RESUMO
Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.
Assuntos
Tolerância Central/genética , Tolerância Central/imunologia , Citidina Desaminase/genética , Ativação Linfocitária/imunologia , Células Precursoras de Linfócitos B/imunologia , Adolescente , Adulto , Idoso , Animais , Apoptose/genética , Apoptose/imunologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Proteínas de Ligação a DNA/genética , Feminino , Genes de Imunoglobulinas/genética , Genes de Imunoglobulinas/imunologia , Humanos , Ativação Linfocitária/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Recombinação Genética/genética , Recombinação Genética/imunologia , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Adulto JovemRESUMO
ZAP70 has a prognostic value in chronic lymphocytic leukemia (CLL), through altered B-cell receptor signaling, which is important in CLL pathogenesis. A good correlation between ZAP70 expression in CLL cells and the occurrence of autoimmune phenomena has been reported. Yet, the great majority of CLL-associated autoimmune cytopenia is due to polyclonal immunoglobulin (Ig) G synthesized by nonmalignant B cells, and this phenomenon is poorly understood. Here, we show, using flow cytometry, that a substantial percentage of CD5- nonmalignant B cells from CLL patients expresses ZAP70 compared with CD5- B cells from healthy subjects. This ZAP70 expression in normal B cells from CLL patients was also evidenced by the detection of ZAP70 mRNA at single-cell level with polyclonal Ig heavy- and light-chain gene transcripts. ZAP70+ normal B cells belong to various B-cell subsets and their presence in the naïve B-cell subset suggests that ZAP70 expression may occur during early B-cell development in CLL patients and potentially before malignant transformation. The presence of ZAP70+ normal B cells is associated with autoimmune cytopenia in CLL patients in our cohort of patients, and recombinant antibodies produced from these ZAP70+ nonmalignant B cells were frequently autoreactive including anti-platelet reactivity. These results provide a better understanding of the implication of ZAP70 in CLL leukemogenesis and the mechanisms of autoimmune complications of CLL.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Autoimunidade , Linfócitos B , Citometria de Fluxo , Prognóstico , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
Assuntos
Reprogramação Celular/genética , Edição de Genes , Genoma Humano/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Autoimunidade/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Masculino , Camundongos , Transplante de Neoplasias , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologiaRESUMO
BACKGROUND: The lack of pathogen-protective, isotype-switched antibodies in patients with common variable immunodeficiency (CVID) suggests germinal center (GC) hypoplasia, yet a subset of patients with CVID is paradoxically affected by autoantibody-mediated autoimmune cytopenias (AICs) and lymphadenopathy. OBJECTIVE: We sought to compare the physical characteristics and immunologic output of GC responses in patients with CVID with AIC (CVID+AIC) and without AIC (CVID-AIC). METHODS: We analyzed GC size and shape in excisional lymph node biopsy specimens from 14 patients with CVID+AIC and 4 patients with CVID-AIC. Using paired peripheral blood samples, we determined how AICs specifically affected B-and T-cell compartments and antibody responses in patients with CVID. RESULTS: We found that patients with CVID+AIC displayed irregularly shaped hyperplastic GCs, whereas GCs were scarce and small in patients with CVID-AIC. GC hyperplasia was also evidenced by an increase in numbers of circulating follicular helper T cells, which correlated with decreased regulatory T-cell frequencies and function. In addition, patients with CVID+AIC had serum endotoxemia associated with a dearth of isotype-switched memory B cells that displayed significantly lower somatic hypermutation frequencies than their counterparts with CVID-AIC. Moreover, IgG+ B cells from patients with CVID+AIC expressed VH4-34-encoded antibodies with unmutated Ala-Val-Tyr and Asn-His-Ser motifs, which recognize both erythrocyte I/i self-antigens and commensal bacteria. CONCLUSIONS: Patients with CVID+AIC do not contain mucosal microbiota and exhibit hyperplastic yet inefficient GC responses that favor the production of untolerized IgG+ B-cell clones that recognize both commensal bacteria and hematopoietic I/i self-antigens.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Imunodeficiência de Variável Comum/imunologia , Centro Germinativo/imunologia , Imunoglobulina G/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Linfócitos B/patologia , Biópsia , Criança , Imunodeficiência de Variável Comum/patologia , Feminino , Centro Germinativo/patologia , Humanos , Hiperplasia , Masculino , Pessoa de Meia-Idade , Linfócitos T/patologiaRESUMO
Humanized mice are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, the existing models cannot support robust adaptive immune responses, especially the generation of class-switched, antigen-specific antibody responses. Here we describe a new mouse strain, in which human interleukin 6 (IL-6) gene encoding the cytokine that is important for B- and T-cell differentiation was knocked into its respective mouse locus. The provision of human IL-6 not only enhanced thymopoiesis and periphery T-cell engraftment, but also significantly increased class switched memory B cells and serum immunoglobulin G (IgG). In addition, immunization with ovalbumin (OVA) induced OVA-specific B cells only in human IL-6 knock-in mice. These OVA-specific antibodies displayed the highest frequency of somatic mutation, further suggesting that human IL-6 is important for efficient B-cell activation and selection. We conclude that human IL-6 knock-in mice represent a novel and improved model for human adaptive immunity without relying on complex surgery to transplant human fetal thymus and liver. These mice can therefore be used to exploit or evaluate immunization regimes that would be unethical or untenable in humans.
