ABSTRACT
Selective expansion of high-affinity antigen-specific B cells in germinal centers (GCs) is a key event in antibody affinity maturation. GC B cells with improved affinity can either continue affinity-driven selection or exit the GC to differentiate into plasma cells (PCs) or memory B cells. Here we found that deleting E3 ubiquitin ligases Cbl and Cbl-b (Cbls) in GC B cells resulted in the early exit of high-affinity antigen-specific B cells from the GC reaction and thus impaired clonal expansion. Cbls were highly expressed in GC light zone (LZ) B cells, where they promoted the ubiquitination and degradation of Irf4, a transcription factor facilitating PC fate choice. Strong CD40 and BCR stimulation triggered the Cbl degradation, resulting in increased Irf4 expression and exit from GC affinity selection. Thus, a regulatory cascade that is centered on the Cbl ubiquitin ligases ensures affinity-driven clonal expansion by connecting BCR affinity signals with differentiation programs.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Germinal Center/immunology , Germinal Center/metabolism , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Animals , Antibody Affinity/ethics , Antibody Affinity/immunology , Antibody Formation/genetics , Antibody Formation/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Clonal Selection, Antigen-Mediated/genetics , Clonal Selection, Antigen-Mediated/immunology , Gene Expression , Gene Knockout Techniques , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , Mutation , Protein Binding , Proteolysis , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/metabolism , UbiquitinationABSTRACT
Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.
Subject(s)
Janus Kinase 2/metabolism , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/physiopathology , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Line, Tumor , Cell Proliferation/genetics , Cytokines/metabolism , Enzyme Stability , Hematopoietic Stem Cells/enzymology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Janus Kinase 2/genetics , Leukemia, Myeloid, Acute/genetics , Membrane Proteins , Mice , Mutation , Proteolysis , Proto-Oncogene Proteins c-cbl/genetics , Signal Transduction/genetics , UbiquitinationABSTRACT
Akt-1 and Akt-2 are the major isoforms of the serine/threonine Akt family that play a key role in controlling immune responses. However, the involvement of Akt-1 and Akt-2 isoforms in antifungal innate immunity is completely unknown. In this study, we show that Akt2 -/-, but not Akt1 -/-, mice are protected from lethal Candida albicans infection. Loss of Akt-2 facilitates the recruitment of neutrophils and macrophages to the spleen and increases reactive oxygen species expression in these cells. Treating C57BL/6 mice with a specific inhibitor for Akt-2, but not Akt-1, provides protection from lethal C. albicans infection. Our data demonstrate that Akt-2 inhibits antifungal innate immunity by hampering neutrophil and macrophage recruitment to spleens and suppressing oxidative burst, myeloperoxidase activity, and NETosis. We thus describe a novel role for Akt-2 in the regulation of antifungal innate immunity and unveil Akt-2 as a potential target for the treatment of fungal sepsis.
Subject(s)
Candida albicans , Candidiasis , Proto-Oncogene Proteins c-akt/metabolism , Animals , Antifungal Agents , Mice , Mice, Inbred C57BL , Neutrophils , Peroxidase/metabolism , Reactive Oxygen Species/metabolism , Serine/metabolism , Threonine/metabolismABSTRACT
The relationship between dendritic cells (DCs) and macrophages is often debated. Here we ask whether steady-state, lymphoid-tissue-resident conventional DCs (cDCs), plasmacytoid DCs (pDCs), and macrophages share a common macrophage-DC-restricted precursor (MDP). Using new clonal culture assays combined with adoptive transfer, we found that MDP fractions isolated by previous strategies are dominated by precursors of macrophages and monocytes, include some multipotent precursors of other hematopoietic lineages, but contain few precursors of resident cDCs and pDCs and no detectable common precursors restricted to these DC types and macrophages. Overall we find no evidence for a common restricted MDP leading to both macrophages and FL-dependent, resident cDCs and pDCs.
