RESUMEN
Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.
Asunto(s)
Separación Celular/métodos , Enfermedad Injerto contra Huésped/prevención & control , Inmunoterapia Adoptiva , Leucemia/terapia , Trasplante de Células Madre , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Supervivencia Celular , Células Cultivadas , Ensayos Clínicos como Asunto , Factores de Transcripción Forkhead/metabolismo , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Humanos , Terapia de Inmunosupresión , Integrina alfa4/metabolismo , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Leucemia/complicaciones , Leucemia/inmunología , Linfocitos T Reguladores/trasplanteRESUMEN
Natural killer (NK) cell therapies are emerging worldwide as promising anticancer treatments, exploiting the fast cytolytic action of NK effectors and their potentially broad applicability against a wide range of malignancies. Until recently, clinical protocols have mainly involved freshly isolated NK cells or short- term activated NK cells or lymphokine-activated killer (LAK) cells. However, overall effector numbers and their anticancer potencies remained restricted, which poses a limiting factor to clinical efficacy. Recent developments in the field aim to improve clinical trial designs by increasing effector to target cell ratios in vivo and by application of superior cytotoxic NK effectors. Large-scale production of clinical grade NK cells through long-term activation in ex vivo cultures are another novel means in achieving these goals. However, such procedures require compliance with the strict Good Manufacturing Practice (GMP) regulations to ensure quality and safety of the NK cell product. Although the overall number of new protocols still remains comparably low, some of the protocols are already translated into clinical use. Also striking is the diversity of the different protocols proposed. We highlight in this review the most recent developments in the NK cell field with a focus on long-term NK cell expansion. Critical issues relating to this novel and promising type of therapy are highlighted and discussed.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/trasplante , Neoplasias/terapia , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Proliferación Celular , Humanos , Inmunoterapia Adoptiva/tendencias , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Neoplasias/inmunologíaRESUMEN
Human blood monocytes can be broadly divided into two distinct subsets: CD14+CD16- and CD14+/lowCD16+ subsets. Perturbation in their proportions in the blood has been observed in several disease conditions. Although numerous phenotypic and functional differences between the two subsets have already been described, the roles contributed by each subset during homeostasis or disease conditions are still largely unclear. To uncover novel differences to aid in elucidating their functions, we perform a global analysis of the two subsets utilizing both proteomics and transcriptomics approaches. From the proteomics and transcriptomics data, the expression of 613 genes by the two subsets is detected at both the protein and mRNA levels. These 613 genes are assessed for up-regulation in each subset at the protein and mRNA levels using a cutoff fold change of > or =|1.5| between subsets. Proteins and mRNAs up-regulated in each subset are then mapped in silico into biological functions. This mapping reveals copious functional differences between the subsets, many of which are seen at both protein and mRNA levels. For instance, expression of genes involved in F(CY) receptor-mediated phagocytosis are up-regulated in the CD14+/lowCD16+ subset, while those involved in antimicrobial function are up-regulated in the CD14+CD16- subset. We uncover novel functional differences between the monocyte subsets from differences in gene expression at the protein and mRNA levels. These functional differences would provide new insights into the different roles of the two monocyte subsets in regulating innate and adaptive immune responses.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Monocitos/fisiología , Proteómica/métodos , Actividad Bactericida de la Sangre , Análisis por Conglomerados , Simulación por Computador , Interpretación Estadística de Datos , Proteínas Ligadas a GPI , Perfilación de la Expresión Génica/métodos , Humanos , Marcaje Isotópico , Receptores de Lipopolisacáridos/sangre , Monocitos/clasificación , Monocitos/metabolismo , Fagocitosis , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/sangre , Reproducibilidad de los ResultadosRESUMEN
A 49-year-old man, who had been diagnosed with chronic lymphocytic leukemia (CLL) in 2002, had a normal karyotype in his bone marrow. Trisomy 8 was demonstrated in his peripheral blood in 2005. Fluorescence in situ hybridization using an LSI CEP 8 probe performed on the archival bone marrow specimen showed three hybridization signals in 40% of 200 interphase cells scored. This confirmed that the trisomy 8 abnormality was present in both the blood and bone marrow samples. Trisomy 8 as the sole chromosomal abnormality in CLL is a very rare finding. The prognostic significance of trisomy 8 in CLL remains to be seen.