Assuntos
Imunidade Adaptativa , Formação de Anticorpos , Técnicas de Introdução de Genes , Switching de Imunoglobulina , Interleucina-6/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Galinhas , Expressão Gênica , Técnicas de Introdução de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunização , Imunoglobulina G/imunologia , Interleucina-6/imunologia , Camundongos , Ovalbumina/imunologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.
Assuntos
Autoantígenos/imunologia , Autoimunidade/imunologia , Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , Doença de Lyme/imunologia , Animais , Afinidade de Anticorpos/imunologia , Borrelia burgdorferi , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Introdução de Genes , Isotipos de Imunoglobulinas/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Ressonância de Plasmônio de SuperfícieRESUMO
The vast majority of IgA production occurs in mucosal tissue following T cell-dependent and T cell-independent Ag responses. To study the nature of each of these responses, we analyzed the gene-expression and Ig-reactivity profiles of T cell-dependent CD27(+)IgA(+) and T cell-independent CD27(-)IgA(+) circulating memory B cells. Gene-expression profiles of IgA(+) subsets were highly similar to each other and to IgG(+) memory B cell subsets, with typical upregulation of activation markers and downregulation of inhibitory receptors. However, we identified the mucosa-associated CCR9 and RUNX2 genes to be specifically upregulated in CD27(-)IgA(+) B cells. We also found that CD27(-)IgA(+) B cells expressed Abs with distinct Ig repertoire and reactivity compared with those from CD27(+)IgA(+) B cells. Indeed, Abs from CD27(-)IgA(+) B cells were weakly mutated, often used Igλ chain, and were enriched in polyreactive clones recognizing various bacterial species. Hence, T cell-independent IgA responses are likely involved in the maintenance of gut homeostasis through the production of polyreactive mutated IgA Abs with cross-reactive anti-commensal reactivity.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Bactérias/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunoglobulinas/imunologia , Memória Imunológica , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Autoimunidade/genética , Análise por Conglomerados , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Imunoglobulinas/genética , Mucosa/imunologia , Mucosa/metabolismo , Receptores CCR/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Immunity to Plasmodium falciparum malaria is naturally acquired in individuals living in malaria-endemic areas of Africa. Abs play a key role in mediating this immunity; however, the acquisition of the components of Ab immunity, long-lived plasma cells and memory B cells (MBCs), is remarkably inefficient, requiring years of malaria exposure. Although long-lived classical MBCs (CD19(+)/CD20(+)/CD21(+)/CD27(+)/CD10(-)) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical MBCs (CD19(+)/CD20(+)/CD21(-)/CD27(-)/CD10(-)). At present, the function of atypical MBCs in malaria is not known, nor are the factors that drive their differentiation. To gain insight into the relationship between classical and atypical IgG(+) MBCs, we compared the Ab H and L chain V gene repertoires of children living in a malaria-endemic region in Mali. We found that these repertoires were remarkably similar by a variety of criteria, including V gene usage, rate of somatic hypermutation, and CDR-H3 length and composition. The similarity in these repertoires suggests that classical MBCs and atypical MBCs differentiate in response to similar Ag-dependent selective pressures in malaria-exposed children and that atypical MBCs do not express a unique V gene repertoire.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Suscetibilidade a Doenças , Região Variável de Imunoglobulina/genética , Memória Imunológica/genética , Malária/genética , Malária/imunologia , África Ocidental , Sequência de Aminoácidos , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Estudos de Coortes , Regiões Determinantes de Complementaridade/genética , Feminino , Rearranjo Gênico do Linfócito B , Variação Genética , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/química , Imunofenotipagem , Malária Falciparum/genética , Malária Falciparum/imunologia , Masculino , Plasmodium falciparum/imunologia , Hipermutação Somática de ImunoglobulinaRESUMO
