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1.
Commun Biol ; 7(1): 250, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38429428

ABSTRACT

Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.


Subject(s)
Biosensing Techniques , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Signal Transduction , Receptor Protein-Tyrosine Kinases/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism
2.
bioRxiv ; 2024 Feb 28.
Article in English | MEDLINE | ID: mdl-38328157

ABSTRACT

Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 uM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.

3.
J Immunother Cancer ; 8(2)2020 10.
Article in English | MEDLINE | ID: mdl-33020243

ABSTRACT

BACKGROUND: Clinical efficacy of T cell-based cancer immunotherapy is limited by the lack of T cell infiltration in the tumor mass, especially in solid tumors. Our group demonstrated previously that leukocyte-specific protein 1 (LSP1), an intracellular signal regulator, negatively regulates T cell infiltration in inflamed tissues. METHODS: To determine the immuno-regulatory effects of LSP1 in T cells on tumor progression, we investigated the growth of B16 melanoma in Lsp1 knockout (KO) mice and T cell-specific Lsp1 transgenic (Tg) mice. The immune cell subpopulation infiltrated into the tumor mass as well as the expression of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in T cells was assessed by flow cytometry and/or immunohistochemistry. Chemotactic migration was assayed with Lsp1 KO and Lsp1 Tg T cells. Adoptive transfer of Lsp1 KO or Lsp1 Tg T cells was performed in B16 melanoma-challenged Rag1 KO mice. RESULTS: Lsp1 KO mice showed decreased growth of B16 melanoma and increased infiltration of T cells in the tumor mass, which were completely reversed in T cell-specific Lsp1 Tg mice. Lsp1 KO CD8+ T cells also exhibited elevated migratory capacity in response to CXCL9 and CXCL10, whereas Lsp1 Tg CD8+ T cells did the opposite. LSP1 expression was increased in tumor-infiltrating T cells and could be induced by T cell receptor activation. Intriguingly, gene expression profiling of Lsp1 KO T cells suggested enhanced cytotoxicity. Indeed, expression of IFN-γ and TNF-α was increased in tumor-infiltrating CD4+ and CD8+ T cells of Lsp1 KO mice, while it was markedly reduced in those of Lsp1 Tg mice. Adoptive transfer of Lsp1 KO T cells to Rag1 KO mice was more effective in suppressing melanoma growth than transfer of Lsp1 Tg T cells. Of note, when treated with antiprogrammed cell death protein 1 (PD-1) antibody, inhibition of melanoma growth was more pronounced in Lsp1 KO mice than in Lsp1-sufficient mice, suggesting that Lsp1 depletion additively increases the antitumor effects of anti-PD-1 antibody. CONCLUSIONS: LSP1 in T cells regulates the growth of B16 melanoma in mice, possibly by affecting migration and infiltration of T cells into the tumor and by modulating production of antitumor effector cytokines by CD8+ T cells. These findings provide evidence that LSP1 can be a target to improve the efficacy of T cell-based immunotherapy.


Subject(s)
Microfilament Proteins/metabolism , T-Lymphocytes/metabolism , Animals , Cell Proliferation , Humans , Mice , Tumor Microenvironment
4.
PLoS Pathog ; 13(12): e1006740, 2017 12.
Article in English | MEDLINE | ID: mdl-29267399

ABSTRACT

Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15)-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15) ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.


Subject(s)
Anti-HIV Agents/pharmacology , CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , Janus Kinase Inhibitors/pharmacology , Virus Latency/drug effects , CD4-Positive T-Lymphocytes/drug effects , Cells, Cultured , HIV-1/drug effects , HIV-1/physiology , Humans , Nitriles , Piperidines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Virus Replication/drug effects
5.
J Immunol ; 197(10): 3927-3935, 2016 11 15.
Article in English | MEDLINE | ID: mdl-27815442

