| Objectives Treatment for advanced lung cancer is limited with poor prognosis. Immunotherapies designed to eliminate tumor cells may be one of the promising approaches due to its specificity. Infusion of γδ T cells is one of the adoptive cellular immunotherapies with the fact that γδ T cells is of low ratio in vivo. The purpose of this study was to find out the mechanism and effect of adoptive cell immunotherapy through γδT cells stimulated by zoledronic acid and dendritic cells.Methods Take zoledronic acid as the stimulating factor to stimulate human monocyte-derived dendritic cells, analyzing correlated phenotypes by flow cytometry and m RNA by realtime PCR, detecting the ratio of γδT cells. Take γδT cell and human lung carcinoma cell(A549ã€H460, H441 å’Œ SPC-A1) as effector cell and target cell,repectively,according to the efffector/target cell ratio( E/T 10:1,20:1). Detection of the content of isopentenyl pyrophosphate in supernatant from different stimulating samples employed high performance liquid chromatography-tandem mass spectrometry, with the purpose of confirming the relationship between isopentenyl pyrophosphate and the way of stimulation. RNA was extracted from the dendritic cells after the co-culture with zoledronic acid. Total RNA from each sample was quantified using the Nano Drop ND-1000 and the RNA integrity was assessed using standard denaturing agarose gel electrophoresis. Total RNA from each sample was amplified and transcribed into fluorescent c RNA with using the manufacturer’s Agilent’s Quick Amp Labeling protocol. The labeled c RNAs were hybridized onto the Whole Human Genome Oligo Microarray.Results The stimulation from zoledronic acid with the concentration of 1 μmol/L can restrain the maturation of dendritic cells, in the form of low ratio of HLA-DR, CD80 and CD86, with no change in CD11 c. Activation of dendritic cells after the co-culture with zoledronic acid led to excessive secretion of inflammatory cytokines such as TNF-α, IL-6, IL-1β, IL-10 and IFN-γ. Also the stimulation prompted the ratio of γδT from 4.99% to 65.7%. In the meantime, the level of corresponding characterization killer gene Perforin, granular enzyme B and CD107 a increased and displayed powerful cytotoxic activity to the four tumour cells. Zoledronate inhibits the farnesyl pyrophosphate synthase,an enzyme of the mevalonate pathway, leading to an accumulation of isopentenyl pyrophosphate. Expanding the concentration of zoledronic acid can augment the concentration of isopentenyl pyrophosphate from peripheral blood and dentritic cells to infuse a large number of highly cytolytic γδT cells. Mevastatin, the HMG-Co A reductase inhibitor, inhibits the mevalonate pathway and isopentenyl pyrophosphate synthesis. RNA was extracted from the dendritic cells after the co-culture with zoledronic acid. The result showed there were 679 up-regulated genes and 796 down-regulated genes, and gene CXCL9 was up-regulated by 253.5 times high with CXCL10 up-regulating 87.5 times. Gene LOC286002 was down-regulated by 20.3 times,and SULT6B1 was down-regulated by 15.4 times. There was a significant enrichment of T cell activation, especially in γδT cell pathway. In the enrichment duration of γδT, Gene STAT5 B and NOD2 were down-regulated to a statistically significant degree.Conclusions Zoledronate can not promote immature dendritic cells maturation and after zoledronate stimulation, the expression levels of various inflammatory factors and IFN-γ expression significantly increased. Induction of γδT cell proliferation can be enhanced by dendritic cells stimulated by zoledronic acid, for zoledronic acid inhibits the farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway, leading to an accumulation of isopentenyl pyrophosphate.The co-culture of dendritic cells and zoledronic acid can up-regulate gene CXCL9 and CXCL10, behaving the antitumor effect by down-regulating gene STAT5 B after the activatin of γδT cells. |
