RESUMEN
A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II-matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos CD58/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Enfermedad de Hodgkin/inmunología , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Formación de Roseta , Antígenos CD2/antagonistas & inhibidores , Antígenos CD58/biosíntesis , Antígenos CD58/genética , Sistemas CRISPR-Cas , Adhesión Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Técnicas de Inactivación de Genes , Humanos , Mapeo de Interacción de ProteínasRESUMEN
Loss of CD58 is a common mechanism for tumor immune evasion in lymphoid malignancies. CD58 loss is known to occur due to both genetic and non-genetic causes; therefore, we hypothesized that restoring CD58 expression in lymphoma cells may be an effective treatment approach. To explore the potential for restoring CD58 expression, we first screened 11 B-cell lymphoma lines and found that 3 had decreased CD58 expression. Among these, CD58 was genetically damaged in two lines but not in the third line. Using the cell line with downregulated CD58 without a genetic abnormality, we performed epigenetic library screening and found that two EZH2 inhibitors, EPZ6438 and GSK126, specifically enhanced CD58 expression. By examining the effect of three EZH2 inhibitors with different selectivity profiles in different B-cell lines, EZH2 inhibition was shown to have a common activity in upregulating CD58 expression. Restoring the expression of CD58 in lymphoma cells using an EZH2 inhibitor was shown to enhance interferon-γ production of T and NK cells against lymphoma cells. H3K27 was shown to be highly trimethylated in the CD58 promoter region, and EZH2 inhibition induced its demethylation and activated transcription of the CD58 gene. These results indicated that EZH2 is involved in the epigenetic silencing of CD58 in lymphoma cells as a mechanism for tumor immune escape, and EZH2 inhibitors are able to restore epigenetically suppressed CD58 expression. Our findings provide a molecular basis for the combination of an EZH2 inhibitor and immunotherapy for lymphoma treatment.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Antígenos CD58/genética , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Epigénesis Genética , Indoles/farmacología , Linfoma de Células B/genética , Piridonas/farmacología , Compuestos de Bifenilo , Antígenos CD58/biosíntesis , Antígenos CD58/inmunología , Línea Celular Tumoral , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Activación de Linfocitos , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Morfolinas , Linfocitos T/inmunologíaRESUMEN
For an effective T-cell activation and response, co-stimulation is required in addition to the antigen-specific signal from their antigen receptors. The CD2/CD58 interaction is considered as one of the most important T-cell co-stimulatory pathways for T-cell activation and proliferation, and its role in regulating intestinal T-cell function in acute and chronic SIV -infected macaques is poorly documented. Here, we demonstrated a significant reduction of CD58 expression in both T- and B-cell populations during acute SIV infection along with high plasma viral load and a loss of intestinal CD4+ T cells compared to SIV-uninfected control macaques. The reduction of CD58 expression in T cells was correlated with the reduced expression of T-cell-mediated IL-2 and TNFα production. Together, these results indicate that reduction in the CD2/CD58 interaction pathway in mucosal lymphocytes might play a crucial role in mucosal T-cell dysfunction during acute SIV/HIV infection.