ABSTRACT

To stimulate a productive T cell response, dendritic cells (DC) must undergo maturation characterized by heightened cell surface expression of MHC and costimulatory molecules as well as cytokine production. Conversely, the inhibition of DC maturation is a central mechanism of immune tolerance. The control of the DC maturation process relies on the integration of several cellular stimulatory or inhibitory signals. The soluble factors and their receptors controlling this central aspect of DC biology are incompletely characterized. We show that murine bone marrow-derived DC (BMDC) maturation induced by LPS, as opposed to polyinosinic:polycytidylic acid or cytosine-phosphate-guanine, is robustly inhibited by vascular endothelial growth factor (VEGF), a previously identified immunosuppressive cytokine. Using BMDC from wild type and conditional knockout mice, we show that neuropilin-1 (NRP-1), a known receptor of VEGF, is necessary to suppress LPS-dependent BMDC maturation. The absence of NRP-1 had no ostensible effects on the biology of BMDC in the absence of VEGF. However, NRP-1-deficient BMDC remained completely insensitive to the VEGF-dependent inhibition of BMDC maturation in culture. In the presence of VEGF, NRP-1 directly interacted with the LPS receptor TLR4 and suppressed downstream signaling through ERK and NF-κß, resulting in a sharp inhibition of MHC class II and costimulatory molecules (CD40, CD86) expression as well as proinflammatory cytokine production. Consequently, we identify NRP-1 as a target to optimize DC maturation within environments that are rich in VEGF, such as tumors.


Subject(s)
Dendritic Cells/drug effects , Dendritic Cells/physiology , Neuropilin-1/immunology , Neuropilin-1/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , B7-2 Antigen/drug effects , B7-2 Antigen/genetics , Bone Marrow Cells/immunology , Bone Marrow Cells/physiology , CD40 Antigens/drug effects , CD40 Antigens/genetics , Cell Differentiation , Cells, Cultured , Cytokines/biosynthesis , Cytokines/drug effects , Cytokines/genetics , Dendritic Cells/immunology , Genes, MHC Class II/drug effects , Genes, MHC Class II/genetics , Immune Tolerance/drug effects , Lipopolysaccharides/immunology , MAP Kinase Signaling System/physiology , Mice , NF-kappa B p50 Subunit/physiology , Neuropilin-1/deficiency , Poly I-C/pharmacology , Signal Transduction/immunology , Signal Transduction/physiology , Toll-Like Receptor 4/metabolism , Vascular Endothelial Growth Factor A/pharmacology
6.
Proc Natl Acad Sci U S A ; 112(47): E6535-43, 2015 Nov 24.
Article in English | MEDLINE | ID: mdl-26554018

ABSTRACT

Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell-dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA.


Subject(s)
Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Calcium-Binding Proteins/metabolism , Cell Movement , Microfilament Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/genetics , Calcium-Binding Proteins/deficiency , Cells, Cultured , Chronic Disease , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Dosage , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Hypersensitivity, Delayed/immunology , Hypersensitivity, Delayed/pathology , Inflammation/pathology , Mice , Microfilament Proteins/genetics , Phosphorylation , Receptors, Antigen, T-Cell/metabolism
7.
Sci Signal ; 7(324): ra43, 2014 May 06.
Article in English | MEDLINE | ID: mdl-24803538

ABSTRACT

Both pro- and anti-inflammatory cytokines activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway; however, they elicit distinct transcriptional programs. Posttranslational modifications of STAT proteins, such as tyrosine phosphorylation, are critical to ensure the differential expression of STAT target genes. Although JAK-STAT signaling is dependent on reversible tyrosine phosphorylation, whether phosphatases contribute to the specificity of STAT-dependent gene expression is unclear. We examined the role of protein tyrosine phosphatase 1B (PTP1B) in regulating the interleukin-10 (IL-10)-dependent, STAT3-mediated anti-inflammatory response. We found that IL-10-dependent STAT3 phosphorylation and anti-inflammatory gene expression were enhanced in macrophages from PTP1B(-/-) mice compared to those in macrophages from wild-type mice. Consistent with this finding, the IL-10-dependent suppression of lipopolysaccharide-induced macrophage activation was increased in PTP1B(-/-) macrophages compared to that in wild-type macrophages, as was the IL-10-dependent increase in the cell surface expression of the anti-inflammatory cytokine receptor IL-4Rα. Furthermore, RNA sequencing revealed the expression of genes encoding proinflammatory factors in IL-10-treated PTP1B(-/-) macrophages, which correlated with increased phosphorylation of STAT1, which is not normally highly activated in response to IL-10. These findings identify PTP1B as a central regulator of IL-10R-STAT3 and IL-10R-STAT1 signaling, and demonstrate that phosphatases can tailor the quantitative and qualitative properties of cytokine-induced transcriptional responses.