Subject(s)
Cell Lineage/immunology , Dendritic Cells/cytology , Lymphoid Tissue/cytology , Macrophages/cytology , Monocyte-Macrophage Precursor Cells/cytology , Adoptive Transfer , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , CX3C Chemokine Receptor 1 , Cell Differentiation/immunology , Cells, Cultured , Cytokines/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocytes/cytology , Granulocytes/immunology , Macrophage Colony-Stimulating Factor/immunology , Mice , Mice, Inbred C57BL , Monocyte-Macrophage Precursor Cells/immunology , Monocytes/cytology , Receptor, Macrophage Colony-Stimulating Factor/immunology , Receptors, Chemokine/immunologyABSTRACT
Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIP(S), which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIP(S), which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis-triggered macrophage cell death.
Subject(s)
Apoptosis/physiology , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Macrophages/physiology , Mycobacterium tuberculosis/physiology , Proto-Oncogene Proteins c-cbl/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Caspase 8/metabolism , Cell Line , Cell Proliferation , Humans , MAP Kinase Kinase Kinase 5/genetics , MAP Kinase Kinase Kinase 5/metabolism , Macrophages/microbiology , Mice , Mice, Knockout , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-cbl/genetics , Signal Transduction , Ubiquitination , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Akt isoforms play key roles in multiple cellular processes; however, the roles of Akt-1 and Akt-2 isoforms in the development of T cell-mediated autoimmunity are poorly defined. In this study, we showed that Akt1-/- mice develop ameliorated experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, whereas Akt2-/- mice develop exacerbated EAE, compared with wild-type mice. At the cellular level, Akt-1 appears to inhibit proliferation of thymus-derived regulatory T cells (tTregs), which facilitates Ag-specific Th1/Th17 responses. In a sharp contrast to Akt-1, Akt-2 potentiates tTreg proliferation in vitro and in vivo and suppresses Ag-specific Th1/Th17 responses. Furthermore, treating mice with established EAE with a specific Akt-1 inhibitor suppressed disease progression. Our data demonstrate that Akt-1 and Akt-2 differentially regulate the susceptibility of mice to EAE by controlling tTreg proliferation. Our data also indicate that targeting Akt-1 is a potential therapeutic approach for multiple sclerosis in humans.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Proto-Oncogene Proteins c-akt/immunology , T-Lymphocytes, Regulatory/immunology , Thymus Gland/immunology , Animals , Cell Proliferation , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-akt/deficiency , Th1 Cells/immunology , Th17 Cells/immunologyABSTRACT
Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Killer Cells, Natural/immunology , Mammary Neoplasms, Experimental/pathology , Melanoma, Experimental/pathology , Neoplasm Metastasis/immunology , Proto-Oncogene Proteins c-cbl/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Female , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Male , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/immunology , Melanoma, Experimental/drug therapy , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/prevention & control , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-cbl/deficiency , Proto-Oncogene Proteins c-cbl/genetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Warfarin/pharmacology , Warfarin/therapeutic use , c-Mer Tyrosine Kinase , Axl Receptor Tyrosine KinaseABSTRACT
Casitas B lymphoma-b (Cbl-b), a RING finger E3 ubiquitin ligase, has been identified as a critical regulator of adaptive immune responses. Cbl-b is essential for establishing the threshold for T cell activation and regulating peripheral T cell tolerance through various mechanisms. Intriguingly, recent studies indicate that Cbl-b also modulates innate immune responses, and plays a key role in host defense to pathogens and anti-tumor immunity. These studies suggest that targeting Cbl-b may represent a potential therapeutic strategy for the management of human immune-related disorders such as autoimmune diseases, infections, tumors, and allergic airway inflammation. In this review, we summarize the latest developments regarding the roles of Cbl-b in innate and adaptive immunity, and immune-mediated diseases.