RESUMO
Romidepsin (FK228), a histone deacetylase inhibitor (HDACi), has anti-tumor effects against several types of solid tumors. Studies have suggested that HDACi could upregulate PD-L1 expression in tumor cells and change the state of anti-tumor immune responses in vivo. However, the influence of enhanced PD-L1 expression in tumor cells induced by romidepsin on anti-tumor immune responses is still under debate. So, the purpose of this study was to explore the anti-tumor effects and influence on immune responses of romidepsin in colon cancer. The results indicated that romidepsin inhibited proliferation, induced G0/G1 cell cycle arrest and increased apoptosis in CT26 and MC38 cells. Romidepsin treatment increased PD-L1 expression in vivo and in vitro via increasing the acetylation levels of histones H3 and H4 and regulating the transcription factor BRD4. In subcutaneous transplant tumor mice and colitis-associated cancer (CAC) mice, romidepsin increased the percentage of FOXP3+ regulatory T cells (Tregs), decreased the ratio of Th1/Th2 cells and the percentage of IFN-γ+ CD8+ T cells in the peripheral blood and the tumor microenvironment. Upon combination with an anti-PD-1 antibody, the anti-tumor effects of romidepsin were enhanced and the influence on CD4+ and CD8+ T cells was partially reversed. Therefore, the combination of romidepsin and anti-PD-1 immunotherapy provides a more potential treatment for colon cancer.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Depsipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Feminino , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Histonas/metabolismo , Imunoterapia/métodos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Targeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines. METHODS: The VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function. RESULTS: The novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells. CONCLUSION: Our results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.
Assuntos
Pontos de Checagem do Ciclo Celular/imunologia , Fase G1/imunologia , Fase de Repouso do Ciclo Celular/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Western Blotting , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Células MCF-7 , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Domínio Único/genéticaRESUMO
It has been shown that psychoneurological disorders are accompanied by different disturbances of immunity. Paper aimed to study the effects of repeated experience of aggression in daily agonistic interactions leading to the development of behavioral psychopathology on the parameters of cellular immunity in the thymus and spleen. There were no found the changes in the weight indexes, the number of cells in the thymus, spleen and blood in aggressive mice. In the spleen of aggressive mice percent of B-lymphocytes--CD19+ and CD16/32+, as well as T-lymphocytes CD4+8-, CD4-8+, and CD4+25(hi) decreased and percent of CD4-25+ increased in comparison with the controls. In the thymus percent of CD4-25+ cells are decreased without changes of other types of lymphocytes. Flow cytometric analysis revealed decreased percentage of apoptotic (A(0)) and resting (G0/G1) cells and increased percentage of proliferating cells in phase S+G2/M in the spleen of aggressive male mice in comparison with the control. The percentage of apoptotic thymocytes is increased and the percentage of thymocytes in S+G2/M phase is decreased under the repeated experience of aggression. Data suggest the possible development of an autoimmune procceses in male mice under the influence of repeated experience of aggression.
Assuntos
Agressão/psicologia , Subpopulações de Linfócitos B/imunologia , Baço/imunologia , Estresse Psicológico/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Autoimunidade , Subpopulações de Linfócitos B/patologia , Proliferação de Células , Fase G1/genética , Fase G1/imunologia , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/imunologia , Expressão Gênica , Imunidade Inata , Imunofenotipagem , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/patologia , Estresse Psicológico/patologia , Subpopulações de Linfócitos T/patologia , Timo/patologiaRESUMO
AIM: To elucidate the immunomodulatory functions of colonic mesenchymal stem cells (MSCs) in the colonic mucosal immune system. METHODS: The colonic MSCs were isolated, enriched and expanded. The immunosuppressive role of colonic MSCs on activated T cells was evaluated. The cell cycle progression of T cells and the expression of FoxP3+ T cells were assessed by fluorescence-activated cell sorting (FACS). The levels of cytokines and PGE2 were measured by ELISA. RESULT: Mouse colonic MSCs can inhibit the proliferation of activated T cells by arresting cells in G0/G1 phase, induce the expression of CD4+CD25+Foxp3+ T cells (8.05%±0.49% in transwell culture vs 8.45%±0.64% in direct contact culture vs 4.30%±0.28% in control, p<0.05), downregulate the levels of the cytokines TNF-α and IFN-γ, and increase the production of IL-10 (p<0.05). The data obtained from transwell culture and direct contact culture showed no difference (p>0.05). PGE2 level was increased when T cells were cultured with colonic MSCs (385.10±19.45 ng/l in transwell culture vs 387.91±19.85 ng/l in direct contact culture vs 276.21±25.49 ng/l in control, p<0.05). Blocking PGE2 partially reversed the immunosuppression of MSCs on activated T cells proliferation (p<0.05). CONCLUSION: Colonic MSCs have the same immunosuppressive property as other MSCs. They performed their functions partially through secreting soluble factor PGE2. The characterization of these colonic MSCs may be helpful for studying the involvement of stromal cell compartment in colon diseases.
