搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

NK cells express different TLRs,such as TLR3,TLR7,and TLR9,but little is known about their role in NK cell stimulation. In this study,we used specific agonists (poly(I:C),loxoribine,and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12,IL-15,or IFN-alpha,and investigated the secretion of IFN-gamma,cytotoxicity,and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine,in conjunction with IL-12,but not IL-15,triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also,secretion of IFN-gamma was dependent on MEK1/ERK,p38 MAPK,p70(S6) kinase,and NF-kappaB,but not on calcineurin. IFN-alpha induced a similar effect,but promoted lesser IFN-gamma secretion. However,cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged,as well as the expression of the NKG2D receptor. Excitingly,IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12,and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12,and this effect can be further enhanced by costimulation through NKG2D. Hence,integration of the signaling cascades that involve TLR3,TLR7,IL-12,and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions,which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer. View Publication

The role of thrombopoietin (Tpo) in promoting hematopoiesis has been extensively studied in late fetal,neonatal,and adult mice. However,the effects of Tpo on early yolk sac hematopoiesis have been largely unexplored. We examined whole embryos or the cells isolated from embryo proper and yolk sacs and identified both Tpo and c-mpl (Tpo receptor) mRNA transcripts in tissues as early as embryonic day 6.5 (E6.5). Presomite whole embryos and somite-staged yolk sac and embryo proper cells were plated in methylcellulose cultures and treated with selected hematopoietic growth factors in the presence or absence of Tpo. Tpo alone failed to promote colony-forming unit (CFU) formation. However,in the presence of other growth factors,Tpo caused a substantial dose-dependent reduction in primitive and definitive erythroid CFU growth in cultures containing E7.5 and E8.0 whole embryos and E8.25 to 9.5 yolk sac-derived cells. Meanwhile,Tpo treatment resulted in a substantial dose-dependent increase in CFU-mixed lineage (CFU-Mix) and CFU-megakaryocyte (CFU-Meg) formation in cultures containing cells from similar staged tissues. Addition of Tpo to cultures of sorted E9.5 yolk sac c-Kit(+)CD34(+) hematopoietic progenitors also inhibited erythroid CFU growth but augmented CFU-Mix and CFU-Meg activity. Effects of Tpo on CFU growth were blocked in the presence of a monoclonal antibody with Tpo-neutralizing activity but not with control antibody. Thus,under certain growth factor conditions,Tpo directly inhibits early yolk sac erythroid CFU growth but facilitates megakaryocyte and mixed lineage colony formation. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

The immune system can act as an extrinsic suppressor of tumors. Therefore,tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here,we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells,and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells,T cells,natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3(-/-) hematopoietic cells,and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell- and NK cell-dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity. View Publication