BACKGROUND: CD19 is a B cell-specific molecule that serves as a major costimulatory molecule for amplifying B-cell receptor (BCR) responses. Biallelic CD19 gene mutations cause common variable immunodeficiency in human subjects. BCR- and Toll-like receptor (TLR) 9-induced B-cell responses are impaired in most patients with common variable immunodeficiency. OBJECTIVE: We sought to analyze whether CD19 is required for TLR9 function in human B cells. METHODS: Expression of surface activation markers was assessed after anti-IgM or CpG stimulation by using flow cytometry on B cells from patients with 1 or 2 defective CD19 alleles, which decrease or abrogate CD19 expression, respectively. The phosphorylation or interaction of signaling molecules was analyzed by using phospho flow cytometry, immunoblotting, or co-immunoprecipitation in CD19-deficient or control B cells and in a B-cell line in which CD19 has been knocked down with lentivirus-transduced short hairpin RNA. RESULTS: B cells from subjects with 1 or 2 defective CD19 alleles showed defective upregulation in vitro of CD86, transmembrane activator and CAML interactor (TACI), and CD23 activation markers after TLR9 stimulation. TLR9 ligands normally induce phosphorylation of CD19 through myeloid differentiation primary response gene-88 (MYD88)/proline-rich tyrosine kinase 2 (PYK2)/LYN complexes, which allows recruitment of phosphoinositide 3-kinase (PI3K) and phosphorylation of Bruton tyrosine kinase (BTK) and AKT in human B cells with a different kinetic than that of BCRs. In addition, inhibition of PI3K, AKT, or BTK, as well as BTK deficiency, also resulted in TLR9 activation defects in B cells similar to those in patients with CD19 deficiency. CONCLUSION: CD19 is required for TLR9-induced B-cell activation. Hence CD19/PI3K/AKT/BTK is an essential axis integrating BCRs and TLR9 signaling in human B cells.
Assuntos
Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Receptor Toll-Like 9/metabolismo , Tirosina Quinase da Agamaglobulinemia , Estudos de Casos e Controles , Quinase 2 de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Heterozigoto , Homozigoto , Humanos , Imunofenotipagem , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptor Toll-Like 9/agonistasRESUMO
BACKGROUND: Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and is regulated by various signaling pathways. However, the molecular mechanisms that negatively regulate these signal transduction pathways remain poorly understood. METHODS AND RESULTS: Here, we characterized Carabin, a protein expressed in cardiomyocytes that was downregulated in cardiac hypertrophy and human heart failure. Four weeks after transverse aortic constriction, Carabin-deficient (Carabin(-/-)) mice developed exaggerated cardiac hypertrophy and displayed a strong decrease in fractional shortening (14.6±1.6% versus 27.6±1.4% in wild type plus transverse aortic constriction mice; P<0.0001). Conversely, compensation of Carabin loss through a cardiotropic adeno-associated viral vector encoding Carabin prevented transverse aortic constriction-induced cardiac hypertrophy with preserved fractional shortening (39.9±1.2% versus 25.9±2.6% in control plus transverse aortic constriction mice; P<0.0001). Carabin also conferred protection against adrenergic receptor-induced hypertrophy in isolated cardiomyocytes. Mechanistically, Carabin carries out a tripartite suppressive function. Indeed, Carabin, through its calcineurin-interacting site and Ras/Rab GTPase-activating protein domain, functions as an endogenous inhibitor of calcineurin and Ras/extracellular signal-regulated kinase prohypertrophic signaling. Moreover, Carabin reduced Ca(2+)/calmodulin-dependent protein kinase II activation and prevented nuclear export of histone deacetylase 4 after adrenergic stimulation or myocardial pressure overload. Finally, we showed that Carabin Ras-GTPase-activating protein domain and calcineurin-interacting domain were both involved in the antihypertrophic action of Carabin. CONCLUSIONS: Our study identifies Carabin as a negative regulator of key prohypertrophic signaling molecules, calcineurin, Ras, and Ca(2+)/calmodulin-dependent protein kinase II and implicates Carabin in the development of cardiac hypertrophy and failure.
