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1.
J Virol ; 96(5): e0168621, 2022 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-34985994

RESUMEN

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore, there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1 domain of the viral L protein to the Na+/taurocholate cotransporting polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/hepatitis D virus (HDV) infection and has been approved as Hepcludex in Europe for the treatment of patients with chronic HDV infection. We established a monoclonal antibody (MAb), N6HB426-20, that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo, administration of the N6HB426-20 MAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84 to 87 in ECL1 and aa 157 to 165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope mapping and the three-dimensional (3D) model of the NTCP structure suggested that the N6HB426-20 MAb may recognize aa 276/277 at the tip of ECL4 and interfere with binding of HBV to the region from aa 84 to 87. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleoside/nucleotide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic MAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these MAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations, and the presence of subviral particles. Although N6HB426-20 requires a higher dose than myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time postadministration in proportion to the half-life of an IgG MAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.


Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Transportadores de Anión Orgánico Sodio-Dependiente , Simportadores , Internalización del Virus , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Células Hep G2 , Hepatitis B/tratamiento farmacológico , Hepatitis B/prevención & control , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Hepatocitos , Humanos , Ratones , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus/efectos de los fármacos
2.
Int Immunol ; 33(2): 79-90, 2021 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-32889526

RESUMEN

In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

4.
J Immunol Methods ; 464: 74-86, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30389576

RESUMEN

Antibody-based therapeutic strategies have become recognized as useful clinical options in several types of cancer, often with the expectation that such therapies will trigger target cell elimination via antibody-dependent cellar cytotoxicity (ADCC) by natural killer cells. The successful development of therapeutic monoclonal antibodies (mAbs) requires an assay system that permits a critical evaluation of their physicochemical and biological characteristics. At present a number of ADCC assay systems have been reported, however, there is still room for improvement in terms of usability, operability and sensitivity. Here we report a novel flow cytometric ADCC assay that uses a human natural killer cell line stably transfected with mouse FcγRIII, and Fc receptor common-γ chain (FcRγ) and a reporter gene as effector cells. This assay relies on discriminating effector and target cells by their differential immunofluorescence, which allows for clear-cut gating and accurate calculation of the number of surviving cells in a target population. This assay is easy and quick to perform and provides reliable data even for low frequency target cells in assay samples and with low concentrations of mAbs. Furthermore, our approach allows us to identify synergistic ADCC activity of mAbs with different epitope specificities on the same target antigen.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antineoplásicos Inmunológicos/farmacología , Citometría de Flujo/métodos , Células Asesinas Naturales/efectos de los fármacos , Leucemia/tratamiento farmacológico , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Técnica del Anticuerpo Fluorescente , Humanos , Células Asesinas Naturales/inmunología , Leucemia/genética , Leucemia/inmunología , Leucemia/patología , Ratones Endogámicos BALB C , Ratones SCID , Receptores de IgG/genética , Receptores de IgG/inmunología
5.
Nat Immunol ; 17(12): 1447-1458, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27798619

RESUMEN

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.


Asunto(s)
Centro Germinal/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Células TH1/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Células Cultivadas , Humanos , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
6.
Int Immunol ; 28(6): 267-82, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26714588

RESUMEN

Memory CD4(+) T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4(+) T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells.


Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos CD4/metabolismo , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Inmunocompetencia , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Transcriptoma
7.
Proc Natl Acad Sci U S A ; 112(43): 13324-9, 2015 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-26460037

RESUMEN

T follicular helper (Tfh) cells are essential providers of help to B cells. The transcription factor B-cell CLL/lymphoma 6 (Bcl6) is a lineage-defining regulator of Tfh cells and germinal center B cells. In B cells, Bcl6 has the potential to recruit distinct transcriptional corepressors through its BTB domain or its poorly characterized middle domain (also known as RDII), but in Tfh cells the roles of the Bcl6 middle domain have yet to be clarified. Mimicked acetylation of the Bcl6 middle domain (K379Q) in CD4 T cells results in significant reductions in Tfh differentiation in vivo. Blimp1 (Prdm1) is a potent inhibitor of Tfh cell differentiation. Although Bcl6 K379Q still bound to the Prdm1 cis-regulatory elements in Tfh cells, Prdm1 expression was derepressed. This was a result of the failure of Bcl6 K379Q to recruit metastasis-associated protein 3 (MTA3). The loss of Bcl6 function in Bcl6 K379Q-expressing CD4 T cells could be partially rescued by abrogating Prdm1 expression. In addition to Prdm1, we found that Bcl6 recruits MTA3 to multiple genes involved in Tfh cell biology, including genes important for cell migration, cell survival, and alternative differentiation pathways. Thus, Bcl6 middle domain mediated repression is a major mechanism of action by which Bcl6 controls CD4 T-cell fate and function.


