ABSTRACT
RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.
Subject(s)
Caspase 8/metabolism , Hereditary Autoinflammatory Diseases/metabolism , Mutation , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Caspase 3/metabolism , Female , Hereditary Autoinflammatory Diseases/genetics , Hereditary Autoinflammatory Diseases/pathology , Humans , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pedigree , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/geneticsABSTRACT
Germinal centers require CD4⺠follicular helper T cells (TFH cells), whose hallmark is expression of the transcriptional repressor Bcl-6, the chemokine receptor CXCR5 and interleukin 21 (IL-21). To track the development and fate of TFH cells, we generated an IL-21 reporter mouse by introducing sequence encoding green fluorescent protein (GFP) into the Il21 locus; these mice had expression of IL-21GFP in CD4âºCXCR5âºPD-1⺠TFH cells. IL-21GFP⺠TFH cells were multifunctional helper cells that coexpressed several cytokines, including interferon-g (IFN-g), IL-2 and IL-4. TFH cells proliferated and gave rise to transferrable memory cells with plasticity, which differentiated after recall into conventional effector helper T cells and TFH cells. Thus, we demonstrated that TFH cells were not terminally differentiated but instead retained the flexibility to be recruited into other helper T cell subsets and nonlymphoid tissues.
Subject(s)
Cell Differentiation/immunology , Cytokines/metabolism , Germinal Center/immunology , Interleukins/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cytokines/genetics , Gene Expression , Germinal Center/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunologic Memory/physiology , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-2/metabolism , Interleukin-4/metabolism , Interleukins/genetics , Interleukins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Programmed Cell Death 1 Receptor , Receptors, CXCR5/genetics , Receptors, CXCR5/metabolism , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/metabolismABSTRACT
The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p16/immunology , Homeodomain Proteins/immunology , Lymphoid Progenitor Cells/immunology , STAT5 Transcription Factor/immunology , Signal Transduction/immunology , Transcription Factors/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Cell Survival/genetics , Cell Survival/immunology , Cyclin-Dependent Kinase Inhibitor p16/genetics , Homeodomain Proteins/genetics , Lymphoid Progenitor Cells/cytology , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/immunology , STAT5 Transcription Factor/genetics , Signal Transduction/genetics , Transcription Factors/geneticsABSTRACT
OBJECTIVES: Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids. METHODS: We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice. RESULTS: Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli. CONCLUSIONS: Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.
Subject(s)
Autoimmunity/immunology , B-Lymphocytes/immunology , Gene Expression Regulation/immunology , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation/immunology , Transcription Factors/immunology , Adjuvants, Immunologic/pharmacology , Animals , B-Lymphocyte Subsets , Gene Expression Regulation/drug effects , Germinal Center/cytology , Glucocorticoids/therapeutic use , Hemocyanins/pharmacology , Histones , In Vitro Techniques , Leukocytes, Mononuclear , Lupus Erythematosus, Systemic/drug therapy , Male , Mice , Mice, Knockout , Nitrophenols/pharmacology , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , T-Lymphocytes , Transcription Factors/genetics , Up-RegulationABSTRACT
In response to antigenic stimulation, mature B cells interact with follicular helper T cells in specialized structures called germinal centers (GCs), which leads to the development of memory B cells and Ab-secreting plasma cells. The transcription factor IFN regulatory factor 4 (IRF4) is essential for the formation of follicular helper T cells and thus GCs, although whether IRF4 plays a distinct role in GC B cells remains contentious. RNAseq analysis on ex vivo-derived mouse B cell populations showed that Irf4 was lowly expressed in naive B cells, highly expressed in plasma cells, but absent from GC B cells. In this study, we used conditional deletion of Irf4 in mature B cells as well as wild-type and Irf4-deficient mixed bone marrow chimeric mice to investigate how and where IRF4 plays its essential role in GC formation. Strikingly, GC formation was severely impaired in mice in which Irf4 was conditionally deleted in mature B cells, after immunization with protein Ags or infection with Leishmania major. This effect was evident as early as day 5 following immunization, before the development of GCs, indicating that Irf4 was required for the development of early GC B cells. This defect was B cell intrinsic because Irf4-deficient B cells in chimeric mice failed to participate in the GC in response to L. major or influenza virus infection. Taken together, these data demonstrate a B cell-intrinsic requirement for IRF4 for not only the development of Ab secreting plasma cells but also for GC formation.
Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Interferon Regulatory Factors/immunology , Plasma Cells/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigens/immunology , B-Lymphocytes/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Flow Cytometry , Gene Expression/immunology , Germinal Center/cytology , Germinal Center/metabolism , Host-Pathogen Interactions/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza A Virus, H3N2 Subtype/physiology , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Leishmania major/immunology , Leishmania major/physiology , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/virology , Plasma Cells/metabolism , Receptors, IgE/genetics , Receptors, IgE/immunology , Receptors, IgE/metabolism , Sequence Analysis, RNA/methods , T-Lymphocytes, Helper-Inducer/metabolismABSTRACT
The response of B cells to Ag targeted to Clec9A on dendritic cells was followed using the hapten nitrophenol (NP) conjugated to rat Ig carrier. Injection of small amounts of NP conjugated to anti-Clec9A in the absence of adjuvants gave high and very prolonged Ab responses, approaching those obtained by high doses of nontargeted NP-protein conjugates with alum adjuvant. The response to NP-anti-Clec9A included the transient formation of germinal centers, maturation of Ab affinity, and some memory B cell formation. Serum Ab titers remained high 35 wk postimmunization, well after the initial follicular response had faded. The results suggest Clec9A-targeting strategies for improving Ab responses to vaccine Ags.
Subject(s)
B-Lymphocytes/immunology , Dendritic Cells/immunology , Lectins, C-Type/immunology , Receptors, Immunologic/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Antibody Formation/immunology , Female , Immunoglobulins/immunology , Immunologic Memory , Mice , Mice, Inbred C57BL , Nitrophenols/immunologyABSTRACT
Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.
Subject(s)
Immunohistochemistry , Necroptosis , Receptor-Interacting Protein Serine-Threonine Kinases , Animals , Humans , Mice , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Immunohistochemistry/methods , Protein Kinases/metabolism , Protein Kinases/genetics , Caspase 8/metabolism , Signal Transduction , Mice, Inbred C57BLABSTRACT
Death by apoptosis shapes tissue homeostasis. Apoptotic mechanisms are so universal that harnessing them for tailored immune intervention would seem challenging; however, the range and different expression levels of pro- and anti-apoptotic molecules among tissues offer hope that targeting only a subset of such molecules may be therapeutically useful. We examined the effects of the drug ABT-737, a mimetic of the killer BH3 domain of the Bcl-2 family of proteins that induces apoptosis by antagonizing Bcl-2, Bcl-X(L), and Bcl-W (but not Mcl-1 and A1), on the mouse immune system. Treatment with ABT-737 reduced the numbers of selected lymphocyte and dendritic cell subpopulations, most markedly in lymph nodes. It inhibited the persistence of memory B cells, the establishment of newly arising bone marrow plasma cells, and the induction of a cytotoxic T cell response. Preexisting plasma cells and germinal centers were unaffected. Notably, ABT-737 was sufficiently immunomodulatory to allow long-term survival of pancreatic allografts, reversing established diabetes in this model. These results provide an insight into the selective mechanisms of immune cell survival and how this selectivity avails a different strategy for immune modulation.
Subject(s)
BH3 Interacting Domain Death Agonist Protein/antagonists & inhibitors , Peptide Fragments/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Animals , Apoptosis/drug effects , Apoptosis/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Biphenyl Compounds/pharmacology , Graft Rejection/prevention & control , Immunity, Humoral/drug effects , Immunologic Factors/pharmacology , Islets of Langerhans Transplantation , Leukocytes/classification , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred NOD , Mice, Knockout , Nitrophenols/pharmacology , Piperazines/pharmacology , Sulfonamides/pharmacology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Transplantation, HomologousABSTRACT
Lyn-deficient (Lyn(-/-)) mice develop an age-dependent autoimmune disease similar to systemic lupus erythematosus, characterized by the production of IgG anti-nuclear Ab. To determine the extent to which this autoimmune phenotype is driven by T cell costimulation, we generated Lyn(-/-) mice expressing a soluble form of the T cell inhibitory molecule, CTLA4 (CTLA4Ig). Surprisingly, although CTLA4Ig prevented myeloid hyperplasia, splenomegaly and IgG anti-nuclear Ab production in Lyn(-/-) mice, it did not inhibit immune complex deposition and tissue destruction in the kidney. In fact, regardless of CTLA4Ig expression, Lyn(-/-) serum contained elevated titers of IgA anti-nuclear Ab, although generally IgA deposition in the kidney was only revealed in the absence of self-reactive IgG. This demonstrated that activation of autoreactive B cell clones in Lyn(-/-) mice can still occur despite impaired costimulation. Indeed, CTLA4Ig did not alter perturbed Lyn(-/-) B cell development and behavior, and plasma cell frequencies were predominantly unaffected. These results suggest that when self-reactive B cell clones are unimpeded in acquiring T cell help, they secrete pathogenic IgG autoantibodies that trigger the fulminant autoimmunity normally observed in Lyn(-/-) mice. The absence of these IgG immune complexes reveals an IgA-mediated axis of autoimmunity that is not sufficient to cause splenomegaly or extramedullary myelopoiesis, but which mediates destructive glomerulonephritis. These findings have implications for the understanding of the basis of Ab-mediated autoimmune diseases and for their treatment with CTLA4Ig.