Asunto(s)
Antígenos CD58/biosíntesis , Expresión Génica , Interleucina-2/metabolismo , Mucosa Intestinal/patología , Linfocitos Intraepiteliales/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Linfocitos B/inmunología , Activación de Linfocitos , Macaca , Plasma/virología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Carga ViralRESUMEN
The purpose of our study was to observe the effects of luteolin on the expression of the genes ICAM-1, LFA-3, and PCNA in H22 hepatoma tissue. Sixty ICR (Institute of Cancer Research) mice with H22 hepatoma were randomly divided into five groups: a normal saline control group, low-, medium-, and high-dose luteolin groups, and a cyclophosphamide group. The mice were euthanized the day after administration withdrawal and subcutaneous tumor tissue was extracted. Quantitative fluorescence RT-PCR was used to detect the expression of ICAM-1, LFA-3, and PCNA in H22 hepatoma tissue in the mice. Luteolin was found to up-regulate the expression of ICAM-1 in H22 hepatoma tissue, of which the middle-dose group had the most obvious effect, showing a significant difference (P < 0.01) as compared to the normal saline group. Each dose group of luteolin significantly down-regulated the expression of LFA-3 in H22 hepatoma tissue, showing significant differences as compared to the saline control group (P < 0.01). The medium- and high-dose luteolin groups significantly reduced the expression of PCNA in H22 hepatoma tissue of ICR mice, where the effect of the high-dose group was the most obvious, and the difference between the two luteolin groups and the normal saline group was statistically significant (P < 0.01). Luteolin may inhibit tumor angiogenesis and tumor cell proliferation by down-regulation of LFA- 3 and PCNA and up-regulation of ICAM-1 in tumor tissue of tumor-bearing mice, thereby achieving its anti-tumor effect.
Asunto(s)
Antígenos CD58/biosíntesis , Carcinoma Hepatocelular/genética , Molécula 1 de Adhesión Intercelular/biosíntesis , Neoplasias Hepáticas/genética , Neovascularización Patológica/genética , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Animales , Antígenos CD58/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fluorouracilo/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Molécula 1 de Adhesión Intercelular/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Luteolina/administración & dosificación , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Antígeno Nuclear de Célula en Proliferación/genéticaRESUMEN
Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.
Asunto(s)
Antígenos CD58/biosíntesis , Animales , Humanos , Inmunoglobulina G , Activación de Linfocitos , Macaca mulatta , Pichia , Plásmidos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Linfocitos TRESUMEN
PreImplantation Factor (PIF(9&15)) secreted by viable embryos exerts an essential transplant acceptance and immune regulatory role in pregnancy. Synthetic PIF replicates endogenous PIF's effect in pregnant and non-pregnant immune disorder models. PIF binds macrophages to regulate CD3/CD28-induced T-cell response. We present evidence that PIF regulates the co-stimulatory T-cell receptor, CD2, which binds to and is activated by phytohemagglutinin (PHA), a potent mitogen, confirming PIF's ability to systemically respond to diverse immune stimulants. PIF's effect on PHA-activated PBMC (male and non-pregnant females) proliferation and cytokine secretion was tested, showing that both PIF(9&15) block PHA-induced PBMC proliferation and promote anti-inflammatory IL10 secretion, while reducing pro-inflammatory IFNγ secretion. Thus favoring a T(H)2 cytokine bias. Surface plasmon resonance spectroscopy, immunocytochemistry and Flex station experiments reveal that PIF effect is direct. PIF targets intracellular targets but does not affect early Ca(2+) mobilization. By promoting the CD2 receptor in activated T-cells and through inhibition of co-ligand CD58 expression, PIF regulates antigen-presenting cell (APC)-T-cell interactions required for PHA action. Structure-based design demonstrated that PIF15 offers improved target specificity as compared to PIF9. Collectively, PIF directly regulates mitogen-induced PBMC activation. Results support PIF translation for therapy of immune disorders.
