目的:观察不成熟CD8α+树突状细胞(DC)体外经同种异基因白血病细胞全抗原冲击后的移植物抗白血病(GVL)效应.方法:C57BL/6(H-2b)小鼠的骨髓细胞以GM-CSF +IL-4 +SCF +Flt3L体外诱导不成熟CD8α+DC,第3 d加入0 mg/L、2.5 mg/L、5 mg/L、10 mg/L和20 mg/L BALB/c(H-2d)小鼠来源的同种异基因EL9611红白血病细胞抗原冲击,冲击后DC与同系(H-2b)T细胞按DC/T 1:1、2:1、4:1比例共培养,MTT法观察T细胞增殖情况,ELISA法检测上清中IFN-γ和IL-10的含量,LDH释放法检测T细胞对EL9611细胞的杀伤活性.以成熟DC为对照,观察同种异基因不成熟CD8α+DC体外对EL9611白血病细胞的GVL效应.结果:同种红白血病全抗原冲击后不成熟CD8α+DC可有效刺激同系T细胞增殖,作用随DC/T比例增加而增加,增殖效应在小剂量抗原(≤5 mg/L)冲击时更明显(P<0.05);Ag冲击后不成熟CD8α+DC刺激T细胞分泌IFN-γ和IL-10明显增高(P<0.05),IL-10的分泌与不成熟CD8α+DC发挥GVL效应存在一定的负相关关系,Ag冲击浓度较低而IL-10分泌少时,T细胞增殖活性较强.杀伤实验结果显示,白血病特异性T细胞对EL9611靶细胞的杀伤活性随抗原冲击浓度的增加而升高,浓度为2.5 mg/L时,杀伤率即可达90%,非特异性杀伤实验显示白血病特异性T细胞对同种异基因正常脾细胞的杀伤活性低于对照组(P<0.05),表明杀伤作用为抗原特异性杀伤,对正常细胞无明显杀伤.结论:经同种异基因白血病抗原冲击后不成熟CD8α+DC体外能刺激同系T细胞产生一定的GVL效应.%ATM: To observe the graft - versus - leukemia ( GVL ) effect of immature CD8α+ dendritic cells ( DC ) plus impulsing of allogeneic leukemia whole - cell antigen in vitro. METHODS: Bone marrow cells of C57BL/6( H - 2b ) mice were induced by GM - CSF +IL-4 + SCF + Flt3L in vitro to prepare immature CD8α+ DC. On day3, different doses of BALB/c( H - 2d ) - derived allogeneic EL9611 leukemia antigen at concentration series of of mg/L, 2. 5 mg/L, 5 mg/L, 10 mg/L and 20 mg/L were added. After impulsing, DCs were co - cultured with syngeneic T cells at DC/T ratio of 1 : 1, 2:1 or 4:1. Proliferation of the T cells was measured by MTT method. The concentrations of IFN - γ and IL - 10 in cultured supernatants were detected by ELISA. Cytotoxicity of T cells on EL9611 cells was measured by LDH releasing assay. Mature DC served as the control and in vitro GVL effect of allogeneic immature CD8α+ DC on EL9611 leukemia cells was observed. RESULTS: Immature CD8α+ DC plus impulsing of allogeneic leukemia antigen ef-fectively stimulated syngeneic T cell proliferation, which increased with DC/T ratio. The effect of proliferation was more obviously, especially in the presence of antigen at low dose ( ≤5 mg/L, P <0. 05 ). The secretions of IFN - y and IL - 10 in the T cells stimulated with antigen plus immature CD8α+ DC became higher significantly ( P < 0. 05 ). There was a certain negative correlation between the secretion of IL - 10 and the GVL effect of CD8α+ immature DC. When the concentration of impulsing antigen and IL - 10 secretion was low, T cell proliferation became higher. The results of cytotoxicity assay showed that the cytotoxicity activity of leukemia specific T cells on EL9611 target cells increased with the antigen concentration, and the killing rate reached 90% when the concentration of the antigen was 2. 5 mg/L. Non - specific cytotoxicity assay showed that cytotoxicity activity of leukemia specific T cells on allogeneic normal spleen cells was lower than that in control group ( P < 0. 05 ), indicating that it was a specific killing, and no obvious cytotoxicity to normal cells was observed. CONCLUSION: CD8α+immature DC plus impulsing of allogeneic leukemia antigen stimulates syngeneic T cells to generate GVL effect.
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