Subject(s)
Antigens, CD/immunology , Autoimmune Diseases/drug therapy , Immunoconjugates/therapeutic use , Immunoglobulin G/therapeutic use , src-Family Kinases/deficiency , Abatacept , Animals , Antigen-Antibody Complex/biosynthesis , Autoantibodies/biosynthesis , Autoimmune Diseases/etiology , Autoimmune Diseases/immunology , B-Lymphocytes/immunology , CTLA-4 Antigen , Clone Cells/immunology , Kidney Diseases , Lupus Erythematosus, Systemic , Mice , Mice, Knockout , T-Lymphocytes/immunologyABSTRACT
High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.
Subject(s)
B-Lymphocytes , Epigenesis, Genetic , Animals , Humans , Mice , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Gene Expression Profiling , Phenotype , Polycomb Repressive Complex 2/metabolismABSTRACT
Immunization with a T cell-dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated V(H) genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1-expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220(+) memory B cells, and this is sufficient to account for the process of affinity maturation.
Subject(s)
B-Lymphocyte Subsets/immunology , Germinal Center/immunology , Immunologic Memory , Plasma Cells/immunology , Adoptive Transfer , Animals , Antigens, CD19/genetics , Antigens, CD19/immunology , Bone Marrow Cells/immunology , Chemokine CXCL13 , Chemokines, CXC/immunology , Flow Cytometry , Germinal Center/cytology , Haptens , Hemocyanins/administration & dosage , Hemocyanins/immunology , Immunization , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin Variable Region/genetics , Leukocyte Common Antigens/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Nitrophenols/immunology , Phenylacetates , Positive Regulatory Domain I-Binding Factor 1 , Repressor Proteins/immunology , Spleen/cytology , Spleen/immunology , Transcription Factors/immunologyABSTRACT
JQ1 is a BET-bromodomain inhibitor that has immunomodulatory effects. However, the precise molecular mechanism that JQ1 targets to elicit changes in antibody production is not understood. Our results show that JQ1 induces apoptosis, reduces cell proliferation, and as a consequence, inhibits antibody-secreting cell differentiation. ChIP-sequencing reveals a selective displacement of Brd4 in response to acute JQ1 treatment (<2 h), resulting in specific transcriptional repression. After 8 h, subsequent alterations in gene expression arise as a result of the global loss of Brd4 occupancy. We demonstrate that apoptosis induced by JQ1 is solely attributed to the pro-apoptotic protein Bim (Bcl2l11). Conversely, cell-cycle regulation by JQ1 is associated with multiple Myc-associated gene targets. Our results demonstrate that JQ1 drives temporal changes in Brd4 displacement that results in a specific transcriptional profile that directly affects B cell survival and proliferation to modulate the humoral immune response.