Asunto(s)
Calcio/metabolismo , Leucocitos Mononucleares/inmunología , Péptidos/farmacología , Fitohemaglutininas/metabolismo , Proteínas Gestacionales/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos CD2/metabolismo , Antígenos CD28/metabolismo , Complejo CD3/metabolismo , Antígenos CD58/biosíntesis , Canales de Calcio/metabolismo , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-10/biosíntesis , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Masculino , Simulación de Dinámica Molecular , Embarazo , Resonancia por Plasmón de Superficie , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1-induced neoplasm with four clinical subtypes: acute, lymphoma, chronic, and smoldering. Although the chronic type is regarded as indolent ATL, about half of the cases progress to acute-type ATL. The molecular pathogenesis of acute transformation in chronic-type ATL is only partially understood. In an effort to determine the molecular pathogeneses of ATL, and especially the molecular mechanism of acute transformation, oligo-array comparative genomic hybridization and comprehensive gene expression profiling were applied to 27 and 35 cases of chronic and acute type ATL, respectively. The genomic profile of the chronic type was nearly identical to that of acute-type ATL, although more genomic alterations characteristic of acute-type ATL were observed. Among the genomic alterations frequently observed in acute-type ATL, the loss of CDKN2A, which is involved in cell-cycle deregulation, was especially characteristic of acute-type ATL compared with chronic-type ATL. Furthermore, we found that genomic alteration of CD58, which is implicated in escape from the immunosurveillance mechanism, is more frequently observed in acute-type ATL than in the chronic-type. Interestingly, the chronic-type cases with cell-cycle deregulation and disruption of immunosurveillance mechanism were associated with earlier progression to acute-type ATL. These findings suggested that cell-cycle deregulation and the immune escape mechanism play important roles in acute transformation of the chronic type and indicated that these alterations are good predictive markers for chronic-type ATL.
Asunto(s)
Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas de Neoplasias/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD58/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Masculino , Persona de Mediana EdadRESUMEN
Human postmortem studies of natural dengue virus (DENV) infection have reported systemically distributed viral antigen. Although it is widely accepted that DENV infects mononuclear phagocytes, the sequence in which specific tissues and cell types are targeted remains uncharacterized. We previously reported that mice lacking alpha/beta and gamma interferon receptors permit high levels of DENV replication and show signs of systemic disease (T. R. Prestwood et al., J. Virol. 82:8411-8421, 2008). Here we demonstrate that within 6 h, DENV traffics to and replicates in both CD169(+) and SIGN-R1(+) macrophages of the splenic marginal zone or draining lymph node, respectively, following intravenous or intrafootpad inoculation. Subsequently, high levels of replication are detected in F4/80(+) splenic red pulp macrophages and in the bone marrow, lymph nodes, and Peyer's patches. Intravenously inoculated mice begin to succumb to dengue disease 72 h after infection, at which time viral replication occurs systemically, except in lymphoid tissues. In particular, high levels of replication occur in CD68(+) macrophages of the kidneys, heart, thymus, and gastrointestinal tract. Over the course of infection, proportionately large quantities of DENV traffic to the liver and spleen. However, late during infection, viral trafficking to the spleen decreases, while trafficking to the liver, thymus, and kidneys increases. The present study demonstrates that macrophage populations, initially in the spleen and other lymphoid tissues and later in nonlymphoid tissues, are major targets of DENV infection in vivo.
Asunto(s)
Virus del Dengue/metabolismo , Dengue/virología , Macrófagos/citología , Bazo/citología , Animales , Transporte Biológico , Médula Ósea/virología , Antígenos CD58/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Dengue/metabolismo , Inmunohistoquímica/métodos , Cinética , Lectinas Tipo C/biosíntesis , Ganglios Linfáticos/virología , Macrófagos/virología , Ratones , Ganglios Linfáticos Agregados/virología , Receptores de Superficie Celular/biosíntesis , Lectina 1 Similar a Ig de Unión al Ácido Siálico/biosíntesis , Bazo/virología , Distribución Tisular , Replicación ViralRESUMEN
Human natural killer (NK) cells express an abundant level of 2B4 and CD2 on their surface. Their counter-receptors, CD48 and CD58, are also expressed on the NK cell surface, raising a question about the functional consequences of potential 2B4/CD48 and CD2/CD58 interactions. Using blocking antibodies specific to each receptor, we demonstrated that both 2B4/CD48 and CD2/CD58 interactions were essential for the development of NK effector functions: cytotoxicity and cytokine secretion. However, only 2B4/CD48, but not CD2/CD58, interactions were shown to be critical for the optimal NK cell proliferation in response to interleukin (IL)-2. IL-2-activated NK cells cultured in the absence of 2B4/CD48 or CD2/CD58 interactions were severely impaired for their ability to induce intracellular calcium mobilization and subsequent ERK activation upon tumor target exposure, suggesting that the early signaling pathway of NK receptors leading to impaired cytolysis and interferon (IFN)-γ secretion was inhibited. Nevertheless, these defects did not fully account for the reduced proliferation of NK cells in the absence of 2B4/CD48 interactions, because anti-CD2 or anti-CD58 monoclonal antibody (mAb)-treated NK cells, showing defective signaling and effector functions, displayed normal proliferation upon IL-2 stimulation. These results propose the signaling divergence between pathways leading to cell proliferation and cytotoxicity/cytokine release, which can be differentially regulated by 2B4 and CD2 during IL-2-driven NK cell activation. Collectively, these results reveal the importance of homotypic NK-to-NK cell cross-talk through 2B4/CD48 and CD2/CD58 pairs and further present their differential and overlapping roles in human NK cells.