Assuntos
Pontos de Checagem do Ciclo Celular/imunologia , Proliferação de Células , Células-Tronco Mesenquimais/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Colo/citologia , Colo/imunologia , Dinoprostona/imunologia , Dinoprostona/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Fase G1/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fase de Repouso do Ciclo Celular/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In this paper, we sought to identify the CD4(+) T-cell dynamics in the course of HIV infection in response to continuous and intermittent intravenous courses of interleukin-2 (IL-2), the principal cytokine responsible for progression of CD4(+) T-lymphocytes from the G1 to the S phase of the cell cycle. Based on multivariate regression models, previous literature has concluded that the increase in survival of CD4(+) T-cell appears to be the critical mechanism leading to sustained CD4(+) T-cell levels in HIV-infected patients receiving intermittent IL-2 therapy. Underscored by comprehensive mathematical modeling, a major finding of the present work is related to the fact that, rather than due to any increase in survival of CD4(+) T-cells, the expressive, selective and sustained CD4(+) T-cell expansions following IL-2 administration may be related to the role of IL-2 in modulating the dynamics of Fas-dependent apoptotic pathways, such as activation-induced cell death (AICD) or HIV-specific apoptotic routes triggered by viral proteins.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/terapia , Linfócitos T CD4-Positivos/imunologia , HIV-1/imunologia , Imunoterapia , Interleucina-2 , Síndrome da Imunodeficiência Adquirida/patologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos T CD4-Positivos/patologia , Feminino , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Humanos , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Masculino , Fase S/efeitos dos fármacos , Fase S/imunologia , Proteínas Virais/imunologia , Receptor fas/imunologiaRESUMO
Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.
Assuntos
Antioxidantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Fibras na Dieta/farmacologia , Polipose Intestinal/tratamento farmacológico , Polipose Intestinal/imunologia , Vitis/química , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Peso Corporal/imunologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/imunologia , Ciclo Celular/genética , Ciclo Celular/imunologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/prevenção & controle , Suplementos Nutricionais , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Fase G1/efeitos dos fármacos , Fase G1/genética , Fase G1/imunologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Polipose Intestinal/genética , Polipose Intestinal/metabolismo , Pólipos Intestinais/tratamento farmacológico , Pólipos Intestinais/genética , Pólipos Intestinais/imunologia , Pólipos Intestinais/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologiaRESUMO
After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G(1) cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G(1)-phase extension and represses CD8 expression in a G(1) extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G(1) extension and CD8 repression. Importantly, forced G(1) extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G(1) progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G(1)-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Fase G1/imunologia , Fatores de Transcrição/fisiologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Células Tumorais CultivadasRESUMO
UNLABELLED: The possibility of antileukemic activity of antithymocyte globulin (ATG) was investigated in 8 human leukemic cell lines and primary leukemic cells from 15 leukemia patients. The study demonstrated that ATG induced apoptosis and reduced proliferation in both cell lines and primary leukemic cells, particularly in lymphatic origin cells, indicating that ATG has broad-spectrum antileukemic activity, especially for cells of lymphatic origin. BACKGROUND: Polyclonal ATGs are currently used to prevent graft-versus-host disease in allogeneic stem cell transplantation patients and to treat patients with severe aplastic anemia. It contains antibodies against antigens expressed on various hematopoietic cells, we hypothesized that it induces cell death not only in healthy cells but also in malignant hematopoietic cells. MATERIALS AND METHODS: In this study, several human leukemic cell lines and primary leukemic cells from 15 patients with leukemia were used to investigate the ability of polyclonal ATGs to induce apoptosis and proliferation. RESULTS: Polyclonal ATGs induced cell apoptosis in primary leukemic cells and in cell lines in a dose-dependent manner, and induced apoptosis in different populations through a variety of targets. Cell proliferation was significantly reduced in the presence of polyclonal ATGs; it arrested cells in the G0-G1 phase by cell cycle analysis. Treatment with polyclonal ATGs plus complement increased cytolysis of the leukemic cells; complement augments polyclonal ATG-induced leukemic cell death. CONCLUSION: These data show that polyclonal ATG has broad-spectrum antileukemic activity, especially for cells of lymphatic origin, as it induced cell death through a variety of targets. This study provides an experimental basis for the application of polyclonal ATGs in allogeneic hematopoietic stem cell transplantation and in patients with lymphatic leukemia.
