RESUMEN
To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/ß genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Genes Codificadores de los Receptores de Linfocitos T , Antígenos de Histocompatibilidad Clase I/fisiología , Inmunoterapia Adoptiva , Leucemia/terapia , Proteínas WT1/inmunología , Animales , Movimiento Celular , Femenino , Ingeniería Genética , Humanos , Leucemia/inmunología , Activación de Linfocitos , Ratones , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Adoptive cell therapy using tumor-specific T cells is a promising strategy for treating patients with malignancy. However, accumulating evidences have demonstrated that optimal function of tumor-reactive T cells is often attenuated by negative regulatory signal(s) delivered through receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and their cognate ligands. Although systemic blocking of these molecules needs careful attention on the risk of uncontrolled immune activation, selective inhibition of negative signals in tumor-specific T cells by their genetic modification is an attractive approach to overcome immunological suppression in cancer patients. Here, we demonstrate the improved effector functions of tumor-specific CD4(+) and CD8(+) human T cells by small interfering RNA (siRNA) -mediated silencing of PD-1 ligands, PD-L1 or PD-L2. Tumor antigen MAGE-A4-specific human T-cell clones upregulated the expression of PD-1 ligands upon activation. siRNA-mediated knockdown of PD-L1 or -L2 enhanced the interferon-γ production and antigen-specific cytotoxicity of these cells. Peripheral blood mononuclear cells transduced with a retroviral vector encoding MAGE-A4-specific T-cell receptor α/ß chains also increased their effector functions by this modification. These results suggest that siRNA-mediated knockdown of PD-1 ligands is an attractive strategy to inhibit a negative regulatory mechanism of tumor-specific T cells resulting in enhanced efficacy of adoptive T-cell therapy of cancer using genetically modified autologous lymphocytes.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Neoplasias/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Línea Celular Tumoral , Citotoxicidad Inmunológica , Vectores Genéticos , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Proteína 2 Ligando de Muerte Celular Programada 1/antagonistas & inhibidores , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Transfección , Regulación hacia ArribaRESUMEN
Adoptive T-cell transfer of in vitro cultured T cells derived from cancer patients with naturally developed immune responses has met with some success as an immunotherapeutic approach, although only a limited number of patients showed spontaneous immune responses. To find alternative ways, such as cancer-specific T-cell receptor (TCR) gene transfer, in preparation for sufficient numbers of antigen-specific T cells is an important issue in the field of adoptive T-cell therapy. Given the inherent disadvantage of alphabeta TCR transfer to other alphabeta T cells, namely the possible formation of mixed TCR heterodimers with endogenous alpha or beta TCR, we employed gammadelta T cells as a target for retroviral transfer of cancer-specific TCR and examined whether gammadelta T cells were useful as an alternative population for TCR transfer. Although retroviral transduction to gammadelta T cells with TCR alphabeta genes alone, isolated from a MAGE-A4(143-151)-specific alphabeta CD8(+) cytotoxic T lymphocyte (CTL) clone, did not provide sufficient affinity to recognize major histocompatibility (MHC)-peptide complexes due to the lack of CD8 co-receptor, gammadelta T cells co-transduced with TCR alphabeta and CD8 alphabeta genes acquired cytotoxicity against tumor cells and produced cytokines in both alphabeta- and gammadelta-TCR-dependent manners. Furthermore, alphabeta TCR and CD8-transduced gammadelta T cells, stimulated either through alphabeta TCR or gammadelta TCR, rapidly responded to target cells compared with conventional alphabeta T cells, reminiscent of gammadelta T cells. We propose alphabeta TCR-transduced gammadelta T cells as an alternative strategy for adoptive T-cell transfer.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Genes Relacionados con las Neoplasias , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Traslado Adoptivo/métodos , Citotoxicidad Inmunológica , Ensayo de Inmunoadsorción Enzimática/métodos , Terapia Genética/métodos , Vectores Genéticos , Humanos , Activación de Linfocitos/inmunología , Transfusión de Linfocitos/métodos , Neoplasias/genética , Neoplasias/patología , Retroviridae/genética , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Transducción Genética/métodos , Células Tumorales CultivadasRESUMEN