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Autoimmune Diseases/immunology , Bacterial Infections/immunology , Hypersensitivity/immunology , Mycoses/immunology , Neoplasms/immunology , Proto-Oncogene Proteins c-cbl/genetics , Virus Diseases/immunology , Adaptive Immunity/drug effects , Adaptor Proteins, Signal Transducing/immunology , Animals , Autoimmune Diseases/drug therapy , Autoimmune Diseases/genetics , Autoimmune Diseases/pathology , Bacterial Infections/drug therapy , Bacterial Infections/genetics , Bacterial Infections/pathology , Cytokines/genetics , Cytokines/immunology , Gene Expression Regulation , Humans , Hypersensitivity/drug therapy , Hypersensitivity/genetics , Hypersensitivity/pathology , Immunity, Innate/drug effects , Immunologic Factors/therapeutic use , Lymphocyte Activation , Molecular Targeted Therapy/methods , Mycoses/drug therapy , Mycoses/genetics , Mycoses/pathology , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Peripheral Tolerance/drug effects , Proto-Oncogene Proteins c-cbl/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Virus Diseases/drug therapy , Virus Diseases/genetics , Virus Diseases/pathologyABSTRACT
CD28 costimulation is essential for the development of thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells ("tTregs"). E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. In this paper, we report that the loss of Cbl-b partially but significantly rescues the defective development of tTregs in Cd28(-/-) mice. This partial rescue is independent of IL-2. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in Cd28(-/-) T cells, the defective development of tTregs in Cd28(-/-) mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Stub1 and Cbl-b. Treating Cd28(-/-) mice with a proteasome inhibitor completely rescues defective tTreg development in these mice. Therefore, Cbl-b, together with Stub1, ubiquitinate Foxp3, and regulate tTreg development.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Forkhead Transcription Factors/immunology , Lymphocyte Activation/immunology , Proto-Oncogene Proteins c-cbl/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Forkhead Transcription Factors/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Thymus Gland/cytology , Thymus Gland/immunology , Transfection , Ubiquitin-Protein Ligases/immunology , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Previously, we demonstrated that CD28 and CTLA-4 signaling control Casitas-B-lineage lymphoma (Cbl)-b protein expression, which is critical for T cell activation and tolerance induction. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b fails to undergo tyrosine phosphorylation upon CD3 stimulation because SHP-1 is recruited to and dephosphorylates Cbl-b, whereas CD28 costimulation abrogates this interaction. In support of this finding, T cells lacking SHP-1 display heightened tyrosine phosphorylation and ubiquitination of Cbl-b upon TCR stimulation, which correlates with decreased levels of Cbl-b protein. The aberrant Th2 phenotype observed in T cell-specific Shp1(-/-) mice is reminiscent of heightened Th2 response in Cblb(-/-) mice. Indeed, overexpressing Cbl-b in T cell-specific Shp1(-/-) T cells not only inhibits heightened Th2 differentiation in vitro, but also Th2 responses and allergic airway inflammation in vivo. Therefore, SHP-1 regulates Cbl-b-mediated T cell responses by controlling its tyrosine phosphorylation and ubiquitination.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/physiology , Proto-Oncogene Proteins c-cbl/metabolism , T-Lymphocytes/immunology , Animals , CD28 Antigens/physiology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Mice , Mice, Inbred C57BL , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 6/chemistry , Th2 Cells/immunology , UbiquitinationABSTRACT
E3 ubiquitin ligase Casitas-B-lineage lymphoma protein-b (Cbl-b) is critical for establishing the threshold for T cell activation and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of CD4(+)CD25(+) inducible regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by naive CD4(+)CD25(-) T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is downregulated in vitro. Using TCR-transgenic Rag(-/-) mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Lymphocyte Activation , Proto-Oncogene Proteins c-cbl/metabolism , T-Lymphocytes, Regulatory/immunology , Ubiquitin-Protein Ligases/metabolism , Animals , CD4 Antigens/metabolism , Cell Differentiation/immunology , Cells, Cultured , Down-Regulation , Forkhead Transcription Factors/metabolism , Homeodomain Proteins/genetics , Interleukin-2 Receptor alpha Subunit/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3(+) multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.
