role for the MAGUK protein, Dlgh1, in polarized T cell synapse assembly and T cell function. We find that Dlgh1 translocates to the immune synapse and lipid rafts in response to T cell receptor (TCR)/CD28 engagement and that LckSH3-mediated interactions with Dlgh1 control its membrane targeting. TCR/CD28 engagement induces the formation of endogenous Lck-Dlgh1-Zap70-Wiskott-Aldrich syndrome protein (WASp) complexes in which Dlgh1 acts to facilitate interactions of Lck with Zap70 and WASp. Using small interfering RNA and overexpression approaches, we show that Dlgh1 promotes antigen-induced actin polymerization, synaptic raft and TCR clustering, nuclear factor of activated T cell activity, and cytokine production. We propose that Dlgh1 coordinates TCR/CD28-induced actin-driven T cell synapse assembly, signal transduction, and effector function. These findings highlight common molecular strategies used to regulate cell polarity, synapse assembly, and transducer organization in diverse cellular systems.


Subject(s)
Actins/metabolism , Lymphocyte Activation , Membrane Microdomains/metabolism , Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , CD28 Antigens/metabolism , Cell Polarity , Cells, Cultured , DNA-Binding Proteins/metabolism , Discs Large Homolog 1 Protein , Guanylate Kinases , Humans , Interferon-gamma/metabolism , Interleukin-2/metabolism , Membrane Proteins , Mice , Mice, Transgenic , NFATC Transcription Factors , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Protein-Tyrosine Kinases/metabolism , Proteins/genetics , Receptor Aggregation , T-Lymphocytes/cytology , Transcription Factors/metabolism , Wiskott-Aldrich Syndrome Protein , ZAP-70 Protein-Tyrosine Kinase
8.
J Immunol ; 174(3): 1479-90, 2005 Feb 01.
Article in English | MEDLINE | ID: mdl-15661907

ABSTRACT

CD45 is dynamically repositioned within lipid rafts and the immune synapse during T cell activation, although the molecular consequences of CD45 repositioning remain unclear. In this study we examine the role of CD45 membrane compartmentalization in regulating murine T cell activation. We find that raft-localized CD45 antagonizes IL-2 production by opposing processive TCR signals, whereas raft-excluded CD45 promotes ERK-dependent polarized synaptic lipid raft clustering and IL-2 production. We propose that these dual CD45 activities ensure that only robust TCR signals proceed, whereas signals meeting threshold requirements are potentiated. Our findings highlight membrane compartmentalization as a key regulator of CD45 function and elucidate a novel signal transduction pathway by which raft-excluded CD45 positively regulates T cell activation.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Interleukin-2/biosynthesis , Leukocyte Common Antigens/physiology , Membrane Microdomains/enzymology , Membrane Microdomains/immunology , Receptors, Antigen, T-Cell/physiology , Signal Transduction/immunology , T-Lymphocytes/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Cell Line, Tumor , Down-Regulation/immunology , Enzyme Activation/immunology , Humans , Hybridomas , Interleukin-2/antagonists & inhibitors , Leukocyte Common Antigens/biosynthesis , Leukocyte Common Antigens/genetics , Lymphocyte Activation/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Son of Sevenless Proteins/genetics , Son of Sevenless Proteins/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Tyrosine/antagonists & inhibitors , Tyrosine/metabolism , Up-Regulation/immunology , src Homology Domains/genetics
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