In adoptive T-cell transfer as an intervention for malignant diseases, retroviral transfer of T-cell receptor (TCR) genes derived from CD8(+) cytotoxic T-lymphocyte (CTL) clones provides an opportunity to generate a large number of T cells with the same antigen specificity. We cloned the TCR-alphabeta genes from a human leukocyte antigen (HLA)-A(*)2402-restricted CTL clone specific for MAGE-A4(143-151). The TCR-alphabeta genes were transduced to 99.2% of non-TCR expressing SupT1, a human T-cell line, and to 12.7-32.6% of polyclonally activated CD8(+) T cells by retroviral transduction. As expected, TCR-alphabeta gene-modified CD8(+) T cells showed cytotoxic activity and interferon-gamma production in response to peptide-loaded T2-A(*)2402 and tumor cell lines expressing both MAGE-A4 and HLA-A(*)2402. A total of 24 clones were established from TCR-alphabeta gene-transduced peripheral blood mononuclear cells and all clones were functional on a transduced TCR-dependent manner. Four clones were kept in culture over 6 months for analyses in detail. The transduced TCR-alphabeta genes were stably maintained phenotypically, functionally and genetically. Our results indicate that TCR-transduced alphabeta T cells by retroviral transduction represent an efficient and promising strategy for adoptive T-cell transfer for long term.
Asunto(s)
Traslado Adoptivo/métodos , Linfocitos T CD8-positivos/inmunología , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Terapia Genética/métodos , Transducción Genética/métodos , Línea Celular Tumoral , Células Cultivadas , Clonación Molecular , Pruebas Inmunológicas de Citotoxicidad , Humanos , Inmunofenotipificación , Interferón gamma/inmunología , Activación de Linfocitos , Melanoma/inmunología , Melanoma/terapia , Retroviridae/genética , Especificidad del Receptor de Antígeno de Linfocitos T , TiempoRESUMEN
We quantitatively assessed the expression of cytokine receptors (interleukin-2 receptor (IL-2R), IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, granulocyte-macrophage colony-stimulating factor R (GM-CSFR), G-CSFR, c-fms, c-mpl, c-kit and FLT3) in cells from 211 adults with acute lymphoblastic leukemia (ALL) by flow cytometry and determined their prevalence and clinical significance. Although all cytokine receptors were expressed to various degrees, the levels of IL-3R alpha-chain (IL-3Ralpha), IL-2Ralpha, IL-2Rbeta, IL-7Ralpha, common-Rgamma(gammac), c-mpl, c-kit and FLT3 exhibited a wide spectrum > or =2000 sites/cell. Among them, IL-3Ralpha, IL-2Ralpha and FLT3 were highly expressed in B-lineage ALL, whereas IL-7Ralpha, gammac and c-kit predominated in T-lineage ALL. Higher levels of IL-3Ralpha, IL-2Ralpha, c-kit and FLT3 correlated with the expression of CD13/33. Increased IL-2Ralpha levels related to the presence of Philadelphia chromosome (Ph), leukocytosis and shorter event-free survival (EFS). C-kit preferred in male. Elevated FLT3 levels correlated with age > or =60 years. Multivariate analysis in B-lineage ALL revealed only IL-2Ralpha (P=0.028) and Ph (P=0.020) as independent factors for EFS. These findings suggest that several cytokine receptors associated with certain cellular and clinical features, but IL-2Ralpha solely had a prognostic value and should be considered as a major prognostic factor for adult ALL that is comparable with Ph.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Adulto , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Receptores de Interleucina/genéticaRESUMEN
Fourteen adult patients with haematological malignancies (eight non-Hodgkin's lymphoma, one multiple myeloma, one chronic lymphocytic leukaemia, two acute lymphoblastic leukaemia and two acute myeloid leukaemia) developed acute interstitial pneumonitis (IP) during the course of chemotherapy. All patients manifested high fever over 38 degrees C, bilateral diffuse pulmonary interstitial infiltrates in the chest radiograph and severe hypoxia without hypercapnia in the arterial blood gas analysis. Pathogenic microorganisms were not detected in repeated examinations in any patient. Chemotherapy given included various anti-neoplastic drugs. Five patients had received granulocyte colony-stimulating factor (G-CSF) for chemotherapy-induced leucopenia. The onset was associated with an increase of leucocytes in 10 patients. All patients were treated with high dose steroid hormone and broad spectrum antibiotics with or without anti-fungal agents, and three required mechanical ventilation. Eleven patients quickly recovered from these situations, whereas three died. Autopsies were done in two patients and disclosed pneumocystis carinii (PC) pneumonitis in one and non-specific pulmonary congestive oedema and fibrosis in the other. In conclusion, IP of unknown cause could develop in patients with various haematological malignancies especially at the recovery phase of chemotherapy-induced leucopenia irrespective of the previous G-CSF administration. High dose steroid hormone should be used as therapy for such patients as soon as possible after exclusion of an infective aetiology.