ABSTRACT
BACKGROUND: The class III NAD-dependent histone deacetylase (HDAC) sirtuin 1 (SIRT1) is an important regulator of senescence, aging, and inflammation. SIRT1de-acetylates chromatin histones, thereby silencing inflammatory gene transcription. We have reported increased steroid-resistant senescent pro-inflammatory CD28nullCD8+ T cells in patients with chronic obstructive pulmonary disease (COPD). We hypothesized that SIRT1 is reduced in these cells in COPD, and that treatment with SIRT1 activators (resveratrol, curcumin) and agents preventing NAD depletion (theophylline) would upregulate SIRT1 and reduce pro-inflammatory cytokine expression in these steroid-resistant cells. METHODS: Blood was collected from n = 10 COPD and n = 10 aged-matched controls. Expression of CD28, SIRT1, and pro-inflammatory cytokines was determined in CD8+ and CD8- T and natural killer T (NKT)-like cells cultured in the presence of ±1 µM prednisolone, ±5 mg/L theophylline, ±1 µM curcumin, ±25 µM resveratrol, using flow cytometry and immunofluorescence. RESULTS: There was an increase in the percentage of CD28nullCD8+ T and NKT-like cells in COPD patients compared with controls. Decreased SIRT1 expression was identified in CD28nullCD8+T and NKT-like cells compared with CD28+ counterparts from both patients and controls (e.g. CD28null 11 ± 3% versus CD28+ 57 ± 9%). Loss of SIRT1 was associated with increased production of IFNγ and TNFα, steroid resistance, and disease severity. SIRT1 expression was upregulated in the presence of all drugs and was associated with a decrease in steroid resistance and IFNγ and TNFα production by CD28nullCD8+T and NKT-like cells. The presence of the SIRT1 inhibitor, EX-527 negated [by 92 ± 12% (median ± SEM)] the effect of the SIRT1 activator SRT720 on the percentage of CD8+ T cells producing IFNγ and TNFα. CONCLUSIONS: Steroid resistance in pro-inflammatory CD28nullCD8+ T and NKT-like cells is associated with decreased SIRT1 expression. Treatment with prednisolone, in combination with theophylline, curcumin or resveratrol increases SIRT1 expression, restores steroid sensitivity, and inhibits pro-inflammatory cytokine production from these cells and may reduce systemic inflammation in COPD. The reviews of this paper are available via the supplemental material section.
Subject(s)
CD8-Positive T-Lymphocytes/enzymology , Cytokines/metabolism , Immunosenescence , Inflammation Mediators/metabolism , Natural Killer T-Cells/enzymology , Pulmonary Disease, Chronic Obstructive/enzymology , Sirtuin 1/metabolism , Aged , Anti-Inflammatory Agents/pharmacology , CD28 Antigens/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Case-Control Studies , Cells, Cultured , Curcumin/pharmacology , Drug Resistance , Female , Glucocorticoids/pharmacology , Humans , Immunosenescence/drug effects , Male , Middle Aged , Natural Killer T-Cells/drug effects , Natural Killer T-Cells/immunology , Phenotype , Prednisolone/pharmacology , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/immunology , Resveratrol/pharmacology , Theophylline/pharmacologyABSTRACT
BACKGROUND: NKT cells play an important role in anti-tumor immunity. Alpha-galactosylceramide (α-GalCer), a synthetic glycolipid is presented to natural killer T (NKT) cells by most antigen-presenting cells through CD1d molecules leading to activation of NKT cells. However, the precise mechanisms of how α-GalCer-activated NKT regulate the polarization of the macrophages and effector T cells in the solid tumor are not studied adequately. METHODS: We induced solid tumor in C57BL/6 mice by subcutaneous injection of B16F10 cell line (1 X 106 cells) and monitored the tumor growth. Animals were given an intraperitoneal injection of α-GalCer (2 µg/injection) in 200 µl PBS on day + 1, + 5, + 10, + 15, and + 20 (with respect to tumor cell injection). Immune cells were characterized using flow cytometry and immunofluorescence staining. NK cells, Gr1+ cells, and F4/80+ macrophages in the mice were depleted by intravenous injection of cell-specific antibodies. Statistical analysis was performed using Student's t-test or one-way ANOVA. RESULTS: Our results showed that intratumoral NKT cells have a lower frequency of CD69, CD25, CD122, and IFN-γR expression; produced less inflammatory cytokines such as IFN-γ, TNF-α, and GM-CSF; higher frequency CD62L+ NKT cells; and also showed reduced proliferation as compared to the splenic NKT cells. Mice treated with α-GalCer showed a significantly increased frequency of IFN-γ-producing NKT cells, CD8+ T cells, and effector Th1 cells. Depletion of NK cells in α-GalCer-treated mice showed a lower frequency of IFN-γ-producing CD4+ and CD8+ T cells in the tumor and prevented the α-GalCer-induced tumor growth. NKT cell activation with α-GalCer treatment significantly increased the iNOS+CD206- M1-macrophages and reduced the iNOS-CD206+ M2-macrophages in the spleen and tumor, and depletion of F4/80+ macrophages prevented the α-GalCer-induced reduction in the tumor growth. CONCLUSIONS: We showed that activation of NKT cell with α-GalCer modulates the frequency of M1-macrophages and effector Th1 cells in the secondary lymphoid tissues and tumor microenvironment and inhibit tumor growth. The finding suggests that activation of NKT cells with α-GalCer may provide an effective anti-cancer outcome.