Subject(s)
Interleukin-10/physiology , Macrophages/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/physiology , Transcription, Genetic/physiology , Animals , Interleukin-4 Receptor alpha Subunit/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Knockout , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , STAT1 Transcription Factor/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
8.
J Leukoc Biol ; 93(5): 713-21, 2013 May.
Article in English | MEDLINE | ID: mdl-23446150

ABSTRACT

4-1BB is a member of the TNFR superfamily, which contributes to the activation of signaling pathways required for the survival of activated and memory T cells. We have shown previously that TRAF1, an adaptor protein recruited to 4-1BB, is required for 4-1BB-mediated CD8 T cell survival in vivo. With the use of a proteomics approach in primary T cells, we have identified LSP1 as a novel protein recruited to the 4-1BB signaling complex in a TRAF1-dependent manner. Further characterization of the interaction between TRAF1 and LSP1 revealed that LSP1 requires the TRAF-N domain of TRAF1 for direct association. Similarly to TRAF1(-/-) T cells, LSP1(-/-) T cells exhibit impaired ERK activation following stimulation through 4-1BB and consequently, are unable to down-modulate expression of the proapoptotic Bcl-2 family member Bim. Moreover, we demonstrate that the absence of LSP1 expression leads to defective expansion and survival of T cells in response to 4-1BB stimulation. Thus, we have identified LSP1 as a new mediator involved in 4-1BB signaling and T cell survival. Collectively, our work shows that TRAF1 and LSP1 cooperate downstream of 4-1BB to activate ERK signaling and down-modulate the levels of Bim leading to enhanced T cell survival.


Subject(s)
Calcium-Binding Proteins/physiology , T-Lymphocytes/physiology , TNF Receptor-Associated Factor 1/physiology , Tumor Necrosis Factor Receptor Superfamily, Member 9/physiology , Animals , Apoptosis Regulatory Proteins/analysis , Bcl-2-Like Protein 11 , Calcium-Binding Proteins/chemistry , Cell Survival , Extracellular Signal-Regulated MAP Kinases/physiology , Membrane Proteins/analysis , Mice , Mice, Inbred C57BL , Microfilament Proteins , Proto-Oncogene Proteins/analysis , Signal Transduction , TNF Receptor-Associated Factor 1/chemistry , TNF Receptor-Associated Factor 2/physiology
9.
Biochem Biophys Res Commun ; 432(1): 129-34, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23376065

ABSTRACT

The Tumour Necrosis Factor (TNF) Receptor-associated factor-1 (TRAF1) adaptor protein is a key component in initiating intracellular signalling pathways downstream of TNF receptors (TNFR). More importantly, TRAF1 has a pattern of expression restricted primarily to lymphoid cells and plays an important role in lymphocyte survival. TRAF1 has been shown to be phosphorylated on Serine 139, consequently inhibiting NF-κB activation downstream of TNFR2 when expressed in HeLa cells. We have previously demonstrated that TRAF1 cooperates with the TNFR family member 4-1BB to mediate signalling in T cells. However, the impact of TRAF1 phosphorylation on events downstream of 4-1BB in T cells remained to be defined. Using a proteomics approach we demonstrate that TANK-binding kinase 1 (TBK1) preferentially associates with the TRAF1 Serine 139 to Alanine (S139A) mutant. TBK1 is a kinase that functions upstream of NIK and IKK in the activation of the NF-κB pathway. When TRAF1-deficient CD8 T cells were reconstituted with the TRAF1 S139A mutant, we observed more sustained levels of IκBα degradation in response to 4-1BB stimulation in contrast to cells expressing either TRAF1 wild-type or TRAF1 S139D phospho-mimetic mutant. Together, these findings define the importance of the basal phosphorylation state of the TRAF1 Serine 139 residue in coordinating signalling events downstream of 4-1BB in primary T cells.


Subject(s)
NF-kappa B/metabolism , T-Lymphocytes/metabolism , TNF Receptor-Associated Factor 1/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Animals , I-kappa B Proteins , Mice , Mice, Mutant Strains , NF-KappaB Inhibitor alpha , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Serine/metabolism , Signal Transduction , TNF Receptor-Associated Factor 1/genetics
11.
J Immunol ; 180(12): 8093-101, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18523273