Assuntos
Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/prevenção & controle , Proteínas Ativadoras de GTPase/biossíntese , Genes ras/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Heterozygous C104R or A181E TNF receptor superfamily member 13b (TNFRSF13B) mutations impair removal of autoreactive B cells, weaken B-cell activation, and convey to patients with common variable immune deficiency (CVID) an increased risk for autoimmunity. How mutant transmembrane activator and CAML interactor (TACI) influences wild-type TACI function is unclear; different models suggest either a dominant negative effect or haploinsufficiency. OBJECTIVE: We investigated potential TACI haploinsufficiency by analyzing patients with antibody-deficient Smith-Magenis syndrome (SMS) who possess only 1 TNFRSF13B allele and antibody-deficient patients carrying one c.204insA TNFRSF13B null mutation. METHODS: We tested the reactivity of antibodies isolated from single B cells from patients with SMS and patients with a c.204insA TNFRSF13B mutation and compared them with counterparts from patients with CVID with heterozygous C104R or A181E TNFRSF13B missense mutations. We also assessed whether loss of a TNFRSF13B allele induced haploinsufficiency in naive and memory B cells and recapitulated abnormal immunologic features typical of patients with CVID with heterozygous TNFRSF13B missense mutations. RESULTS: We found that loss of a TNFRSF13B allele does not affect TACI expression, activation responses, or establishment of central B-cell tolerance in naive B cells. Additionally, patients with SMS and those with a c.204insA TNFRSF13B mutation display normal regulatory T-cell function and peripheral B-cell tolerance. The lack of a TNFRSF13B allele did result in decreased TACI expression on memory B cells, resulting in impaired activation and antibody secretion. CONCLUSION: TNFRSF13B hemizygosity does not recapitulate autoimmune features of CVID-associated C104R and A181E TNFRSF13B mutations, which likely encode dominant negative products, but instead reveals selective TACI haploinsufficiency at later stages of B-cell development.
Assuntos
Linfócitos B/imunologia , Imunodeficiência de Variável Comum/imunologia , Síndrome de Smith-Magenis/imunologia , Linfócitos T Reguladores/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Formação de Anticorpos/genética , Autoimunidade , Criança , Feminino , Haploinsuficiência , Hemizigoto , Humanos , Memória Imunológica , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Adulto JovemRESUMO
Central B cell tolerance is believed to be regulated by B cell receptor signaling induced by the recognition of self-antigens in immature B cells. Using humanized mice with defective MyD88, TLR7, or TLR9 expression, we demonstrate that TLR9/MYD88 are required for central B cell tolerance and the removal of developing autoreactive clones. We also show that CXCL4, a chemokine involved in systemic sclerosis (SSc), abrogates TLR9 function in B cells by sequestering TLR9 ligands away from the endosomal compartments where this receptor resides. The in vivo production of CXCL4 thereby impedes both TLR9 responses in B cells and the establishment of central B cell tolerance. We conclude that TLR9 plays an essential early tolerogenic function required for the establishment of central B cell tolerance and that correcting defective TLR9 function in B cells from SSc patients may represent a novel therapeutic strategy to restore B cell tolerance.