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Hematológicas/tratamiento farmacológico , Enfermedades Pulmonares Intersticiales/etiología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Antiinfecciosos/uso terapéutico , Antiinflamatorios/uso terapéutico , Femenino , Humanos , Huésped Inmunocomprometido , Recuento de Leucocitos , Pulmón/diagnóstico por imagen , Enfermedades Pulmonares Intersticiales/diagnóstico por imagen , Enfermedades Pulmonares Intersticiales/tratamiento farmacológico , Masculino , Metilprednisolona/uso terapéutico , Persona de Mediana Edad , Radiografía , Resultado del TratamientoRESUMEN
BACKGROUND: Colonoscopic and clinical differences between primary ileocolonic mucosa-associated lymphoid tissue (MALT) lymphoma and mantle cell lymphoma (MCL) have not been defined. METHODS: We reviewed colonoscopic and clinical features in eight patients with primary MALT lymphoma and eight patients with MCL in the terminal ileum and/or colorectum. All cases were examined for CD5 and/or cyclin D1 expression. RESULTS: Endoscopic features of MALT lymphoma were characterized as protrusions that were covered with normal-appearing mucosa with or without ulceration. The gross appearances of MALT lymphomas were categorized as solitary (4 patients), multiple (3 patients), and multiple lymphomatous polyposis (MLP) (1 patient). The gross features of MCL at endoscopy were categorized as multiple protrusions (2 patients), and MLP (6 patients). The clinical stages of patients with MCL were more advanced than in patients with MALT lymphoma. CONCLUSIONS: Solitary or multiple protrusions at an early clinical stage is the most common presentation pattern of patients with MALT lymphoma, but an MLP appearance at an early stage is also possible. On the other hand, MLP appearance with an advanced clinical stage is the main presentation pattern in patients with MCL, although multiple protrusions with an early clinical stage is also possible. Histological and immunohistochemical investigation including that of cyclin D1 and CD5 expression is essential to make the final diagnosis.
Asunto(s)
Neoplasias del Colon/patología , Neoplasias del Íleon/patología , Válvula Ileocecal , Linfoma de Células B de la Zona Marginal/patología , Linfoma de Células del Manto/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/mortalidad , Colonoscopía , Ciclina D1/metabolismo , Femenino , Humanos , Neoplasias del Íleon/metabolismo , Neoplasias del Íleon/mortalidad , Linfoma de Células B de la Zona Marginal/metabolismo , Linfoma de Células B de la Zona Marginal/mortalidad , Linfoma de Células del Manto/metabolismo , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tasa de SupervivenciaAsunto(s)
Duodenitis/cirugía , Hemorragia Gastrointestinal/cirugía , Coagulación con Láser/métodos , Traumatismos por Radiación/cirugía , Radioterapia/efectos adversos , Anciano , Carcinoma Hepatocelular/radioterapia , Duodenitis/etiología , Endoscopía del Sistema Digestivo , Femenino , Hemorragia Gastrointestinal/etiología , Humanos , Neoplasias Hepáticas/radioterapiaRESUMEN
T(11;18)(q21;q21) results in a chimeric transcript between API2 at 11q21 and MALT1 at 18q21 and is a characteristic chromosomal aberration of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). API2-MALT1 chimeric transcripts are present in approximately one-third of all cases of MALT lymphoma. MALT lymphoma is also known to have variations in histological features and tumor cell proportions. Real-time polymerase chain reaction (PCR) was used to examine number of API2-MALT1 copies in clinical samples for further investigation of the pathophysiology of MALT lymphoma. A total of 13 samples of MALT lymphoma contained API2-MALT1 transcripts from 1.7 x 10(-2) to 1.0 copies/beta-actin copy. These findings were compared to the proportions of tumor cells in genomic VDJ PCR products determined by Southern blotting. Tumor cell ratios varied widely among the patients' samples, and no significant correlation was found between transcript copy number and tumor cell ratio. These results suggest that copy numbers of API2-MALT1 do not reflect tumor cell proportions, and that the number of copies of API2-MALT1 in a tumor cell is different for each clinical sample.
