γδT细胞杀肿瘤细胞过程中溶酶体运动及肌动蛋白纤维、T细胞受体重排特征研究

Trafficking of lysosomes and reorganization of actin filament together with the redistribution of T cell receptors inγδT lymphocytes while killing tumor cells

目的观察γδT细胞溶解肿瘤靶细胞过程中溶酶体运动特征,了解T细胞受体、肌动蛋白纤维在效应／靶细胞接触区域的分布特征,探讨γδT细胞抗肿瘤生物学活性的可能机理。方法Lysotracker Red-DND99标记的γδT细胞与人恶性黑色素瘤MeWo细胞共同培养,激光共聚焦显微镜实时观察γδT细胞杀伤肿瘤细胞过程中溶酶体运动过程及其特点。γδT细胞与MeWo细胞共同培养后甲醛固定,以rhodamine phalloidin标记肌动蛋白纤维或anti—TCRδ1-FITC抗体标记T细胞受体,激光共聚焦显微镜观察γδT细胞及肿瘤细胞之间免疫突触形成特征。结果γδT细胞可快速识别并集聚于肿瘤细胞周围。在与肿瘤细胞直接接触的10 min内,将胞内溶酶体成分“倾吐”于肿瘤细胞后随之离开。肿瘤细胞受大量γδT细胞攻击后约60 min,出现细胞膜皱缩、变圆,约80 min死亡。在上述效应／靶细胞接触作用过程中,肌动蛋白纤维及T细胞受体均匀分布于γδT表面,不出现在效应／靶细胞接触区域富集现象。结论γδT细胞通过与肿瘤细胞直接接触,释放其细胞内溶酶体内含物直接杀伤肿瘤细胞。与细胞毒性T淋巴细胞不同,γδT细胞与肿瘤细胞之间不形成典型的免疫突触。

Objective To visualize the kinetic of lysosomal compartment transport and the actin cytoskeleton reorganization together with the T cell receptor redistribution during the course of γδT cells recognize and kill the tumor cells. Methods Alive lysotracker marked γδT cells were co-cultured with human melanoma MeWo cell and examined by confocal microscopy with sequential images taken every minute up to 2 hours. Rhodamine phalloidin and and-TCRδ1-FTTC were used to stain fixed cell samples to monitor the actin cytoskeleton and T cell receptors reorganization as characteristics of the forming immunological synapse between target/effector cells. Results γδT cells recognized and reached MeWo cells shortly after the co-culture . During the short course of contact, γδT cells trafficked the lysosomes to and ＂poured＂ their contents into MeWo cells before they fled away. There were distributions of actin filaments and T cell receptors in the cell-cell contact area but they were not enriched. Conclusion The anti-tumor biosctivity of γδT cells is associated with direct cell-cell interaction and quick releasing of lysosomal contents. There is no typical immunological synapse formed between γδT cells tumor cells that imdicates a different mechaanism involoved in the bioactivity of γδT cells to other cytotoocic T cell, eg. CD8^＋ T and NK cell.