Assuntos
Soro Antilinfocitário/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Leucemia/tratamento farmacológico , Animais , Anticorpos/imunologia , Soro Antilinfocitário/imunologia , Apoptose/imunologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Morte Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Células HL-60 , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Células Jurkat , Células K562 , Leucemia/imunologia , Leucemia/patologia , Leucemia/cirurgia , Coelhos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/imunologia , Células U937RESUMO
Complement system activation plays an important role in both innate and acquired immunity. Activation of the complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on the cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis.
Assuntos
Apoptose/imunologia , Membrana Celular/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Quinase CDC2 , Caspase 8/imunologia , Caspase 8/metabolismo , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Ciclina B/imunologia , Ciclina B/metabolismo , Quinase 2 Dependente de Ciclina/imunologia , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Fase G1/imunologia , Humanos , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Musculares/imunologia , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase S/imunologia , Proteína de Morte Celular Associada a bcl/imunologia , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
AIM: Bacillus Calmette-Guerin (BCG) is one of therapeutic options for urothelial carcinoma (UC). The objectives of this study were to determine the direct effect of viable or heat-killed BCG and BCG cell wall skeleton (BCG-CWS) on UC cells in vitro. MATERIALS AND METHODS: UC cell lines were co-cultured with viable or heat-killed BCG Immunobladder® (Tokyo 172 strain) and BCG-CWS. Viability of the cells, apoptosis and BrdU incorporation were estimated. RESULTS: BCG induced cell growth retardation in highly malignant UC bearing integrin α5ß1 (VLA5). VLA5-blocking antibody partially abrogated this effect. BCG treatment induced a modest increase in the sub-G(1) fraction of cells and a decrease of BrdU incorporation. Cell growth retardation effect of viable BCG was reproduced by both heat-killed BCG and BCG-CWS. CONCLUSION: The results indicate that VLA5 may be a biomarker of UC with sensitivity to BCG. Moreover, BCG-CWS is a promising substance which might replace BCG, preventing life-threatening complications of viable BCG treatment.
Assuntos
Vacina BCG/farmacologia , Mycobacterium bovis/imunologia , Neoplasias da Bexiga Urinária/terapia , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Esqueleto da Parede Celular/imunologia , Esqueleto da Parede Celular/farmacologia , Quinase 1 de Adesão Focal/biossíntese , Quinase 1 de Adesão Focal/imunologia , Fase G1/imunologia , Humanos , Integrina alfa5beta1/biossíntese , Integrina alfa5beta1/imunologia , Transdução de Sinais , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.
Assuntos
Diversidade de Anticorpos/genética , Ciclo Celular/genética , Ciclo Celular/imunologia , Genes de Imunoglobulinas/imunologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Galinhas , Células Clonais , Citidina Desaminase/biossíntese , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Fase G1/genética , Fase G1/imunologia , Rearranjo Gênico de Cadeia Leve de Linfócito B/imunologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Linfoma/enzimologia , Linfoma/genética , Linfoma/imunologia , Fatores de TempoRESUMO
Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I-IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2-dependent yet is gp91phox-independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue-derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum-starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.