Asunto(s)
Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/patología , Proteínas de Fusión Oncogénica/genética , Adulto , Anciano , Northern Blotting , Southern Blotting , Colon/patología , Femenino , Dosificación de Gen , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Pulmón/patología , Linfoma de Células B de la Zona Marginal/fisiopatología , Masculino , Persona de Mediana Edad , Membrana Mucosa/patología , Especificidad de Órganos , Cavidad Pleural/patología , ARN Mensajero , Proteínas Recombinantes de Fusión/genética , Estómago/patologíaRESUMEN
t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), especially in FAB M2. Clinically, this type of AML often shows eosinophilia and has a high complete remission rate with conventional chemotherapy. t(8;21) AML is also frequently associated with additional karyotypic aberrations, such as a loss of the sex chromosome; however, it is unclear whether these aberrations change the biological and clinical characteristics of t(8;21) AML. To investigate this issue, 94 patients with t(8;21) AML were categorized according to their additional karyotypic aberrations, which were detected in more than three cases, and then morphologic features, phenotypes, expression of cytokine receptors, and clinical features were compared to t(8;21) AML without other additional aberrant karyotypes. t(8;21) AML with loss of the sex chromosome and abnormality of chromosome 9 were found in 27 cases (29.3%) and 10 cases (10.6%), respectively; however, no differences were observed from the t(8;21) AML without other additional karyotypes in terms of morphological and phenotypic features. There was also no significant difference in the clinical outcome among these three groups. On the other hand, trisomy 4 was found in three cases (3.2%) and these cells showed low expressions of CD19 (P=0.06) and IL-7 receptor (P=0.05), and high expressions of CD33 (P=0.13), CD18 (P=0.03), and CD56 (P=0.03) when compared to t(8;21) AML without additional karyotypes. Moreover, all three t(8;21) AML cases with trisomy 4 did not show eosinophilia in their bone marrow and died within 2.4 years. These observations suggest that additional karyotypic aberration, t(8;21) with trisomy 4 is rare, but it may constitute a distinctive subtype of t(8;21) AML.
Asunto(s)
Cromosomas Humanos Par 21/ultraestructura , Cromosomas Humanos Par 4 , Cromosomas Humanos Par 8/ultraestructura , Leucemia Mieloide/genética , Translocación Genética , Trisomía , Adolescente , Anciano , Antígenos CD19/análisis , Antígenos de Neoplasias/análisis , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Aberraciones Cromosómicas , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Japón , Cariotipificación , Leucemia Mieloide/clasificación , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/mortalidad , Tablas de Vida , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Proteínas de Fusión Oncogénica/análisis , Estudios Prospectivos , Proteína 1 Compañera de Translocación de RUNX1 , Receptores de Interleucina-7/análisis , Análisis de Supervivencia , Factores de Transcripción/análisisRESUMEN
The Notch pathway is a widely utilized, evolutionarily conserved regulatory system that plays a central role in the fate decisions of multipotent precursor cells. Notch often acts by inhibiting differentiation along a particular pathway while permitting or promoting self-renewal or differentiation along alternative pathways. Haematopoietic cells and stromal cells express Notch receptors and their ligands, and Notch signalling affects the survival, proliferation, and fate choices of precursors at various stages of haematopoietic development, including whether haematopoietic stem cells self-renew or differentiate, common lymphoid precursors undergo T or B cell differentiation, or monocytes differentiate into macrophage or dendritic cells. These findings suggest that the Notch pathway plays a fundamental role in regulating haematopoietic development.
