抗AML特异性T细胞的诱导、扩增及功能研究

Induction,proliferation and functional study of specific anti-acute myeloid leukemia(AML) T cells

目的:体外培养抗急性髓性白血病(AML)细胞的特异性T细胞,并研究其生物学特性和功能。方法:取12例AML患者的外周血或骨髓,分离单个核细胞(MNCs)后接种于96孔板,加入组合细胞因子,诱导AML细胞来源的树突细胞(DC)生成,培养过程中用倒置显微镜进行形态学观察,并在7天后用流式细胞术检测免疫学表型。培养第7天时将组合细胞因子培养液换为高IL-2因子培养液,促进抗AML细胞的特异性T细胞生长,培养4～5周后测定T细胞表型,并采用乳酸脱氢酶(LDH)释放法进行T细胞杀伤活性测定。结果:AML细胞经组合细胞因子诱导后,大部分细胞呈现树突状细胞的典型形态,而且CD80、CD86和HLA-DR较培养前明显上调(P<0.01);高IL-2因子培养液培养4～5周后,82.5%的培养孔内CD3+ T细胞占孔内细胞总数99%以上;LDH释放法进行T细胞杀伤试验表明培养出的T细胞对自体AML有较强的杀伤作用,而对异体AML细胞杀伤力弱。结论:在体外可以成功诱导出抗AML细胞的特异性T细胞,且培养出的T细胞对自体AML细胞有特异性杀伤作用。

Objective：To develop specific anti-AML T cells in vitro, and to study their biological characteristics and functions. Methotis： Peripheral blood or bone marrow mononuclear cells （MNCs） were isolated from 12 patients with AML, and co-cultured with cytokine combinations in 96 well plates to be induced into dendritic cells （DCs）. Cell morphology was observed under an inverted microscope during the first week and immunophenotype was detected by flow cytometry at day 7. Cytokine combination was replaced with high dose IL-2 at day 7 to promote specific anti-AML T cells. T cell phenotype was detected after 4 to 5 weeks. Lactate dehydrogenase （LDH） release assay was used to determine T cell killing activity. Results： After being cultured with cytokine combinations, the typical dendritic appearance with delicate membrane projections was observed. CD80, CD86 and HLA-DR markers were significantly upregulated（ P 〈 0.01 ）. 82.5 % of the wells contained more than 99% of CD3^＋T cells. LDH release showed that these T cells caused specific lysis of autologous AML cells but not allogeneic AML cells. Conclusion： Specific anti-AML T cells can be deduced in vitro, and these T cells can kill autologous AML cells specifically.