RESUMEN
Thrombopoietin (TPO), through activation of its cognate receptor Mpl, is the major regulator of platelet production. However, residual platelets observed in TPO- and Mpl-loss-of-function (LOF) mice suggest the existence of an additional factor to TPO in platelet production. As erythropoietin (EPO) exhibited both in vitro megakaryocytic potential, in association with other early-acting cytokines, and in vivo platelet activation activity, we sought to investigate its role in this setting. Here, we used multiple LOF models to decipher the reciprocal role of EPO and TPO in the regulation of platelet production in TPO-LOF and Mpl-LOF mice and of platelet size heterogeneity in wild-type mice. We first identified EPO as the major thrombopoietic factor in the absence of the TPO-Mpl pathway. Based on the study of several mouse models we found that the EPO-EPO receptor pathway acts on late-stage megakaryopoiesis and is responsible for large-sized platelet production, while the TPO-Mpl pathway promotes small-sized platelet production. On the basis of our data, EPO might be used for thrombocytopenia supportive therapy in congenital amegakaryocytopoiesis. Furthermore, as a distribution skewed toward large platelets is an independent risk factor and a poor prognosis indicator in atherothrombosis, the characterization of EPO's role in the production of large-sized platelets, if confirmed in humans, may open new perspectives in the understanding of the role of EPO-induced platelets in atherothrombosis.
Asunto(s)
Plaquetas/metabolismo , Eritropoyetina/metabolismo , Megacariocitos/microbiología , Trombopoyesis , Trombopoyetina/metabolismo , Animales , Eritropoyetina/genética , Femenino , Ratones , Ratones Noqueados , Trombopoyetina/genéticaRESUMEN
Most diseases have multifactorial origins and their study requires complex in vivo validation strategies selected for their particular relevance. Most of the in vivo models used to date have been selected according to their availability and the accessibility of the corresponding technology platform. With the rapid development of new technologies, an increasing number of relevant systems for in vivo target validation are now available. In this review, we present in vivo loss-of-function tools acting at three biological levels (the gene, the messenger RNA and the protein); we discuss the specificity of each strategy and how the three techniques can be combined during the validation process in order to overcome the limitations of each one. Thus, combination will broaden the spectrum of the available validation systems and will enable target validation that predicts the situation in humans as accurately as possible.
Asunto(s)
Anticuerpos , Bioensayo , Descubrimiento de Drogas/métodos , Técnicas de Inactivación de Genes , Proteínas/genética , Proteínas/inmunología , Interferencia de ARN , Animales , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Humanos , Modelos Animales , Fenotipo , Proteínas/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los ResultadosRESUMEN
We identified an antigen recognized on a human non-small-cell lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide is presented by HLA-A2 and is encoded by the CALCA gene, which codes for calcitonin and for the alpha-calcitonin gene-related peptide. The peptide is derived from the carboxy-terminal region of the preprocalcitonin signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing. Processing occurs within the endoplasmic reticulum of all tumoral and normal cells tested, including dendritic cells, and it involves signal peptidase and the aspartic protease, signal peptide peptidase. The CALCA gene is overexpressed in medullary thyroid carcinomas and in several lung carcinomas compared with normal tissues, leading to recognition by the T cell clone. This new epitope is, therefore, a promising candidate for cancer immunotherapy.
Asunto(s)
Antígenos de Neoplasias/genética , Calcitonina/genética , Calcitonina/inmunología , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Señales de Clasificación de Proteína/genética , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , ADN Complementario/genética , ADN de Neoplasias/genética , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
CD158k molecules belong to the family of killer cell immunoglobulin-like receptors (KIR) that are expressed on a minor population of circulating NK and CD8+ T lymphocytes. Here, we report a strong positive correlation between the percentage of CD158k+ blood lymphocytes analyzed by flow cytometry and the percentage of atypical circulating cells (Sezary cells) determined by cytomorphology in a large group of patients with Sezary syndrome. Moreover, we show that circulating CD4+CD158k+ lymphocytes correspond to the malignant clonal cell population. Our findings suggest that the CD158k marker could be a useful tool for the evaluation of the circulating tumoral burden and the follow-up of patients with Sezary syndrome.
