ABSTRACT
Nutritional status potentially influences immune responses; however, how nutritional signals regulate cellular dynamics and functionality remains obscure. Herein, we report that temporary fasting drastically reduces the number of lymphocytes by â¼50% in Peyer's patches (PPs), the inductive site of the gut immune response. Subsequent refeeding seemingly restored the number of lymphocytes, but whose cellular composition was conspicuously altered. A large portion of germinal center and IgA+ B cells were lost via apoptosis during fasting. Meanwhile, naive B cells migrated from PPs to the bone marrow during fasting and then back to PPs during refeeding when stromal cells sensed nutritional signals and upregulated CXCL13 expression to recruit naive B cells. Furthermore, temporal fasting before oral immunization with ovalbumin abolished the induction of antigen-specific IgA, failed to induce oral tolerance, and eventually exacerbated food antigen-induced diarrhea. Thus, nutritional signals are critical in maintaining gut immune homeostasis.
Subject(s)
B-Lymphocytes/physiology , Immunity, Mucosal , Animals , Antigens/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Bone Marrow/immunology , Bone Marrow/metabolism , Chemokine CXCL13/genetics , Chemokine CXCL13/metabolism , Fasting , Gene Expression Regulation , Glycolysis , Immunoglobulin A/metabolism , Male , Mice , Mice, Inbred BALB C , Nutritional Status , Ovalbumin/immunology , Peyer's Patches/immunology , Peyer's Patches/metabolism , Peyer's Patches/pathology , Receptors, CXCR5/genetics , Receptors, CXCR5/metabolism , Signal Transduction , Stromal Cells/cytology , Stromal Cells/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.
Subject(s)
Antibodies, Monoclonal/immunology , B-Lymphocytes/physiology , Hemorrhagic Fever, Ebola/immunology , Adult , Amino Acid Sequence/genetics , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , B-Lymphocytes/metabolism , Chlorocebus aethiops , Ebola Vaccines/immunology , Ebolavirus/genetics , Ebolavirus/metabolism , Ebolavirus/pathogenicity , Epitopes/blood , Female , Glycoproteins/genetics , Hemorrhagic Fever, Ebola/metabolism , Hemorrhagic Fever, Ebola/virology , Humans , Immunoglobulin G/immunology , Jurkat Cells , Longitudinal Studies , Male , Mice , Mice, Inbred BALB C , Survivors , Vero Cells , Viral Envelope Proteins/geneticsABSTRACT
Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.
Subject(s)
B-Lymphocytes/physiology , Germinal Center/pathology , Histone Demethylases/metabolism , Lymphoma/metabolism , Proto-Oncogene Proteins c-bcl-6/metabolism , Animals , CRISPR-Cas Systems , Carcinogenesis , DNA, Intergenic/genetics , Germinal Center/immunology , Histone Demethylases/genetics , Hyperplasia , Immunological Synapses/genetics , Introns/genetics , Lymphoma/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-bcl-6/geneticsABSTRACT
Most adult B cell lymphomas originate from germinal center (GC) B cells, but it is unclear to what extent B cells in overt lymphoma retain the functional dynamics of GC B cells or are blocked at a particular stage of the GC reaction. Here we used integrative single-cell analysis of phenotype, gene expression and variable-region sequence of the immunoglobulin heavy-chain locus to track the characteristic human GC B cell program in follicular lymphoma B cells. By modeling the cyclic continuum of GC B cell transitional states, we identified characteristic patterns of synchronously expressed gene clusters. GC-specific gene-expression synchrony was lost in single lymphoma B cells. However, distinct follicular lymphoma-specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B cell state but might adopt new dynamic modes of functional diversity, which opens the possibility of novel definitions of lymphoma identity.
Subject(s)
B-Lymphocyte Subsets/physiology , B-Lymphocytes/physiology , Germinal Center/physiology , Immunoglobulin Variable Region/genetics , Lymphoma, B-Cell/genetics , Adult , Cell Differentiation , Cells, Cultured , Female , Gene Expression Regulation, Neoplastic , Germinal Center/pathology , Humans , Lymphoma, B-Cell/pathology , Male , Middle Aged , Single-Cell Analysis , Transcriptome/geneticsABSTRACT
Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.
