[用白血病细胞总RNA脉冲处理的树突状细胞诱导针对自体白血病细胞的高效T细胞免疫]
[Induction of efficient T-cell immunity against autologous leukemia cells by dendritic cells pulsed with the leukemia cell total RNA].
作者信息
Ge Wei, You Sheng-Guo, Wang Ya-Fei, Li Chang-Hong, Liu Xiao-Fan, He Xue-Peng, Ma Shuang, Qiu Lugui
机构信息
The State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS and PUMC, Tianjin, China.
出版信息
Zhonghua Xue Ye Xue Za Zhi. 2005 Aug;26(8):461-4.
OBJECTIVE
To assess the feasibility and efficiency of eliciting leukemia-specific T cell responses in acute myeloid leukemia patients in complete remission (AML-CR) in vitro by dendritic cells (DC) pulsed with the leukemia cells total RNA.
METHODS
The immature DCs were generated from the adherent bone marrow mononuclear cell in vitro in the presence of combined cytokines (GM-CSF 100 ng/ml, IL-4 500 U/ml), and pulsed with total RNA isolated from autologous leukemic cells by cationic lipid 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP) at day 5 of culture. Then the cells were incubated for another 24 h in a medium containing 10 ng/ml of TNF-alpha for maturation of DC. After the total 7 days culture, the cells were harvested as the mRNA-DC and the expression of mature DC markers were determined by FACS. The proliferative capacity of T cell activated by mRNA-DC was determined by MTT assay. Meanwhile, the mRNA-DC was co-cultured with T lymphocytes at a ratio of 1:3 for 7 days. The activated T lymphocytes were harvested, the secretion of IFN-gamma was determined by ELISPOT assay, and the cytotoxicity was analyzed in vitro by LDH release assay.
RESULTS
After culture, the BMMNC from 14 AML-CR patients developed morphologic and phenotypic characteristics of mature DC. At a stimulator/reactor ratio of 1:16, auto-T lymphocytes primed with mRNA-DC exhibited significant proliferative activity compared with T lymphocyte primed with non-pulsed DC [(36.84 +/- 5.68)% vs (12.20 +/- 3.16)%, (P < 0.05)]. An expansion of mRNA reacted T cell secreting IFN-gamma could be observed on ELISPOT assay. At an effector/target ratio of 20:1, the auto-T lymphocytes primed with mRNA-DC exhibited significant killing activity to auto-AML cells (45.46 +/- 6.34 )% as compared with that stimulated by IL-2 alone (13.26 +/- 2.28)% or primed by non-pulsed DC (12.32 +/- 1.32)% (P < 0.05).
CONCLUSION
Immunization with DC-leukemia cell RNA vaccines may be a simple, rapid and potent approach to elicitation of T cell-mediated anti-leukemia immunity.
目的
评估用白血病细胞总RNA脉冲树突状细胞（DC）在体外诱导完全缓解的急性髓系白血病患者（AML-CR）产生白血病特异性T细胞反应的可行性和效率。
方法
在联合细胞因子（GM-CSF 100 ng/ml，IL-4 500 U/ml）存在的情况下，从贴壁骨髓单个核细胞体外生成未成熟DC，并在培养第5天用阳离子脂质1,2-二油酰氧基-3-三甲基氯化铵丙烷（DOTAP）将从自体白血病细胞中分离的总RNA脉冲到DC中。然后将细胞在含有10 ng/ml TNF-α的培养基中再孵育24小时以使DC成熟。总共培养7天后，收获细胞作为mRNA-DC，并通过流式细胞术测定成熟DC标志物的表达。通过MTT法测定mRNA-DC激活的T细胞的增殖能力。同时，将mRNA-DC与T淋巴细胞以1:3的比例共培养7天。收获激活的T淋巴细胞，通过ELISPOT法测定IFN-γ的分泌，并通过乳酸脱氢酶释放法体外分析细胞毒性。
结果
培养后，14例AML-CR患者的骨髓单个核细胞呈现出成熟DC的形态学和表型特征。在刺激物/反应细胞比例为1:16时，与用未脉冲DC刺激的T淋巴细胞相比，用mRNA-DC刺激的自身T淋巴细胞表现出显著的增殖活性[（36.84±5.68）%对（12.20±3.16）%，（P<0.05）]。在ELISPOT试验中可观察到分泌IFN-γ的mRNA反应性T细胞的扩增。在效应细胞/靶细胞比例为20:1时，与单独用IL-2刺激（13.26±2.28）%或用未脉冲DC刺激（12.32±1.32）%相比，用mRNA-DC刺激的自身T淋巴细胞对自身AML细胞表现出显著的杀伤活性（45.46±6.34）%（P<0.05）。
结论
用DC-白血病细胞RNA疫苗免疫可能是一种简单、快速且有效的诱导T细胞介导的抗白血病免疫的方法。
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