Asunto(s)
Antígenos CD/química , Antígenos CD2/química , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/química , Animales , Antígenos CD/biosíntesis , Antígeno CD48 , Antígenos CD58/biosíntesis , Proliferación Celular , Humanos , Inmunidad Innata , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Células K562 , Leucocitos Mononucleares/citología , Activación de Linfocitos , Ratones , Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
To establish methods and requirements for quality control of rhLFA3-IgG1, biological potency of rhLFA3-IgG1 was determined by CD2 molecule competitive binding assay on Jurkat cell surface. Purity of rhLFA3-IgG1 was analyzed by SEC-HPLC and IEC-HPLC. Peptide mapping was preformed by tryptic digestion and RP-HPLC after sample reduced and carboxymethylation by DTT and indoacetic acid, respectively. CHO host cell protein and Protein A residual were detected by ELISA separately. The quality control methods and requirements, such as biological potency, the physical-chemical characteristic of rhLFA3-IgG1 had been established. The methods and requirements for quality control of rhLFA3-IgG1 showed advantages of assuring the products safety and efficacy, which can be used for routine quality control of rhLFA3-IgG1.
Asunto(s)
Biotecnología/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Alefacept , Unión Competitiva , Antígenos CD2/metabolismo , Antígenos CD58/biosíntesis , Antígenos CD58/química , Cromatografía Líquida de Alta Presión , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Células Jurkat , Peso Molecular , Mapeo Peptídico , Control de Calidad , Proteínas Recombinantes de Fusión/químicaRESUMEN
The CD2 receptor on T lymphocytes is essential for T cell adhesion and stimulation by antigen presenting cells (APCs). Blockade of CD2 function is immunosuppressive in both model systems and humans, indicating the importance of CD2 for the cellular immune response. Although the affinity of the molecular interaction between CD2 and its counter-receptor, CD58, is relatively low when measured in solution, this interaction mediates tight adhesion within the 2D cell-cell interface. To understand the mechanisms responsible for regulating the avidity of the CD2-CD58 interaction, we measured the number, affinity, and lateral mobility of CD2 molecules on resting and activated T cells. Cell activation caused a 1.5-fold increase in the number of CD2 sites on the cell surface, and the 2D affinity of CD2 for CD58 increased by 2.5-fold. The combination of T cell activation and CD2 ligation to CD58 decreased the laterally mobile fraction of the ligated CD2. Together, these changes would substantially enhance CD2 avidity and strengthen T cell-APC adhesion. The change in CD2 mobile fraction suggests that the cell uses cytoskeletal regulators to immobilize the receptor selectively at the site of contact with surfaces expressing CD58. Our observations are consistent with a model in which T cell activation initially induces increased CD2 2D affinity, cell surface receptor expression, and lateral mobility, allowing the CD2 molecules to diffuse to sites of contact with CD58-bearing APCs. Subsequently, T cell activation causes the CD58-bound CD2 to be recognized and immobilized at sites of cell-cell contact, thereby strengthening T cell-APC adhesion.
