Jožef Stefan Institute, Department of Biotechnology, Ljubljana, Slovenia.
University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia.
Radiol Oncol. 2019 Mar 3;53(1):57-68. doi: 10.2478/raon-2019-0007.
Background Cystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential. Materials and methods Levels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates. Results Two models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated. Conclusions Cystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.
背景 胱抑素 F 是一种半胱氨酸蛋白酶抑制剂蛋白，主要在免疫细胞中表达，并定位于内体/溶酶体隔室中。在细胞毒性免疫细胞中，胱抑素 F 抑制两种主要的前颗粒酶转化酶，即组织蛋白酶 C 和 H，它们激活颗粒酶，以及组织蛋白酶 L，它作为穿孔素激活剂。由于穿孔素和颗粒酶是细胞毒性淋巴细胞杀伤靶细胞的关键分子，因此颗粒酶或穿孔素激活缺陷会影响其细胞毒性潜力。
材料和方法 通过 Western blot 评估胱抑素 F 的水平，并通过免疫沉淀和共聚焦显微镜分析胱抑素 F 与组织蛋白酶 C、H 和 L 的相互作用。在 TALL-104 细胞中，使用肽底物测定组织蛋白酶和颗粒酶 B 的特异性活性。
结果 建立了两种 TALL-104 细胞系 T 细胞毒性降低的模型，一种是通过离子霉素处理，另一种是通过免疫抑制转化生长因子 β。细胞毒性降低与胱抑素 F 水平升高以及组织蛋白酶 C、H 和 L 及颗粒酶 B 的活性降低相关。证明了胱抑素 F 与组织蛋白酶 C、H 和 L 的共定位以及胱抑素 F 与组织蛋白酶 C 和 H 的相互作用。
结论 胱抑素 F 被指定为 T 细胞毒性的可能调节剂，类似于其在自然杀伤细胞中的作用。
