RESUMO
BACKGROUND: Human B-cell lymphoma 6 (BCL6) gene, usually coding protein of 706 amino acids, is closely associated with large B cell lymphoma. Researches showed that protein mutation or change of expression levels usually happened in the mounting non-hodgkin lymphoma (NHL). Thus BCL6 is considered to be involved in germinal center (GC)-derived lymphoma. RESULTS: The BCL61-350 gene codons were optimized for prokaryotic system. After expression of BCL61-350 in E. coli, the BCL61-350 protein was purified with Ni column. Then the BCL61-350 protein, mixing with QuickAntibody-Mouse5W adjuvant, was injected into Balb/c mice. After immunization and cell fusion, a stable cell line named 1E6A4, which can secrete anti-BCL6 antibody, was obtained. The isotype of 1E6A4 mAb was determined as IgG2a, and the affinity constant reached 5.12×1010 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the 1E6A4 mAb was able to detect BCL6 specifically and sensitively. CONCLUSIONS: BCL61-350 antigen has been successfully generated with an effective and feasible method, and a highly specific antibody named 1E6A4 against BCL6 has been screened and characterized in this study, which was valuable in clinical diagnosis.
Assuntos
Anticorpos Monoclonais Murinos , Imunoglobulina G , Linfoma de Células B/diagnóstico , Linfoma de Células B/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imuno-Histoquímica , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-6/biossínteseRESUMO
In response to acute viral infection, activated naive T cells give rise to effector T cells that clear the pathogen and memory T cells that persist long-term and provide heightened protection. T cell factor 1 (Tcf1) is essential for several of these differentiation processes. Tcf1 is expressed in multiple isoforms, with all isoforms sharing the same HDAC and DNA-binding domains and the long isoforms containing a unique N-terminal ß-catenin-interacting domain. In this study, we specifically ablated Tcf1 long isoforms in mice, while retaining expression of Tcf1 short isoforms. During CD8+ T cell responses, Tcf1 long isoforms were dispensable for generating cytotoxic CD8+ effector T cells and maintaining memory CD8+ T cell pool size, but they contributed to optimal maturation of central memory CD8+ T cells and their optimal secondary expansion in a recall response. In contrast, Tcf1 long isoforms were required for differentiation of T follicular helper (TFH) cells, but not TH1 effectors, elicited by viral infection. Although Tcf1 short isoforms adequately supported Bcl6 and ICOS expression in TFH cells, Tcf1 long isoforms remained important for suppressing the expression of Blimp1 and TH1-associated genes and for positively regulating Id3 to restrain germinal center TFH cell differentiation. Furthermore, formation of memory TH1 and memory TFH cells strongly depended on Tcf1 long isoforms. These data reveal that Tcf1 long and short isoforms have distinct, yet complementary, functions and may represent an evolutionarily conserved means to ensure proper programming of CD8+ and CD4+ T cell responses to viral infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Fator 1 de Transcrição de Linfócitos T/química , Fator 1 de Transcrição de Linfócitos T/imunologia , Animais , Diferenciação Celular , Testes Imunológicos de Citotoxicidade , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Vírus da Coriomeningite Linfocítica/isolamento & purificação , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Fator 1 de Transcrição de Linfócitos T/deficiência , Fator 1 de Transcrição de Linfócitos T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Di-(2-ethylhexyl) phthalate (DEHP) has been considered as a widespread environmental persistent organic pollutant and its potential concern on human health is raised by previous studies. In particular, children are more likely to be exposed to DEHP through gastrointestinal route and consequently are more susceptible to DEHP hazards. Some reports have uncovered a positive association between DEHP exposure and an increased prevalence of allergic diseases in infants and juveniles. Allergy is a hypersensitive reaction rooted in imbalanced humoral immunity. T follicular helper cell (Tfh), an important CD4(+) Th cell subset, until recently has been identified as a key player in humoral immune response by modifying B cells functions. Tfh cells are therefore perceived as the therapeutic target of immune disorders. In the present study, focusing on the newly confirmed Tfh cells, we examined the effects of DEHP on humoral immunity and investigated the underlying mechanisms. Using ovalbumin (OVA) sensitization weanling mice model under the condition of gastrointestinal exposure to DEHP, we found that DEHP acted as an immunoadjuvant to augment OVA-specific IgE and IgG1 production, amplified germinal center formation in lymphoid nodule, as well as stimulated the expansion of CD4(+)CXCR5(+)ICOS(+)/CD4(+)CXCR5(+)PD-1(+) Tfh cells and CD19(+)CD138(+)GL7(+) plasma cells. Based on the results of immune adoptive transfusion, DEHP-related anaphylactic response was ascribed to Tfh cells hyperfunction directly. We further proved that DEHP gavage together with OVA sensitization adjuvantly promoted the synthesis of cytokines IL-21 and IL-4 and the expression of transcription factors Bcl-6 and c-Maf in Tfh cells. In conclusion, our study demonstrates that DEHP has adjuvant toxic effects on Tfh cells by synthesizing an excess of cytokines IL-21 and IL-4 via over-expression of transcription factors Bcl-6 and c-Maf, leading to an increasing secretion of allergy-related IgE and IgG1.