ABSTRACT

During an acute immune response, CD8 T cells undergo rapid expansion followed by a contraction phase during which the majority of activated T cells die, leaving a few survivors to persist as memory cells. The regulation of T cell survival is critical at each stage of this response. 4-1BB, a TNFR family member, has been implicated in prolonging the survival of activated and memory CD8 T cells; however, the precise mechanisms by which 4-1BB sustains T cell survival are incompletely understood. Upon aggregation on T cells, 4-1BB associates with two TNFR-associated factors (TRAF), TRAF1 and TRAF2. TRAF2 is essential for downstream signaling from 4-1BB; however, the role of TRAF1 in 4-1BB signaling has not been elucidated and there have been conflicting data as to whether TRAF1 provides a positive or a negative signal in T cells. In this study, we report that TRAF1 plays a critical role in survival signaling downstream of 4-1BB during CD8 T cell expansion in response to viral infection in vivo. Further analysis reveals that TRAF1-deficient cells are impaired in their ability to up-regulate the prosurvival Bcl-2 family member Bcl-x(L) and show increased levels of the proapoptotic Bcl-2 family member Bim following 4-1BB signaling. TRAF1-deficient CD8 T cells fail to activate ERK in response to 4-1BB ligation and inhibition of ERK signaling downstream of 4-1BB in wild-type cells leads to increased Bim levels. Thus, TRAF1 has a prosurvival effect in CD8 T cells via the 4-1BB-mediated up-regulation of Bcl-x(L) and ERK-dependent Bim down-modulation.


Subject(s)
Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/physiology , Apoptosis/immunology , CD8-Positive T-Lymphocytes/immunology , Extracellular Signal-Regulated MAP Kinases/physiology , MAP Kinase Signaling System/immunology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/physiology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/physiology , TNF Receptor-Associated Factor 1/physiology , Tumor Necrosis Factor Receptor Superfamily, Member 9/physiology , Animals , Apoptosis/genetics , Bcl-2-Like Protein 11 , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/virology , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , MAP Kinase Signaling System/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/immunology , TNF Receptor-Associated Factor 1/deficiency , TNF Receptor-Associated Factor 1/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/deficiency , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics , bcl-X Protein/biosynthesis
12.
J Clin Invest ; 117(12): 3833-45, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17992258

ABSTRACT

TNF-alpha antagonists are widely used in the treatment of inflammatory and autoimmune diseases, but their use is associated with reactivation of latent infections. This highlights the importance of TNF-alpha in immunity to certain pathogens and raises concerns that critical aspects of immune function are impaired in its absence. Unfortunately, the role of TNF-alpha in the regulation of T cell responses is clouded by a myriad of contradictory reports. Here, we show a role for TNF-alpha and its receptors, TNFR1 and TNFR2, specifically in antitumor immunity. TNF-alpha-deficient mice exhibited normal antiviral responses associated with strong inflammation. However, TNF-alpha/TNFR1-mediated signals on APCs and TNF-alpha/TNFR2 signals on T cells were critically required for effective priming, proliferation, and recruitment of tumor-specific T cells. Furthermore, in the absence of TNF-alpha signaling, tumor immune surveillance was severely abrogated. Finally, treatment with a CD40 agonist alone or in combination with TLR2 stimuli was able to rescue proliferation of TNF-alpha-deficient T cells. Therefore, TNF-alpha signaling may be required only for immune responses in conditions of limited immunostimulatory capacity, such as tumor surveillance. Importantly, these results suggest that prolonged continuous TNF-alpha blockade in patients may have long-term complications, including potential tumor development or progression.


Subject(s)
Immunologic Surveillance , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Neoplasms/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Antigen-Presenting Cells/immunology , CD40 Ligand/genetics , CD40 Ligand/immunology , Cell Proliferation , Cells, Cultured , Humans , Immunologic Surveillance/genetics , Inflammation/genetics , Inflammation/immunology , Lymphocytic Choriomeningitis/genetics , Mice , Mice, Knockout , Neoplasms/genetics , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/immunology , Receptors, Tumor Necrosis Factor, Type II/genetics , Receptors, Tumor Necrosis Factor, Type II/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunology , Tumor Necrosis Factor-alpha/genetics
13.
Trends Immunol ; 28(8): 333-9, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17597006

ABSTRACT

Immunological memory is a critical feature of the adaptive immune system and the underlying principal behind vaccination. The mechanisms that maintain memory T cell survival between the initial and subsequent encounter with antigen remain incompletely defined. Although the cytokines IL-15 and IL-7 are important in memory T cell homeostasis, additional signals by way of TNFR family members are required for maximal maintenance of T cell memory. Here we propose a unifying model in which subsets of TNF family ligands distinguish the competitive niches for maintenance of CD4 versus CD8 T cell memory. Understanding the unique 'memory niches' defined by TNF family ligand expression will provide new insights into the mechanisms of memory T cell maintenance.


