Shouval D, Rager-Zisman B, Quan P, Shafritz D A, Bloom B R, Reid L M
J Clin Invest. 1983 Aug;72(2):707-17. doi: 10.1172/jci111020.
The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 10(6) cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 10(6) cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 10(6) PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response. The nature of the response was delineated by treating nude mice challenged with tumor cells with sheep anti-mouse interferon globulin (anti-IFN). When 2 X 10(6) cells were injected, tumor growth occurred in 75% of anti-IFN-treated mice, whereas controls injected with the same number of cells, but not receiving anti-IFN, failed to develop tumors. The tumors in the anti-IFN-treated mice were highly invasive and the latency period until tumor appearance was reduced to 3-5 d. An inverse correlation was found between susceptibility of the hepatoma cells to natural killer (NK) activity in vitro and resistance to tumor growth in vivo. In vitro cytotoxicity for PLC/PRF/5 cells was eliminated by anti-NK 1.1 and complement, establishing the effector cell as an NK cell. NK cell activity 14 d after inoculation of mice with PLC/PRF/5 cells was augmented against PLC/PRF/5 target cells but not against SK-HEP-1 cells. Treatment of mice with ALS, irradiation, or anti-IFN abolished NK activity against PLC/PRF/5 cells. Co-cultivation of nude mouse spleen cells with PLC/PRF/5 but not with HBsAg or SK-HEP-1 cells induced secretion of murine IFNalpha. These results suggest that the IFN/NK cell system may play a role in limiting tumorigenicity and invasiveness of HBV-infected human hepatocellular carcinoma cells by a mechanism similar to that found for other cells persistently infected with viruses.
人肝癌细胞系PLC/PRF/5持续感染乙型肝炎病毒（HBV），已整合HBV-DNA，分泌HBV表面抗原（HBsAg），且在先天性无胸腺（nu/nu）小鼠中不易生长。本研究旨在确定该细胞系低致瘤性是否受宿主免疫反应调控和/或与HBsAg表达有关。将4 - 5×10⁶个细胞皮下注射到BALB/c裸鼠体内，25%的动物在29 - 40天内产生了具有原发性肝细胞癌形态特征的局部包囊化肿瘤。较低细胞接种量时未观察到肿瘤生长。相比之下，HBV阴性的人肝癌细胞系SK-HEP-1，接种1 - 5×10⁶个细胞时，66%的动物产生了肿瘤。用抗淋巴细胞血清（ALS）或照射对小鼠进行免疫抑制，可使接种1×10⁶个PLC/PRF/5细胞的小鼠肿瘤发生率增加至近100%，并产生局部侵袭性。免疫抑制还缩短了潜伏期，即肿瘤出现的时间，并增加了平均肿瘤重量。这些结果表明致瘤性受宿主免疫反应限制。通过用羊抗小鼠干扰素球蛋白（抗IFN）处理受肿瘤细胞攻击的裸鼠来明确反应的性质。注射2×10⁶个细胞时，75%接受抗IFN处理的小鼠出现肿瘤生长，而注射相同数量细胞但未接受抗IFN的对照小鼠未发生肿瘤。接受抗IFN处理的小鼠中的肿瘤具有高度侵袭性，肿瘤出现的潜伏期缩短至3 - 5天。发现肝癌细胞体外对自然杀伤（NK）活性的敏感性与体内对肿瘤生长的抗性呈负相关。抗NK 1.1和补体消除了对PLC/PRF/5细胞的体外细胞毒性，确定效应细胞为NK细胞。接种PLC/PRF/5细胞14天后，小鼠针对PLC/PRF/5靶细胞的NK细胞活性增强，但对SK-HEP-1细胞无增强作用。用ALS、照射或抗IFN处理小鼠可消除针对PLC/PRF/5细胞的NK活性。将裸鼠脾细胞与PLC/PRF/5共培养可诱导小鼠IFNα分泌，而与HBsAg或SK-HEP-1细胞共培养则不能。这些结果表明，IFN/NK细胞系统可能通过与其他持续感染病毒的细胞类似的机制，在限制HBV感染的人肝癌细胞的致瘤性和侵袭性方面发挥作用。