Subject(s)
Bcl-2-Like Protein 11/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/drug effects , Azepines/pharmacology , B-Lymphocytes/metabolism , Bcl-2-Like Protein 11/physiology , Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/genetics , Transcription Factors/physiology , Triazoles/pharmacology , Xenograft Model Antitumor AssaysSubject(s)
Hospice Care , Palliative Care , Pulmonary Disease, Chronic Obstructive , Humans , Pulmonary Disease, Chronic Obstructive/therapy , Pulmonary Disease, Chronic Obstructive/ethnology , Male , Female , Aged , Middle Aged , United States/epidemiology , Healthcare Disparities/ethnology , White People , Black or African AmericanABSTRACT
Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in mammalian cells, regulating many important functions including cell signalling, proliferation and differentiation. Here we show the role of PRMT1 in B-cell activation and differentiation. PRMT1 expression and activity in human and mouse peripheral B cells increases in response to in vitro or in vivo activation. Deletion of the Prmt1 gene in mature B cells establishes that although the frequency and phenotype of peripheral B cell subsets seem unaffected, immune responses to T-cell-dependent and -independent antigens are substantially reduced. In vitro activation of Prmt1-deficient B cells with a variety of mitogens results in diminished proliferation, differentiation and survival, effects that are correlated with altered signal transduction from the B cell receptor. Thus PRMT1 activity in B cells is required for correct execution of multiple processes that in turn are necessary for humoral immunity.PRMT1 is an arginine methyltransferase involved in a variety of cell functions. Here the authors delete PRMT1 specifically in mature B cells to show the importance of arginine methylation for B cell proliferation, differentiation and survival, and thereby for humoral immunity.
Subject(s)
Arginine/metabolism , B-Lymphocytes/immunology , Immunity, Humoral/genetics , Lymphocyte Activation/genetics , Protein-Arginine N-Methyltransferases/genetics , Repressor Proteins/genetics , Animals , B-Lymphocytes/metabolism , Cell Differentiation/genetics , Humans , Immunity, Humoral/immunology , Lymphocyte Activation/immunology , Methylation , Mice , Protein Processing, Post-Translational/geneticsABSTRACT
Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation. PU.1 and SpiB regulate the expression of many components of the B cell receptor signaling pathway and the receptors for CD40L, BAFF and TLR ligands. Thus, PU.1 and SpiB enable B cells to appropriately respond to environmental cues.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Proto-Oncogene Proteins c-ets/immunology , Proto-Oncogene Proteins/immunology , Trans-Activators/immunology , Animals , B-Lymphocytes/cytology , CD40 Antigens/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Female , Germinal Center/cytology , Germinal Center/immunology , Germinal Center/metabolism , Immunity, Humoral/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Plasma Cells/cytology , Plasma Cells/immunology , Plasma Cells/metabolism , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Signal Transduction , Toll-Like Receptors/metabolism , Trans-Activators/deficiency , Trans-Activators/geneticsABSTRACT
Histone deacetylase inhibitors (HDACi) are approved for treating certain haematological malignancies, however, recent evidence also illustrates they are modulators of the immune system. In experimental models, HDACi are particularly potent against malignancies originating from the B-lymphocyte lineage. Here we examine the ability of this class of compounds to modify both protective and autoimmune antibody responses. In vitro, HDACi affect B-cell proliferation, survival and differentiation in an HDAC-class-dependent manner. Strikingly, treatment of lupus-prone Mrl/lpr mice with the HDACi panobinostat significantly reduces autoreactive plasma-cell numbers, autoantibodies and nephritis, while other immune parameters remain largely unaffected. Immunized control mice treated with panobinostat or the clinically approved HDACi vorinostat have significantly impaired primary antibody responses, but these treatments surprisingly spare circulating memory B cells. These studies indicate that panobinostat is a potential therapy for B-cell-driven autoimmune conditions and HDACi do not induce major long-term detrimental effects on B-cell memory.
Subject(s)
B-Lymphocytes/drug effects , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Drug Evaluation, Preclinical , Female , Germinal Center/drug effects , Histone Deacetylase Inhibitors/therapeutic use , Hydroxamic Acids/therapeutic use , Immunologic Memory/drug effects , Indoles/therapeutic use , Lupus Erythematosus, Systemic/drug therapy , Male , Mice, Inbred C57BL , PanobinostatABSTRACT
CD19 is a co-stimulatory surface protein expressed exclusively on B cells and serves to reduce the threshold for signalling via the B-cell receptor (BCR). Co-ligation of CD19 with the BCR synergistically enhances mitogen-activated protein (MAP) kinase activity, calcium release and proliferation. We recently found that these parameters were also enhanced in CD19-null primary murine B cells following BCR ligation, suggesting a regulatory role for CD19 in BCR signalling. In this study, we demonstrate that the enhanced BCR signalling in the absence of CD19 was not dependent on the src kinase Lyn, but linked to phosphoinositide 3-kinase (PI3K) activity. Consistent with this, we detect PI3K associated with CD19 outside the lipid raft in resting B cells. Pre-ligation of CD19 to restrict its translocation with the BCR into lipid rafts attenuated BCR-induced PI3K and MAP kinase activation and subsequent B-cell proliferation. Thus, we propose that CD19 can modulate BCR signalling in both a positive and negative manner depending on the receptor/ligand interaction in vivo.
