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BACKGROUND: Several commercial and academic autologous chimeric antigen receptor T-cell (CAR-T) products targeting CD19 have been approved in Europe for relapsed/refractory B-cell acute lymphoblastic leukemia, high-grade B-cell lymphoma and mantle cell lymphoma. Products for other diseases such as multiple myeloma and follicular lymphoma are likely to be approved by the European Medicines Agency in the near future. DESIGN: The European Society for Blood and Marrow Transplantation (EBMT)-Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association collaborated to draft best practice recommendations based on the current literature to support health care professionals in delivering consistent, high-quality care in this rapidly moving field. RESULTS: Thirty-six CAR-T experts (medical, nursing, pharmacy/laboratory) assembled to draft recommendations to cover all aspects of CAR-T patient care and supply chain management, from patient selection to long-term follow-up, post-authorisation safety surveillance and regulatory issues. CONCLUSIONS: We provide practical, clinically relevant recommendations on the use of these high-cost, logistically complex therapies for haematologists/oncologists, nurses and other stakeholders including pharmacists and health sector administrators involved in the delivery of CAR-T in the clinic.
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Hematología , Receptores Quiméricos de Antígenos , Acreditación , Adulto , Médula Ósea , Humanos , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos TRESUMEN
Although the landscape for treating acute myeloid leukemia (AML) patients has changed substantially in recent years, the majority of patients will eventually relapse and succumb to their disease. Allogeneic stem cell transplantation provides the best anti-AML treatment strategy, but is only suitable in a minority of patients. In contrast to B-cell neoplasias, chimeric antigen receptor (CAR) T-cell therapy in AML has encountered challenges in target antigen heterogeneity, safety, and T-cell dysfunction. We established a Fab-based adapter CAR (AdCAR) T-cell platform with flexibility of targeting and control of AdCAR T-cell activation. Utilizing AML cell lines and a long-term culture assay for primary AML cells, we were able to demonstrate AML-specific cytotoxicity using anti-CD33, anti-CD123, and anti-CLL1 adapter molecules in vitro and in vivo. Notably, we show for the first time the feasibility of sequential application of adapter molecules of different specificity in primary AML co-cultures. Importantly, using the AML platform, we were able to demonstrate that chronic T-cell stimulation and exhaustion can be counteracted through introduction of treatment-free intervals. As T-cell exhaustion and target antigen heterogeneity are well-known causes of resistance, the AdCAR platform might offer effective strategies to ameliorate these limitations.
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Leucemia Mieloide Aguda , Agotamiento de Células T , Humanos , Línea Celular Tumoral , Leucemia Mieloide Aguda/metabolismo , Inmunoterapia Adoptiva , Linfocitos TRESUMEN
PURPOSE: Early clinical trials are the first step into clinical therapies for new drugs. Within the six Bavarian university-based hospitals (Augsburg, Erlangen, Regensburg, Munich (LMU and TU), Würzburg) we have enrolled a virtual network platform for patient discussion. METHODS: The virtual Early Clinical Trial Unit Tumor Board (ECTU Tumor Board) is a secured web-based meeting to evaluate early clinical trial options for patients, where representatives from local ECTUs participate. We retrospectively analyzed patient cases discussed between November 2021 and November 2022. RESULTS: From November 2021 to November 2022, a total of 43 patients were discussed in the ECTU Tumor Board. Median age at diagnosis was 44.6 years (range 10-76 years). The median number of previous lines of therapies was 3.7 (range 1-9 therapies) including systemic treatment, surgery, and radiation therapy. A total of 27 different tumor entities were presented and 83.7% (36/43) patients received at least one trial recommendation. In total, 21 different active or shortly recruiting clinical trials were recommended: ten antibody trials, four BiTE (bispecific T cell engager) trials, six CAR (chimeric antigen receptor) T-cell trials, and one chemotherapy trial. Only six trials (28.6%) were recommended on the basis of the previously performed comprehensive genetic profiling (CGP). CONCLUSION: The ECTU Tumor Board is a feasible and successful network, highlighting the force of virtual patient discussions for improving patient care as well as trial recruitment in advanced diseases. It can provide further treatment options after local MTB presentation, aiming to close the gap to access clinical trials.
