Zychlinsky A, Joag S, Liu C C, Young J D
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021.
Cell Immunol. 1990 Apr 1;126(2):377-90. doi: 10.1016/0008-8749(90)90329-p.
A highly purified population of murine lymphokine-activated killer (LAK) cells was obtained by selecting plastic-adherent splenocytes after incubation in high doses of recombinant IL-2. The population obtained was shown to be more than 95% positive for the cell marker asialo-GM1, and negative for both Lyt-1 (CD5) and Lyt-2 (CD8). The cells presented typical large granular lymphocyte morphology, and killed NK-susceptible target cells in an exclusively calcium-dependent fashion. A target cell DNA fragmentation activity of LAK cells could be detected even before target cell death. The presence of Hanukkah Factor/granzyme A/serine esterase 1, CTLA-1/granzyme B/serine esterase 2, and pore-forming protein (PFP/perforin) in these LAK cells was demonstrated by Northern blot analysis, suggesting that these markers are not exclusively associated with cytotoxic T lymphocytes. On immunoblots, antibodies specific for a lymphocyte PFP/perforin reacted with a 70-kDa protein of LAK cells. PFP/perforin was localized by immunofluorescence to the cell granules. A 50-kDa protein antigenically related to the macrophage cytokine tumor necrosis factor (TNF) was detected by immunoblotting and localized by immunofluorescence to both the cell granules and the cytosol. No RNA for TNF, however, could be detected using TNF-specific probes, suggesting that LAK cells may contain a cytotoxic factor which is related to, but distinct from, TNF. The work presented here demonstrates that cytotoxic mediators identified in cell lines are also present in primary cell cultures.
通过在高剂量重组白细胞介素-2中孵育后选择贴壁脾细胞，获得了高度纯化的小鼠淋巴因子激活的杀伤（LAK）细胞群体。所获得的细胞群体显示，细胞标志物脱唾液酸GM1的阳性率超过95%，而Lyt-1（CD5）和Lyt-2（CD8）均为阴性。这些细胞呈现典型的大颗粒淋巴细胞形态，并以完全依赖钙的方式杀伤对NK敏感的靶细胞。甚至在靶细胞死亡之前就能检测到LAK细胞的靶细胞DNA片段化活性。通过Northern印迹分析证明这些LAK细胞中存在哈努卡因子/颗粒酶A/丝氨酸酯酶1、CTLA-1/颗粒酶B/丝氨酸酯酶2和成孔蛋白（PFP/穿孔素），这表明这些标志物并非仅与细胞毒性T淋巴细胞相关。在免疫印迹中，针对淋巴细胞PFP/穿孔素的特异性抗体与LAK细胞的一种70 kDa蛋白发生反应。通过免疫荧光将PFP/穿孔素定位到细胞颗粒中。通过免疫印迹检测到一种与巨噬细胞细胞因子肿瘤坏死因子（TNF）抗原相关的50 kDa蛋白，并通过免疫荧光将其定位到细胞颗粒和细胞质中。然而，使用TNF特异性探针未检测到TNF的RNA，这表明LAK细胞可能含有一种与TNF相关但不同的细胞毒性因子。本文所展示的工作表明，在细胞系中鉴定出的细胞毒性介质在原代细胞培养物中也存在。