Subject(s)
Lectins, C-Type/immunology , Macrophages/immunology , Mannose-Binding Lectins/immunology , Natural Killer T-Cells/immunology , Nitric Oxide Synthase Type II/immunology , Receptors, Cell Surface/immunology , Animals , Lectins, C-Type/metabolism , Lymphocyte Activation , Macrophages/enzymology , Macrophages/metabolism , Male , Mannose Receptor , Mannose-Binding Lectins/metabolism , Melanoma, Experimental/enzymology , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/metabolism , Nitric Oxide Synthase Type II/metabolism , Receptors, Cell Surface/metabolism , Tumor Microenvironment/immunologyABSTRACT
Invariant natural killer T cells (iNKT cells) develop through an incompletely understood process that requires positive selection by CD4+CD8+ double-positive thymocytes and SLAM family receptors (SFRs). Here we found that SFRs promoted the development of iNKT cells by reducing the strength of the T cell antigen receptor (TCR) signal after positive selection. This effect improved the survival of iNKT cells and their responses to antigen. Loss of SFRs upregulated the expression of inhibitory receptors, including PD-1, on iNKT cells to mitigate the deleterious effect of SFR deficiency. The role of SFRs could be mimicked by expression of SLAMF6 alone in SFR-deficient mice, and this involved the adaptor SAP-kinase Fyn complex and the phosphatase SHP-1. Thus, SFRs foster iNKT cell development by attenuating TCR signal strength after positive selection.
Subject(s)
Natural Killer T-Cells/immunology , Receptors, Antigen, T-Cell/metabolism , Signaling Lymphocytic Activation Molecule Family/physiology , Animals , Cell Proliferation , Cell Survival , Costimulatory and Inhibitory T-Cell Receptors/metabolism , Humans , Mice , Mice, Knockout , Natural Killer T-Cells/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-fyn/metabolism , Receptors, Antigen, T-Cell/immunology , Signal Transduction , Signaling Lymphocytic Activation Molecule Associated Protein/genetics , Signaling Lymphocytic Activation Molecule Family/genetics , Signaling Lymphocytic Activation Molecule Family/metabolismABSTRACT
CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Subject(s)
Antigens, CD1d/immunology , Immunity, Innate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , rho-Associated Kinases/immunology , Amides/pharmacology , Cell Differentiation , Dendritic Cells/enzymology , Dendritic Cells/immunology , Enzyme Inhibitors/pharmacology , Fusion Proteins, bcr-abl/immunology , Humans , Imatinib Mesylate/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Myeloid Cells/enzymology , Myeloid Cells/immunology , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/immunology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
BACKGROUND: Histone acetyltransferases (HAT) and histone deacetylases (HDAC) are enzymes that upregulate and down-regulate pro-inflammatory gene transcription respectively. HDAC2 is required by corticosteroids to switch off activated inflammatory genes and is reduced in lung macrophages in COPD. We have shown that COPD patients have increased steroid resistant CD28null (senescent) pro-inflammatory T and NKT-like peripheral blood cells (particularly CD8+ subsets) and we hypothesized that these changes would be associated with a loss of HDAC2 from these senescent pro-inflammatory lymphocytes. METHODS: Blood was collected from 10 COPD and 10 aged-matched controls. Intracellular pro-inflammatory cytokines, IFNγ and TNFα, and expression of CD28, HDAC2 and HAT, were determined in lymphocyte subsets in the presence of ± 5 mg/ml theophylline (HDAC2 activator), 10 µM prednisolone and 2.5 ng/ml cyclosporine A (immunosuppressant), using flow cytometry. RESULTS: There was a loss of HDAC2 from CD28null CD8+ T and NKT-like cells in COPD. There was a significant negative correlation between HDAC2 expression and the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -.763, p < 0.001 for T-cell IFNγ). There was a synergistic upregulation of HDAC2 and associated decrease in pro-inflammatory cytokine production in CD28nullCD8+ T and NKT-like cells in the presence of 5 mg/L theophylline + 10(-6) M prednisolone or 2.5 ng/mL cyclosporine A (CsA). CONCLUSIONS: Lymphocyte senescence in COPD is associated with loss of HDAC2 in CD28nullCD8+ T and NKT-like cells. Alternative treatment options such as combined theophylline with low-dose CsA, that inhibit these pro-inflammatory cells, may reduce systemic inflammation in COPD.