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BACKGROUND: Pulmonary metastasis (M1-PUL) as first site of dissemination in pancreatic ductal adenocarcinoma (PDAC) is a rare event and may define a distinct biological subgroup. PATIENTS AND METHODS: Arbeitsgemeinschaft Internistische Onkologie-Young Medical Oncologists-Pankreas-0515 study (AIO-YMO-PAK-0515) was a retrospective German multicenter study investigating clinical and molecular characteristics of M1-PUL PDAC patients; 115 M1-PUL PDAC patients from 7 participating centers were included. Clinical characteristics and potential prognostic factors were defined within the M1-PUL cohort. Archival tumor samples were analyzed for Her2/neu, HNF1A and KRT81 expression. Additionally, messenger RNA (mRNA) expression analysis (using a 770-gene immune profiling panel) was carried out in the M1-PUL and in a control cohort (M1-ANY). RESULTS: Median overall survival in the entire M1-PUL cohort was 20 months; the most favorable prognosis (median survival: 28 months) was observed in the subgroup of 66 PDAC patients with metachronous lung metastases after previous curative-intent surgery. The number of metastatic lesions, uni- or bilateral lung involvement as well as metastasectomy were identified as potential prognostic factors. Her2/neu expression and PDAC subtyping (by HNF1A and KRT81) did not differ between the M1-PUL and the M1-ANY cohort. mRNA expression analysis revealed significant differentially expressed genes between both cohorts: CD63 and LAMP1 were among the top 20 differentially expressed genes and were identified as potential mediators of organotropism and favorable survival outcome of M1-PUL patients. CONCLUSION: M1-PUL represents a clinically favorable cohort in PDAC patients. Site of relapse might already be predetermined at the time of surgery and could potentially be predicted by gene expression profiling.
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Neoplasias Pulmonares , Neoplasias Pancreáticas , Biología , Humanos , Neoplasias Pulmonares/genética , Recurrencia Local de Neoplasia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pronóstico , Estudios RetrospectivosRESUMEN
Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.
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Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Proteínas de la Matriz Viral/inmunología , Apoptosis/inmunología , Linfocitos B , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada , Reacciones Cruzadas , Humanos , Necrosis , Fagocitosis/inmunología , Latencia del VirusRESUMEN
The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8(+) cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4(+) T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4(+) T cells, EBNA1 is preferentially recognized. We present evidence that the CD4(+) response may provide a protective role, including interferon gamma secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II-mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4(+) T cell lines to recognize autologous B-LCL-expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4(+) T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4(+) T cell immunity be enhanced to prevent and treat EBV-associated malignancies.
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Linfocitos T CD4-Positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Adulto , Presentación de Antígeno , Linfocitos B/inmunología , Línea Celular , Células Dendríticas/inmunología , Epítopos/química , Epítopos/genética , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Técnicas In Vitro , Interferón gamma/biosíntesis , Activación de Linfocitos , Estructura Terciaria de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-Ab MHC class II presenting a peptide derived from I-Ealpha. When immature DCs from I-Ab mice were cultured for 5-20 h with activated I-E+ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DRalpha includes the same peptide sequence as mouse I-Ealpha. Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-E- bearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self.
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Presentación de Antígeno , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Fagocitosis/inmunología , Animales , Células Dendríticas/citología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML (n = 356) and normal bone marrow samples (n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.
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Antígenos de Neoplasias/metabolismo , Antineoplásicos/uso terapéutico , Médula Ósea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Terapia Molecular Dirigida , Células Madre Neoplásicas/metabolismo , Proteoma/análisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Estudios de Casos y Controles , Células Cultivadas , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Pronóstico , Adulto JovenRESUMEN
The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.
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Leucemia Mieloide Aguda/terapia , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Secuencias Repetidas en Tándem/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Humanos , Inmunoterapia/métodos , Leucemia Mieloide Aguda/metabolismo , Mutación/efectos de los fármacos , Mutación/genética , Pronóstico , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ⩾1 mutation during remission at a variant allele frequency of ⩾2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (P<0.0001) and, in multivariate analyses adjusting for age, genetic risk, and allogeneic transplantation, with inferior relapse-free survival (hazard ratio, 2.34; P=0039) and overall survival (hazard ratio, 2.14; P=036). Patients with persisting mutations had a higher cumulative incidence of relapse before, but not after allogeneic stem cell transplantation. Our work underlines the relevance of mutation persistence during first remission as a novel risk factor in AML. Persistence of pre-leukemic clones may contribute to the inferior outcome of elderly AML patients. Allogeneic transplantation abrogated the increased relapse risk associated with persisting pre-leukemic clones, suggesting that mutation persistence may guide postremission treatment.Leukemia accepted article preview online, 18 December 2017. doi:10.1038/leu.2017.350.
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Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.
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Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Escape del Tumor/efectos de los fármacos , Animales , Antígeno B7-H1/análisis , Antígeno B7-H1/fisiología , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Ratones , Receptor de Muerte Celular Programada 1/análisis , Receptor de Muerte Celular Programada 1/fisiología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/análisisRESUMEN
In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.
