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1.
Nat Immunol ; 25(6): 1073-1082, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38816615

RESUMEN

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.


Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Proteína gp41 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , Macaca mulatta , Animales , Humanos , Proteína gp41 de Envoltorio del VIH/inmunología , Anticuerpos Anti-VIH/inmunología , Ratones , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Vacunación , Anticuerpos ampliamente neutralizantes/inmunología , Linfocitos B/inmunología , Nanopartículas/química , Femenino , Regiones Determinantes de Complementariedad/inmunología , Epítopos/inmunología
3.
Cell ; 166(6): 1459-1470.e11, 2016 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-27610570

RESUMEN

Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Vacunas Sintéticas/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/genética , Antígenos Virales/inmunología , Femenino , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación , Alineación de Secuencia , Vacunas Sintéticas/administración & dosificación
4.
Nat Immunol ; 19(9): 942-953, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30111894

RESUMEN

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.


Asunto(s)
Células Dendríticas/fisiología , Endosomas/metabolismo , Exonucleasas/metabolismo , Hepatitis/genética , Macrófagos/fisiología , Glicoproteínas de Membrana/metabolismo , Fosfolipasa D/metabolismo , Enfermedad de Alzheimer/genética , Animales , Artritis Reumatoide/genética , ADN de Cadena Simple/inmunología , Exonucleasas/genética , Estudio de Asociación del Genoma Completo , Humanos , Interferón gamma/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa D/genética , Esclerodermia Sistémica/genética , Transducción de Señal , Receptor Toll-Like 9/metabolismo
5.
Immunity ; 48(1): 133-146.e6, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29287996

RESUMEN

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Centro Germinal/inmunología , VIH-1/inmunología , Animales , Anticuerpos ampliamente neutralizantes , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Anticuerpos Anti-VIH , Masculino , Ratones , Ratones Transgénicos
6.
Proc Natl Acad Sci U S A ; 117(37): 22920-22931, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32873644

RESUMEN

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/farmacología , Antígenos CD4/inmunología , Técnicas de Sustitución del Gen/métodos , Centro Germinal/inmunología , Antígenos VIH , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Ratones , Ratones Endogámicos , Ratones Transgénicos , Células Precursoras de Linfocitos B/inmunología , Vacunación/métodos
7.
J Immunol ; 191(6): 3186-3191, 2013 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-23940276

RESUMEN

A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated knock-in mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the companion article (Ota et al. 2013. J. Immunol. 191: 3179-3185), 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms, including receptor editing, clonal deletion, and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1⁻/⁻ background, 4E10HL mice had virtually no serum Ig of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of a membrane proximal external region-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly described bNAb 10E8.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Tolerancia Inmunológica/inmunología , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Sustitución del Gen , Anticuerpos Anti-VIH/sangre , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos
8.
Sci Transl Med ; 16(748): eadn0223, 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38753806

RESUMEN

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.


Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Animales , Humanos , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Ratones , Vacunación , Inmunización Secundaria , VIH-1/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Anticuerpos ampliamente neutralizantes/inmunología
9.
J Immunol ; 185(2): 1015-27, 2010 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-20554953

RESUMEN

During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered gamma2a-reactive superantigen (gamma2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Fas(lpr) lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.


Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Receptor fas/inmunología , Traslado Adoptivo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Línea Celular , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Endogámicos , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Superantígenos/genética , Superantígenos/inmunología , Superantígenos/metabolismo , Receptor fas/genética , Receptor fas/metabolismo
10.
Front Cell Infect Microbiol ; 12: 962945, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36968243

RESUMEN

The ability to efficiently isolate antigen-specific B cells in high throughput will greatly accelerate the discovery of therapeutic monoclonal antibodies (mAbs) and catalyze rational vaccine development. Traditional mAb discovery is a costly and labor-intensive process, although recent advances in single-cell genomics using emulsion microfluidics allow simultaneous processing of thousands of individual cells. Here we present a streamlined method for isolation and analysis of large numbers of antigen-specific B cells, including next generation antigen barcoding and an integrated computational framework for B cell multi-omics. We demonstrate the power of this approach by recovering thousands of antigen-specific mAbs, including the efficient isolation of extremely rare precursors of VRC01-class and IOMA-class broadly neutralizing HIV mAbs.


Asunto(s)
Anticuerpos Neutralizantes , VIH-1 , Linfocitos B , Anticuerpos Anti-VIH , Antígenos , Anticuerpos Monoclonales
11.
J Exp Med ; 201(5): 817-28, 2005 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-15738053

RESUMEN

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.


