Guan Xin, Leng Xi-sheng, Peng Ji-run, Wei Yu-hua, Wang Hui, Yuan Lan
Center of Hepatobiliary Surgery, Peking University People's Hospital, Beijing 100044, China.
Zhonghua Yi Xue Za Zhi. 2005 Dec 14;85(47):3332-6.
To fuse human hepatocellular carcinoma (HCC) cells with mature monocyte-derived dendritic cells (FastDC) and to observe in vitro the function of the fused cells in stimulating autologous T cells proliferation and inducing HCC-specific cytotoxic T lymphocyte (CTL) response.
CD14(+) cells were isolated and purified from the peripheral blood of a healthy HLA-A2 blood donor and cultured in fresh dendritic cell complete medium for 24 h, then proinflammatory mediators were supplemented for another 24 h, thus generating mature dendritic cell (FastDCs). The FastDCs were fused with human HCC cells of the line HCCLM3 to generate novel dendritoma. T cells were isolated from selected CD14(-) cells and then divided into 4 groups to be stimulated with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells respectively for 96 hours. 18 hours before the end of cultivation (3)H-TdR was added into the culture fluid. Scintillation counter was used to measure the cpm values. CD8(+)T cells were isolated from CD14(-) cells, and added with different stimulating cells radiated by (60)Co and IL-2, IL-6, and IL-7. The values of IFN-gamma in the supernatants of the culture fluid of CD8(+)T cells with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells was measured. HCCLM3, K562, HLE, self monocytes labeled with Na(2)(51)CrO(4) were added with effector cells, gamma-scintillation counter was used to measure the cpm value so as to calculate the killing ability of CTL.
The CTLs activated by dendritoma cells specifically killed the HCCLM3 cells in the context of MHC class I and acted less vigorously against the control target cells. The CTLs activated by dendritoma cells were stronger in killing HCCLM3 cells than DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells (all P < 0.05). The killing activity was decreased on the HCCLM3 cells incubated with anti-HLA-ABC antibody. Three, five, and seven days after co-cultivation the value of IFN-gamma in the supernatants of the culture fluid of CD8(+)T cells with fused cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells increased gradually, especially in the supernatants of the culture fluid of CD8(+)T cells with dendritoma cells (400 pg/ml +/- 60 pg/ml 3 days after, 1030 pg/ml +/- 160 pg/ml 5 days after, and 1260 pg/L +/- 180 pg/L 7 days after).
The novel dendritomas formed with HCCLM3 cells and mature FastDCs from healthy human peripheral blood CD14(+) monocytes are potent stimulators for CD8(+)T cells in inducing HCCLM3 cell-specific lysis. With shorter time required for in vitro DC development, the rapid method of generation of dendritoma is more economic and may represent a new strategy for immunotherapy of hepatocellular carcinoma.
将人肝癌细胞与成熟的单核细胞来源的树突状细胞（FastDC）融合，体外观察融合细胞刺激自体T细胞增殖及诱导肝癌特异性细胞毒性T淋巴细胞（CTL）反应的功能。
从健康的HLA - A2血型供者外周血中分离纯化CD14(+)细胞，在新鲜的树突状细胞完全培养基中培养24 h，然后补充促炎介质再培养24 h，从而生成成熟的树突状细胞（FastDCs）。将FastDCs与人肝癌细胞系HCCLM3融合，生成新型树突状细胞瘤。从筛选出的CD14(-)细胞中分离T细胞，然后分为4组，分别用树突状细胞瘤细胞、DCs、HCCLM3细胞和DCs - HCCLM3细胞混合刺激96小时。培养结束前18小时，向培养液中加入(3)H - TdR。用闪烁计数器测量cpm值。从CD14(-)细胞中分离CD8(+)T细胞，并加入经(60)Co照射的不同刺激细胞以及IL - 2、IL - 6和IL - 7。测量用树突状细胞瘤细胞、DCs、HCCLM3细胞和DCs - HCCLM3细胞刺激的CD8(+)T细胞培养液上清中IFN - γ的值。将HCCLM3、K562、HLE、用Na(2)(51)CrO(4)标记的自体单核细胞加入效应细胞，用γ闪烁计数器测量cpm值，以计算CTL的杀伤能力。
树突状细胞瘤细胞激活的CTL在MHC I类背景下特异性杀伤HCCLM3细胞，对对照靶细胞的作用较弱。树突状细胞瘤细胞激活的CTL杀伤HCCLM3细胞的能力强于DCs、HCCLM3细胞和DCs - HCCLM3细胞混合组（均P < 0.05）。用抗HLA - ABC抗体孵育HCCLM3细胞后，杀伤活性降低。共培养3、5和7天后，用融合细胞、DCs、HCCLM3细胞和DCs - HCCLM3细胞刺激的CD8(+)T细胞培养液上清中IFN - γ的值逐渐升高，尤其是用树突状细胞瘤细胞刺激的CD8(+)T细胞培养液上清（3天后为400 pg/ml±60 pg/ml，5天后为1030 pg/ml±160 pg/ml，7天后为1260 pg/L±180 pg/L）。
由HCCLM3细胞与健康人外周血CD14(+)单核细胞来源的成熟FastDCs形成的新型树突状细胞瘤是诱导HCCLM3细胞特异性裂解的CD8(+)T细胞的有效刺激物。由于体外DC发育所需时间较短，快速生成树突状细胞瘤的方法更经济，可能代表了一种肝癌免疫治疗的新策略。
