急性髓系白血病细胞总RNA冲击的树突细胞诱导抗自身白血病的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：葛薇[1,2] 尤胜国[1,2] 王亚非[1,2] 李长虹[1,2] 刘晓帆[1,2] 何学鹏 马双[1,2] 邱录贵[1,2] 

机构地区：[1]中国医学科学院,中国协和医科大学血液学研究所 [2]实验血液学国家重点实验室,天津300020

出　　处：《中华血液学杂志》2005年第8期461-464,共4页Chinese Journal of Hematology

基　　金：天津市自然科学基金资助项目(993700711)

摘　　要：目的探讨白血病细胞RNA冲击的完全缓解期的急性髓系白血病(AML-CR)患者骨髓单个核细胞(BMMNC)衍生的树突细胞(mRNA-DC)体外刺激自体T细胞抗白血病免疫的可行性及有效性。方法应用联合细胞因子(GM-CSF、IL-4)培养法从AML-CR患者贴壁BMMNC诱导DC,在诱导的第5天用脂质体DOTAP介导法以自体白血病细胞的总RNA冲击DC,然后以含10 ng/m l的TNF-α的培养基继续培养24 h促进其成熟,培养7 d后收获细胞,作为mRNA-DC。以流式细胞术检测DC特征性表型的表达;以MTT法测定mRNA-DC对自体T细胞的促增殖活性;将mRNA-DC与T细胞按1∶3混合,共培养7 d,收获活化的T细胞,采用酶联免疫斑点法(ELISPOT)测定分泌γ干扰素(IFN-γ)阳性细胞数;乳酸脱氢酶(LDH)释放法测定细胞毒活性。结果14例AML-CR患者BMMNC均可在体外分化为具有特征性形态和表型的成熟DC;当刺激细胞与反应细胞比例为1∶16时,mRNA-DC可刺激自体T细胞产生明显的增殖活性[(36.84±5.68)%],与未冲击DC组[(12.20±3.16)%]比较差异具有统计学意义(P<0.05,n=9);ELISPOT分析显示分泌IFN-γ的mRNA反应性的T细胞得到明显扩增;体外活化的T细胞在效靶比为20∶1时,对自体白血病细胞显示明显的杀伤活性[(45.46±6.34)%],与未冲击的DC组[(12.32±1.32)%]及单纯IL-2组[(13.26±2.28)%]比较,差异有统计学意义(P<0.05,n=5)。结论应用白血病细胞RNA冲击的DC疫苗免疫可以作为一种可行、有效的防治微量残留白血病的方法。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-α 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 proli-(ferative) 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-γ 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-γ 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.

关 键 词：急性髓系白血病 树突细胞 T细胞 细胞免疫 

分 类 号：R733.7[医药卫生—肿瘤]