Subject(s)
CD8-Positive T-Lymphocytes/enzymology , Cellular Senescence , Histone Deacetylase 2/blood , Pulmonary Disease, Chronic Obstructive/enzymology , Adult , Aged , CD28 Antigens/blood , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Case-Control Studies , Cellular Senescence/drug effects , Cyclosporine/pharmacology , Down-Regulation , Enzyme Activation , Enzyme Activators/pharmacology , Female , Histone Acetyltransferases/blood , Humans , Immunosuppressive Agents/pharmacology , Interferon-gamma/blood , Male , Middle Aged , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/immunology , Phenotype , Prednisolone/pharmacology , Pulmonary Disease, Chronic Obstructive/blood , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/immunology , Theophylline/pharmacology , Tumor Necrosis Factor-alpha/bloodABSTRACT
ADP-ribosyltransferases comprise a family of enzymes originally discovered as bacterial toxins and later characterised also in mammals. In mice, the ADP-ribosyltransferase ARTC2.2 is expressed at the surface of T lymphocytes and has been studied extensively. In the presence of extracellular NAD(+), ARTC2.2 ADP-ribosylates several cell surface target proteins and thereby regulates their function. P2X7, an ATP-gated cation channel, has been discovered as a prominent ARTC2.2 target at the surface of mouse T cells. ADP-ribosylation of P2X7 in the presence of low micromolar extracellular NAD(+) induces long-lasting P2X7 activation and triggers cell death. Regulatory T cell subsets (Tregs and NKT cells) are remarkably sensitive to NAD(+)-induced cell death (NICD). Thus, liberation of endogenous NAD(+) by stressed cells is now viewed as a danger signal promoting immune responses by hindering regulatory T cells. This review will highlight the recent discoveries on the in vivo role of the ARTC2.2/P2X7 pathway triggered by the endogenous release of extracellular NAD(+), the relative sensitivity of lymphocytes subsets to this regulatory pathway and its pharmacological manipulation using camelid-derived ARTC2.2-blocking nanobodies.
Subject(s)
ADP Ribose Transferases/metabolism , Adenosine Diphosphate Ribose/metabolism , Natural Killer T-Cells/cytology , Natural Killer T-Cells/metabolism , Receptors, Purinergic P2X7/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , ADP Ribose Transferases/genetics , Animals , Cell Death , Humans , Mice , NAD/metabolism , Natural Killer T-Cells/enzymology , Receptors, Purinergic P2X7/genetics , T-Lymphocytes, Regulatory/enzymologyABSTRACT
Invariant natural killer T (iNKT) cells play important roles in bridging innate and adaptive immunity via rapidly producing a variety of cytokines. A small subset of iNKT cells produces IL-17 and is generated in the thymus during iNKT-cell ontogeny. The mechanisms that control the development of these IL-17-producing iNKT-17 cells (iNKT-17) are still not well defined. Diacylglycerol kinase ζ (DGKζ) belongs to a family of enzymes that catalyze the phosphorylation and conversion of diacylglycerol to phosphatidic acid, two important second messengers involved in signaling from numerous receptors. We report here that DGKζ plays an important role in iNKT-17 development. A deficiency of DGKζ in mice causes a significant reduction of iNKT-17 cells, which is correlated with decreased RORγt and IL-23 receptor expression. Interestingly, iNKT-17 defects caused by DGKζ deficiency can be corrected in chimeric mice reconstituted with mixed wild-type and DGKζ-deficient bone marrow cells. Taken together, our data identify DGKζ as an important regulator of iNKT-17 development through iNKT-cell extrinsic mechanisms.