Subject(s)
Benzothiazoles/pharmacology , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/metabolism , Phenylurea Compounds/pharmacology , Signal Transduction/drug effects , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/metabolism , Animals , Benzothiazoles/administration & dosage , Cell Cycle/drug effects , Cell Proliferation , Disease Models, Animal , Female , Humans , Leukocyte Count , Leukocytes/drug effects , Liver/pathology , Lung/pathology , Male , Membrane Proteins/metabolism , Mice , Mice, Knockout , Multipotent Stem Cells/drug effects , Multipotent Stem Cells/metabolism , Mutation , Myeloid Cells/pathology , Myeloproliferative Disorders/drug therapy , Phenylurea Compounds/administration & dosage , Proto-Oncogene Proteins c-cbl/genetics , Splenomegaly/drug therapyABSTRACT
c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-Cbl(YF/YF) knock-in mice. c-Cbl KO and c-Cbl(YF/YF) platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl(YF/YF) platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.
Subject(s)
Blood Platelets/physiology , Clot Retraction/physiology , Hemostasis/physiology , Proto-Oncogene Proteins c-cbl/metabolism , Signal Transduction/physiology , Animals , Blood Platelets/cytology , Humans , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-cbl/genetics , Tyrosine/metabolismABSTRACT
E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or autoimmunity.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Proto-Oncogene Proteins c-cbl/physiology , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/physiology , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Autoimmune Diseases/enzymology , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Cell Line, Tumor , Clonal Anergy/genetics , Enzyme Activation/genetics , Enzyme Activation/immunology , Female , Gene Knock-In Techniques , Immunophenotyping , Lymphocyte Activation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Point Mutation , Proto-Oncogene Proteins c-cbl/deficiency , Proto-Oncogene Proteins c-cbl/genetics , RING Finger Domains/genetics , T-Lymphocyte Subsets/pathology , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/pathology , Ubiquitin-Protein Ligases/geneticsABSTRACT
The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4(+) T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4(+) T cells in prediabetic 3A9-TCR × insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4(+) cells or the numbers of islet-specific CD4(+) or CD4(+)Foxp3(+) T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-ß-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4(+) cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4(+) cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Diabetes Mellitus, Type 1/immunology , Genetic Predisposition to Disease , Islets of Langerhans/immunology , Proto-Oncogene Proteins c-cbl/physiology , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Autoantibodies/biosynthesis , CD4-Positive T-Lymphocytes/pathology , Cells, Cultured , Clonal Anergy/genetics , Clonal Anergy/immunology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/pathology , Disease Progression , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/physiology , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Organ Specificity/genetics , Organ Specificity/immunology , Pancreas/immunology , Pancreas/metabolism , Pancreas/pathology , Proto-Oncogene Proteins c-cbl/deficiency , Proto-Oncogene Proteins c-cbl/genetics , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
Caspase-11 (Casp-11) is known to induce pyroptosis and defends against cytosol-invading bacterial pathogens, but its regulation remains poorly defined. Here, we identified extended synaptotagmin 1 (E-Syt1), an endoplasmic reticulum protein, as a key regulator of Casp-11 oligomerization and activation. Macrophages lacking E-Syt1 exhibited reduced production of interleukin-1ß (IL-1ß) and impaired pyroptosis upon cytosolic lipopolysaccharide (LPS) delivery and cytosol-invasive bacterial infection. Moreover, cleavage of Casp-11 and its downstream substrate gasdermin D were significantly diminished in ESyt1-/- macrophages. Upon LPS stimulation, E-Syt1 underwent oligomerization and bound to the p30 domain of Casp-11 via its synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. E-Syt1 oligomerization and its interaction with Casp-11 facilitated Casp-11 oligomerization and activation. Notably, ESyt1-/- mice were susceptible to infection by cytosol-invading bacteria Burkholderia thailandensis while being resistant to LPS-induced endotoxemia. These findings collectively suggest that E-Syt1 may serve as a platform for Casp-11 oligomerization and activation upon cytosolic LPS sensing.