Subject(s)
Immunologic Memory , Receptors, Tumor Necrosis Factor/physiology , T-Lymphocytes/immunology , 4-1BB Ligand/physiology , Animals , CD11c Antigen/physiology , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/immunology , Cytokines/physiology , Humans , Ki-1 Antigen/physiology , Receptors, OX40/physiology , TNF Receptor-Associated Factor 1/physiology
14.
Proc Natl Acad Sci U S A ; 103(49): 18703-8, 2006 Dec 05.
Article in English | MEDLINE | ID: mdl-17116875

ABSTRACT

The mechanisms that allow the maintenance of immunological memory remain incompletely defined. Here we report that tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 1, a protein recruited in response to several costimulatory TNFR family members, is required for maximal CD8 T cell responses to influenza virus in mice. Decreased recovery of CD8 T cells in vivo occurred under conditions where cell division was unimpaired. In vitro, TRAF1-deficient, antigen-activated T cells accumulated higher levels of the proapoptotic BH3-only family member Bim, particularly the most toxic isoform, Bim(S). In the presence of excess IL-15, memory phenotype T cells with similar surface phenotype and comparable levels of Bcl-2 family members could be generated from WT or TRAF1-deficient T cell receptor transgenic OT-I T cells. However, when the memory CD8 T cells were allowed to compete for survival signals in the absence of antigen in vivo, the TRAF1-deficient T cells showed decreased recovery compared with TRAF1-sufficient T cells. This defect in T cell recovery in vivo was alleviated by introduction of siRNA to down-modulate Bim in TRAF1-deficient memory T cells. These studies identify the TRAF1 signaling axis and Bim down-regulation as critical for CD8 memory T cell survival in vivo.


Subject(s)
Apoptosis Regulatory Proteins/antagonists & inhibitors , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory , Membrane Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , TNF Receptor-Associated Factor 1/physiology , Animals , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , CD8-Positive T-Lymphocytes/cytology , Cell Survival/genetics , Cell Survival/immunology , Immunologic Memory/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Signal Transduction/genetics , Signal Transduction/immunology , TNF Receptor-Associated Factor 1/deficiency , TNF Receptor-Associated Factor 1/genetics
15.
J Immunol ; 176(9): 5438-45, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16622011

ABSTRACT

The onset of activation in Th cells is triggered by localized co-engagement of TCRs and the coreceptor CD4. A CD4 crystal suggested that CD4 may form dimers in some circumstances. In this study, we use live-cell fluorescence resonance energy transfer imaging to demonstrate that CD4 dimers are present at a basal level on the cell surface and accumulate at the synapse. Mechanistically, we reveal two conditions under which dimers are highly relevant. First, CD4 dimers are more proficient in mediating prolonged cell contacts with APCs in the presence or absence of Ag. This is consistent with a model whereby the dimer functions to increase T-APC avidity. Second, we show that dimer mutations result in an increased level of an inactive lckTyr(505) bound to the CD4 molecule relative to dimer-competent CD4. We also find a consistent defect in signaling onset in these cells. This supports a role for CD4 dimerization in maintaining active signaling machinery. We suggest that modulation of the dimer/monomer ratio may permit tuning of activation thresholds during initial engagement.


Subject(s)
CD4 Antigens/immunology , CD4 Antigens/metabolism , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , CD4 Antigens/genetics , Calcium/metabolism , Cell Line , Dimerization , Fluorescence Resonance Energy Transfer , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mice , Mutation/genetics , Phosphorylation , Protein Binding , Signal Transduction
16.
DNA Cell Biol ; 25(2): 104-15, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16460234

ABSTRACT

Several studies have shown that the levels of caspase-3 are upregulated under different conditions of apoptosis. Previously, we have shown that activation of T cells through the TCR leads to the upregulation of caspase-3 levels. These findings highlight the importance of regulating the expression of caspase-3 in order to prevent premature cell death. To better understand the regulation of the caspase-3 gene, a portion of the 5'- untranslated region was cloned, sequenced, and characterized. The segment of the 5'-flanking region of the caspase-3 gene was also cloned upstream of a luciferase reporter gene, demonstrating that this fragment contains promoter activity. Higher luciferase expression was found with several of the promoter deletion constructs in Jurkat T cells but not the mouse Neuro-2A neuroblastoma cell line, suggesting the presence of a T-cell-specific regulated region. The importance of these sequences is further supported by the genomic organization of the human and mouse caspase-3 promoter regions. These findings demonstrated that the -2245/+14 region of the caspase-3 promoter shows constitutive levels of expression, and that several regions of the promoter play a role in basal regulation. Finally, some of the conserved transcription factor binding sites identified between the human and mouse promoters appear to play an important role in lymphoid cells.