Subject(s)
Antigens, CD19/metabolism , B-Lymphocytes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Antigen, B-Cell/metabolism , Animals , Antigens, CD19/genetics , Antigens, CD19/immunology , Enzyme Activation , Lymphocyte Activation , Mice, Inbred C57BL , Receptors, Antigen, B-Cell/immunology , Signal Transduction , src-Family Kinases/metabolismABSTRACT
Activated B cells undergo immunoglobulin class-switch recombination (CSR) and differentiate into antibody-secreting plasma cells. The distinct transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors: those that maintain the B cell program, including BCL6 and PAX5, and plasma cell-promoting factors, such as IRF4 and BLIMP-1. We show that the complex of IRF8 and PU.1 controls the propensity of B cells to undergo CSR and plasma cell differentiation by concurrently promoting the expression of BCL6 and PAX5 and repressing AID and BLIMP-1. As the PU.1-IRF8 complex functions in a reciprocal manner to IRF4, we propose that concentration-dependent competition between these factors controls B cell terminal differentiation.
Subject(s)
Cell Differentiation/genetics , Interferon Regulatory Factors/genetics , Plasma Cells/cytology , Plasma Cells/metabolism , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Animals , Cell Line , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation , Humans , Immunoglobulin Class Switching/genetics , Interferon Regulatory Factors/metabolism , Mice , Mice, Transgenic , Plasma Cells/immunology , Protein Binding , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolismABSTRACT
Maintenance of an appropriate number of plasma cells, long-lived antibody-producing cells that are derived from B cells, is essential for maintaining immunological memory while limiting disease. Plasma cell survival relies on extrinsic factors, the limited availability of which determines the size of the plasma cell population. Mice deficient in the nonreceptor tyrosine kinase Lyn are prone to an autoimmune disease that is characterized by inflammation and an excess of plasma cells (plasmacytosis). We demonstrated that the plasmacytosis was intrinsic to B cells and independent of inflammation. We also showed that Lyn attenuated signaling by signal transducer and activator of transcription 3 (STAT3) and STAT5 in response to the cytokines interleukin-6 (IL-6) and IL-3, respectively, in two previously uncharacterized plasma cell signaling pathways. Thus, in the absence of Lyn, the survival of plasma cells was improved, which enabled the plasma cells to become established in excess numbers in niches in vivo. These data identify Lyn as a key regulator of survival signaling in plasma cells, limiting plasma cell accumulation and autoimmune disease susceptibility.
Subject(s)
Cell Survival/physiology , Cytokines/metabolism , Immunologic Memory/immunology , Plasma Cells/physiology , Signal Transduction/immunology , src-Family Kinases/metabolism , Animals , Blotting, Western , Bromodeoxyuridine , Enzyme-Linked Immunospot Assay , Flow Cytometry , Mice , Plasma Cells/immunology , Plasma Cells/metabolism , Real-Time Polymerase Chain Reaction , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolismABSTRACT
Signaling by the serine and threonine kinase Akt (also known as protein kinase B), a pathway that is common to all eukaryotic cells, is central to cell survival, proliferation, and gene induction. We sought to elucidate the mechanisms underlying regulation of the kinase activity of Akt in the immune system. We found that the four-transmembrane protein CD37 was essential for B cell survival and long-lived protective immunity. CD37-deficient (Cd37(-/-)) mice had reduced numbers of immunoglobulin G (IgG)-secreting plasma cells in lymphoid organs compared to those in wild-type mice, which we attributed to increased apoptosis of plasma cells in the germinal centers of the spleen, areas in which B cells proliferate and are selected. CD37 was required for the survival of IgG-secreting plasma cells in response to binding of vascular cell adhesion molecule 1 to the α(4)ß(1) integrin. Impaired α(4)ß(1) integrin-dependent Akt signaling in Cd37(-/-) IgG-secreting plasma cells was the underlying cause responsible for impaired cell survival. CD37 was required for the mobility and clustering of α(4)ß(1) integrins in the plasma membrane, thus regulating the membrane distribution of α(4)ß(1) integrin necessary for activation of the Akt survival pathway in the immune system.