Subject(s)
B-Lymphocytes/physiology , Gastrointestinal Microbiome/immunology , Germinal Center/physiology , Mutation/genetics , Phosphatidylinositol 3-Kinases/genetics , T-Lymphocytes, Helper-Inducer/physiology , Animals , Autoantibodies/blood , Cells, Cultured , Class I Phosphatidylinositol 3-Kinases/genetics , Disease Models, Animal , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolism , Humans , Immunity, Humoral/genetics , Immunoglobulin Class Switching/genetics , Immunologic Deficiency Syndromes/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response-activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.
Subject(s)
B-Lymphocytes/physiology , Mitochondria/metabolism , Receptors, Antigen, B-Cell/metabolism , T-Lymphocytes, Helper-Inducer/immunology , Toll-Like Receptor 9/metabolism , Animals , Apoptosis , Calcium/metabolism , Calcium Channels/metabolism , Cytokines/metabolism , Glycolysis , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NIH 3T3 Cells , Oxidative Phosphorylation , Receptors, Antigen, B-Cell/genetics , Signal Transduction , Toll-Like Receptor 9/geneticsABSTRACT
IgE is an ancient and conserved immunoglobulin isotype with potent immunological function. Nevertheless, the regulation of IgE responses remains an enigma, and evidence of a role for IgE in host defense is limited. Here we report that topical exposure to a common environmental DNA-damaging xenobiotic initiated stress surveillance by γδTCR+ intraepithelial lymphocytes that resulted in class switching to IgE in B cells and the accumulation of autoreactive IgE. High-throughput antibody sequencing revealed that γδ T cells shaped the IgE repertoire by supporting specific variable-diversity-joining (VDJ) rearrangements with unique characteristics of the complementarity-determining region CDRH3. This endogenous IgE response, via the IgE receptor FcεRI, provided protection against epithelial carcinogenesis, and expression of the gene encoding FcεRI in human squamous-cell carcinoma correlated with good disease prognosis. These data indicate a joint role for immunosurveillance by T cells and by B cells in epithelial tissues and suggest that IgE is part of the host defense against epithelial damage and tumor development.
Subject(s)
B-Lymphocytes/physiology , Carcinoma, Squamous Cell/immunology , Epithelial Cells/physiology , Immunoglobulin E/metabolism , Intraepithelial Lymphocytes/physiology , Neoplasms, Experimental/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Receptors, IgE/metabolism , Animals , Anthracenes/toxicity , Carcinoma, Squamous Cell/diagnosis , Cell Death , Cells, Cultured , Complementarity Determining Regions/genetics , DNA Damage , Female , High-Throughput Nucleotide Sequencing , Immunoglobulin Class Switching , Immunoglobulin E/genetics , Immunologic Surveillance , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms, Experimental/chemically induced , Piperidines/toxicity , Prognosis , Receptors, Antigen, T-Cell, gamma-delta/geneticsABSTRACT
Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.
Subject(s)
B-Lymphocytes/physiology , Biomarkers, Tumor/genetics , HLA Antigens/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class I/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Carcinogenesis/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Line, Tumor , Cell Lineage , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Gene Expression Regulation, Neoplastic , Genetic Testing , Genome-Wide Association Study , HLA Antigens/metabolism , Humans , Immunologic Surveillance , Lymphoma, Large B-Cell, Diffuse/metabolism , Tumor Escape/geneticsABSTRACT
T lymphocytes and B lymphocytes integrate activating signals to control the size of their proliferative response. Here we report that such control was achieved by timed changes in the production rate of cell-cycle-regulating proto-oncoprotein Myc, with division cessation occurring when Myc levels fell below a critical threshold. The changing pattern of the level of Myc was not affected by cell division, which identified the regulating mechanism as a cell-intrinsic, heritable temporal controller. Overexpression of Myc in stimulated T cells and B cells did not sustain cell proliferation indefinitely, as a separate 'time-to-die' mechanism, also heritable, was programmed after lymphocyte activation and led to eventual cell loss. Together the two competing cell-intrinsic timed fates created the canonical T cell and B cell immune-response pattern of rapid growth followed by loss of most cells. Furthermore, small changes in these timed processes by regulatory signals, or by oncogenic transformation, acted in synergy to greatly enhance cell numbers over time.