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Antineoplásicos/farmacología , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Sinergismo Farmacológico , Reordenamiento Génico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Imidazoles/farmacología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolinas/farmacología , Transducción de Señal , Sirolimus/farmacología , Análisis de Supervivencia , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In acute myeloid leukemia (AML), assessment of minimal residual disease (MRD) by flow cytometry (flow MRD) after induction and consolidation therapy has been shown to provide independent prognostic information. However, data on the value of earlier flow MRD assessment are lacking. Therefore, the value of flow MRD detection was determined during aplasia in 178 patients achieving complete remission after treatment according to AMLCG (AML Cooperative Group) induction protocols. Flow MRD positivity during aplasia predicted poor outcome (5-year relapse-free survival (RFS) 16% vs 43%, P<0.001) independently from age and cytogenetic risk group (hazard ratio for MRD positivity 1.71; P=0.009). Importantly, the prognosis of patients without detectable MRD was neither impacted by morphological blast count during aplasia nor by MRD status postinduction. Early flow MRD was also evaluated in the context of existing risk factors. Flow MRD was prognostic within the intermediate cytogenetic risk group (5-year RFS 15% vs 37%, P=0.016) as well as for patients with normal karyotype and NPM1 mutations (5-year RFS 13% vs 49%, P=0.02) or FLT3-ITD (3-year RFS rates 9% vs 44%, P=0.016). Early flow MRD assessment can improve current risk stratification approaches by prediction of RFS in AML and might facilitate adaptation of postremission therapy for patients at high risk of relapse.
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Células de la Médula Ósea/citología , Médula Ósea/anomalías , Leucemia Mieloide Aguda/sangre , Leucocitos Mononucleares/citología , Recurrencia Local de Neoplasia/sangre , Neoplasia Residual/diagnóstico , Anciano , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo , Humanos , Cariotipificación , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Nucleofosmina , Pronóstico , Modelos de Riesgos Proporcionales , Inducción de Remisión , Factores de Riesgo , Resultado del TratamientoRESUMEN
Alveolar macrophages (AMs) harvested from 32 HIV-infected patients with respiratory problems (opportunistic pulmonary infections, n = 12; other lung disease, n = 20) and 13 healthy controls were stained with a panel of 15 monoclonal antibodies directed against surface antigens implicated in cell function. Antigen expression was quantified by flow cytometry and expressed as relative linear median fluorescence intensity (RLMFI). On AMs of patients, as compared with controls, there was a significant enhancement of HLA DP (12.1 +/- 1.5 vs 6.5 +/- 0.9, p = 0.01, M +/- SEM, RLMFI units), CD11b (3.4 +/- 0.5 vs 1.7 +/- 0.4, p = 0.014), CD11c (8.9 +/- 1.0 vs 4.8 +/- 0.8, p = 0.0046), CD14 (2.1 +/- 0.3 vs 1.0 +/- 0.2, p = 0.0009), and CD33 (1.7 +/- 0.1 vs 1.0 +/- 0.2, p = 0.0093). No significant differences could be established for HLA-DR (36.9 +/- 5.8 vs 30.9 +/- 7.5, NS), HLA-DQ (3.4 +/- 0.3 vs 3.1 +/- 0.6, NS), CD54 (1.9 +/- 0.3 vs 1.2 +/- 0.1, NS), CD13 (2.5 +/- 0.6 vs 1.5 +/- 0.3, NS), CD36 (1.4 +/- 0.2 vs 0.9 +/- 0.3, NS), CD71 (10.3 +/- 1.9 vs 8.9 +/- 1.8, NS), CD25 (0.8 +/- 0.0 vs 0.9 +/- 0.1, NS), 27E10 (1.1 +/- 0.1 vs 0.8 +/- 0.3, NS), RM3/1 (1.9 +/- 0.4 vs 1.5 +/- 0.4, NS), and CD4 (1.5 +/- 0.3 vs 1.0 +/- 0.0, NS). The expression of CD14 and CD11b, but not of HLA class II antigens and CD71, was increased in the smaller cell population compared with the larger, thus suggesting monocyte recruitment. The increased expression of HLA-DP, CD11c, CD14, and CD33 on the patients' AMs was independent of smoking habits. The degree of immunodeficiency as indicated by the absolute peripheral CD4 count, the character of HIV-related pulmonary disease, and the prophylactic use of pentamidine or zidovudine did not significantly modify the antigen expression pattern. It is concluded that HIV infection may lead, most probably indirectly, to enhanced expression of surface antigens by local upregulation and/or recruitment of monocytes from the peripheral circulation. The functional significance of enhanced marker expression requires further clarification.
