HLA-A匹配的人胃癌细胞系诱生胃癌特异性T淋巴细胞

Induction of tumor specific cytotoxic T lymphocytes from human peripheral blood by stimulation with HLA-A matched allogeneic gastric tumor cell lines

目的 用HLA A匹配的人胃癌细胞系诱导对胃癌细胞具有特异性杀伤作用的T淋巴细胞。方法 分离胃癌患者及正常人外周血淋巴细胞 (PBL) ,用HLAⅠ类型别已知、经照射的异体胃癌细胞系刺激 ,进行筛选性的混合淋巴细胞肿瘤细胞培养 (MLTC) ,初步获得HLA A可能匹配的淋巴细胞供体 ,血清法测定其HLA A、B分子确认匹配后 ,进一步进行MLTC ,3H掺入法评估其增殖能力 ,直接免疫荧光法分析其细胞表型 ,5 1 Cr释放试验检测效应细胞的杀伤效应。结果 从 9例 (共分离 11次 )胃癌患者及 10例 (共分离 16次 )正常人PBL中 ,初筛发现有 2例胃癌患者及 1例正常人PBL得以大量增殖 ,检测出其中 1例胃癌患者及正常人的HLA A抗原均为HLA A2、A2 4,与用作刺激的异体胃癌细胞系相匹配 ,1例则不相匹配。对匹配的PBL进一步实施MLTC ,经 3～ 4次刺激后 ,CD3+ 淋巴细胞达到 98% ,其对HLA A匹配胃癌细胞的杀伤表现出明显的特异性。结论 利用HLA A2 (A2 4)匹配的异体胃癌细胞系作为刺激细胞反复诱导胃癌患者或健康人PBL ,有可能产生胃癌特异性的CTL。推测在刺激及培养条件适宜时 ,人外周血中较少的CTL前体细胞 (CTLp)是可能捕捉到的。

Objective To generate tumor specific cytotoxic T lymphocytes from human peripheral blood by stimulation with HLA A matched allogeneic gastric tumor cell lines. Methods Peripheral blood lymphocytes (PBL) were isolated from 9 patients for 11 times with gastric carcinoma and from 10 healthy subjects for 16 times. Preliminary mixed lymphocyte tumor cell cultures (MLTCs) were performed to screen the HLA A matched lymphocytes by using irradiated known HLA A allogeneic gastric tumor cell lines in the presence of 50U/ml of IL 2. The selected proliferative lymphocytes were typed for HLA A,B antigens. Lymphocyte proliferation in response to tumor cells was studied by 3H thymidine incorporation. Phenotype was characterized by direct immunofluorescence using flow cytometer. The 51 Cr release assay was used for determination of lymphocyte cytotoxic activity. Results Lymphocytes from 2 patients and 1 healthy subject had marked increase in proliferation after the culture. Their HLA A antigens were found to be matched with known HLA A antigens of the allogeneic gastric tumor cell lines in one healthy subject and one patient. MLTCs were further cultured (with 3 4 pulse of the tumor cells) to obtain cytotoxic lymphocytes. An increased expansion of the lymphocytes which contained 98% CD3 + cells was achieved (up to 20 100 folds). These T lymphocytes showed specific cytotoxicity to the HLA A matched gastric cancer cells. Conclusions Gastric cancer specific cytotoxic T lymphocytes could be obtained from PBL by stimulation with allogeneic gastric tumor cell lines, which suggests that PBL from patients with gastric carcinoma and healthy subjects contain CTL precursors (CTLp) for generating antitumor CTL.