Asunto(s)
Hematopoyesis/fisiología , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Ligandos , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores NotchRESUMEN
Ikaros, a zinc finger transcription factor, is essential for lymphoid development. Mutant mice expressing dominant-negative Ikaros gene (Ikaros) isoforms develop an aggressive form of lymphoid malignancies. We examined the expression of Ikaros isoforms in 11 leukemic cell lines and adult acute lymphoblastic leukemia cells from 36 patients with B-precursor acute lymphoblastic leukemia (pre-B ALL) and nine with T-precursor acute lymphoblastic leukemia (pre-T ALL), using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In one pre-B ALL cell line, INC cells, and primary leukemic cells from 16 patients with pre-B ALL, we found the predominant expression of a non-DNA-binding Ikaros isoform, Ik-6. However, Ik-6 was not detected in pre-T ALL cells. All of pre-B ALL cells expressing Ik-6 were CD10(+), whereas CD10(-) pre-B ALL cells did not express Ik-6. The expression of Ik-6 was not related to karyotype abnormalities such as t(9;22) and t(4;11). Proteins from the cells that expressed Ik-6 alone failed to bind to the Ikaros protein-specific binding sequence in DNA. Ikaros proteins lacking the DNA binding sequences were detected in the cytoplasm but not in the nucleus of the cells. When INC and primary pre-B ALL cells that express Ik-6 alone were irradiated and cultured in the absence of serum, these cells produced functional Ikaros isoforms, Ik-1 and Ik-2. Purified CD19(+) CD10(-) and CD19(+) CD10(+) cells from normal human bone marrow did not express Ik-6. The predominant expression of Ik-6, which is the result of post-transcription dysregulation, is characteristic of adult pre-B ALL, especially CD10(+) pre-B ALL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Factores de Transcripción/genética , Enfermedad Aguda , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Humanos , Factor de Transcripción Ikaros , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Unión Proteica , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Procesamiento Postranscripcional del ARN , Factores de Transcripción/biosíntesis , Células Tumorales CultivadasRESUMEN
We prepared a plasmid encoding 147 amino acid residues from the N terminus of c-erbB-2/HER2/neu (HER2), which included both a cytotoxic T lymphocyte (CTL) epitope (HER2p63) and a helper epitope (HER2p1), using the mammalian expression vector pCAGGS-New (pCAGGS147HER2). In a parallel analysis with a Tetramer assay and CTL assay, good specificity and sensitivity of a quantitative enzyme-linked immunospot (ELISPOT) assay to detect functional HER2p63-specific CD8(+) T cells were demonstrated after intramuscular immunization of pCAGGS147HER2. In an ELISPOT assay for HER2p63, spots of IFN gamma-producing cells were first detected 10 days after the first immunization, and additional immunizations increased the number of spots. HER2p63-specific CD8(+) T cells were detected over a period of more than 10 months after the last immunization. In hosts receiving more than three immunizations, surprisingly high numbers of specific CD8(+) T cells were persistently detectable. HER2 protein-specific antibodies of IgG class with dominance of IgG2a remain detectable 6 months after single or multiple immunizations. The antibodies however, were not reactive with cell surface HER2 antigens. Total suppression of tumor growth was observed when syngeneic HER2(+) tumor cells (2 x 10(6)) were injected subcutaneously 14 days after a single immunization with pCAGGS147HER2. Furthermore, the number of pulmonary metastases decreased significantly when DNA vaccination was initiated on the day of, or 3 days after, intravenous injection (1 x 10(6) cells).