Assuntos
Dietilexilftalato/toxicidade , Poluentes Ambientais/toxicidade , Centro Germinativo/efeitos dos fármacos , Hipersensibilidade/etiologia , Imunidade Humoral/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ovalbumina , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Administração Oral , Transferência Adotiva , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dietilexilftalato/administração & dosagem , Modelos Animais de Doenças , Poluentes Ambientais/administração & dosagem , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interleucina-4/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Proto-Oncogênicas c-maf/metabolismo , Medição de Risco , Fatores de Risco , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/transplanteRESUMO
About half of patients with diffuse large B-cell lymphoma (DLBCL) do not respond to or relapse soon after the standard chemotherapy, indicating a critical need to better understand the specific pathways perturbed in DLBCL for developing effective therapeutic approaches. Mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal centre or post-germinal centre B cells. Here we show that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal centre transcription factor. Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, Smurf2 expression is significantly decreased in primary human DLBCL samples, and low levels of Smurf2 expression correlate with inferior survival in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting pharmaceutical targeting of Smurf2 as a new therapeutic paradigm for DLBCL.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , Ubiquitina-Proteína Ligases/genética , Fator de Transcrição YY1/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Proliferação de Células , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Ativação Linfocitária , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Transcrição Gênica , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Fator de Transcrição YY1/metabolismoRESUMO
We utilized gene expression profiling of a comprehensive panel of purified developmentally defined normal murine B cells to identify unique transcriptional signatures for each subset. To elucidate transcription factor activities that function in a stage-specific fashion, we used gene sets that share transcription factor targets and found that germinal center B cells had a robust enrichment of up-regulated and down-regulated signatures compared with the other B-cell subsets. Notably, we found Yy1 and its targets to be central regulators of the germinal center B (GCB)-specific transcriptional program with binding of Yy1 to select signature genes in GCB cells, and translation of the Yy1 signatures to human GCB cells. We then tested whether our newly generated, stage-specific transcriptional signatures could be used to link murine lymphoma models to stages of normal B-cell development. Although each of the molecularly defined murine lymphoma models conserved certain stage-specific features of normal B-cell development, there was a significant alteration of the normal differentiation signature following malignant transformation. These findings offer important tools and insights for elucidating differences between normal and malignant B cells.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Regulação da Expressão Gênica/imunologia , Centro Germinativo/metabolismo , Linfoma/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/imunologia , Imunoprecipitação da Cromatina , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , CamundongosRESUMO
CONTEXT: Gene expression profiling of diffuse large B-cell lymphoma using complementary DNA microarrays has revealed 2 major prognostic groups in Western countries: germinal center B-cell-like and nongerminal center B-cell-like lymphomas. Immunohistochemical analysis using antibodies specific for CD10, BCL6, and MUM1 has been proposed as a surrogate for gene expression profiling. OBJECTIVE: To study the immunohistochemical features of diffuse large B-cell lymphoma cases from northern China because geographic differences for this disease are known to exist. DESIGN: Morphologic, immunohistochemical, and fluorescence in situ hybridization analyses of 63 cases of diffuse large B-cell lymphoma from northern China. RESULTS: There were 38 men and 25 women with a median age of 57 years (range, 12-87 years). CD10 was positive in 19 cases (30%), BCL6 was positive in 22 cases (35%), and MUM1 was positive in 32 cases (51%). Twenty-one (33%) cases were germinal center B-cell-like lymphoma, and 42 (67%) were nongerminal center B-cell-like lymphoma. BCL2 was expressed more often in nongerminal center B-cell-like disease versus germinal center B-cell-like disease (60% versus 24%, P = .01) and in nodal versus extranodal (64% versus 30%, P = .01) cases. Fluorescence in situ hybridization analysis showed BCL6, MYC , and BCL2 rearrangements in 11 of 32 (34%), 8 of 27 (30%), and 11 of 50 (22%) cases, respectively. CONCLUSIONS: These results add to what is known about the geographic variation of diffuse large B-cell lymphomas. In northern China, the frequency of the germinal center B-cell-like type and BCL6 expression and/or BCL6 rearrangement is less and the frequency of MYC rearrangement is greater than have been reported in Western countries.
Assuntos
Linfócitos B/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Centro Germinativo/metabolismo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização In Situ , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-Idade , Neprilisina/genética , Neprilisina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Serial de TecidosRESUMO
Using the method of luminescent microscopy, the catecholamine (CA) and serotonin (S) content was examined in spleen structures, and the interrelations between them were traced in rats before and 15 minutes, 1, 2 and 4 hours after the acupuncture in LI 4 and GV 14 points. It was found that after acupuncture, neurotransmitter content in splenic red and white pulp was changed already in 15 minutes. The changes of CA and S content in granular luminescent cells (GLC) of the germinal centers of spleen lymphoid nodules had a wavy character. At the same time, the correlations between monoamine content in the central arteriole wall and in germinal center GLCs, as well as that one between GLCs and germinal center lymphocytes, became strong positive. The data obtained suggest the presence of an immune-stimulating component in acupuncture, which became apparent during the first hour and persisted for 4 hours after a single acupuncture in GV 14 and LI 4 points.
Assuntos
Pontos de Acupuntura , Catecolaminas , Centro Germinativo , Serotonina , Baço , Animais , Catecolaminas/imunologia , Catecolaminas/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Masculino , Ratos , Serotonina/imunologia , Serotonina/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Fatores de TempoRESUMO
BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.