RESUMO
NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56(bright)CD16(+) subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.
Assuntos
Ligante 4-1BB/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Interleucinas/biossíntese , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Ligante 4-1BB/genética , ADP-Ribosil Ciclase 1/biossíntese , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígeno CD56/biossíntese , Linhagem Celular Tumoral , Técnicas de Cocultura , Proteínas Ligadas por GPI/biossíntese , Células HeLa , Células Hep G2 , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoterapia Adotiva , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucinas/genética , Interleucinas/farmacologia , Lectinas Tipo C/biossíntese , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Subfamília D de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Neoplasias/terapia , Receptores de IgG/biossínteseRESUMO
AIMS/INTRODUCTION: To investigate the ability of human amniotic fluid stem cells (hAFSCs) to differentiate into insulin-producing cells. MATERIALS AND METHODS: hAFSCs were induced to differentiate into pancreatic cells by a multistep protocol. The expressions of pancreas-related genes and proteins, including pancreatic and duodenal homeobox-1, insulin, and glucose transporter 2, were detected by polymerase chain reaction and immunofluorescence. Insulin secreted from differentiated cells was tested by enzyme-linked immunosorbent assay. RESULTS: hAFSCs were successfully isolated from amniotic fluid that expressed the pluripotent markers of embryonic stem cells, such as Oct3/4, and mesenchymal stem cells, such as integrin ß-1 and ecto-5'-nucleotidase. Here, we first obtained the hAFSCs that expressed pluripotent marker stage-specific embryonic antigen 1. Real-time polymerase chain reaction analysis showed that pancreatic and duodenal homeobox-1, paired box gene 4 and paired box gene 6 were expressed in the early phase of induction, and then stably expressed in the differentiated cells. The pancreas-related genes, such as insulin, glucokinase, glucose transporter 2 and Nkx6.1, were expressed in the differentiated cells. Immunofluorescence showed that these differentiated cells co-expressed insulin, C-peptide, and pancreatic and duodenal homeobox-1. Insulin was released in response to glucose stimulation in a manner similar to that of adult human islets. CONCLUSIONS: The present study showed that hAFSCs, under selective culture conditions, could differentiate into islet-like insulin-producing cells, which might be used as a potential source for transplantation in patients with type 1 diabetes mellitus.