RESUMEN
During an organism's ontogeny and in the adult, each B and T lymphocyte generates a unique antigen receptor, thereby creating the organism's ability to respond to a vast number of different antigens. The antigen receptor loci are organized into distinct regions that contain multiple variable (V), diversity (D), and/or joining (J) and constant (C) coding elements that are scattered across large genomic regions. In this review, we discuss the epigenetic modifications that take place in the different antigen receptor loci, the chromatin structure adopted by the antigen receptor loci to allow recombination of elements separated by large genomic distances, and the relationship between epigenetics and chromatin structure and how they relate to the generation of antigen receptor diversity.
Asunto(s)
Cromatina/química , Receptores de Antígenos/metabolismo , Animales , Epigénesis Genética , Sitios Genéticos , Variación Genética/inmunología , Humanos , Receptores de Antígenos/química , Receptores de Antígenos/genética , Transcripción Genética , Recombinación V(D)JRESUMEN
Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage-specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.
Asunto(s)
Linfocitos B/inmunología , Proliferación Celular , ADN Helicasas/inmunología , Elementos de Facilitación Genéticos/inmunología , Proteínas Nucleares/inmunología , Factores de Transcripción/inmunología , Animales , Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/inmunología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Elementos de Facilitación Genéticos/genética , Citometría de Flujo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Hibridación Fluorescente in Situ , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage-specific transcription signature.
Asunto(s)
Linfocitos B/fisiología , Linaje de la Célula , Núcleo Celular/genética , Linfopoyesis , Células Precursoras de Linfocitos B/fisiología , Animales , Linfocitos B/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factor de Unión a CCCTC , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Lámina Nuclear/metabolismo , Células Precursoras de Linfocitos B/citología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
Why does a beneficial treatment effect on a longitudinal biomarker not translate into overall treatment benefit on survival, when the biomarker is in fact a prognostic factor of survival? In a recent exploratory data analysis in oncology, we were faced with this seemingly paradoxical result. To address this problem, we applied a theoretically principled methodology called dynamic path analysis, which allows us to perform mediation analysis with a longitudinal mediator and survival outcome. The aim of the analysis is to decompose the total treatment effect into a direct treatment effect and an indirect treatment effect mediated through a carefully constructed mediation path. The dynamic nature of the underlying methodology enables us to describe how these effects evolve over time, which can add to the mechanistic understanding of the underlying processes. In this paper, we present a detailed description of the dynamic path analysis framework and illustrate its application to survival mediation analysis using simulated and real data. The use case analysis provides clarity on the specific exploratory question of interest while the methodology generalizes to a wide range of applications in drug development where time-to-event is the primary clinical outcome of interest.
RESUMEN
Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.
Asunto(s)
Silenciador del Gen , División Celular/genética , Células Cultivadas , Metilación de ADN , Células HEK293 , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Linfocitos T/metabolismo , Efectores Tipo Activadores de la Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Immunoglobulin heavy chain (IgH) variable region exons are assembled from V(H), D and J(H) gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the V(H) and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of D(H)-proximal V(H) gene segments and promoting rearrangement of distal V(H) segments. IGCR1 maintains ordered and lineage-specific V(H)(D)J(H) recombination by suppressing V(H) joining to D segments not joined to J(H) segments, and V(H) to DJ(H) joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal V(H)-to-DJ(H) recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.