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Antígenos de Superficie/análisis , Infecciones por VIH/inmunología , Macrófagos Alveolares/inmunología , Adulto , Antígenos CD/análisis , Líquido del Lavado Bronquioalveolar/citología , Femenino , Citometría de Flujo , Infecciones por VIH/complicaciones , Infecciones por VIH/diagnóstico , Antígenos HLA/análisis , Humanos , Inmunofenotipificación , Masculino , Enfermedades Respiratorias/complicaciones , Enfermedades Respiratorias/inmunologíaAsunto(s)
Enfermedades Hematológicas/terapia , Neoplasias Hematológicas/terapia , Anticoagulantes/uso terapéutico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sistemas de Liberación de Medicamentos , Enfermedades Hematológicas/diagnóstico , Neoplasias Hematológicas/diagnóstico , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma Folicular/diagnóstico , Linfoma Folicular/terapia , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/terapia , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/terapia , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/terapia , Macroglobulinemia de Waldenström/diagnóstico , Macroglobulinemia de Waldenström/terapiaRESUMEN
Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell-depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-alpha, IL-1beta, IL-6, and interferon-alpha. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction-stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.
Asunto(s)
Citocinas/farmacología , Células Dendríticas/citología , Monocitos/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Humanos , Monocitos/citologíaRESUMEN
Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL), provides resistance to Epstein-Barr virus (EBV), as is demonstrated by the occurrence of posttransplant lymphoproliferative disease in immunosuppressed patients. We set out to use dendritic cells (DCs) to elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8(+) donors, monocyte-derived mature DCs were pulsed with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The cultured cells specifically lysed EBNA-3A peptide-pulsed, HLA-B8(+), B-lymphoblastoid cell lines in a 5-hour (51)Cr-release assay. The generation of CTLs did not require the addition of interleukin-2. In comparison, monocytes were weak antigen-presenting cells. DCs were then infected with recombinant vaccinia-EBNA-3A. Vaccinia infection significantly decreased the viability of immature DCs after 3 days of culture (to 25% to 45%) but had a smaller effect on mature DC recovery (40% to 70%). To decrease these cytopathic effects and to expand the potential use of vaccinia vectors for DC therapy in immunocompromised patients, we successfully used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility complex class I products can even be charged with UV-inactivated recombinant vaccinia.
Asunto(s)
Presentación de Antígeno , Antígenos Virales/inmunología , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/inmunología , Proteínas Virales/inmunología , ADN Recombinante , Células Dendríticas/virología , Antígeno HLA-B8/inmunología , Humanos , Rayos Ultravioleta , Virus Vaccinia/genética , Virus Vaccinia/efectos de la radiación , Proteínas Virales/genéticaRESUMEN
Epstein-Barr virus (EBV) infects more than 95 % of the human population and causes an asymptomatic life-long infection in the majority of EBV carriers. Cell-mediated immunity provides resistance to EBV, as demonstrated by the occurrence of EBV-induced post-transplant lymphoproliferative disease in immunosuppressed patients. Here we looked for IFN-gamma-producing T lymphocytes in the blood of healthy donors with a rapid enzyme-linked immunospot (ELISPOT) assay, comparing as antigen presenting cells monocytes and dendritic cells (DC) infected with recombinant vaccinia virus (rVV). We found a strong CD8(+) ELISPOT response to one or more of the EBNA 3A, 3B and 3C antigens in the PBMC from 14 / 18 donors. The sensitivity of the overnight ELISPOT assay was increased using DC as antigen-presenting cells, including 3 / 3 individuals who lacked ELISPOT in PBMC. In addition, DC could markedly expand EBV-specific spots after a 7-day culture. In a smaller number of donors, we documented recognition of the subdominant LMP 1, LMP 2 and EBNA 1 antigens that are expressed in a variety of EBV-associated malignancies. Therefore our data provide more evidence for the efficacy of DC in eliciting rapid responses to EBV latency antigens in circulating CD8(+) T cells.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Herpesvirus Humano 4/inmunología , Latencia del Virus/inmunología , Células Dendríticas/inmunología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Recombinación Genética , Virus Vaccinia/genética , Virus Vaccinia/inmunologíaRESUMEN
Vaccinia virus employs multiple mechanisms to evade the immune system, yet is highly immunogenic. We studied the interaction between vaccinia and human dendritic cells (DCs), potent APCs. DCs develop from precursor cells in two stages: an immature stage in which Ag uptake and processing occur, and a mature stage in which there is up-regulation of costimulatory and HLA molecules and efficient T cell activation. Vaccinia virus undergoes an abortive replication in both stages of DCs and induces apoptotic cell death. Furthermore, maturation of immature DCs and consequently T cell activation are inhibited. Obstruction of DC maturation may constitute a novel mechanism by which vaccinia attempts to evade the immune response.