人工抗原呈递细胞诱导CML28特异性T淋巴细胞杀伤急性白血病细胞的研究

In vitro cytotoxic effects of CML28 specific T cells on acute leukemia cells

目的探讨以人工抗原呈递细胞模拟树突细胞（DC）体外诱导特异性T淋巴细胞活化、增殖，观察特异性T淋巴细胞杀伤白血病细胞的效应。方法用慢性粒细胞白血病细胞特异性抗原肽（CML28）作为目标肽，并以磁珠同时负载人工合成的HLA-A2-Ig二聚体-抗原肽（CML28）复合物以及B7-1分子，作为诱导T淋巴细胞的活化、增殖的双信号分子，模拟抗原呈递细胞的生物功能，建立一种人工抗原呈递细胞；从HLA-A2健康骨髓捐献者骨髓或外周血中分离出单个核细胞作为效应细胞，并与人工抗原呈递细胞共孵育，以流式细胞术检测特异性T淋巴细胞活化、增殖程度。以初治急性白血病细胞作为靶细胞，将特异性T淋巴细胞与白血病细胞分别以5：1，10：1，20：1，40：1，80：1比例共孵育4h，用乳酸脱氢酶（LDH）释放实验检测杀伤急性白血病细胞的效果。结果以该人工抗原呈递细胞刺激T淋巴细胞后，其CML28特异性T淋巴细胞比例明显增高，实验组中抗原肽ALFCGVACA、FLQGDTSVL、DLMSSTKGL、VLTFALDSV所诱导的特异性T淋巴细胞比例分别为（13．5±1．6）％、（15．2±1．5）％、（14．7±1．8）％、（34．3±3．5）％，与对照组[（2．2±0．4）％]比较，差异均有统计学意义（P〈0．01）。当效应细胞与靶细胞分别以5：1，10：1，20：1，40：1，80：1混合后，随着效应细胞的增加，诱导杀伤急性白血病细胞效率也明显增强，与对照组比较，差异均有统计学意义（P〈0．05）。结论以磁珠为平台的人工抗原呈递细胞能有效模拟人抗原呈递细胞，诱导T淋巴细胞特异性杀伤白血病细胞，为白血病的免疫治疗提供了一种新的途径。

Objective To explore the activation and proliferation of specific T cells induced by artificial antigen-presenting cells （aAPCs） simulated dendritic cells （DCs） and to observed the effect of these T cells on leukemic cell killing. Methods aAPCs were developed by coating a human leukocyte antigen-immu- noglobulin fusion protein （ HLA-lg）, which was connected each one of the four CML28 antigen epitopes （DLMSSTKGL, DLMSSTKGL, ALFCGVACA, VLTFALDSV）, and CD28-specific antibody, to magnetbeads CML cell specific peptides （CML28） served as target peptides. Bone marrow （BM） or peripharal blood （PB） mononuclear cells （MNCs） were isolated from HLA-A2 healthy volunteers, and co-cultured with aAPCs. Specific T lymphocyte were detected by flow cytometry. The fresh acute leukemic cells were used as target cells. The specific T cells incubated with leukemic cells for 4 h at ratios of 5： 1,10： 1,20： 1,40： 1,80： 1, respectively. The effect of leukemic cells killing was detected by lactate dehydrogenase release test. Resuits The average ratio of CML-28 specific T lymphocyte in control group was （2.2 ± 0.4 ） % and in experimental groups （ DLMSSTKGL, DLMSSTKGL, ALFCGVACA, VLTFALDSV） were （ 13. 5 ± 1. 6 ）%, （ 15.2 ± 1.5 ） %, （ 14.7 ± 1.8 ） % and （ 34.3 ± 3.5 ） %, respectively, being significantly higher than that in control group （P 〈 0.01 ）. Induction efficiencies of acute leukemic cells killing were significantly enhanced by increase of effector cells. The cytotoxic activity of specific T lymphocyte in one experimental group （VLTFALDSV） was much higher than that in other three experimental group （P 〈 0.05）. Conclusion This ＂prime and expand ＂ regimen should be an alternative method for large scale amplification of rare tumor-specific CTLs and aAPCs might be a useful tool for leukemia immunotherapy.