Asunto(s)
Diferenciación Celular/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Inmunoprecipitación de Cromatina , Clonación Molecular , Cartilla de ADN/genética , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-6 , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/inmunología , Factores de Transcripción/metabolismo
8.
J Immunol ; 194(12): 5599-603, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-25957170

RESUMEN

T follicular helper (Tfh) cells are essential for germinal centers (GCs) and most long-term humoral immunity. Differentiation of Tfh cells depends on the transcriptional repressor B cell CLL/lymphoma 6 (Bcl6). Bcl6 mediates gene repression via the recruitment of corepressors. Currently, it is unknown how Bcl6 recruits corepressors to regulate gene expression of Tfh cells. In this article, we demonstrate, using a mutant form of Bcl6 with two BTB (bric-a-brac, tramtrack, broad-complex) mutations that abrogate corepressor binding, that the Bcl6 BTB domain is required for proper differentiation of Tfh and GC-Tfh cells in vivo. Importantly, we also observe a significant defect in GC B cell development. These results are consistent in multiple contexts, including a novel lymphocytic choriomeningitis virus nucleoprotein-specific TCR-transgenic mouse model. Taken together, these data suggest that the Bcl6 BTB domain is a key mediator of the differentiation of Tfh cells.


Asunto(s)
Diferenciación Celular , Centro Germinal/inmunología , Centro Germinal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-6/química , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología
9.
Eur J Immunol ; 44(5): 1258-64, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24610726

RESUMEN

Germinal centers (GCs) are generally considered to be the sole site of memory B-cell generation. However, recent studies demonstrate that memory B cells can also develop in response to a T-cell dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B-cell types requires classical T-cell help, the generation of GC-dependent memory B cells requires TFH -cell help, while the generation of GC-independent memory cells does not. These findings led to the conclusion that B-cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC-independent pathway of memory B-cell development, and discuss how the unique features of memory B cells are maintained in the GC-independent pathway.


Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular/fisiología , Centro Germinal/inmunología , Memoria Inmunológica/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/citología , Centro Germinal/citología , Humanos , Linfocitos T Colaboradores-Inductores/citología
10.
Int Immunol ; 25(12): 683-95, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24021876

RESUMEN

High-affinity memory B cells are preferentially selected during secondary responses and rapidly differentiate into antibody-producing cells. However, it remains unknown whether only high-affinity, mutated memory B cells simply expand to dominate the secondary response or if in fact memory B cells with a diverse VH repertoire, including those with no mutations, accumulate somatic mutations to create a new repertoire through the process of affinity maturation. In this report, we took a new approach to address this question by analyzing the VH gene repertoire of IgG1(+) memory B cells before and after antigen re-exposure in a host unable to generate IgG(+) B cells. We show here that both mutated and unmutated IgG1(+) memory B cells respond to secondary challenge and expand while accumulating somatic mutations in their VH genes in a stepwise manner. Both types of memory cells subsequently established a VH gene repertoire dominated by two major clonotypes, which are distinct from the original repertoire before antigen re-exposure. In addition, heavily mutated memory B cells were excluded from the secondary repertoire. Thus, both mutated and unmutated IgG1(+) memory cells equally contribute to establish a new antibody repertoire through a dynamic process of mutation and selection, becoming optimally adapted to the recall challenge.


Asunto(s)
Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Memoria Inmunológica , Mutación , Traslado Adoptivo , Animales , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Antígenos/inmunología , Linfocitos B/citología , Diferenciación Celular/inmunología , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Noqueados
11.
Immunity ; 39(1): 136-47, 2013 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-23850379

RESUMEN

Memory B cells are essential for generating rapid and robust secondary antibody responses. It has been thought that the unique cytoplasmic domain of IgG causes the prompt activation of antigen-experienced IgG memory B cells. To assess this model, we have generated a mouse containing IgG1 B cells that have never encountered antigen. We found that, upon challenge, antigen-experienced IgG1 memory B cells rapidly differentiated into plasma cells, whereas nonexperienced IgG1 B cells did not, suggesting the importance of the stimulation history. In addition, our results suggest that repression of the Bach2 transcription factor, which results from antigen experience, contributes to predisposition of IgG1 memory B cells to differentiate into plasma cells.