Asunto(s)
Biomarcadores de Tumor/sangre , Linfocitos/química , Receptores Inmunológicos/sangre , Síndrome de Sézary/diagnóstico , Neoplasias Cutáneas/diagnóstico , Citometría de Flujo , Estudios de Seguimiento , Humanos , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Receptores KIR , Receptores KIR2DL2 , Receptores KIR3DL2RESUMEN
T lymphocytes infiltrating a human lung carcinoma stimulated in vitro with autologous tumor cell line showed a TCRVbeta13.6(+) T-cell expansion. This subset was isolated using TCRVbeta-specific antibody and several T-cell clones were generated. All these clones expressed a unique Vbeta13.6-Jbeta2.7 TCR with the same junctional region strongly suggesting that they derived from the same cell. They were CD8(+)/CD28(-) and expressed the MHC class I binding killer cell Ig-like receptor (KIR)3DL2/p140, but not KIR3DL1/p70, KIR2DL1/p58.1 and KIR2DL2/3/p58.2. Sequence analysis indicated that KIR3DL2/p140 cDNA was identical to the previously reported 3DL2*002 allele except for two nucleic acid substitutions. Functional studies showed that KIR3DL2/p140(+) CTL secrete a significant level of IFNgamma and mediate an HLA-A2-restricted cytotoxicity against the autologous and some allogeneic tumor cells but not towards the autologous EBV-B cells. Strikingly, both the lytic and the cytokine secretion activities induced upon specific cell interactions were unaffected by anti-KIR3DL2/p140 antibody. In addition, crosslinking KIR3DL2/p140 molecules on CTL did not result into the modification of cytotoxicity and cytokine production triggered by anti-CD3 antibody. These results strongly suggest that, as opposed to distinct KIR expressed by CTL, the in vitro KIR3DL2/p140 engagement does not result into inhibitory (nor activatory) effects on tumor-specific CTL.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T Citotóxicos/fisiología , Antígenos CD/análisis , Carcinoma de Pulmón de Células no Pequeñas/patología , Células Clonales , Citotoxicidad Inmunológica , Técnica del Anticuerpo Fluorescente , Humanos , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/patología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores Inmunológicos/genética , Receptores KIR , Receptores KIR2DL1 , Receptores KIR2DL2 , Receptores KIR2DL3 , Receptores KIR3DL1 , Receptores KIR3DL2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Células Tumorales CultivadasRESUMEN
We described previously a CTL clone able to lyse the autologous carcinoma cell line IGR-Heu after specific recognition of an HLA-A2/mutated alpha-actinin-4 peptide complex. Here, we used IGR-Heu, cultured either as standard two-dimensional monolayers or as three-dimensional spheroids, to further analyze the influence of target architecture on CTL reactivity. Interestingly, we found that changes in the tumor structure from two- to three-dimensional induced a dramatic decrease in its capacity to activate autologous CTL, as measured by IFN-gamma and tumor necrosis factor-alpha secretion. These functional alterations were attributable neither to MHC class I expression nor to tumor antigen (Ag) down-regulation, because IGR-Heu, cultured as two- or three-dimensional, expressed similar levels of HLA-A2 and alpha-actinin-4. More importantly, incubation of three-dimensional cells with synthetic epitope completely restored cytokine release by CTL. This defective Ag presentation correlated with a decrease in heat shock protein (hsp)70 expression by three-dimensional tumors compared with two-dimensional cells. Furthermore, transfection of the tumor cells with hsp70 cDNA completely restored the Ag-presenting potential of spheroids and, therefore, cytokine production by T cells. These data strongly suggest that hsp70 down-regulation in three-dimensional cells may result in tumor resistance to the immune response.
