RESUMEN
BACKGROUND: Immune-targeted therapies are being successfully implemented into cancer clinical practice. In particular checkpoint inhibitors are employed to modulate the immune microenvironment of solid tumors. We sought to determine the expression of PD-L1, HVEM, and B7H3 in human and canine osteosarcoma, and correlate expression with clinical features and tumor infiltrating lymphocytes in naturally-occurring canine osteosarcoma. METHODS: Flow cytometry was used to measure ligand surface expression of five human and three canine cell lines. Immunohistochemistry was utilized for expression of ligands and lymphocyte markers in thirty-seven treatment-naïve canine osteosarcoma patients. RESULTS: All cell lines expressed all three ligands at variable levels in both species. Metastatic lesions were associated with higher expression of all three ligands in patient tumor samples. PD-L1 expression strongly correlated with B7H3 and HVEM expression, while HVEM and B7H3 were weakly correlated. Whereas peritumoral T-cell expression positively correlated with PD-L1 and HVEM tumor expression, the presence of T-cells intratumorally were rare. Furthermore, intratumor penetration by T-cells was greatest in metastatic lesions, despite log-fold increases in peritumoral T-cells. In summary, PD-L1, HVEM, and B7H3 are expressed in osteosarcoma, with metastatic disease lesions expressing higher levels. We show for the first time that these ligands expressed on osteosarcoma cells positively correlate with each other and the presence of peritumoral T cell infiltration. Furthermore, osteosarcoma appears to be an intratumoral immune desert with significant resistance to effector T cells. Multiple agents targeting checkpoints are in clinical practice, and may have immune modulating benefit in osteosarcoma.
Asunto(s)
Neoplasias Óseas/veterinaria , Enfermedades de los Perros/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Osteosarcoma/veterinaria , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos B7/biosíntesis , Antígeno B7-H1/biosíntesis , Western Blotting/veterinaria , Neoplasias Óseas/inmunología , Neoplasias Óseas/secundario , Línea Celular , Perros , Femenino , Citometría de Flujo , Humanos , Masculino , Osteosarcoma/inmunología , Osteosarcoma/secundario , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesisRESUMEN
Objectives: Herpes virus entry mediator (HVEM) is a costimulatory molecule, and has been proved to play an important role in airway inflammatory and remodeling processes of asthma. We aimed to investigate the expression of HVEM gene in patients with asthma as a means of assessing disease severity.Methods: This study was carried out on 59 subjects, 16 patients with mild persistent asthma, 11 patients with moderate persistent asthma, 13 patients with severe persistent asthma, and 19 age and gender matched healthy controls. The HVEM mRNA expressions of all subjects were determined by real time PCR. Correlations between HVEM mRNA expression and fractional exhaled nitric oxide (FeNO), pulmonary function test values, total blood white cell count and differential, total immunoglobulin E (IgE) level, and Asthma Control Test (ACT) score were analyzed, respectively. The discrimination abilities of HVEM mRNA between different groups were tested using receiver operating characteristics (ROC) curve analyses.Results: This study showed the expressions of HVEM mRNA were significantly higher in the patients with severe and moderate persistent asthma than in patients with mild persistent asthma and healthy subjects (2.97 ± 1.23 vs. 1.17 ± 0.42 vs. 0.62 ± 0.38 vs. 0.46 ± 0.18/NAPDH, p < 0.001), but there was no significant difference between patients with mild persistent asthma and health controls (0.62 ± 0.38 vs. 0.46 ± 0.18/NAPDH, p = 0.557). HVEM mRNA expression at cut off point [1.01/NAPDH, area under the ROC curve (AUC) = 0.99] is sufficient to discriminate severe patients from mild-to-moderate patients, and at cut off point (0.93/NAPDH, AUC = 0.91) for discrimination of moderate-to-severe patients from mild ones, while at cut off point (0.76/NAPDH, AUC = 0.75) for discrimination of asthmatic patients from controls. Furthermore, HVEM mRNA expression was positively correlated with FeNO level (r = 0.524, p = 0.015), and total lymphocyte count (r = 0.426, p = 0.017) in patients with persistent asthma.Conclusions: HVEM gene expressions can be used as a potential biomarker for evaluating the severity of patients with persistent asthma.