Subject(s)
B-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , Cell Division , Cell Proliferation/genetics , Immunity, Cellular , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Death/genetics , Cell Division/genetics , Cells, Cultured , Gene Expression Regulation , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction , Transgenes/geneticsABSTRACT
B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.
Subject(s)
B-Lymphocytes/physiology , Germinal Center/immunology , Glycogen Synthase Kinase 3 beta/metabolism , Animals , Antigens, CD19/genetics , Antigens, CD19/metabolism , Apoptosis/genetics , CD40 Ligand/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Glycogen Synthase Kinase 3 beta/genetics , Glycolysis , Interleukin-4/metabolism , Mice , Mice, Knockout , Oxidative Stress , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.
Subject(s)
B-Lymphocytes/physiology , Clonal Selection, Antigen-Mediated , Germinal Center/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation , Adaptive Immunity , Animals , Cell Differentiation , Cell Lineage , Cells, Cultured , GRB2 Adaptor Protein/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Phospholipase C gamma/metabolism , Receptors, Antigen, B-Cell/metabolism , Self Tolerance , src-Family Kinases/metabolismABSTRACT
The FcµR receptor for the crystallizable fragment (Fc) of immunoglobulin M (IgM) can function as a cell-surface receptor for secreted IgM on a variety of cell types. We found here that FcµR was also expressed in the trans-Golgi network of developing B cells, where it constrained transport of the IgM-isotype BCR (IgM-BCR) but not of the IgD-isotype BCR (IgD-BCR). In the absence of FcµR, the surface expression of IgM-BCR was increased, which resulted in enhanced tonic BCR signaling. B-cell-specific deficiency in FcµR enhanced the spontaneous differentiation of B-1 cells, which resulted in increased serum concentrations of natural IgM and dysregulated homeostasis of B-2 cells; this caused the spontaneous formation of germinal centers, increased titers of serum autoantibodies and excessive accumulation of B cells. Thus, FcµR serves as a critical regulator of B cell biology by constraining the transport and cell-surface expression of IgM-BCR.
Subject(s)
B-Lymphocytes/physiology , Immunoglobulin M/metabolism , Precursor Cells, B-Lymphoid/physiology , Receptors, Antigen, B-Cell/metabolism , Receptors, Fc/metabolism , Animals , Cell Differentiation , Cells, Cultured , Cytokines/metabolism , Female , Gene Expression Regulation , Immunoglobulin M/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, B-Cell/genetics , Signal Transduction , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3-/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.
Subject(s)
ADAM10 Protein/metabolism , Adaptive Immunity , Amyloid Precursor Protein Secretases/metabolism , B-Lymphocytes/physiology , Cell Differentiation , Cell Lineage , Germinal Center/immunology , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , ADAM10 Protein/genetics , Amyloid Precursor Protein Secretases/genetics , Animals , Cells, Cultured , Clonal Selection, Antigen-Mediated , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Receptor, Notch2/metabolism , Receptors, Antigen, B-Cell/metabolism , Signal TransductionABSTRACT
Despite the importance of memory B cells in protection from reinfection, how such memory cells are selected and generated during germinal-center (GC) reactions remains unclear. We found here that light-zone (LZ) GC B cells with B cell antigen receptors (BCRs) of lower affinity were prone to enter the memory B cell pool. Mechanistically, cells in this memory-prone fraction had higher expression of the transcriptional repressor Bach2 than that of their counterparts with BCRs of higher affinity. Haploinsufficiency of Bach2 resulted in reduced generation of memory B cells, independently of suppression of the gene encoding the transcription factor Blimp-1. Bach2 expression in GC cells was inversely correlated with the strength of help provided by T cells. Thus, we propose an instructive model in which weak help from T cells maintains relatively high expression of Bach2, which predisposes GC cells to enter the memory pool.
Subject(s)
B-Lymphocytes/physiology , Basic-Leucine Zipper Transcription Factors/metabolism , Germinal Center/immunology , Immunologic Memory , T-Lymphocytes, Helper-Inducer/immunology , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Cell Differentiation , Cells, Cultured , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Positive Regulatory Domain I-Binding Factor 1 , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.