Asunto(s)
Presentación de Antígeno/inmunología , Carcinoma de Células Grandes/inmunología , Proteínas HSP70 de Choque Térmico/genética , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor , Linfocitos T Citotóxicos/inmunología , Secuencia de Bases , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patología , Cartilla de ADN , Humanos , Procesamiento de Imagen Asistido por Computador , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.
Asunto(s)
Citotoxicidad Inmunológica/genética , Silenciador del Gen/inmunología , Proteínas de Neoplasias/fisiología , Linfocitos T Citotóxicos/inmunología , Proteína p53 Supresora de Tumor/fisiología , Adenoviridae/genética , Apoptosis/genética , Apoptosis/inmunología , Células Clonales , Pruebas Inmunológicas de Citotoxicidad , Vectores Genéticos , Granzimas , Humanos , Inmunidad Innata/genética , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores del Factor de Necrosis Tumoral/biosíntesis , Serina Endopeptidasas/fisiología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Transactivadores/genética , Transactivadores/metabolismo , Transactivadores/fisiología , Activación Transcripcional/inmunología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Receptor fas/biosíntesis , Receptor fas/fisiologíaRESUMEN
We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a lung carcinoma of a patient with long survival. These clones showed a CD3+, CD8+, CD4-, CD28- phenotype and expressed a T-cell receptor (TCR) encoded either by Vbeta8-Jbeta1.5 or Vbeta22-Jbeta1.4 rearrangements. Functional studies indicated that these clones mediated a high human leukocyte antigen (HLA)-A2.1-restricted cytotoxic activity against the autologous tumor cell line. Interestingly, TCRbeta chain gene usage indicated that CTL clones identified in vitro were selectively expanded in vivo at the tumor site as compared to autologous peripheral blood lymphocytes (PBL). These findings provide evidence that an immune response may take place in non-small cell lung carcinoma and that effector T cells may contribute to tumor regression. Further study indicated that the CTL clones recognized the same decamer peptide encoded by a mutated alpha-actinin-4 gene. Using tetramers of soluble HLA-A2 molecules loaded with the mutated antigenic peptide, we have derived several anti-alpha-actinin-4 T-cell clones from patient PBL. These CTL, recognizing a truly tumor-specific antigen, may play a role in the clinical evolution of this lung cancer patient. Adoptive transfer of CTL clones in a SCID/NOD mice model transplanted with autologous tumor supported their antitumor effect in vivo.
Asunto(s)
Actinina/inmunología , Antígenos de Neoplasias/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas de Microfilamentos , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Actinina/química , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Carcinoma de Pulmón de Células no Pequeñas/patología , Células Clonales/inmunología , Citotoxicidad Inmunológica , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Antígeno HLA-A2/inmunología , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Linfocitos T Reguladores/inmunología , Células Tumorales Cultivadas/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the present report, we have investigated TRAIL/APO2 ligand (APO2L) expression, regulation, and function in human lung carcinoma tumor-infiltrating lymphocytes. Using a panel of non-small cell lung carcinoma cell lines, we first showed that most of them expressed TRAIL-R1/DR4, TRAIL-R2/DR5, but not TRAIL-R3/DcR1 and TRAIL-R4/DcR2, and were susceptible to APO2L/TRAIL-induced cell death. Two APO2L/TRAIL-sensitive tumor cell lines (MHC class I(+)/II(+) or I(+)/II(-)) were selected and specific CD4(+) HLA-DR- or CD8(+) HLA-A2-restricted CTL clones were respectively isolated from autologous tumor-infiltrating lymphocytes. Interestingly, although the established T cell clones did not constitutively express detectable levels of APO2L/TRAIL, engagement of their TCR via activation with specific tumor cells selectively induced profound APO2L/TRAIL expression on the CD4(+), but not on the CD8(+), CTL clones. Furthermore, as opposed to the CD8(+) CTL clone which mainly used granule exocytosis pathway, the CD4(+) CTL clone lysed the specific target via both perforin/granzymes and APO2L/TRAIL-mediated mechanisms. The latter cytotoxicity correlated with APO2L/TRAIL expression and was significantly enhanced in the presence of IFN-alpha. More interestingly, in vivo studies performed in SCID/nonobese diabetic mice transplanted with autologous tumor and transferred with the specific CD4(+) CTL clone in combination with IFN-alpha resulted in an important APO2L/TRAIL-mediated tumor growth inhibition, which was prohibited by soluble TRAIL-R2. Our findings suggest that APO2L/TRAIL, specifically induced by autologous tumor and up-regulated by IFN-alpha, may be a key mediator of tumor-specific CD4(+) CTL-mediated cell death and point to a potent role of this T cell subset in tumor growth control.