Asunto(s)
ADN Intergénico/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Recombinación Genética/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/metabolismo , Exones VDJ/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Factor de Unión a CCCTC , Linaje de la Célula/genética , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Retroalimentación Fisiológica , Células Germinativas/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Mutación/genética , Timo/citología , Transcripción Genética/genéticaRESUMEN
Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eµ), creating a D(H)-J(H)-Eµ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Proteínas Represoras/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , ADN sin Sentido/genética , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos/genética , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Interferencia de ARN , ARN sin Sentido/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , CohesinasRESUMEN
PURPOSE: Effective treatments for resectable non-small-cell lung cancer (NSCLC) are limited and relapse rates are high. The interleukin (IL)-1ß pathway has been linked with tumor development and progression, including in the Canakinumab Anti-Inflammatory Thrombosis Outcomes cardiovascular study in which IL-1ß pathway inhibition with canakinumab reduced lung cancer incidence and mortality in an exploratory analysis. METHODS: CANOPY-A (ClinicalTrials.gov identifier: NCT03447769) is a phase III, randomized, double-blind, multicenter study of canakinumab versus placebo for adult patients with stage II-IIIA or IIIB (T >5 cm, N2-positives II-IIIB; American Joint Committee on Cancer/Union for International Cancer Control version 8), completely resected NSCLC who had received adjuvant cisplatin-based chemotherapy. The primary end point was disease-free survival (DFS) and the key secondary end point was overall survival (OS). RESULTS: In total, 1,382 patients were randomized to 200 mg canakinumab (n = 693) or placebo (n = 689) once every 3 weeks for 18 cycles. Grade ≥3 adverse events (AEs) were reported in 20.8% and 19.6% of patients receiving canakinumab and placebo, respectively; AEs led to discontinuation in 4.3% and 4.1% of patients in these groups, respectively. This study did not meet its primary end point. Median DFS was 35.0 months (canakinumab arm) and 29.7 months (placebo arm; hazard ratio, 0.94; 95% CI, 0.78 to 1.14; one-sided P = .258). DFS subgroup analyses did not show any meaningful differences between arms. As expected, because of canakinumab-driven IL-1ß pathway inhibition, C-reactive protein and IL-6 levels decreased in the canakinumab arm versus placebo arm, but had no correlation with differential clinical outcomes. OS was not formally tested as DFS was not statistically significant. CONCLUSION: CANOPY-A did not show a DFS benefit of adding canakinumab after surgery and adjuvant cisplatin-based chemotherapy in patients with resected, stage II-III NSCLC. No new safety signals were identified with canakinumab.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Adulto , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/cirugía , Cisplatino , Recurrencia Local de Neoplasia/tratamiento farmacológico , Quimioterapia Adyuvante , Método Doble CiegoRESUMEN
B-cell-activating factor of the TNF family (BAFF)/BLyS contributes to B-cell homeostasis and function in the periphery. BAFF is expressed as a membrane-bound protein or released by proteolytic cleavage, but the functional importance of this processing event is poorly understood. Mice expressing BAFF with a mutated furin consensus cleavage site, i.e. furin-mutant BAFF (fmBAFF), were not different from BAFF-deficient mice with regard to their B-cell populations and responses to immunization. It is however noteworthy that an alternative processing event releases some soluble BAFF in fmBAFF mice. Mild overexpression (â¼ 5-fold) of fmBAFF alone generated intermediate levels of B cells without improving humoral responses to immunization. Processed BAFF was however important for B-cell homeostasis, as peripheral B-cell populations and antibody responses were readily restored by administration of soluble BAFF trimers in BAFF-deficient mice. However, the rescue of CD23 expression in B cells of BAFF-deficient mice required both soluble BAFF trimers and fmBAFF, or a polymeric form of soluble BAFF (BAFF 60-mer). These results point to a predominant role of processed BAFF for B-cell homeostasis and function, and indicate possible accessory roles for membrane-bound BAFF.
Asunto(s)
Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Linfocitos B/inmunología , Mutación , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Factor Activador de Células B/química , Factor Activador de Células B/deficiencia , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , Furina/química , Células HEK293 , Homeostasis , Humanos , Inmunoglobulinas/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , SolubilidadRESUMEN
Common variable immunodeficiency (CVID) is the most prevalent form of symptomatic primary immunodeficiency in humans. The genetic cause of CVID is still unknown in about 70% of cases. Ten percent of CVID patients carry heterozygous mutations in the tumor necrosis factor receptor superfamily member 13B gene (TNFRSF13B), encoding TACI. Mutations in TNFRSF13B alone may not be sufficient for the development of CVID, as 1% of the healthy population carry these mutations. The common hypothesis is that TACI mutations are not fully penetrant and additional factors contribute to the development of CVID. To determine these additional factors, we investigated the perturbations of transcription factor (TF) binding and the transcriptome profiles in unstimulated and CD40L/IL21-stimulated naïve B cells from CVID patients harboring the C104R mutation in TNFRSF13B and compared them to their healthy relatives with the same mutation. In addition, the proteome of stimulated naïve B cells was investigated. For functional validation, intracellular protein concentrations were measured by flow cytometry. Our analysis revealed 8% less accessible chromatin in unstimulated naïve B cells and 25% less accessible chromatin in class-switched memory B cells from affected and unaffected TACI mutation carriers compared to healthy donors. The most enriched TF binding motifs in TACI mutation carriers involved members from the ETS, IRF, and NF-κB TF families. Validation experiments supported dysregulation of the NF-κB and MAPK pathways. In steady state, naïve B cells had increased cell death pathways and reduced cell metabolism pathways, while after stimulation, enhanced immune responses and decreased cell survival were detected. Using a multi-omics approach, our findings provide valuable insights into the impaired biology of naïve B cells from TACI mutation carriers.