Subject(s)
Diacylglycerol Kinase/physiology , Interleukin-17/metabolism , Natural Killer T-Cells/cytology , Natural Killer T-Cells/enzymology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Receptors, Interleukin/metabolism , Animals , Blotting, Western , Bone Marrow/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Diglycerides/metabolism , Flow Cytometry , Interleukin-17/genetics , Mice , Mice, Congenic , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Phosphatidic Acids/metabolism , Phosphorylation , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Antigen, T-Cell, alpha-beta/physiology , Receptors, Interleukin/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Spleen/cytology , Spleen/metabolism , Thymocytes/cytology , Thymocytes/metabolismABSTRACT
Critically ill patients are routinely exposed to high concentrations of supplemental oxygen for prolonged periods of time, which can be life-saving in the short term, but such exposure also causes severe lung injury and increases mortality. To address this therapeutic dilemma, we studied the mechanisms of the tissue-damaging effects of oxygen in mice. We show that pulmonary invariant natural killer T (iNKT) cells are unexpectedly crucial in the development of acute oxygen-induced lung injury. iNKT cells express high concentrations of the ectonucleotidase CD39, which regulates their state of activation. Both iNKT cell-deficient (Jα18(-/-)) and CD39-null mice tolerate hyperoxia, compared with wild-type control mice that exhibit severe lung injury. An adoptive transfer of wild-type iNKT cells into Jα18(-/-) mice results in hyperoxic lung injury, whereas the transfer of CD39-null iNKT cells does not. Pulmonary iNKT cell activation and proliferation are modulated by ATP-dependent purinergic signaling responses. Hyperoxic lung injury can be induced by selective P2X7-receptor blockade in CD39-null mice. Our data indicate that iNKT cells are involved in the pathogenesis of hyperoxic lung injury, and that tissue protection can be mediated through ATP-induced P2X7 receptor signaling, resulting in iNKT cell death. In conclusion, our data suggest that iNKT cells and purinergic signaling should be evaluated as potential novel therapeutic targets to prevent hyperoxic lung injury.
Subject(s)
Acute Lung Injury/immunology , Hyperoxia/immunology , Natural Killer T-Cells/immunology , Acute Lung Injury/etiology , Acute Lung Injury/pathology , Adoptive Transfer , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Apoptosis , Apyrase/genetics , Apyrase/metabolism , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Hyperoxia/complications , Hyperoxia/pathology , Lung/immunology , Lung/pathology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/physiology , Neutrophil Infiltration , Neutrophils/immunology , Neutrophils/metabolismABSTRACT
Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.
Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Globosides/physiology , Natural Killer T-Cells/immunology , Trihexosylceramides , Animals , Carbohydrate Sequence , Dendritic Cells/enzymology , Dendritic Cells/metabolism , Globosides/deficiency , Liver/cytology , Liver/enzymology , Liver/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/metabolism , Spleen/cytology , Spleen/enzymology , Spleen/metabolism , Thymus Gland/cytology , Thymus Gland/enzymology , Thymus Gland/metabolism , Trihexosylceramides/deficiency , Trihexosylceramides/physiology , alpha-Galactosidase/genetics , alpha-Galactosidase/physiologyABSTRACT
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem-cell malignancy characterized by the presence of the chimeric BCR-ABL oncoprotein with deregulated tyrosine-kinase (TK) activity. Although conventional T cells are acknowledged as important players in the control of CML, a possible modification of invariant NKT (iNKT) cells, known for their antitumoral activity, has not been established as yet. Here, we showed that the expression of perforin, CD95L, and promyelocytic leukemia zinc finger, a transcription factor required for maintenance of iNKT cell functions, was reduced or suppressed in CML patients at diagnosis, as compared with healthy individuals. The proliferation rate of blood iNKT cells in response to their cognate ligand was likewise diminished. These functional deficiencies were corrected in patients having achieved complete cytogenetic remission following TK inhibitor or IFN-α therapy. iNKT cells from CML patients in the chronic phase did not display increased TK activity, which argued against a direct autonomous action of BCR-ABL. Instead, we found that their anergic status originated from both intrinsic and APC-dependent dysfunctions. Our data demonstrate that chronic phase CML is associated with functional deficiencies of iNKT cells that are restored upon remission. These results suggest a possible contribution to disease control by TK inhibitor therapies.