Assuntos
Fator Plaquetário 4 , Escleroderma Sistêmico , Receptor Toll-Like 9 , Animais , Humanos , Camundongos , Linfócitos B , Ligantes , Fator 88 de Diferenciação Mieloide/metabolismo , Fator Plaquetário 4/metabolismo , Escleroderma Sistêmico/metabolismo , Receptor 7 Toll-Like , Receptor Toll-Like 9/metabolismoRESUMO
Fibroblast-like synoviocytes (FLSs) are important actors in rheumatoid arthritis (RA) pathogenesis. The autoimmune nature of RA is attributed to autoantibody production, which confers to B cells a predominant role in RA. Several arguments support an induction of class switch recombination (CSR) in RA synovium, causing--in conjunction with somatic hypermutation--the production of potentially pathogenic IgG. To determine whether RA FLSs can directly promote CSR and to analyze the role of external factors like TLR signals and BAFF (B cell activating factor) family cytokines in this FLS-B cell crosstalk, we performed cocultures of blood B cells (from normal individuals or RA patients) with RA FLSs and analyzed CSR induction by quantification of AICDA (encoding activation-induced cytidine deaminase, AID) and switch circular transcripts expression, and IgG secretion. RA FLSs--and to a lesser extent osteoarthritis or control FLSs--promoted CSR, and TLR3 stimulation potentialized it. In addition, induction of CSR by RA FLSs was totally dependent on cell-cell contact in basal conditions, and partially dependent in the case of TLR3 stimulation. Finally, we showed that the mechanism by which RA FLSs induce CSR is mostly BAFF-dependent. Our results support the hypothesis that CSR can be induced outside the ectopic lymphoid structures in RA.
Assuntos
Artrite Reumatoide/imunologia , Fator Ativador de Células B/metabolismo , Switching de Imunoglobulina , Imunoglobulina G/biossíntese , Membrana Sinovial/citologia , Artrite Reumatoide/patologia , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citidina Desaminase/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Humanos , Imunoglobulina G/metabolismo , Região de Troca de Imunoglobulinas , Osteoartrite/imunologia , Osteoartrite/patologia , Reação em Cadeia da Polimerase , Hipermutação Somática de Imunoglobulina , Membrana Sinovial/metabolismo , Receptor 3 Toll-Like/metabolismoRESUMO
Although negative selection of developing B cells in the periphery is well described, yet poorly understood, evidence of naive B cell positive selection remains elusive. Using 2 humanized mouse models, we demonstrate that there was strong skewing of the expressed immunoglobulin repertoire upon transit into the peripheral naive B cell pool. This positive selection of expanded naive B cells in humanized mice resembled that observed in healthy human donors and was independent of autologous thymic tissue. In contrast, negative selection of autoreactive B cells required thymus-derived Tregs and MHC class II-restricted self-antigen presentation by B cells. Indeed, both defective MHC class II expression on B cells of patients with rare bare lymphocyte syndrome and prevention of self-antigen presentation via HLA-DM inhibition in humanized mice resulted in the production of autoreactive naive B cells. These latter observations suggest that Tregs repressed autoreactive naive B cells continuously produced by the bone marrow. Thus, a model emerged, in which both positive and negative selection shaped the human naive B cell repertoire and that each process was mediated by fundamentally different molecular and cellular mechanisms.
Assuntos
Apresentação de Antígeno , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunodeficiência Combinada Severa/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Autoimmune regulator (AIRE) mutations result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome characterized by defective central T cell tolerance and the production of many autoantibodies targeting tissue-specific antigens and cytokines. By studying CD3- and AIRE-deficient patients, we found that lack of either T cells or AIRE function resulted in the peripheral accumulation of autoreactive mature naïve B cells. Proteomic arrays and Biacore affinity measurements revealed that unmutated antibodies expressed by these autoreactive naïve B cells recognized soluble molecules and cytokines including insulin, IL-17A, and IL-17F, which are AIRE-dependent thymic peripheral tissue antigens targeted by autoimmune responses in APECED. AIRE-deficient patients also displayed decreased frequencies of regulatory T cells (Tregs) that lacked common TCRß clones found instead in their conventional T cell compartment, thereby suggesting holes in the Treg TCR repertoire of these patients. Hence, AIRE-mediated T cell/Treg selection normally prevents the expansion of autoreactive naïve B cells recognizing peripheral self-antigens.