Asunto(s)
Inmunodeficiencia Variable Común , FN-kappa B , Linfocitos B , Cromatina/metabolismo , Humanos , Mutación , FN-kappa B/metabolismoRESUMEN
T cell engagers represent a novel promising class of cancer-immunotherapies redirecting T cells to tumor cells and have some promising outcomes in the clinic. These molecules can be associated with a mode-of-action related risk of cytokine release syndrome (CRS) in patients. CRS is characterized by the rapid release of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6 and IL-1ß and immune cell activation eliciting clinical symptoms of fever, hypoxia and hypotension. In this work, we investigated the biological mechanisms triggering and amplifying cytokine release after treatment with T cell bispecific antibodies (TCBs) employing an in vitro co-culture assay of human PBMCs or total leukocytes (PBMCs + neutrophils) and corresponding target antigen-expressing cells with four different TCBs. We identified T cells as the triggers of the TCB-mediated cytokine cascade and monocytes and neutrophils as downstream amplifier cells. Furthermore, we assessed the chronology of events by neutralization of T-cell derived cytokines. For the first time, we demonstrate the contribution of neutrophils to TCB-mediated cytokine release and confirm these findings by single-cell RNA sequencing of human whole blood incubated with a B-cell depleting TCB. This work could contribute to the construction of mechanistic models of cytokine release and definition of more specific molecular and cellular biomarkers of CRS in the context of treatment with T-cell engagers. In addition, it provides insight for the elaboration of prophylactic mitigation strategies that can reduce the occurrence of CRS and increase the therapeutic index of TCBs.
Asunto(s)
Anticuerpos Biespecíficos , Citocinas , Síndrome de Liberación de Citoquinas , Humanos , Neutrófilos , Linfocitos TRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a central regulator of immune homeostasis. STAT3 levels are strictly controlled, and STAT3 impairment contributes to several diseases including the monogenic autosomal-dominant hyper-immunoglobulin E (IgE) syndrome (AD-HIES). We investigated patients of four consanguineous families with an autosomal-recessive disorder resembling the phenotype of AD-HIES, with symptoms of immunodeficiency, recurrent infections, skeletal abnormalities, and elevated IgE. Patients presented with reduced STAT3 expression and diminished T helper 17 cell numbers, in absence of STAT3 mutations. We identified two distinct homozygous nonsense mutations in ZNF341, which encodes a zinc finger transcription factor. Wild-type ZNF341 bound to and activated the STAT3 promoter, whereas the mutant variants showed impaired transcriptional activation, partly due to nuclear translocation failure. In summary, nonsense mutations in ZNF341 account for the STAT3-like phenotype in four autosomal-recessive kindreds. Thus, ZNF341 is a previously unrecognized regulator of immune homeostasis.
Asunto(s)
Inmunocompetencia/genética , Síndrome de Job/genética , Factor de Transcripción STAT3/genética , Células Th17/inmunología , Factores de Transcripción/genética , Adolescente , Adulto , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Núcleo Celular/metabolismo , Niño , Codón sin Sentido , Consanguinidad , Exones/genética , Femenino , Genes Recesivos/genética , Genes Recesivos/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Lactante , Síndrome de Job/sangre , Síndrome de Job/inmunología , Masculino , Linaje , Regiones Promotoras Genéticas/genética , Factor de Transcripción STAT3/inmunología , Células Th17/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Adulto Joven , Dedos de Zinc/genéticaRESUMEN
There is now substantial evidence that the eukaryotic nucleus consists of highly organized structures. Among such structures are transcription factories that consist of an ensemble of genes recruited by the RNA polymerase machinery. Here we suggest that antigen receptor variable regions are similarly organized. Specifically, we propose that the immunoglobulin heavy chain locus variable gene segments are anchored to the base of rosettes, wrapping around a cavity that contains the recombination machinery. We suggest that the folding of the chromatin fiber into rosettes underpins a crucial mechanism by which antigen receptor diversity is generated.