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Citotoxicidad Inmunológica , Interferón-alfa/fisiología , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/toxicidad , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/toxicidad , Traslado Adoptivo , Anciano , Animales , Proteínas Reguladoras de la Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/prevención & control , Células Clonales/inmunología , Células Clonales/metabolismo , Células Clonales/trasplante , Citotoxicidad Inmunológica/inmunología , Femenino , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/toxicidad , Humanos , Inyecciones Intralesiones , Interferón-alfa/administración & dosificación , Interferón-alfa/toxicidad , Ligandos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/trasplante , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplante , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/trasplante , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
We have previously identified an antigen (Ag) recognized on a human large cell carcinoma of the lung by a tumor-specific cytotoxic T lymphocyte clone derived from autologous tumor infiltrating lymphocytes (TILs). The antigenic peptide is presented by HLA-A2 molecules and is encoded by a mutated alpha-actinin-4 (ACTN4) gene. In the present report, we have isolated two anti-alpha-actinin-4 T cell clones from the same patient TIL and from his peripheral blood lymphocytes (PBLs) by using tetramers of soluble HLA-A2 molecules loaded with the mutated peptide. Although all of the clones displayed similar tetramer labeling, those isolated from PBL showed lower avidity of Ag recognition and killed the specific target much less efficiently, indicating that tetramer staining does not correlate with clone avidity/tumor reactivity. T cell receptor (TCR) analysis revealed that alpha-actinin-4-reactive clones used distinct alpha and beta chain rearrangements, demonstrating TCR repertoire diversity. Interestingly, TCR beta chain gene usage indicated that only Ag-specific clones with high functional avidity were expanded at the tumor site, whereas a low-avidity clone was exclusively amplified in patient peripheral blood. Our results point to the existence of distinct but overlapping antitumor TCR repertoires in TIL and PBL and suggest a selective in situ expansion of tumor-specific cytotoxic T lymphocyte with high avidity/tumor reactivity.
Asunto(s)
Actinina/genética , Actinina/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antígeno HLA-A2/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas de Microfilamentos , Linfocitos T Citotóxicos/inmunología , Carcinoma de Células Grandes/inmunología , Línea Celular , Regiones Determinantes de Complementariedad/genética , Antígeno HLA-A2/química , Humanos , Neoplasias Pulmonares/inmunología , Mutación , Estructura Cuaternaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Distribución TisularRESUMEN
Immunotherapy of cancer has always represented a very attractive fourth-modality therapeutic approach. Over the past few years, advances in the identification of tumor antigens have opened new perspectives and provided new opportunities for a more accurate immunotherapy of cancer. However, when applied to patients with established tumors, it rarely leads to an objective response. This is in part due to the fact that tumors evade host immunity at both the induction and effector phases. In this regard, several different functional defects in T-lymphocytes that infiltrate cancers have been reported. Indeed, lymphocytes of patients with advanced malignancies are hyporeactive and functionally compromised. Furthermore, it has become clear that immunotherapeutic and gene therapeutic approaches aimed at the induction of anti-tumor cytotoxic responses should consider the resistance of tumor cells to cytotoxic mechanisms. Thus, understanding of tumor escape mechanisms may be the key to a successful immunotherapy for cancer. How tumors escape immunological destruction following the acquisition of resistance to cell death and the potential role the tumor suppressor p53 protein in immunosensitization of tumor cells will be discussed.