Subject(s)
5' Untranslated Regions , Caspases/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Caspase 3 , Cell Line, Tumor , Cloning, Molecular , Genes, Reporter , Humans , Mice , Molecular Sequence Data
17.
Mol Immunol ; 42(11): 1345-54, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15950730

ABSTRACT

Activation-induced cell death (AICD) in T lymphocytes depends on the expression of Fas-ligand, which triggers the apoptotic process after binding to its receptor Fas. This leads to the activation of cysteine proteases of the caspase family and especially of caspase-3, a critical effector protein during AICD. We have previously observed the up-regulation of caspase-3 expression in effector but not memory T cells stimulated in vivo. In this study, we further characterized the regulation of caspase expression following T cell receptor (TCR) signaling and demonstrate that a three-fold increase in caspase-3 mRNA levels was observed by semi-quantitative and real-time RT-PCR analysis. Caspase-3 expression was selectively increased among five different caspases following TCR stimulation, as assessed by RNase protection assay. Real-time RT-PCR analysis demonstrated that a three-fold up-regulation in caspase-3 mRNA levels was observed following TCR triggering, whereas caspase-8 mRNA levels remained unchanged. The increase in caspase-3 mRNA levels occurred before cleavage and activation of caspase-3 and in the absence of apoptosis. TCR-mediated induction in caspase-3 expression was not dependent on STAT1 activation, since following stimulation of KOX-14 cells the transcription factor was not phosphorylated. Together, these results show that TCR activation triggers the selective increase in caspase-3 mRNA levels, independently of caspase activity and the induction of apoptosis.


Subject(s)
Caspases/genetics , Receptors, Antigen, T-Cell/metabolism , Animals , Apoptosis , Base Sequence , Caspase 3 , Caspases/metabolism , DNA/genetics , DNA-Binding Proteins/metabolism , Fas Ligand Protein , Gene Expression Regulation, Enzymologic , Hybridomas/cytology , Hybridomas/enzymology , Hybridomas/immunology , In Vitro Techniques , Lymphocyte Activation , Membrane Glycoproteins/metabolism , Mice , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , STAT1 Transcription Factor , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Trans-Activators/metabolism , Up-Regulation
18.
J Immunol ; 173(9): 5425-33, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15494489

ABSTRACT

Caspases play a central role in T lymphocyte activation and death. We have demonstrated previously that caspase-3, an effector molecule for activation-induced cell death (AICD), is processed following T cell activation in the absence of apoptosis. We report in this study that caspase-3 mRNA levels were selectively increased in peripheral T cells, following Ag receptor-mediated activation. The up-regulation of caspase-3 mRNA was confined to cells in the early phases of the cell cycle (G0/G1) and was independent of IL-2 signaling. This increase led to the renewal of procaspase-3 as evidenced by a 6-fold up-regulation of the zymogen in nonapoptotic stimulated T cells. The increase of mRNA levels and of both the zymogen and the cleaved forms of caspase-3 was observed in in vivo stimulated Ag-specific effector, but not memory T cells, correlating with the enhanced susceptibility of effector T cells to AICD. Furthermore, we confirm that caspase-3 levels directly influence the sensitivity of activated T cells to apoptosis, as shown using T lymphocytes isolated from caspase-3 heterozygous and knockout mice. These findings indicate that the selective up-regulation of caspase-3 transcription is required to maintain the cytoplasmic levels of this protease, which control AICD and T cell homeostasis.


Subject(s)
Apoptosis/immunology , Caspases/biosynthesis , Immunologic Memory , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/enzymology , T-Lymphocytes, Regulatory/immunology , Up-Regulation/immunology , Animals , Apoptosis/genetics , Caspase 3 , Caspases/deficiency , Caspases/genetics , Caspases/metabolism , Enzyme Activation/immunology , Epitopes, T-Lymphocyte/immunology , G1 Phase/immunology , Immunity, Innate/genetics , Immunologic Memory/genetics , Interleukin-2/physiology , Lymphocyte Activation/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , RNA, Messenger/biosynthesis , Receptors, Antigen, T-Cell/immunology , Resting Phase, Cell Cycle/immunology , T-Lymphocyte Subsets/cytology , T-Lymphocytes, Regulatory/cytology
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