Asunto(s)
Receptores de Antígenos/genética , Recombinación Genética , Transcripción Genética , Animales , Núcleo Celular/química , Núcleo Celular/genética , Cromatina/química , Cromatina/genética , Humanos , Región Variable de Inmunoglobulina/genéticaRESUMEN
The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células del Estroma/citología , Células del Estroma/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Animales , Factor Activador de Células B/inmunología , Factor Activador de Células B/metabolismo , Línea Celular , Supervivencia Celular , Regulación de la Expresión Génica , Humanos , Ratones , Unión Proteica , Proteoglicanos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.
Asunto(s)
Factor Activador de Células B/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Activación de Linfocitos , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Factor Activador de Células B/química , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/metabolismo , Linfocitos B/inmunología , Línea Celular , Proliferación Celular , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Ligandos , Ratones , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Alineación de Secuencia , Transducción de Señal , Bazo/inmunología , Proteína Activadora Transmembrana y Interactiva del CAML/deficiencia , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Regulación hacia ArribaRESUMEN
Monoclonal antibody (mAb) 5D10 is directed against the human breast cancer cell line MCF-7. Biochemical characterization of the antibody epitope was attempted and revealed a complex, most likely carbohydrate-linked nature, which prevented isolation and further studies of the interaction. A major goal of this work was to generate structural mimics of the 5D10 epitope to serve as putative substitutes in such studies. A peptide library displayed on filamentous phage was used to select for mimotope peptide sequences. All positive phage clones selected from the library displayed the amino acid sequence H(2)N-QMNPMYYR-CO(2)H. This peptide sequence, as well as a branched form of the peptide, was found to bind mAb 5D10. Moreover, both peptide sequences were able to inhibit the binding of 5D10 to the MCF-7 cells in a concentration-dependent manner, with an EC(50) value in the range of 65 microM. According to these results, random phage peptide libraries can serve to identify mimotopic peptides for unknown complex cell surface epitopes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos/inmunología , Imitación Molecular , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Western Blotting , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatografía en Gel , ADN Viral/análisis , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos , Femenino , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Humanos , Oligopéptidos/síntesis química , Oligopéptidos/química , Biblioteca de Péptidos , Análisis de Secuencia de ADNRESUMEN
BAFF, APRIL and their receptors play important immunological roles, especially in the B cell arm of the immune system. A number of splice isoforms have been described for both ligands and receptors in this subfamily, some of which are conserved between mouse and human, while others are species-specific. Structural and mutational analyses have revealed key determinants of receptor-ligand specificity. BAFF-R has a strong selectivity for BAFF; BCMA has a higher affinity for APRIL than for BAFF, while TACI binds both ligands equally well. The molecular signaling events downstream of BAFF-R, BCMA and TACI are still incompletely characterized. Survival appears to be mediated by upregulation of Bcl-2 family members through NF-kappaB activation, degradation of the pro-apototic Bim protein, and control of subcellular localization of PCKdelta. Very little is known about other signaling events associated with receptor engagement by BAFF and APRIL that lead for example to B cell activation or to CD40L-independent Ig switch.
Asunto(s)
Factor Activador de Células B/fisiología , Receptor del Factor Activador de Células B/fisiología , Antígeno de Maduración de Linfocitos B/fisiología , Linfocitos B/inmunología , Proteína Activadora Transmembrana y Interactiva del CAML/fisiología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/fisiología , Empalme Alternativo , Animales , Presentación de Antígeno/fisiología , Apoptosis/fisiología , Factor Activador de Células B/química , Factor Activador de Células B/genética , Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/química , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/inmunología , Linfocitos B/citología , Humanos , Cambio de Clase de Inmunoglobulina/fisiología , Activación de Linfocitos , Subgrupos Linfocitarios/inmunología , Ratones , Modelos Moleculares , FN-kappa B/fisiología , Subunidad p50 de NF-kappa B/fisiología , Conformación Proteica , Mapeo de Interacción de Proteínas , Proteína Quinasa C-delta/antagonistas & inhibidores , Transducción de Señal , Relación Estructura-Actividad , Especificidad por Sustrato , Proteína Activadora Transmembrana y Interactiva del CAML/química , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Proteína Activadora Transmembrana y Interactiva del CAML/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Necrosis Tumoral/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.