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/genética , Genes erbB-2 , Terapia Genética/métodos , Vacunas de ADN/administración & dosificación , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Esquemas de Inmunización , Inmunoglobulina G/inmunología , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Receptor ErbB-2/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de TiempoRESUMEN
Scanning electrochemical microscopy has been firstly used to map the enzymatic activity in natural plant tissues. The peroxidase (POD) was maintained in its original state in the celery (Apium graveolens L.) tissues and electrochemically visualized under its native environment. Ferrocenemethanol (FMA) was selected as a mediator to probe the POD in celery tissues based on the fact that POD catalyzed the oxidation of FMA by H(2)O(2) to increase FMA(+) concentration. Two-dimensional reduction current profiles for FMA(+) produced images indicating the distribution and activity of the POD at the surface of the celery tissues. These images showed that the POD was widely distributed in the celery tissues, and larger amounts were found in some special regions such as the center of celery stem and around some vascular bundles.
Asunto(s)
Microscopía Electrónica de Rastreo/métodos , Peroxidasa/metabolismo , Plantas/enzimología , Apium/citología , Apium/enzimología , Apium/ultraestructura , Electroquímica , Compuestos Ferrosos/metabolismo , Microelectrodos , Microscopía Electrónica de Rastreo/instrumentación , Sondas Moleculares/metabolismo , Células Vegetales , Plantas/ultraestructura , Distribución TisularRESUMEN
Recognition of altered self-antigens in tumor cells by lymphocytes forms the basis for antitumor immune responses. The effector cells in most experimental tumor systems are CD8(+) T cells that recognize MHC class I binding peptides derived from molecules with altered expression in tumor cells. Although the need for CD4(+) helper T cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumor responses remain unclear. We examined whether broadly expressed wild-type molecules in murine tumor cells eliciting humoral immunity contributed to the generation of CD8(+) T cells and protective antitumor immune responses to unrelated tumor-specific antigens [mutated ERK2 (mERK2) and c-erbB2/HER/neu (HER2)]. The immunogenic wild-type molecules, presumably dependent on recognition by CD4(+) helper T cells, were defined by serological analysis of recombinant cDNA expression libraries (SEREX) using tumor-derived lambda phage libraries screened with IgG antibodies of hosts bearing transplanted 3-methylchoranthrene-induced tumors. Coimmunization of mice with plasmids encoding SEREX-defined murine wild-type molecules and mERK2 or HER2 led to a profound increase in CD8(+) T cells specific for mERK2 or HER2 peptides. This heightened response depended on CD4(+) T cells and copresentation of SEREX-defined molecules and CD8(+) T cell epitopes. In tumor protection assays, immunization with SEREX-defined wild-type molecules and mERK2 resulted in an inhibition of pulmonary metastasis, which was not achieved by immunization with mERK2 alone.
Asunto(s)
Neoplasias Experimentales/inmunología , Animales , Antígenos de Neoplasias/genética , Autoantígenos/genética , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos/genética , Femenino , Expresión Génica , Inmunización , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Mutación , Neoplasias Experimentales/genética , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Células Tumorales CultivadasRESUMEN
We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.
Asunto(s)
Células Dendríticas/citología , Células Madre Hematopoyéticas/citología , Antígenos CD1/análisis , Antígenos CD34/sangre , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Medios de Cultivo/química , Medios de Cultivo/farmacología , Interacciones Farmacológicas , Sustancias de Crecimiento/farmacología , Neoplasias Hematológicas/terapia , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunofenotipificación , Receptores de Lipopolisacáridos/análisis , Prueba de Cultivo Mixto de LinfocitosRESUMEN
We measured the plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) activity and antigen in patients with disseminated intravascular coagulation (DIC) to examine the relationship between hypofibrinolysis and the pathogenesis of DIC. TAFI activity and antigen levels in the plasma were both significantly low in patients with DIC. TAFI activity in plasma was correlated with TAFI antigen, indicating that activity and antigen correspond well. The decrease of TAFI activity in DIC may be due to enhanced consumption. Since the plasma thrombin-antithrombin III complex (TAT) level was found to be elevated in DIC, increase of thrombomodulin-thrombin complex generation is suggested in this state. TAFI activity and antigen levels were negatively correlated with TAT and D-dimer, suggesting that the plasma levels of TAFI are reduced by thrombin generation. Since TAFI was not correlated with fibrinogen, plasma-alpha(2)plasmin inhibitor complex (PPIC) and tissue type plasminogen activator/plasminogen activator inhibitor-1 (tPA/PAI-1) complex, TAFI might be a secondary modulator of fibrinolysis. The TAFI activity in plasma was significantly low in patients with infection and in those with organ failure, suggesting that TAFI may play an important role in the mechanism of organ failure in DIC-associated sepsis. In brief, TAFI may play an important role in the pathogenesis of DIC and organ failure.