Asunto(s)
Antígenos CD2/biosíntesis , Antígenos CD58/biosíntesis , Linfocitos T/citología , Afinidad de Anticuerpos , Células Presentadoras de Antígenos/metabolismo , Adhesión Celular , Fluoresceína-5-Isotiocianato/farmacología , Colorantes Fluorescentes/farmacología , Humanos , Inmunosupresores/farmacología , Células Jurkat , Cinética , Membrana Dobles de Lípidos/química , Unión Proteica , Resonancia por Plasmón de Superficie , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To express the TSO45W-4BX of Taenia solium in combination with CD58 as a molecular adjuvant for improving the protective efficacy of the TSO45W-4BX recombinant vaccine. METHODS: TSO45W-4BX and porcine CD58 genes were amplified by PCR, using recombinant plasmids pGEM-4B and pGEM-CD58 as template respectively. The CD58 fragment was inserted into the recombinant plasmid pGEX-4T-1 with directly ligated TSO45W-4BX. The transformant was induced with IPTG and followed by identifying the integrity of the recombinant containing TS045W-4BX and porcine CD58 with PCR and sequencing. The products were analyzed by SDS-PAGE and Western blotting. RESULTS: The expression products of Mr 69,000 GST-4BX/CD58 and Mr 41,000 GST-4BX were present mainly in the form of inclusion bodies and soluble substance respectively, and both were recognized by sera of cysticercosis patients. CONCLUSIONS: The TSO45W-4BX co-expressed with porcine CD58 conserves its immune reactivity.
Asunto(s)
Antígenos de Protozoos/biosíntesis , Antígenos CD58/biosíntesis , Cisticercosis/sangre , Cisticercosis/parasitología , Escherichia coli/metabolismo , Taenia solium/inmunología , Animales , Antígenos de Protozoos/genética , Escherichia coli/genética , Humanos , Plásmidos , Reacción en Cadena de la Polimerasa , Porcinos , Taenia solium/genéticaRESUMEN
The influence of the age of the red blood cell (RBC) within its 120-day lifecycle at the time of blood donation on the RBC storage lesion is not well understood. Expression of cell adhesion molecules (CAMs) (CD44, CD47, CD58 and CD147), glycophorin A (GPA) and phosphatidylserine (PS) on young and old RBCs density separated prior to storage of the RBC concentrate was determined by flow cytometry. Older RBCs showed significantly reduced expression of GPA throughout storage and CD44 and CD147 from Day 28 onwards compared to young RBCs. Storage in the presence of leukocytes caused a significant decline in the expression of CD44, CD58, CD147 and GPA, whereas RBCs that were pre-storage leukocyte depleted maintained a relatively consistent level of expression throughout storage. PS was not detected at the external RBC membrane of young or old RBCs during storage. Increased levels of annexin V were detected in the supernatant of RBCs stored in the presence of leukocytes, with significantly greater supernatant levels found for old RBCs compared to young RBCs. These findings provide new insight into the RBC storage lesion and indicate that RBC age at the time of donation impacts upon the quality of stored RBC concentrates.