Subject(s)
Interferon-alpha/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Natural Killer T-Cells/immunology , Protein-Tyrosine Kinases/immunology , Benzamides , Fas Ligand Protein/blood , Flow Cytometry , Humans , Imatinib Mesylate , Kruppel-Like Transcription Factors/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Natural Killer T-Cells/enzymology , Perforin/blood , Piperazines/pharmacology , Promyelocytic Leukemia Zinc Finger Protein , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacologyABSTRACT
It is well established that dendritic cells (DCs) take up, process, and present lipid Ags in complex with CD1d molecules to invariant NKT cells. The lipid-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), has previously been shown to regulate CD1d expression in human monocyte-derived DCs, providing a link between lipid metabolism and lipid Ag presentation. We report that PPARγ regulates the expression of a lysosomal protease, cathepsin D (CatD), in human monocyte-derived DCs. Inhibition of CatD specifically reduced the expansion of invariant NKT cells and furthermore resulted in decreased maturation of saposins, a group of lipid transfer proteins required for lysosomal lipid Ag processing and loading. These results reveal a novel mechanism of lipid Ag presentation and identify CatD as a key component of this machinery and firmly place PPARγ as the transcriptional regulator linking lipid metabolism and lipid Ag processing.
Subject(s)
Antigen Presentation/immunology , Cathepsin D/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Lipoproteins/metabolism , PPAR gamma/physiology , Antigens, CD1d/immunology , Antigens, CD1d/metabolism , Cathepsin D/biosynthesis , Cathepsin D/physiology , Cells, Cultured , Coculture Techniques , Humans , Lipid Metabolism/immunology , Lipoproteins/immunology , Lysosomes/enzymology , Lysosomes/metabolism , Monocytes/immunology , Monocytes/metabolism , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Saposins/metabolism , Saposins/physiology , Signal Transduction/immunology , Up-Regulation/immunologyABSTRACT
iNKT cells derive from CD4(+)CD8(+) DP thymocytes, and are selected by thymocyte-thymocyte interactions through signals from their invariant Vα14-Jα18 TCR and from the costimulatory molecules SLAMF1 and SLAMF6. Genetic studies have demonstrated the contribution of different signaling pathways to this process. Surprisingly, current models imply that the Ras/MAPK pathway, one of the critical mediators of conventional αß T cell positive selection, is not necessary for iNKT cell development. Using mice defective at different levels of this pathway our results refute this paradigm, and demonstrate that Ras, and its downstream effectors Egr-1 and Egr-2 are required for positive selection of iNKT cells. Interestingly our results also show that there are differences in the contributions of several of these molecules to the development of iNKT and conventional αß T cells.
Subject(s)
MAP Kinase Signaling System , Natural Killer T-Cells/cytology , Natural Killer T-Cells/enzymology , ras Proteins/metabolism , Animals , Antigens, CD/metabolism , Antigens, CD1d/metabolism , Early Growth Response Protein 1/metabolism , Early Growth Response Protein 2/metabolism , Integrases/metabolism , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Cell Surface/metabolism , Signaling Lymphocytic Activation Molecule Family , Signaling Lymphocytic Activation Molecule Family Member 1 , Thymus Gland/cytologyABSTRACT
The Tec family tyrosine kinase interleukin-2 inducible T-cell kinase (Itk) is predominantly expressed in T cells and has been shown to be critical for the development, function and differentiation of conventional αß T cells. However, less is known about its role in nonconventional T cells such as NKT and γδ T cells. In this minireview, we discuss evidence for a role for Itk in the development of invariant NKT αß cells, as well as a smaller population NKT-like γδ T cells. We discuss how these cells take what could be the same signaling pathway regulated by Itk, and interpret it to give different outcomes with regards to development and function.