Asunto(s)
Carboxipeptidasa B2/sangre , Coagulación Intravascular Diseminada/etiología , Antígenos/sangre , Antitrombina III , Biomarcadores/sangre , Carboxipeptidasa B2/inmunología , Estudios de Casos y Controles , Coagulación Intravascular Diseminada/sangre , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Hemostasis , Humanos , Péptido Hidrolasas/sangre , Trombofilia/sangre , Trombofilia/etiologíaRESUMEN
We examined the expression of hybrid phenotype in 236 adults with acute lymphoblastic leukemia (ALL; 188 B-lineage ALL and 48 T-lineage ALL). In B-lineage ALL, myeloid antigen (mAg) CD15 was concentrated in CD10-CD20- cases (49%); CD13 (42%); and CD33 (43%) in CD10+CD20- cases. This trend had no correlation with the presence of Ph1 or t(4;11) chromosomal abnormality. T-cell antigen CD2, CD4, and CD7 was seen in four, four, and two cases, respectively, and CD4+ and CD7+ cases commonly expressed CD13 and/or CD33 (CD13/CD33). In T-lineage ALL, expression of mAg, CD11b (47%), CD13 (38%), CD15 (28%), and CD33 (51%) was restricted to CD3- cases. B-cell antigen CD19 was found in two cases with CD7 solely as T-cell antigen, and these cases possessed CD13/CD33. CD21 was detected in three cases with CD3. In whole ALL, CD13/CD33 was associated closely with the presence of stem-cell antigen CD34, and in T-lineage ALL, CD13/CD33 had a significant correlation with additional stem-cell features, such as HLA-DR, multidrug resistance 1 (MDR1) and c-kit gene expression. Our results suggest that immature ALL cells frequently express B+M+, T+M+, and occasionally B+T+M+ phenotype; that B+T+M- phenotype is extremely rare; and that mAg expression in B-lineage ALL is complicated as compared to T-lineage ALL.
Asunto(s)
Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Antígenos de Neoplasias/análisis , Linfoma de Burkitt/inmunología , Leucemia-Linfoma de Células T del Adulto/inmunología , Adolescente , Adulto , Anticuerpos Monoclonales , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Antígenos CD13/análisis , Resistencia a Múltiples Medicamentos , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Antígeno de Macrófago-1/análisis , Fenotipo , Proteínas Proto-Oncogénicas c-kit/análisis , Proteínas Proto-Oncogénicas c-kit/genética , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
Dendritic cells (DC) with the potential to induce anti-tumour immunity represent one of the promising candidates for cancer vaccines. Efficiency of ex vivo DC generation depends on culture conditions, especially protein components in the plasma or serum used. Using human serum albumin (HSA), we devised a constant and reproducible culture method for DC generation from peripheral blood CD14+ cells. The number of DC obtained with 2% HSA-supplemented cultures containing granulocyte-macrophage colony-stimulating factor and interleukin 4 were consistently higher than in cultures with various concentrations of autologous plasma or serum. The concentrations and time points tested for plasma or serum considerably affected the number of DC recovered. DC prepared with HSA acquired the ability to uptake dextran, and expressed high levels of major histocompatibility (MHC) and co-stimulatory molecules similar to DC cultured with autologous plasma or serum. Although DC cultured with autologous plasma or serum consisted of CD1a+ and CD1a- populations, DC differentiated in the presence of HSA expressed CD1a. DC obtained with HSA primed and induced immunogenic peptide-specific cytotoxic T lymphocytes against a tumour rejection antigen, HER2. These findings suggest that our method for preparation of DC with HSA should prove valuable in DC generation for immunotherapy.