Asunto(s)
Linfocitos B/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Diferenciación Celular/inmunología , Células Plasmáticas/inmunología , Animales , Linfocitos B/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Citometría de Flujo , Expresión Génica/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Memoria Inmunológica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/inmunología , Factor de Transcripción PAX5/metabolismo , Células Plasmáticas/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina-Treonina Quinasas TOR/inmunología , Serina-Treonina Quinasas TOR/metabolismo
12.
Vaccine ; 31(17): 2184-90, 2013 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-23434386

RESUMEN

Infection with single strand RNA (ssRNA) viruses, such as influenza A virus, is known to induce protective acquired immune responses, including the production of neutralizing antibodies. Vaccination also causes a reduction in the number of peripheral blood leukocytes (PBL) shortly after inoculation, a result which may have undesirable adverse effects. The cellular mechanisms for this response have not been elucidated so far. Here we report that formalin-inactivated influenza A whole virus vaccine (whole virion) induces a significant decrease in PBL in mice 5-16 h after administration, whereas an ether-split vaccine (HA split) made from the same influenza virus strain does not induce a similar loss of PBL. Concordant with this reduction in the number of PBL, a rapidly induced and massive production of interferon (IFN)-α is observed when mice are injected with whole virion, but not with HA split vaccines. The role of Toll-like receptors (TLR), which are involved in signal transduction of influenza virus, and the subsequent induction of IFNα were confirmed using mice lacking TLR7, MyD-88, or IFNα/ß receptor. We further demonstrated that the observed PBL loss is caused by apoptosis in an IFNα-dependent manner, and not by leukocyte redistribution due to chemokine signaling failure. These findings indicate that RNA-encapsulated whole virion vaccines can rapidly induce a loss of leukocytes from peripheral blood by apoptosis, which may modulate the subsequent immune response.


Asunto(s)
Apoptosis/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Interferón-alfa/inmunología , Virión/inmunología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Vacunas contra la Influenza/administración & dosificación , Interferón-alfa/biosíntesis , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal , Factores de Tiempo , Receptor Toll-Like 7/deficiencia , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/inmunología , Vacunación , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología
13.
J Exp Med ; 209(11): 2079-97, 2012 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-23027924

RESUMEN

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.


Asunto(s)
Anticuerpos/inmunología , Linfocitos B/inmunología , Células Germinativas/inmunología , Centro Germinal/inmunología , Memoria Inmunológica/inmunología , Transducción de Señal/inmunología , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Células Germinativas/metabolismo , Centro Germinal/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-6 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/genética
14.
J Immunol ; 189(7): 3472-9, 2012 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-22942428

RESUMEN

Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn(-/-)) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn(-/-) mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenyl-chicken γ-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp(33)-to-Leu mutation in the IgV(H)-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Centro Germinal/inmunología , Tejido Linfoide/inmunología , Proteínas Nucleares/biosíntesis , Fosfoproteínas/biosíntesis , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunología , Familia-src Quinasas/fisiología , Animales , Subgrupos de Linfocitos B/citología , Adhesión Celular/genética , Adhesión Celular/inmunología , Supervivencia Celular/inmunología , Células Cultivadas , Centro Germinal/metabolismo , Centro Germinal/patología , Humanos , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Transducción de Señal/genética , Regulación hacia Arriba/genética , Familia-src Quinasas/deficiencia , Familia-src Quinasas/genética
15.
Int Immunol ; 23(7): 433-41, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21642447

RESUMEN

The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef. However, the interpretation is complicated by Nef expression early in the development of T cells in these animal models. Here, we analyzed the consequence of Nef expression in ovalbumin-specific/CD4(+) peripheral T cells by using a novel mouse model and demonstrate that Nef inhibits antigen-specific T-cell proliferation and multiple functions required for immune response in vivo, which includes T-cell helper activity for the primary and memory B-cell response. However, Nef does not completely abrogate T-cell activity, as defined by low levels of cytokine production, which may afford the virus a replicative advantage. These results support a model, in which Nef expression does not cause T-cell hyperresponse in immune reaction, but instead reduces the T-cell activity, that may contribute to a low level of virus spread without viral cytopathic effects.