Asunto(s)
Asma/genética , Asma/fisiopatología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesis , Adulto , Biomarcadores , Pruebas Respiratorias , Femenino , Humanos , Inmunoglobulina E/sangre , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Óxido Nítrico/análisis , ARN Mensajero , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Pruebas de Función Respiratoria , Índice de Severidad de la EnfermedadRESUMEN
Investigation of the genetic lesions underlying classical Hodgkin lymphoma (CHL) has been challenging due to the rarity of Hodgkin and Reed-Sternberg (HRS) cells, the pathognomonic neoplastic cells of CHL. In an effort to catalog more comprehensively recurrent copy number alterations occurring during oncogenesis, we investigated somatic alterations involved in CHL using whole-genome sequencing-mediated copy number analysis of purified HRS cells. We performed low-coverage sequencing of small numbers of intact HRS cells and paired non-neoplastic B lymphocytes isolated by flow cytometric cell sorting from 19 primary cases, as well as two commonly used HRS-derived cell lines (KM-H2 and L1236). We found that HRS cells contain strikingly fewer copy number abnormalities than CHL cell lines. A subset of cases displayed nonintegral chromosomal copy number states, suggesting internal heterogeneity within the HRS cell population. Recurrent somatic copy number alterations involving known factors in CHL pathogenesis were identified (REL, the PD-1 pathway, and TNFAIP3). In eight cases (42%) we observed recurrent copy number loss of chr1:2,352,236-4,574,271, a region containing the candidate tumor suppressor TNFRSF14. Using flow cytometry, we demonstrated reduced TNFRSF14 expression in HRS cells from 5 of 22 additional cases (23%) and in two of three CHL cell lines. These studies suggest that TNFRSF14 dysregulation may contribute to the pathobiology of CHL in a subset of cases.
Asunto(s)
Enfermedad de Hodgkin/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Línea Celular Tumoral , Separación Celular , Citometría de Flujo , Enfermedad de Hodgkin/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesis , Miembro 14 de Receptores del Factor de Necrosis Tumoral/deficiencia , Células de Reed-SternbergRESUMEN
Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.
Asunto(s)
Factores de Transcripción NFATC/fisiología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesis , Linfocitos T/metabolismo , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulación hacia Abajo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.
Asunto(s)
Animales , Ratones , Secuencia de Bases , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulación hacia Abajo , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Factores de Transcripción NFATC/fisiología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesis , Linfocitos T/metabolismoRESUMEN
PURPOSE: Herpes virus entry mediator (HVEM) plays a critical role in the regulation of inflammation through interaction with its natural ligands LIGHT and lymphotoxin alpha and also serves as one of the entry receptors of herpes simplex virus (HSV). The purpose of this study was to better understand the expression of HVEM in the cornea and trigeminal ganglia (TG), which are important targets of HSV infection. MATERIALS AND METHODS: Immunohistochemistry was used to define HVEM expression in the cornea and TG of normal and HSV-1 infected mice euthanized 2 to 5 days or 7 months following corneal inoculation of virus. RESULTS: We found that HVEM is widely expressed in the normal corneal epithelium and endothelium, is weakly and focally expressed in the corneal stroma, and is expressed in a portion of neurons and non-neuronal cells in the TG. Acute HSV-1 keratitis and ganglionitis were associated with increased HVEM expression in the corneal epithelium and stroma and in neurons and non-neuronal cells of TG, and many inflammatory cells in these tissues also expressed HVEM. TG derived from mice 7 months after virus inoculation demonstrated latent HSV-1 infection that was associated with increased HVEM expression in neurons and non-neuronal cells relative to uninfected control tissues. Latent TG also contained focal infiltrates of mononuclear inflammatory cells, many of which expressed HVEM. Corneas derived from latently infected mice demonstrated chronic keratitis, with no evidence of virus replication or increased HVEM expression in the corneal epithelium, and inflammatory cells present showed only weak HVEM expression. CONCLUSIONS: HVEM is expressed in the cornea and TG and therefore may serve as an HSV entry receptor in these tissues. Furthermore, these findings raise the possibility that changes in HVEM expression following ocular HSV-1 infection can modulate HSV spread and infection-induced inflammation in the cornea and TG.
Asunto(s)
Córnea/metabolismo , Regulación de la Expresión Génica , Herpesvirus Humano 1/fisiología , Queratitis Herpética/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesis , Ganglio del Trigémino/metabolismo , Enfermedad Aguda , Animales , Línea Celular , Córnea/patología , Córnea/virología , Cricetinae , Femenino , Humanos , Inflamación/metabolismo , Inflamación/virología , Queratitis Herpética/patología , Queratitis Herpética/virología , Ratones , Ratones Endogámicos BALB C , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Ganglio del Trigémino/patología , Ganglio del Trigémino/virología , Internalización del Virus , Latencia del Virus , Replicación ViralRESUMEN
B- and T-lymphocyte attenuator (BTLA) is a lymphoid receptor that inhibits lymphocyte activation on interaction with its ligand, herpesvirus entry mediator (HVEM). We developed monoclonal antibodies against BTLA and HVEM to study their expression using immunohistochemical and flow cytometric analyses in human tissues. In reactive lymph nodes, they were both expressed in interfollicular T cells and in B cells from mantle and marginal zones. Within germinal centers, B cells were negative, whereas T follicular helper (TFH) cells were BTLA+ and follicular dendritic cells were HVEM+. BTLA was strongly expressed in chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, 19 of 19 positive) when compared with other small B-cell lymphomas, including follicular lymphoma (0 of 24 positive), mantle cell lymphoma (0 of 10 positive), and marginal zone lymphoma (0 of 5 positive). Our results suggest that down-regulation of the BTLA-HVEM pathway may be involved in germinal center B-cell activation. The specific high expression of BTLA in B-CLL/SLL represents a new potential diagnostic tool. The BTLA positivity of TFH cells may be a basis for designing future immunotherapies.