Assuntos
Glicoproteínas/imunologia , Canais Iônicos/imunologia , Macrófagos Peritoneais/imunologia , Glicoproteínas de Membrana/imunologia , Mitocôndrias/imunologia , Proteínas Mitocondriais/imunologia , NADPH Oxidases/imunologia , Superóxidos/imunologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Movimento Celular/genética , Movimento Celular/imunologia , Transporte de Elétrons/imunologia , Fase G1/genética , Fase G1/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Glicoproteínas/genética , Canais Iônicos/genética , Macrófagos Peritoneais/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Necrose/genética , Necrose/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Fatores de Tempo , Proteína Desacopladora 2RESUMO
The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Memória Imunológica/imunologia , Transdução de Sinais/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Antígenos Virais/imunologia , Comunicação Celular/genética , Núcleo Celular/imunologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/imunologia , Fase G1/imunologia , Memória Imunológica/genética , Interferon gama/imunologia , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação/genética , Fosforilação/imunologia , Fase de Repouso do Ciclo Celular/imunologia , Transdução de Sinais/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Estomatite Vesicular/imunologia , Vesiculovirus/imunologiaRESUMO
Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK(-/-) mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27(kip1) expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27(kip1) induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7-dependent proliferation and survival of pre-B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre-B-cell leukemia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Janus Quinase 3/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Apoptose/imunologia , Ciclo Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação para Baixo/imunologia , Fase G1/imunologia , Regulação Leucêmica da Expressão Gênica , Interleucina-7/genética , Interleucina-7/metabolismo , Camundongos , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT5/genética , Transdução de Sinais/imunologiaRESUMO
Human primary melanoma cells (T1) were found to be more susceptible to lysis by a Melan-A/MART-1-specific CTL clone (LT12) than their metastatic derivative (G1). We show that this differential susceptibility does not involve antigen presentation by target cells, synapse formation between the metastatic target and CTL clone, or subsequent granzyme B (GrB) polarization. Although PI-9, an inhibitor of GrB, was found to be overexpressed in metastatic G1 cells, knockdown of the PI-9 gene did not result in the attenuation of G1 resistance to CTL-induced killing. Interestingly, we show that whereas T1 cells express high levels of intercellular adhesion molecule-1 (ICAM-1), a dramatically reduced expression was noted on G1 cells. We also showed that sorted ICAM-1+ G1 cells were highly sensitive to CTL-induced lysis compared with ICAM-1- G1 cells. Furthermore, incubation of metastatic G1 cells with IFN-gamma resulted in the induction of ICAM-1 and the potentiation of their susceptibility to lysis by LT12. More importantly, we found that the level of ICAM-1 expression by melanoma cells correlated with decreased PTEN activity. ICAM-1 knockdown in T1 cells resulted in increased phosphorylation of PTEN and the subsequent activation of AKT. We have additionally shown that inhibition of the phosphatidylinositol (3,4,5)-triphosphate kinase (PI3K)/AKT pathway by the specific inhibitor wortmannin induced a significant potentiation of susceptibility of G1 and ICAM-1 small interfering RNA-treated T1 cells to CTL-induced lysis. The present study shows that a shift in ICAM-1 expression, which was associated with an activation of the PI3K/AKT pathway, can be used by metastatic melanoma cells to escape CTL-mediated killing.
