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1.
Nat Immunol ; 23(4): 543-555, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35288714

RESUMO

Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-γ levels 1 d following secondary immunization. Notably, we found that natural killer cells and CD8+ T cells in the draining lymph nodes are the major producers of this circulating IFN-γ. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8+ T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.


Assuntos
Linfócitos T CD8-Positivos , Vacinas , Imunidade Adaptativa , Animais , Vacina BNT162 , Humanos , Imunidade Inata , Camundongos , Vacinas Sintéticas , Vacinas de mRNA
2.
Immunity ; 57(5): 973-986.e7, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38697117

RESUMO

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.


Assuntos
Endorribonucleases , Quinase I-kappa B , Inflamação , Macrófagos , Proteínas Serina-Treonina Quinases , Receptores Toll-Like , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Citocinas/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Receptores Toll-Like/metabolismo , Ubiquitina/metabolismo
3.
Cell ; 166(6): 1445-1458.e12, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27610569

RESUMO

A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/administração & dosagem , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunização , Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos B/imunologia , Clonagem Molecular , Primers do DNA/química , Epitopos/imunologia , Técnicas de Introdução de Genes , Infecções por HIV/imunologia , Camundongos , Mutação , Alinhamento de Sequência
4.
Cell ; 161(7): 1505-15, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26091035

RESUMO

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.


Assuntos
Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Técnicas de Introdução de Genes , HIV-1/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Antígenos Virais , Linfócitos B/imunologia , Antígenos CD4/metabolismo , Infecções por HIV/imunologia , Humanos , Camundongos , Mutação , Baço/citologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
5.
Nature ; 587(7833): 275-280, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32971525

RESUMO

Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.


Assuntos
Caspase 8/metabolismo , Citocinas/imunologia , Imunidade Inata/imunologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Citocinas/antagonistas & inibidores , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
6.
Immunity ; 44(4): 769-81, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-26944202

RESUMO

Somatic hypermutation (SHM) and class-switch recombination (CSR) increase the affinity and diversify the effector functions of antibodies during immune responses. Although SHM and CSR are fundamentally different, their independent roles in regulating B cell fate have been difficult to uncouple because a single enzyme, activation-induced cytidine deaminase (encoded by Aicda), initiates both reactions. Here, we used a combination of Aicda and antibody mutant alleles that separate the effects of CSR and SHM on polyclonal immune responses. We found that class-switching to IgG1 biased the fate choice made by B cells, favoring the plasma cell over memory cell fate without significantly affecting clonal expansion in the germinal center (GC). In contrast, SHM reduced the longevity of memory B cells by creating polyreactive specificities that were selected against over time. Our data define the independent contributions of SHM and CSR to the generation and persistence of memory in the antibody system.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/genética , Switching de Imunoglobulina/imunologia , Memória Imunológica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular/imunologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Switching de Imunoglobulina/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipermutação Somática de Imunoglobulina/genética
7.
Immunity ; 45(3): 483-496, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27617678

RESUMO

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Polissacarídeos/imunologia , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Epitopos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunização/métodos , Camundongos , Camundongos Knockout , Mutação/imunologia , Alinhamento de Sequência , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
8.
Biochem J ; 479(10): 1103-1119, 2022 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-35608338

RESUMO

Apoptosis, pyroptosis, and necroptosis are distinct forms of programmed cell death that eliminate infected, damaged, or obsolete cells. Many proteins that regulate or are a part of the cell death machinery undergo ubiquitination, a post-translational modification made by ubiquitin ligases that modulates protein abundance, localization, and/or activity. For example, some ubiquitin chains target proteins for degradation, while others function as scaffolds for the assembly of signaling complexes. Deubiquitinases (DUBs) are the proteases that counteract ubiquitin ligases by cleaving ubiquitin from their protein substrates. Here, we review the DUBs that have been found to suppress or promote apoptosis, pyroptosis, or necroptosis.


Assuntos
Inflamação , Ubiquitina , Morte Celular , Enzimas Desubiquitinantes/metabolismo , Humanos , Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
9.
Nature ; 509(7502): 637-40, 2014 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-24805232

RESUMO

During immune responses, B lymphocytes clonally expand and undergo secondary diversification of their immunoglobulin genes in germinal centres (GCs). High-affinity B cells are expanded through iterative interzonal cycles of division and hypermutation in the GC dark zone followed by migration to the GC light zone, where they are selected on the basis of affinity to return to the dark zone. Here we combine a transgenic strategy to measure cell division and a photoactivatable fluorescent reporter to examine whether the extent of clonal expansion and hypermutation are regulated during interzonal GC cycles. We find that both cell division and hypermutation are directly proportional to the amount of antigen captured and presented by GC B cells to follicular helper T cells in the light zone. Our data explain how GC B cells with the highest affinity for antigen are selectively expanded and diversified.


Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Seleção Clonal Mediada por Antígeno , Centro Germinativo/citologia , Centro Germinativo/imunologia , Hipermutação Somática de Imunoglobulina/genética , Animais , Afinidade de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Movimento Celular , Proliferação de Células , Seleção Clonal Mediada por Antígeno/imunologia , Células Clonais/citologia , Células Clonais/imunologia , Células Clonais/metabolismo , Genes Reporter/genética , Masculino , Camundongos , Fase S , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Tempo
11.
Proc Natl Acad Sci U S A ; 110(41): 16538-43, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-24043801

RESUMO

Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1-infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.


Assuntos
Antirretrovirais/imunologia , Anticorpos Neutralizantes/imunologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Imunoterapia/métodos , Animais , Antirretrovirais/farmacologia , Anticorpos Neutralizantes/farmacologia , Primers do DNA/genética , DNA Viral/metabolismo , Dependovirus , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Carga Viral/efeitos dos fármacos
12.
Proc Natl Acad Sci U S A ; 109(14): 5364-9, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22434908

RESUMO

Under the instruction of cell-fate-determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic α- and ß-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.


Assuntos
Cromatina/metabolismo , Pâncreas/efeitos dos fármacos , Transcrição Gênica , Linhagem Celular , Regulação para Baixo , Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Pâncreas/citologia , Pâncreas/metabolismo , Regulação para Cima
13.
Elife ; 92020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32515732

RESUMO

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.


Assuntos
Adjuvantes Imunológicos , Formação de Anticorpos , Linfócitos T CD8-Positivos/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Esqualeno/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Emulsões , Imunidade Inata , Linfonodos/citologia , Linfonodos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polissorbatos , Proteínas Repressoras/metabolismo
14.
J Exp Med ; 214(11): 3435-3448, 2017 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-28939548

RESUMO

The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T-B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh-B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells.


Assuntos
Antígenos CD/imunologia , Linfócitos B/imunologia , Moléculas de Adesão Celular/imunologia , Comunicação Celular/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos CD/genética , Antígenos CD/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Clonais/imunologia , Células Clonais/metabolismo , Citometria de Fluxo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo
15.
Science ; 358(6360)2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28935768

RESUMO

B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.


Assuntos
Apoptose/imunologia , Linfócitos B/citologia , Divisão Celular , Centro Germinativo/citologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Apoptose/genética , Linfócitos B/enzimologia , Linfócitos B/imunologia , Citidina Desaminase/metabolismo , Centro Germinativo/enzimologia , Centro Germinativo/imunologia , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo
16.
J Exp Med ; 214(9): 2573-2590, 2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28847869

RESUMO

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.


Assuntos
Anticorpos Neutralizantes/imunologia , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Cristalografia por Raios X , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Multimerização Proteica/imunologia , Estrutura Terciária de Proteína
17.
Nat Protoc ; 11(10): 1908-1923, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27658009

RESUMO

Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.

18.
Nat Commun ; 7: 10618, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26907590

RESUMO

VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos B/imunologia , Técnicas de Introdução de Genes , HIV-1/genética , Humanos , Imunização , Fragmentos Fab das Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
19.
Science ; 349(6248): 643-6, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26184917

RESUMO

The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression, while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles.


Assuntos
Linfócitos B/citologia , Ciclo Celular/imunologia , Replicação do DNA/imunologia , Centro Germinativo/citologia , Imunidade Humoral/genética , Linfócitos T/imunologia , Animais , Ciclo Celular/genética , Proliferação de Células , Replicação do DNA/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fase S/genética , Fase S/imunologia
20.
Science ; 345(6200): 1058-62, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25170154

RESUMO

T follicular helper (T(FH)) cells select high-affinity, antibody-producing B cells for clonal expansion in germinal centers (GCs), but the nature of their interaction is not well defined. Using intravital imaging, we found that selection is mediated by large but transient contacts between T(FH) and GC B cells presenting the highest levels of cognate peptide bound to major histocompatibility complex II. These interactions elicited transient and sustained increases in T(FH) intracellular free calcium (Ca(2+)) that were associated with T(FH) cell coexpression of the cytokines interleukin-4 and -21. However, increased intracellular Ca(2+) did not arrest TFH cell migration. Instead, T(FH) cells remained motile and continually scanned the surface of many GC B cells, forming short-lived contacts that induced selection through further repeated transient elevations in intracellular Ca(2+).


Assuntos
Linfócitos B/imunologia , Sinalização do Cálcio/imunologia , Centro Germinativo/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Proteínas de Fluorescência Verde/metabolismo , Interleucina-4/imunologia , Interleucinas/imunologia , Camundongos , Camundongos Knockout , Imagem Molecular
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