Asunto(s)
Inmunidad Adaptativa/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , Linfocitos T/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Linfocitos B/inmunología , Línea Celular , Movimiento Celular/genética , Movimiento Celular/inmunología , Proliferación Celular , Regulación de la Expresión Génica/inmunología , Infecciones por VIH/inmunología , Humanos , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Linfocitos T/citología , Linfocitos T/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética
16.
Clin Immunol ; 139(1): 65-74, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21300571

RESUMEN

The aims of this study were to examine the therapeutic effects of sublingual immunotherapy (SLIT) and to identify potential biomarkers that would predict the therapeutic response in a randomized, double-blind, placebo-controlled clinical trial. The trial was carried out over two pollinosis seasons in 2007 and 2008. Carry-over therapeutic effects were analyzed in 2009. SLIT significantly ameliorated the symptoms of pollinosis during the 2008 and 2009 pollen seasons. Cry j 1-specific cytokine production in a subgroup of patients with mild disease in the SLIT group was significantly attenuated. The ratio of specific IgE to total IgE before treatment correlated with the symptom-medication score in the SLIT group in 2008. Patients with increased Cry j 1-iTreg in the SLIT group had significantly improved QOL and QOL-symptom scores. In summary, the specific IgE to total IgE ratio and upregulation of Cry j 1-iTreg are candidates for biomarker of the clinical response to SLIT.


Asunto(s)
Cryptomeria/fisiología , Inmunoglobulina E/sangre , Extractos Vegetales/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/terapia , Linfocitos T Reguladores/metabolismo , Administración Sublingual , Adolescente , Adulto , Anciano , Especificidad de Anticuerpos , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Método Doble Ciego , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Masculino , Persona de Mediana Edad , Extractos Vegetales/administración & dosificación , Adulto Joven
17.
Int Arch Allergy Immunol ; 153(4): 378-87, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20559004

RESUMEN

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergies in Japan. Recently, two reports described the positive effects of sublingual immunotherapy (SLIT) against Japanese cedar pollinosis. However, the therapeutic biomarkers for SLIT are still unclear. We performed this unblinded, nonrandomized, open-label study to identify therapeutic biomarkers for SLIT against Japanese cedar pollinosis. METHODS: We performed an open-label study during one pollinosis season in 2007, enrolling 19 patients from in-house volunteers suffering from Japanese cedar pollinosis. Peripheral blood was obtained from all participants before SLIT treatment as well as before and after the pollen season. The plasma levels of an immunoglobulin specific to a major allergen (Cry j 1) were determined. We analyzed the induction of regulatory T cells (iTregs), namely IL-10(+)Foxp3(+) cells in CD25(+)CD4(+) leukocytes, by flow cytometry. The Th2-type responses were analyzed by cytokine production from peripheral blood mononuclear cells after stimulation with Cry j 1. Clinical symptoms were estimated using a quality of life questionnaire in the middle of the pollen season. RESULTS: The difference in numbers of iTregs between the medium-only control cell culture and cells stimulat- ed with Cry j 1 was significantly decreased in the non-SLIT group but was unchanged in the SLIT group after the pollen season. The subgroup of the SLIT group with increased iTregs showed more attenuated Th2-type cytokine profiles, and symptom scores in the subgroup with increased iTregs were significantly lower than those in the subgroup with decreased iTregs. CONCLUSION: The antigen-specific iTreg level is a potential therapeutic biomarker that correlates with clinical pollinosis symptoms and may be involved in the therapeutic mechanisms of SLIT.