Assuntos
Molécula 1 de Adesão Intercelular/imunologia , Melanoma/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Citotóxicos/imunologia , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Fase G1/imunologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Interferon gama/farmacologia , Antígeno MART-1 , Melanoma/enzimologia , Melanoma/secundário , Proteínas de Neoplasias/imunologia , PTEN Fosfo-Hidrolase/metabolismo , RNA Interferente Pequeno/genética , Serpinas/biossíntese , Serpinas/imunologiaRESUMO
Infection with Schistosoma mansoni induces a wide range of effects on the immune responses of the host. In the present study we investigated the influence of soluble egg antigens (SEA) on the cell cycle of peripheral blood mononuclear cells (PBMC) from infected and non-infected individuals with S. mansoni resident in an endemic area and blood donors from non-endemic area. The cell cycle, the expression of activation markers and cyclin D(+)(1,2,3) CD3(+) frequency was assessed by flow cytometry. Stimulation of PBMC from infected patients with SEA resulted in a lower frequency of CD3(+) T cells in S phase when compared with the non-infected group. In addition, infected patients presented a decrease of activation marker expression (CD69(+), HLA-DR(+) and CD28(-) on CD4(+) cells and CD25(+), HLA-DR(+) on CD8(+) cells). A reduced frequency was observed of cyclin D(1,2,3) expression in SEA-stimulated T cells from infected individuals when compared with those from the non-infected group. The decreased expression of activation markers and frequency of cyclin D(1,2,3) in T cells may result in arrest of T cells in the G(0)/G(1) phase of the cell cycle, thus explaining the down-regulation observed in chronic schistosomiasis.
Assuntos
Antígenos de Helmintos/imunologia , Ciclina D1/metabolismo , Ciclinas/metabolismo , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Schistosoma mansoni/imunologia , Animais , Complexo CD3/metabolismo , Células Cultivadas , Doença Crônica , Ciclina D2 , Ciclina D3 , Regulação para Baixo , Fezes/parasitologia , Fase G1/imunologia , Humanos , Contagem de Ovos de Parasitas , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Linfócitos T/imunologiaRESUMO
Angiogenesis contributes to a wide range of neoplastic, ischemic, and inflammatory disorders. Definition of the intrinsic molecular controls in angiogenic vessel growth promises novel therapeutic approaches for angiogenesis-related diseases. CD148 (also named DEP-1/PTP eta) is a receptor-like protein tyrosine phosphatase that is abundantly expressed in vascular endothelial cells. To explore a role of CD148 in endothelial vessel formation, we generated a monoclonal antibody, Ab1, against the ectodomain sequence of CD148 and examined its effects on endothelial-cell growth and vessel formation. Here we report that a bivalent, but not a monovalent, form of the Ab1 antibody inhibits endothelial-cell growth and blocks angiogenesis in mouse cornea in vivo. We further demonstrate that (1) bivalent Ab1 arrests cell-cycle progression of CD148-transfected CHO cells at G(0)/G(1) phase, (2) coexpression of catalytically inactive CD148 mutants attenuates the Ab1-cell growth inhibition, and (3) bivalent Ab1 suppresses phosphorylation of ERK1/2 kinases and Met tyrosine kinase as activated CD148 does, with an increase in CD148-associated tyrosine phosphatase activity. Taken together, these findings demonstrate that Ab1-induced ectodomain oligomerization arrests endothelial-cell growth through catalytic activity of the CD148 cytoplasmic domain. The present study defines CD148 as a valuable molecular target for antiangiogenesis therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Córnea/irrigação sanguínea , Córnea/imunologia , Córnea/metabolismo , Córnea/patologia , Cricetinae , Cricetulus , Células Endoteliais/patologia , Inibidores Enzimáticos/imunologia , Fase G1/efeitos dos fármacos , Fase G1/genética , Fase G1/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/imunologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/imunologiaRESUMO
Synthetic type B phosphorothioate oligodeoxyribonucleotides (ODN) activate mouse B cells via Toll-like receptor 9 (TLR9). Starting with closely related 15-mer prototype ODN, the sequence requirements for stimulatory (ST-) and inhibitory (IN-) activity were contrasted, by measuring apoptosis protection, G(1) entry and interleukin-6 secretion. ST-ODN and IN-ODN differ in that (1) ST-ODN require a 5' T, (2) the central CG is obligatory, (3) CG must be flanked 3' specifically by TT at the position where IN-ODN have GG, and (4) IN-ODN tolerate truncation of the 3' end better than ST-ODN. Features shared by ST-ODN and IN-ODN include (1) requiring CC adjacent to the 5' end, and (2) avoiding CC immediately 5' to the CG. This pattern is used to create a model of how ST-ODN binding might function to aggregate TLR9 so as to initiate the signal, and how the 5' ends of ST-ODN and IN-ODN compete for binding. Further justification for considering TLR9 to be the ODN receptor was provided by a demonstration that in HEK293 cells transfected with TLR9, the potency of a panel of ODN for activating NF-kappaB roughly parallels that seen in the biological assays in mouse B cells.