Asunto(s)
Linfocitos B/inmunología , Sistema Inmunológico/inmunología , Tolerancia Inmunológica/inmunología , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Animales , Autoantígenos/genética , Autoantígenos/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Línea Celular Tumoral , Genes RAG-1/genética , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología
12.
J Exp Med ; 216(10): 2331-2347, 2019 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-31345930

RESUMEN

Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies.


Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Células Precursoras de Linfocitos B/inmunología , Adulto , Animales , Femenino , Infecciones por VIH/genética , VIH-1/genética , Humanos , Masculino , Ratones , Ratones Transgénicos
13.
J Exp Med ; 214(9): 2573-2590, 2017 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-28847869

RESUMEN

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.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Animales , Cristalografía por Rayos X , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Ratones , Multimerización de Proteína/inmunología , Estructura Terciaria de Proteína
14.
Science ; 349(6244): 156-61, 2015 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-26089355

RESUMEN

A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env to unmutated precursors of bnAbs, including those of the VRC01 class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short CDRL3 (light-chain complementarity-determining region 3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Afinidad de Anticuerpos , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Epítopos/genética , Epítopos/inmunología , Anticuerpos Anti-VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Ratones Noqueados
15.
Keio J Med ; 53(3): 151-8, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15477728

RESUMEN

This lecture discusses two interrelated topics, B cell tolerance in the peripheral immune system and BAFF. Using the 3-83 antibody transgenic mouse bred to mice carrying cognate antigen in the liver, we previously found that clonal elimination drastically reduced the precursor frequency of autoreactive cells. The consensus model to explain this tolerance is the 2-signal hypothesis, which proposes that in the absence of T cell help BCR stimulation is a negative signal for B cells. However, this model fails to explain how these same B cells can respond to T-independent type II (TI-2) antigens, raising the question of how they distinguish TI-2 antigens from multimeric self determinants. We propose that B cells use NK-like missing self recognition to provide the needed specificity, as foreign antigens are unlikely to carry self markers. The model has implications for the evolution of the immune system, B lymphocyte signaling, tissue specificity of autoimmunity, and microbial subversion of the immune system. Overexpression of the critical B cell survival cytokine BAFF/BLyS has been associated with autoimmunity. We have discovered a novel splice isoform that regulates BAFF activity and may play a role in limiting B cell activity. The novel form, called DBAFF, is able to heteromultimerize with normal BAFF and can suppress receptor binding and proteolytic release from the cell surface. Preliminary studies from transgenic mice overexpressing wild type or DBAFF are consistent with a possible regulatory role for DBAFF, raising the possibility that the relative expression levels of BAFF and DBAFF regulates tolerance.


Asunto(s)
Linfocitos B/inmunología , Tolerancia Inmunológica/inmunología , Proteínas de la Membrana/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Factor Activador de Células B , Humanos , Proteínas de la Membrana/genética , Factor de Necrosis Tumoral alfa/genética
16.
J Exp Med ; 204(12): 2853-64, 2007 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-17967905

RESUMEN

The initial B cell repertoire contains a considerable proportion of autoreactive specificities. The first major B cell tolerance checkpoint is at the stage of the immature B cell, where receptor editing is the primary mode of eliminating self-reactivity. The cells that emigrate from the bone marrow have a second tolerance checkpoint in the transitional compartment in the spleen. Although it is known that the second checkpoint is defective in lupus, it is not clear whether there is any breakdown in central B cell tolerance in the bone marrow. We demonstrate that receptor editing is less efficient in the lupus-prone strain MRL/lpr. In an in vitro system, when receptor-editing signals are given to bone marrow immature B cells by antiidiotype antibody or after in vivo exposure to membrane-bound self-antigen, MRL/lpr 3-83 transgenic immature B cells undergo less endogenous rearrangement and up-regulate recombination activating gene messenger RNA to a lesser extent than B10 transgenic cells. CD19, along with immunoglobulin M, is down-regulated in the bone marrow upon receptor editing, but the extent of down-regulation is fivefold less in MRL/lpr mice. Less efficient receptor editing could allow some autoreactive cells to escape from the bone marrow in lupus-prone mice, thus predisposing to autoimmunity.


Asunto(s)
Lupus Eritematoso Sistémico/genética , Edición de ARN/genética , Receptores de Antígenos de Linfocitos B/genética , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/fisiología , Células Cultivadas , Predisposición Genética a la Enfermedad , Interleucina-7/fisiología , Ratones , Ratones Endogámicos MRL lpr , Ratones Endogámicos , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos B/fisiología
17.
J Immunol ; 178(10): 6332-41, 2007 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-17475862