Subject(s)
Natural Killer T-Cells/enzymology , Natural Killer T-Cells/immunology , Protein-Tyrosine Kinases/immunology , Animals , Cell Differentiation/immunology , Humans , Mice , Models, Immunological , Natural Killer T-Cells/classification , Natural Killer T-Cells/cytology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Signal Transduction/immunology , T-Lymphocyte Subsets/classification , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunologyABSTRACT
NKT cells are known to rapidly produce a large amount of cytokines upon activation. Although a number of signaling pathways that regulate the development of NKT cells have been identified, the signaling pathways involved in the regulation of NKT cell cytokine production remain unclear. In this study, we show that the p38 MAPK pathway is dispensable for the development of NKT cells. However, NKT cell cytokine production and NKT-mediated liver damage are highly dependent on activation of this pathway. p38 MAPK does not substantially affect cytokine gene expression in NKT cells, but it regulates the synthesis of cytokines through the Mnk-eIF4E pathway. Thus, in addition to gene expression, translational regulation by p38 MAPK could be a novel mechanism that contributes to the overall production of cytokine by NKT cells.
Subject(s)
Cytokines/biosynthesis , Cytokines/genetics , MAP Kinase Signaling System/immunology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Protein Modification, Translational/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Enzyme Activation/genetics , Enzyme Activation/immunology , Liver Diseases/enzymology , Liver Diseases/genetics , Liver Diseases/immunology , MAP Kinase Kinase 3/deficiency , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/physiology , MAP Kinase Kinase 6/deficiency , MAP Kinase Kinase 6/genetics , MAP Kinase Kinase 6/physiology , MAP Kinase Signaling System/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Natural Killer T-Cells/enzymologyABSTRACT
Killer cell Ig-like receptor (KIR) with two Ig-like domains and a long cytoplasmic domain 4 (2DL4; CD158d) is a unique KIR expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated the cytokine-producing function of 2DL4, whereas Triad3A short hairpin RNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes.
Subject(s)
NF-kappa B/antagonists & inhibitors , NF-kappa B/physiology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Receptors, KIR2DL4/metabolism , Signal Transduction/immunology , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/immunology , Cell Line , Cell Line, Tumor , Endocytosis/immunology , Gene Expression Regulation/immunology , HEK293 Cells , Humans , Natural Killer T-Cells/enzymology , Protein Interaction Mapping , Protein Structure, Tertiary , Signal Transduction/genetics , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/physiologyABSTRACT
Natural Killer T (NKT) cells are lipid-reactive, CD1d-restricted T lymphocytes important in infection, cancer, and autoimmunity. In addition to foreign antigens, NKT cells react with endogenous self lipids. However, in the face of stimulating self antigen, it remains unclear how overstimulation of NKT cells is avoided. We hypothesized that constantly degraded endogenous antigen only accumulates upon inhibition of alpha-galactosidase A (alpha-Gal-A) in lysosomes. Here, we show that alpha-Gal-A deficiency caused vigorous activation of NKT cells. Moreover, microbes induced inhibition of alpha-Gal-A activity in antigen-presenting cells. This temporary enzyme block depended on Toll-like receptor (TLR) signaling and ultimately triggered lysosomal lipid accumulation. Thus, we present TLR-dependent negative regulation of alpha-Gal-A as a mechanistic link between pathogen recognition and self lipid antigen induction for NKT cells.
Subject(s)
Autoantigens/immunology , Lipids/immunology , Lysosomes/immunology , Natural Killer T-Cells/immunology , alpha-Galactosidase/immunology , Animals , Antigen Presentation , Cells, Cultured , Coculture Techniques , Dendritic Cells/immunology , Homeostasis , Lymphocyte Activation , Lysosomes/enzymology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Natural Killer T-Cells/enzymology , Signal Transduction , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism , alpha-Galactosidase/metabolismABSTRACT
Proteases of the serine and cysteine protease families are involved in many processes crucial to the lytic functions of cytotoxic T lymphocytes and natural killer cells. In this study we describe those functions and attempt to place them in the pathophysiological context of defence to pathogen invasion. In particular, we stress that the co-evolution of pathogens with the immune systems of higher organisms over evolutionary time has ensured that redundancy, flexibility and polymorphism of the proteases can be identified, both within the protease repertoire of a given species, and by comparing orthologous protease functions across species.