Assuntos
Apoptose/imunologia , Ilhas de CpG/imunologia , Oligodesoxirribonucleotídeos/imunologia , Animais , Linfócitos B/imunologia , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Relação Dose-Resposta Imunológica , Feminino , Fase G1/imunologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/genética , Relação Estrutura-Atividade , Receptor Toll-Like 9/imunologiaRESUMO
Interleukin-3 (IL-3) mediates hematopoietic cell survival and proliferation via several signaling pathways such as the Janus kinase/signal transducer and activator of transcription pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphoinositide-3 kinase (PI-3K) pathway. Mammalian target of rapamycin (mTOR) is one of the downstream targets of the PI-3K pathway, and it plays an important role in hematopoiesis and immune cell function. To better elucidate how mTOR mediates proliferation signals from IL-3, we assessed the role of S6 kinase 2 (S6K2), one of the downstream targets of mTOR, in IL-3 signaling. We show that S6K2 is activated by IL-3 in the IL-3-dependent Ba/F3 cell line and that this is mediated by mTOR and its upstream activator PI-3K but not by the MAPK kinase/extracellular signal-regulated kinase pathway. S6K2 is also activated in primary mouse bone marrow-derived mast cells upon IL-3 stimulation. Expression of a rapamycin-resistant form of S6K2, T388E, in Ba/F3 cells provides a proliferation advantage in the absence or presence of rapamycin, indicating that S6K2 can potentiate IL-3-mediated mitogenic signals. In cells expressing T388E, rapamycin still reduces proliferation at all doses of rapamycin, showing that mTOR targets other than S6K2 play an important role in IL-3-dependent proliferation. Cell-cycle analysis shows that T388E-expressing Ba/F3 cells enter S phase earlier than the control cells, indicating that the proliferation advantage may be mediated by a shortened G1 phase. This is the first indication that S6K2 plays a role in IL-3-dependent cell proliferation.
Assuntos
Células da Medula Óssea/enzimologia , Proliferação de Células/efeitos dos fármacos , Interleucina-3/metabolismo , Mastócitos/enzimologia , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Fase G1/efeitos dos fármacos , Fase G1/imunologia , Humanos , Interleucina-3/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Fase S/efeitos dos fármacos , Fase S/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
BACKGROUND & AIMS: Although Helicobacter pylori is recognized by the human immune system, the bacteria are not eliminated and lead to a chronic inflammation of the gastric mucosa. METHODS: We investigated the interaction of H. pylori with human lymphocytes. T and B lymphocytes were isolated from H. pylori-infected patients and stimulated with anti-CD3/CD28 or interleukin-6. RESULTS: Proliferation of lymphocytes was abolished on co-incubation with different H. pylori strains (1-5 bacteria/cell) or with protein extracts of culture supernatants. Inhibition of proliferation was independent of known virulence factors. The factor is a protein or protein complex with an apparent molecular weight between 30 and 60 kilodaltons, clearly distinct from VacA. Although antigen-specific activation of T cells (as shown by nuclear factor of activated T cells [NFAT]-activation, interferon-gamma production, and CD25 or CD69 up-regulation) remained intact, cell-cycle analysis showed that S-phase entry of T cells was inhibited completely by H. pylori. Consequently, stimulated T cells arrested in the G1 phase of the cell cycle. Western blot analysis showed markedly reduced phosphorylation of the retinoblastoma protein (pRb), suggesting inhibition of G1 cyclin-dependent kinase activity. In line with this, activities of cyclin D3 and cyclin E were down-regulated, and levels of the cyclin-dependent kinase inhibitor p27Kip1 were increased. Mouse embryonic fibroblasts deficient in p27 showed a decrease in H. pylori-induced inhibition of cell proliferation, suggesting a central role for p27 in mediating H. pylori-induced G1 arrest. CONCLUSIONS: Induction of cell-cycle arrest in lymphocytes may be of major significance for the chronic persistence of bacteria in the human stomach.