Assuntos
Autoanticorpos/imunologia , Autoimunidade/genética , Linfócitos B/imunologia , Poliendocrinopatias Autoimunes/imunologia , Fatores de Transcrição/deficiência , Adolescente , Autoanticorpos/sangue , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Linfócitos B/metabolismo , Complexo CD3/deficiência , Complexo CD3/genética , Complexo CD3/imunologia , Criança , Pré-Escolar , Citocinas/imunologia , Feminino , Humanos , Tolerância Imunológica/genética , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Mutação , Poliendocrinopatias Autoimunes/sangue , Poliendocrinopatias Autoimunes/genética , Análise Serial de Proteínas , Proteômica/métodos , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Proteína AIRERESUMO
Patients with rheumatoid arthritis (RA) may display atypical CD21-/lo B cells in their blood, but the implication of this observation remains unclear. We report here that the group of patients with RA and elevated frequencies of CD21-/lo B cells shows decreased ataxia telangiectasia-mutated (ATM) expression and activation in B cells compared with other patients with RA and healthy donor controls. In agreement with ATM involvement in the regulation of V(D)J recombination, patients with RA who show defective ATM function displayed a skewed B cell receptor (BCR) Igκ repertoire, which resembled that of patients with ataxia telangiectasia (AT). This repertoire was characterized by increased Jκ1 and decreased upstream Vκ gene segment usage, suggesting improper secondary recombination processes and selection. In addition, altered ATM function in B cells was associated with decreased osteoprotegerin and increased receptor activator of nuclear factor κB ligand (RANKL) production. These changes favor bone loss and correlated with a higher prevalence of erosive disease in patients with RA who show impaired ATM function. Using a humanized mouse model, we also show that ATM inhibition in vivo induces an altered Igκ repertoire and RANKL production by immature B cells in the bone marrow, leading to decreased bone density. We conclude that dysregulated ATM function in B cells promotes bone erosion and the emergence of circulating CD21-/lo B cells, thereby contributing to RA pathophysiology.
Assuntos
Artrite Reumatoide/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/metabolismo , Reabsorção Óssea/imunologia , Animais , Artrite Reumatoide/fisiopatologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Densidade Óssea , Reabsorção Óssea/fisiopatologia , Sobrevivência Celular/imunologia , Humanos , Imunoglobulinas/imunologia , Articulações/patologia , Contagem de Linfócitos , Camundongos , Pessoa de Meia-Idade , Osteogênese , Osteoprotegerina/metabolismo , Fenótipo , Ligante RANK/metabolismo , Receptores de Complemento 3d/metabolismo , Recombinação Genética/genéticaRESUMO
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RESUMO
Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.
Assuntos
Antígeno CTLA-4/metabolismo , Proteínas de Ligação a DNA/deficiência , Fatores de Troca do Nucleotídeo Guanina/deficiência , Doenças da Imunodeficiência Primária/genética , Antígeno B7-1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Homeostase , Humanos , Células Jurkat , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
B cells play a central role in systemic lupus erythematosus (SLE) pathophysiology but dysregulated pathways leading to a break in B cell tolerance remain unclear. Since Toll-like receptor 9 (TLR9) favors the elimination of autoreactive B cells in the periphery, we assessed TLR9 function in SLE by analyzing the responses of B cells and plasmacytoid dendritic cells (pDCs) isolated from healthy donors and patients after stimulation with CpG, a TLR9 agonist. We found that SLE B cells from patients without hydroxychloroquine treatment displayed defective in vitro TLR9 responses, as illustrated by the impaired upregulation of B cell activation molecules and the diminished production of various cytokines including antiinflammatory IL-10. In agreement with CD19 controlling TLR9 responses in B cells, decreased expression of the CD19/CD21 complex on SLE B cells was detected as early as the transitional B cell stage. In contrast, TLR7 function was preserved in SLE B cells, whereas pDCs from SLE patients properly responded to TLR9 stimulation, thereby revealing that impaired TLR9 function in SLE was restricted to B cells. We conclude that abnormal CD19 expression and TLR9 tolerogenic function in SLE B cells may contribute to the break of B cell tolerance in these patients.
Assuntos
Antígenos CD19/imunologia , Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Complemento 3d/imunologia , Receptor Toll-Like 9/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/metabolismo , Autoimunidade , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Humanos , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/sangue , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/farmacologia , Cultura Primária de Células , Receptores de Complemento 3d/metabolismo , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Regulação para Cima , Adulto JovemRESUMO
The germline immunoglobulin (Ig) variable heavy chain 4-34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34-expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27+IgG+ B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34-expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34-encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34-encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.