Subject(s)
Cysteine Proteases/physiology , Immune System/enzymology , Killer Cells, Natural/enzymology , Killer Cells, Natural/physiology , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/physiology , Serine Proteases/physiology , Animals , Cysteine Proteases/genetics , Evolution, Molecular , Humans , Immune System/physiology , Immune System/physiopathology , Polymorphism, Genetic , Serine Proteases/geneticsABSTRACT
Invariant natural killer T (iNKT) cells are CD1d-restricted, glycolipid-reactive lymphocytes with potent immunoregulatory characteristics. Although recent years have witnessed intensified interest in iNKT cells, little is known about intracellular signaling pathways that control iNKT cell responses, including those mediated by mitogen-activated protein kinases (MAPKs). We employed selective inhibitors of ERK1/2, JNK and p38 to examine the importance of these MAPKs in iNKT cell responses to the prototype glycolipid antigen alpha-galactosylceramide (alpha GC). Activation of DN32.D3 iNKT cells in the presence of PD98059 led to decreased interleukin (IL)-2 production, indicating a role for ERK in mouse iNKT cell responses. In contrast, addition of the JNK inhibitor SP600125 to cultures did not significantly affect cytokine production, suggesting that JNK is not critically needed for iNKT cell responses. Interestingly, selective inhibition of p38 by either SB203580 or SK&F 86002 resulted in augmented IL-2 production by DN32.D3 cells after stimulation with alpha GC. This was also evident when iNKT cells were stimulated with an anti-CD3 monoclonal antibody thus bypassing the requirement for CD1d-mediated antigen presentation, indicating that p38 inhibition affects signal transduction downstream of iNKT cells' T cell receptors. Primary splenic iNKT cells similarly exhibited enhanced cytokine response to alpha GC when cultured in the presence of p38 inhibitors. Importantly, in vivo administration of SB203580 resulted in higher IL-4 and interferon-gamma secretion in alpha GC-treated mice. These results demonstrate that MAPKs play distinct signaling roles in iNKT cells and that both in vitro and in vivo iNKT cell responses to glycolipid antigens can be negatively modulated by p38.
Subject(s)
Antigens/immunology , Galactosylceramides/immunology , Natural Killer T-Cells/immunology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Anthracenes/pharmacology , Antibodies/immunology , Antigen Presentation , Antigens, CD1d/immunology , CD3 Complex/immunology , Cytokines/immunology , Down-Regulation , Enzyme Inhibitors/pharmacology , Flavonoids/immunology , Imidazoles/pharmacology , Interferon-gamma/immunology , Interleukin-2/analysis , Interleukin-4/immunology , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/drug effects , Natural Killer T-Cells/enzymology , Protein Kinase Inhibitors , Pyridines/pharmacology , Receptors, Antigen, T-Cell/immunology , Spleen/immunology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG uses cis-regulatory elements with cell type-restricted function.
Subject(s)
Cell Lineage/genetics , Cell Lineage/immunology , Gene Expression Regulation/immunology , Genetic Loci/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Animals , Cells, Cultured , Conserved Sequence/genetics , Conserved Sequence/immunology , Core Binding Factor Alpha 3 Subunit/metabolism , Humans , Killer Cells, Natural/enzymology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Protein Transport/genetics , Protein Transport/immunology , RNA Polymerase II/metabolism , Regulatory Elements, Transcriptional/immunology , Th1 Cells/enzymology , Th1 Cells/immunology , Th1 Cells/metabolism , Transcription Initiation SiteABSTRACT
Human Vgamma9Vdelta2 T cells, a major innate-like peripheral T cell subset, are thought to play in vivo an important role in innate and adaptive immune responses to infection agents and tumors. However, the mechanisms regulating their broad effector functions, such as cytotoxicity and cytokine responses, remain poorly understood. In this study, we used single-cell calcium video imaging to analyze the early intracellular events associated with TCR-induced Vgamma9Vdelta2 T cell functional responses. When compared with other human T cell subsets, including NKT and Vdelta2(neg) gammadelta T cells, TCR/CD3-activated Vgamma9Vdelta2 T cells displayed an unusually delayed and sustained intracellular calcium mobilization, which was dramatically quickened and shortened on costimulation by NKG2D, a main activating NKR regulating gammadelta T cell tumor cytolysis. Importantly, the protein kinase C transduction pathway was identified as a main regulator of the NKG2D-mediated costimulation of antitumor Vgamma9Vdelta2 cytolytic responses. Therefore, this study identifies a new mechanism regulating Vgamma9Vdelta2 T cell functional plasticity through fine-tuning of early signal transduction events.