RESUMEN

PI3K plays key roles in cell growth, differentiation, and survival by generating the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). PIP3 activates numerous enzymes, in part by recruiting them from the cytosol to the plasma membrane. We find that in immature B lymphocytes carrying a nonautoreactive Ag receptor, PI3K signaling suppresses RAG expression and promotes developmental progression. Inhibitors of PI3K signaling abrogate this positive selection. Furthermore, immature primary B cells from mice lacking the p85alpha regulatory subunit of PI3K suppress poorly RAG expression, undergo an exaggerated receptor editing response, and, as in BCR-ligated cells, fail to progress into the G1 phase of cell cycle. Moreover, immature B cells carrying an innocuous receptor have sustained elevation of PIP3 levels and activation of the downstream effectors phospholipase C (PLC)gamma2, Akt, and Bruton's tyrosine kinase. Of these, PLCgamma2 appears to play the most significant role in down-regulating RAG expression. It therefore appears that when the BCR of an immature B cell is ligated, PIP3 levels are reduced, PLCgamma2 activation is diminished, and receptor editing is promoted by sustained RAG expression. Taken together, our results provide evidence that PI3K signaling is an important cue required for fostering development of B cells carrying a useful BCR.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Homeodominio/biosíntesis , Fosfatidilinositol 3-Quinasas/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/inmunología , Animales , Antígenos CD19/fisiología , Subgrupos de Linfocitos B/citología , Diferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Ligandos , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/deficiencia , Fosfatidilinositol 3-Quinasas/genética , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Transducción de Señal/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología
18.
Eur J Immunol ; 36(4): 985-96, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16511898

RESUMEN

In mice carrying a synthetic Igkappa-reactive superantigen ("kappa macroself antigen"), low level expression induced split peripheral B cell tolerance in the sIgkappa+ compartment, with striking reductions in follicular and marginal zone (MZ) B cells and the retention of significant numbers of sIgkappa+ B-1a but not B-1b cells in the peritoneum. Here, we characterize the transgenic line pKkappa with this split tolerance phenotype and assess the effects of B cell competition and the survival cytokine BAFF (B cell activating factor belonging to the TNF family) on peripheral tolerance. In pKkappa mice the surviving peritoneal and splenic kappa+ B cells were largely lost in mice carrying one copy of the human Ckappa exon in place of the mouse version, a maneuver that generates additional antigen non-reactive competitor B cells in this model. Furthermore, overexpression of BAFF suppressed kappa-macroself antigen-induced deletion and promoted production of both IgM,kappa and IgA,kappa antibodies in mice with normal Igkappa alleles but not in mice carrying one copy of the human Ckappa allele. These findings suggest that BAFF overexpression has minimal effects on the survival of autoreactive B cells in a polyclonal immune system and that B cell:B cell competition plays a potent role in suppressing the survival of B-1 and splenic B cells with excessive autoreactivity.


Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Comunicación Celular/inmunología , Tolerancia Inmunológica/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Proteínas de la Membrana/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Factor Activador de Células B , Supervivencia Celular , Citometría de Flujo , Humanos , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Factor de Necrosis Tumoral alfa/inmunología
19.
J Immunol ; 176(2): 939-48, 2006 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-16393979

RESUMEN

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Tolerancia Inmunológica , Cadenas kappa de Inmunoglobulina/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciación Celular , Supresión Clonal , Genes de Inmunoglobulinas , Ligandos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Quimera por Radiación/genética , Quimera por Radiación/inmunología , Transducción de Señal , Bazo/citología , Bazo/inmunología
20.
Immunity ; 22(4): 519-31, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15845455

RESUMEN

In developing B cells, expression of surface immunoglobulin is an important signal to terminate recombinase activator gene (RAG) expression and V(D)J recombination. However, autoreactive antigen receptors instead promote continued gene rearrangement and receptor editing. The regulation by B cell receptor (BCR) signaling of RAG expression and editing is poorly understood. We report that in editing-competent cells BCR ligand-induced RAG mRNA expression is regulated at the level of RAG transcription, rather than mRNA stability. In immature B cells carrying innocuous receptors, RAG expression appears to be under rapidly reversible negative regulation. Studies involving transduction of a superrepressive (sr) I kappa B alpha protein indicate that NF-kappaB/Rel proteins promote RAG transcription. Interestingly, NF kappa B1-deficient cells overexpress RAG and undergo an exaggerated receptor editing response. Our data implicate NF kappa B transcription factors in the BCR-mediated regulation of RAG locus transcription. Rapidly activated NF kappa B pathways may facilitate prompt antigen receptor-regulated changes in RAG expression important for editing and haplotype exclusion.


Asunto(s)
Proteínas de Unión al ADN/genética , Genes RAG-1/genética , Cadenas kappa de Inmunoglobulina/genética , FN-kappa B/fisiología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Secuencia de Bases , Línea Celular , Elementos de Facilitación Genéticos , Cadenas kappa de Inmunoglobulina/metabolismo , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/metabolismo , Regulación hacia Arriba
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