Asunto(s)
Anexina A5/metabolismo , Conservación de la Sangre , Eritrocitos/citología , Eritrocitos/patología , Glicoforinas/metabolismo , Leucocitos/metabolismo , Envejecimiento , Basigina/biosíntesis , Antígenos CD58/biosíntesis , Adhesión Celular , Eritrocitos/metabolismo , Citometría de Flujo , Humanos , Receptores de Hialuranos/biosíntesis , Concentración de Iones de Hidrógeno , Fosfatidilserinas/metabolismo , Control de Calidad , Manejo de Especímenes , Tiempo , Factores de TiempoRESUMEN
Dendritic cells (DCs) are the most potent of the antigen-presenting cells (APCs). Preparation of sufficient numbers of mature DCs, however, is both costly and time-consuming. We have examined here the possibility of using an alternative source of APCs that would be easier to obtain, would not require extensive culture, and thus would be more applicable to human immunotherapy protocols. We show here that freshly isolated human B cells can be efficiently infected by a replication-defective fowlpox recombinant vector, designated rF-TRICOM (TRIad of COstimulatory Molecules), to markedly increase surface expression of the human costimulatory molecule B7-1 and moderately increase expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-3 (LFA-3). Peptide-pulsed rF-TRICOM-infected B cells were highly efficient in activating antigen-specific human T cells and shown to be superior to the use of CD40L in enhancing APC potency. Moreover, when infection of freshly isolated B cells with rF-TRICOM was combined with CD40L, a still further marked enhancement of the antigen-presenting potency was observed. Ex vivo-generated antigen-specific T cells activated in this manner might be applied to experimental protocols or used for adoptive transfer in immunotherapy protocols.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos B/virología , Antígeno B7-1/biosíntesis , Antígenos CD58/biosíntesis , Virus de la Viruela de las Aves de Corral/inmunología , Molécula 1 de Adhesión Intercelular/biosíntesis , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/virología , Linfocitos B/metabolismo , Antígeno B7-1/inmunología , Ligando de CD40/inmunología , Antígenos CD58/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno Carcinoembrionario/inmunología , Línea Celular , Virus de la Viruela de las Aves de Corral/genética , Expresión Génica , Vectores Genéticos/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/inmunología , Interferón gamma/biosíntesis , Leucocitos Mononucleares/citología , Activación de Linfocitos/inmunología , Papillomaviridae/inmunología , Péptidos/inmunología , Péptidos/metabolismo , Fenotipo , Antígeno Prostático Específico/inmunología , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4(+) T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4(+) T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8(+) CTL. In contrast, CD4(+) T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8(+) CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8(+) CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8(+) T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4(+) T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8(+) CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4(+) T cell contact was prevented, indicated a major role for CD4(+) T cell cytokines in the differentiation of LCL.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Línea Celular Transformada/citología , Transformación Celular Viral/inmunología , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Herpesvirus Humano 4/inmunología , Activación de Linfocitos , Linfocitos T Citotóxicos/inmunología , Adulto , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/virología , Linfocitos T CD4-Positivos/virología , Antígenos CD58/biosíntesis , Antígenos CD58/fisiología , Moléculas de Adhesión Celular/biosíntesis , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Línea Celular Transformada/inmunología , Línea Celular Transformada/metabolismo , Línea Celular Transformada/virología , Técnicas de Cocultivo , Pruebas Inmunológicas de Citotoxicidad , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Antígenos HLA/biosíntesis , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Linfocitos T Citotóxicos/virología , Latencia del Virus/genética , Latencia del Virus/inmunologíaRESUMEN
The beta2 integrin LFA-1 (CD11a/CD18) mediates adhesion of lymphocytes to cells expressing ICAM. The strength of this adhesion is regulated by different signals delivered by cytokines and chemokines, and by the TCR in the case of T cells. To determine the receptor-ligand interactions required for adhesion of resting NK cells, Drosophila cells expressing different combinations of ligands of human NK cell receptors were generated. Expression of ICAM-1 alone was sufficient for an adhesion of resting NK cells that was sensitive to inhibitors of src family kinase and of phosphatidylinositol 3-kinase. Binding of resting NK cells to solid-phase ICAM-1 showed similar signaling requirements. A pulse of either IL-2 or IL-15 to resting NK cells resulted in strongly enhanced, actin-dependent adhesion to insect cells expressing ICAM-1 alone. Coexpression of either LFA-3 (CD58) or CD48 with ICAM-1 resulted in strong adhesion by resting NK cells, even in the absence of cytokines. Therefore, receptors for LFA-3 and CD48 on resting NK cells strengthen the adhesion mediated by LFA-1.