Subject(s)
B-Lymphocytes/physiology , CD4-Positive T-Lymphocytes/physiology , Germinal Center/immunology , Inducible T-Cell Co-Stimulator Protein/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Antibody Formation/genetics , Cell Differentiation/genetics , Cells, Cultured , Inducible T-Cell Co-Stimulator Protein/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Protein Binding , Protein Serine-Threonine Kinases/genetics , Signal Transduction , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 3/geneticsABSTRACT
Aberrant production of IgE antibodies can lead to allergic diseases. Normally, IgE(+) B cells rarely differentiate into memory B cells (Bmem) or long-lived plasma cells (LLPCs), as they only transiently participate in the germinal center (GC), but the mechanism behind this remains elusive. We found that membrane IgE (mIgE) autonomously triggered rapid plasma-cell differentiation and apoptosis independently of antigen or cellular context, predominantly through the mutually independent CD19-PI3K-Akt-IRF4 and BLNK-Jnk/p38 pathways, respectively, and we identified the ectodomains of mIgE as being responsible. Accordingly, deregulated GC IgE(+) B cell proliferation and prolonged IgE production with exaggerated anaphylaxis were observed in CD19- and BLNK-deficient mice. Our findings reveal an autonomous mIgE signaling mechanism that normally prevents IgE(+) Bmem and LLPC formation, providing insights into the molecular pathogenesis of allergic diseases.
Subject(s)
Anaphylaxis/immunology , B-Lymphocytes/physiology , Cell Membrane , Germinal Center/immunology , Hypersensitivity/immunology , Immunoglobulin E/metabolism , Immunologic Memory , Plasma Cells/physiology , 3T3 Cells , Animals , Antigens, CD19/genetics , Apoptosis , Calcium Signaling , Cell Differentiation , Cell Membrane/metabolism , Cell Proliferation , Enzyme-Linked Immunospot Assay , Immunoglobulin Class Switching , Immunoglobulin G/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, B-Cell/metabolismABSTRACT
The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.
Subject(s)
B-Lymphocytes/physiology , DNA Methylation , NF-kappa B/metabolism , Plasma Cells/physiology , Transcription Factor AP-1/metabolism , Animals , Binding Sites/genetics , Cell Differentiation/genetics , Cell Division/genetics , Cells, Cultured , CpG Islands/genetics , Epigenesis, Genetic , Female , Gene Expression Regulation , Immunity, Humoral/genetics , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Male , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , Octamer Transcription Factor-2/genetics , Octamer Transcription Factor-2/metabolism , Transcription Factor AP-1/geneticsABSTRACT
Inactivating mutations of the CREBBP and EP300 acetyltransferases are among the most common genetic alterations in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Here, we examined the relationship between these two enzymes in germinal center (GC) B cells, the normal counterpart of FL and DLBCL, and in lymphomagenesis by using conditional GC-directed deletion mouse models targeting Crebbp or Ep300. We found that CREBBP and EP300 modulate common as well as distinct transcriptional programs implicated in separate anatomic and functional GC compartments. Consistently, deletion of Ep300 but not Crebbp impaired the fitness of GC B cells in vivo. Combined loss of Crebbp and Ep300 completely abrogated GC formation, suggesting that these proteins partially compensate for each other through common transcriptional targets. This synthetic lethal interaction was retained in CREBBP-mutant DLBCL cells and could be pharmacologically targeted with selective small molecule inhibitors of CREBBP and EP300 function. These data provide proof-of-principle for the clinical development of EP300-specific inhibitors in FL and DLBCL.
Subject(s)
B-Lymphocytes/physiology , CREB-Binding Protein/genetics , E1A-Associated p300 Protein/genetics , Epigenesis, Genetic/genetics , Germinal Center/physiology , Lymphoma, Follicular/etiology , Lymphoma, Large B-Cell, Diffuse/genetics , Acetyltransferases/genetics , Animals , Cell Line , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Sequence Deletion/genetics , Transcription, Genetic/geneticsABSTRACT
HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.
Subject(s)
B-Lymphocytes/physiology , DNA End-Joining Repair/genetics , DNA-Binding Proteins/genetics , DNA/genetics , Immunoglobulin Class Switching/genetics , Animals , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Damage/genetics , Mice , Mice, Inbred C57BL , Translocation, Genetic/geneticsABSTRACT
Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.