Asunto(s)
Desensibilización Inmunológica , Factores de Transcripción Forkhead/biosíntesis , Rinitis Alérgica Estacional/diagnóstico , Rinitis Alérgica Estacional/tratamiento farmacológico , Linfocitos T Reguladores/metabolismo , Administración Sublingual , Adulto , Antígenos de Plantas/inmunología , Biomarcadores Farmacológicos/metabolismo , Antígenos CD4/biosíntesis , Recuento de Células , Células Cultivadas , Cryptomeria , Progresión de la Enfermedad , Femenino , Humanos , Inmunoglobulina E/sangre , Interleucina-10/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Masculino , Persona de Mediana Edad , Proteínas de Plantas/inmunología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/patología , Rinitis Alérgica Estacional/fisiopatología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología
18.
J Immunol ; 185(1): 211-9, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20505144

RESUMEN

Although CD40 signaling is required for activation and differentiation of B cells, including germinal center (GC) formation and generation of memory B cells, in vivo generation of CD40 signaling augments plasma cell differentiation but disrupts GCs. Thus, CD40 signaling is thought to direct B cells to extrafollicular plasma cell fate rather than GC formation. In this study, we analyzed CD40L transgenic (CD40LTg) mice that constitutively express CD40L on B cells. After immunization, activation of B cells, but not dendritic cells, was augmented, although dendritic cells can be activated by CD40 ligation. Bone marrow chimera carrying CD40LTg and nontransgenic B cells showed increased Ab production from transgenic, but not from coexisting nontransgenic, B cells, suggesting that CD40L on a B cell preferentially stimulates the same B cell through an autocrine pathway, thereby augmenting Ab production. Although GCs rapidly regressed after day 5 of immunization and failed to generate late-appearing high-affinity Ab, CD40LTg mice showed normal GC formation up to day 5, as well as normal generation of long-lived plasma cells and memory B cell responses. This observation suggests that CD40 signaling does not block GC formation or differentiation of GC B cells, but it inhibits sustained expansion of GC B cells and augments B cell differentiation.


Asunto(s)
Adyuvantes Inmunológicos/genética , Subgrupos de Linfocitos B/inmunología , Ligando de CD40/genética , Diferenciación Celular/inmunología , Centro Germinal/inmunología , Inhibidores de Crecimiento/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Adyuvantes Inmunológicos/fisiología , Animales , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/patología , Ligando de CD40/fisiología , Diferenciación Celular/genética , Células Cultivadas , Femenino , Centro Germinal/citología , Centro Germinal/metabolismo , Inhibidores de Crecimiento/fisiología , Haptenos/administración & dosificación , Haptenos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Datos de Secuencia Molecular , Nitrofenoles/administración & dosificación , Nitrofenoles/inmunología , Fenilacetatos/administración & dosificación , Fenilacetatos/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología
19.
J Exp Med ; 206(3): 681-9, 2009 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-19273623

RESUMEN

Resting antigen-experienced memory B cells are thought to be responsible for the more rapid and robust antibody responses after antigen reencounter, which are the hallmark of memory humoral responses. The molecular basis for the development and survival of memory B cells remains largely unknown. We report that phospholipase C (PLC) gamma2 is required for efficient formation of germinal center (GC) and memory B cells. Moreover, memory B cell homeostasis is severely hampered by inducible loss of PLC-gamma2. Accordingly, mice with a conditional deletion of PLC-gamma2 in post-GC B cells had an almost complete abrogation of the secondary antibody response. Collectively, our data suggest that PLC-gamma2 conveys a survival signal to GC and memory B cells and that this signal is required for a productive secondary immune response.


Asunto(s)
Linfocitos B/enzimología , Linfocitos B/inmunología , Memoria Inmunológica/inmunología , Fosfolipasa C gamma/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Eliminación de Gen , Centro Germinal/citología , Centro Germinal/efectos de los fármacos , Centro Germinal/enzimología , Centro Germinal/inmunología , Inmunización , Inmunoglobulina G/inmunología , Memoria Inmunológica/efectos de los fármacos , Ratones , Ratones Noqueados , Nitrofenoles/farmacología , Fenilacetatos/farmacología , Fosfolipasa C gamma/deficiencia
20.
PLoS One ; 3(10): e3455, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18941623

RESUMEN

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection.


Asunto(s)
Inmunocompetencia , Neutrófilos/inmunología , Streptococcus pyogenes/patogenicidad , Factores de Transcripción , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/biosíntesis , Glicoproteínas/biosíntesis , Proteínas de Choque Térmico/genética , Humanos , Interleucina-8/análisis , Ratones , Proteínas Mutantes , Receptores Depuradores de Clase A/genética , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Estreptolisinas/biosíntesis , Activación Transcripcional , Regulación hacia Arriba
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