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antígenos CD/biosíntesis , Antígenos CD58/biosíntesis , Molécula 1 de Adhesión Intercelular/fisiología , Interfase/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/fisiología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos CD/fisiología , Antígeno CD48 , Antígenos CD58/fisiología , Adhesión Celular/inmunología , Línea Celular , Drosophila melanogaster , Sinergismo Farmacológico , Humanos , Interleucina-15/farmacología , Interleucina-2/farmacología , Células Asesinas Naturales/inmunología , Transducción de Señal/inmunología , TransfecciónRESUMEN
OBJECTIVE: We hypothesized that acute hyperglycemia (an independent cardiovascular risk factor) increases the expression of proatherogenic leukocyte adhesion molecule in type 2 diabetes and controls and that the expression of these adhesion molecules would be antioxidant sensitive. METHODS AND RESULTS: Twenty-three type 2 diabetes patients and 13 control patients underwent two oral glucose tolerance tests 14 days apart and took placebo or 800 IU daily of oral alpha tocopherol between tests. Monocyte and neutrophil expression of adhesion molecules Mac-1, LFA-1 and 3, ICAM-1, and VLA-4 were measured at 0, 120, and 240 minutes by using laser flow cytometry. Baseline adhesion molecule expression did not differ between groups, but there was a rapid, highly significant increase (P<0.0001) in the intensity of monocyte Mac-1 expression after a glucose load in both groups. Alpha-tocopherol supplementation reduced only Mac-1 expression in the diabetes group (P=0.03). CONCLUSIONS: Acute glycemic excursions of any degree cause highly significant, rapid increases in monocyte Mac-1 expression in type 2 diabetes patients and controls. Mac-1 mediates leukocyte vascular infiltration and is prothrombotic. These data suggest a mechanism for the link between glycemic excursions and increased vascular event rates.
Asunto(s)
Antioxidantes/uso terapéutico , Moléculas de Adhesión Celular/biosíntesis , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hiperglucemia/sangre , Monocitos/metabolismo , Neutrófilos/metabolismo , Enfermedad Aguda , Administración Oral , Adulto , Anciano , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Antígenos CD58/biosíntesis , Antígenos CD58/sangre , Moléculas de Adhesión Celular/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Hiperglucemia/complicaciones , Integrina alfa4beta1 , Integrinas/biosíntesis , Integrinas/sangre , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/sangre , Interleucina-6/sangre , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Antígeno-1 Asociado a Función de Linfocito/sangre , Antígeno de Macrófago-1/biosíntesis , Antígeno de Macrófago-1/sangre , Masculino , Persona de Mediana Edad , Monocitos/patología , Neutrófilos/patología , Receptores Mensajeros de Linfocitos/biosíntesis , Receptores Mensajeros de Linfocitos/sangre , Solubilidad , Factor de Necrosis Tumoral alfa/metabolismo , alfa-Tocoferol/administración & dosificación , alfa-Tocoferol/sangre , alfa-Tocoferol/uso terapéuticoRESUMEN
We examined the influence of the gram-positive cell wall products peptidoglycan (PepG) and lipoteichoic acid (LTA), compared to lipopolysaccharide (LPS), on the monocyte expression of receptors involved in antigen presentation (HLA-DR, B7.1, and B7.2), cell adhesion (intercellular adhesion molecule-1 [ICAM-1] and lymphocyte function associated antigen-3 [LFA-3]), phagocytosis (Fc gamma RI), and cell activation (CD14). We also evaluated possible influences of the immunosuppressive drugs cyclosporine A, tacrolimus, and sirolimus on the expression of these receptors. Pretreatment of whole blood for 4 h with the immunosuppressive drugs did not influence the expression of the surface receptors in normal or stimulated blood. Stimulation with both PepG and LTA caused significant up-regulation of the surface expression of ICAM-1 and HLA-DR on whole blood monocytes, similar to that obtained with LPS, whereas B7.1, B7.2, LFA-3, and Fc gamma RI were not modulated. PepG and LTA also caused increased expression of CD14, whereas LPS down-regulated this molecule. In contrast, we did not detect any significant influence of any of the bacterial products on the plasma concentration of soluble CD14. We hypothesized that the increased expression of surface CD14 in blood stimulated with PepG would prime for cellular activation by LPS. Indeed, we show that PepG and the partial PepG structure muramyl dipeptide acted in synergy with LPS to cause the release of tumor necrosis factor-alpha. The results suggest that PepG and LPS provoke partly different responses on monocyte phenotype and that CD14 may play different roles in the innate response to gram-positive and gram-negative bacteria.