Subject(s)
Caspases , Lipopolysaccharides , Animals , Mice , Caspase 1/metabolism , Caspases/metabolism , Cytosol/metabolism , Inflammasomes/metabolism , Lipopolysaccharides/pharmacology , Lipopolysaccharides/metabolism , Macrophages/metabolism , Synaptotagmin I/metabolismABSTRACT
Repeated injection of alpha-galactosylceramide, an agonistic ligand for natural killer T (NKT) cells, results in long-term unresponsiveness or anergy, which severely limits its clinical application. However, the molecular mechanisms leading to NKT anergy induction remain unclear. We show here that the decreased IFN-gamma production and failed tumor rejection observed in anergized NKT cells are rescued by Cbl-b deficiency. Cbl-b E3 ligase activity is critical for the anergy induction, as revealed by the similarity between Cbl-b(-/-) and its RING finger mutant NKT cells. Cbl-b binds and promotes monoubiquitination to CARMA1, a critical signaling molecule in NFkappaB activation. Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability. In addition, CARMA1(-/-) NKT cells are defective in IFN-gamma production. The study identifies an important signaling pathway linking Cbl-b-induced monoubiquitination to NFkappaB activation in NKT cell anergy induction, which may help design approaches for human cancer therapy.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CARD Signaling Adaptor Proteins/metabolism , Clonal Anergy , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , CARD Signaling Adaptor Proteins/deficiency , CARD Signaling Adaptor Proteins/genetics , Galactosylceramides/immunology , Humans , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Melanoma, Experimental/secondary , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , NF-kappa B/metabolism , Proto-Oncogene Proteins c-cbl/deficiency , Proto-Oncogene Proteins c-cbl/genetics , Signal Transduction/immunology , UbiquitinationABSTRACT
Receptor tyrosine kinases (RTKs) serve as transmembrane receptors that participate in a broad spectrum of cellular processes including cellular growth, motility, differentiation, proliferation, and metabolism. Hence, elucidating the regulatory mechanisms of RTKs involved in an assortment of diseases such as cancers attracts increasing interest from researchers. Members of the Cbl family ubiquitin ligases (c-Cbl, Cbl-b and Cbl-c in mammals) have emerged as negative regulators of activated RTKs. Upon activation of RTKs by growth factors, Cbl binds to RTKs via its tyrosine kinase binding (TKB) domain and targets them for ubiquitination, thus facilitating their degradation and negative regulation of RTK signaling. RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGF), fibroblast growth factor receptor (FGFR) and hepatocyte growth factor receptor (HGFR) undergo ubiquitination upon interaction with Cbl family members. In this review, we summarize the current knowledge related to the negative regulation of RTKs by Cbl family proteins.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin , Animals , Mammals/metabolism , Signal Transduction/physiology , Tyrosine/metabolism , Ubiquitin/metabolism , UbiquitinationABSTRACT
Recurrent mutations in RNA splicing proteins and epigenetic regulators contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in ~50% of patients and TET2 mutations in ~60%. Clonal analysis indicates that either mutation can arise as the founder lesion. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly in hematopoietic stem cells. At 20-24 weeks post mutation induction, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelomonocytic hyperplasia), with increased immature promonocytes and monoblasts and binucleate promonocytes. Exome analysis of progressed disease demonstrated mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis, and splenomegaly. We reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML.
Subject(s)
Dioxygenases , Leukemia, Myelomonocytic, Chronic , Leukemia, Myelomonocytic, Juvenile , Myelodysplastic Syndromes , Serine-Arginine Splicing Factors , Animals , Humans , Mice , Dioxygenases/genetics , DNA-Binding Proteins/genetics , Hematopoiesis/genetics , Leukemia, Myelomonocytic, Chronic/genetics , Leukemia, Myelomonocytic, Chronic/pathology , Mutation , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors/geneticsABSTRACT
E3 ubiquitin ligase Cbl-b is involved in the maintenance of a balance between immunity and tolerance. Mice lacking Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis (EAE), a Th17-mediated autoimmune disease. However, how Cbl-b regulates Th17 cell responses remains unclear. In this study, utilizing adoptive transfer and cell type-specific Cblb knockout strains, we show that Cbl-b expression in macrophages, but not T cells or dendritic cells (DCs), restrains the generation of pathogenic Th17 cells and the development of EAE. Cbl-b inhibits IL-6 production by macrophages that is induced by signaling from CARD9-dependent C-type lectin receptor (CLR) pathways, which directs T cells to generate pathogenic Th17 cells. Therefore, our data unveil a previously unappreciated function for Cbl-b in the regulation of pathogenic Th17 responses.