Asunto(s)
Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Peptidoglicano/farmacología , Ácidos Teicoicos/farmacología , Presentación de Antígeno/efectos de los fármacos , Antígenos CD58/biosíntesis , Adhesión Celular/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Monocitos/fisiología , Fagocitosis/efectos de los fármacos , Receptores de IgG/biosíntesisRESUMEN
To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.
Asunto(s)
Timo/citología , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos CD58/biosíntesis , Línea Celular Transformada , Células Cultivadas , Citocinas/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Lactante , Molécula 1 de Adhesión Intercelular/biosíntesis , Interferón gamma/inmunología , Interferón gamma/farmacología , Ratones , Ratones Endogámicos BALB C , Fenotipo , Receptores Colinérgicos/biosíntesis , Células del Estroma/clasificación , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiologíaRESUMEN
T-cell activation usually requires at least two signals. The first signal is antigen-specific, and the second signal(s) involves the interaction of a T-cell costimulatory molecule(s) on the antigen-presenting cell (APC) with its ligand on the T cell. Dendritic cells (DCs) are the most potent APCs, attributable, in part, to their expression of several T-cell costimulatory molecules. Human DCs generated in vitro, however, will vary in methods of generation and maturation and in terms of expression of different phenotypic markers-including costimulatory molecules-among different donors. We report here that a recombinant avipox (fowlpox, rF) vector has been constructed that can efficiently express the transgenes for three human T-cell costimulatory molecules (B7-1, ICAM-1, and LFA-3) as a result of individual early avipox promoters driving the expression of each transgene. This triad of costimulatory molecules (designated TRICOM) was selected because each has an individual ligand on T cells and each has been shown previously to prime a unique signaling pathway in T cells. We report here that rF-TRICOM can efficiently infect human DCs of different states of maturity and hyperexpress each of the three costimulatory molecules on the DC surface without affecting other DC phenotypic markers. Infection of influenza or human papilloma virus 9-mer peptide-pulsed DCs from different individuals, or at different stages of maturity with rF-TRICOM, resulted in enhanced activation of T cells from peripheral blood mononuclear cells of autologous donors after 24 h of incubation with DCS: This enhanced activation was analyzed by both titrating the peptide and differing the DC:effector cell ratios. No effect was observed using the control wild-type avipox vector. No increase in apoptosis was observed in T cells hyperstimulated with the TRICOM vector, and no decrease in interleukin-12 production was seen in lipopolysaccharide-stimulated DCs infected with rF-TRICOM. Antibody-blocking experiments demonstrated that enhanced T-cell activation by TRICOM was attributed to each of the three costimulatory molecules. Peptide-pulsed, rF-TRICOM-infected DCs were also shown to be more effective than peptide-pulsed DCs in activating T cells to 9-mer peptides derived from two relatively weak "self" immunogens, i.e., human prostate-specific antigen and human carcinoembryonic antigen. These studies thus demonstrate for the first time that a vector that can simultaneously hyperexpress three costimulatory molecules can be used to efficiently infect human DCs, leading to enhanced peptide-specific T-cell activation. The use of this approach for in vitro studies and clinical applications in immunotherapy is discussed.