Non-cytotoxic functions of lymphoycte granule exocytosis

淋巴细胞颗粒胞吐作用的非细胞毒性功能

• 批准号: 7070841
• 负责人: Pierre A Henkart
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7070841
• 关键词: T cell receptor; cell line; cell population study; chemokine; cytotoxic T lymphocyte; exocytosis; fluorescence microscopy; granule; helper T lymphocyte; histology; intracellular transport; lymphocyte; protein localization; protein transport; secretion; vesicle /vacuole

Upon antigen recognition, effector T lymphocytes secrete a variety of mediators that influence, creating a network of responsive cells. Most of these mediators are newly synthesized proteins that are immediately exported from the T cells. However, cytotoxic lymphocytes have a hallmark "regulated" secretory pathway in which pre-formed cytotoxic mediators (perforin and granzymes) are stored in cytoplasmic granules until antigen signaling triggers their rapid exocytosis. Another laboratory studying cloned cytotoxic T cells reported that the chemokine RANTES was also stored in cytotoxic granules. When we examined activation-induced RANTES secretion from purified memory and effector subpopulations of CD8+ human T cells from blood as well as short term cultured blasts, we found an early burst of preformed RANTES secretion with more rapid kinetics than the exocytosis of the lysosomal/cytotoxic granules. Deconvolution fluorescence microscopy revealed that intracellular RANTES is present in vesicles that do not colocalize with lysosomal markers, perforin or granzymes, a result confirmed by immunogold staining of ultrathin EM sections. Like these CD8+ lymphocytes, CD4+ blasts and NK cells also contain preformed RANTES in highly mobilizable non-lysosomal cytoplasmic vesicles, providing a rapid burst of local chemokine release that can recruit other inflammatory cells after antigen recognition. As a follow-up project, we have examined the intracellular localization and surface expression of the negative regulator CTLA-4 in T cell blasts and in the CD4+CD25+ "T regulatory" cells. This protein binds avidly to co-stimulatory molecules on antigen-presenting cells and negatively signals T cells, allowing for a termination of antigen-induced activation. In both types of T cells, deconvolution microscopy shows that CTLA-4 is present in vesicles that do not significantly colocalize with lysosomal or endosomal markers. In mouse lymph node CD4+CD25+ cells, activation-induced CTLA-4 surface expression is rapid, and resistant to protein synthesis inhibitors for the first hour. Using Rab27a-defective ashen mice that fail to exocytose lysosomal/cytotoxic granules after TcR crosslinking, this stimulation triggers normal CTLA-4 surface expression in both CD8+ T cell blasts and in the CD4+CD25+ "T regulatory" cells. The Rab27a-independent exocytosis of CTLA-4 indicates that intracellular CTLA-4 in T cells is stored in yet another distinct T cell compartment that undergoes rapid TcR-induced exocytosis.

在抗原识别后，效应T淋巴细胞分泌多种影响的介质，产生应答细胞的网络。这些介质中的大多数是新合成的蛋白质，它们立即从T细胞输出。然而，细胞毒性淋巴细胞具有标志性的“调节”分泌途径，其中预先形成的细胞毒性介质（穿孔素和颗粒酶）储存在细胞质颗粒中，直到抗原信号传导触发它们的快速胞吐作用。另一个研究克隆细胞毒性T细胞的实验室报告说，趋化因子RANTES也储存在细胞毒性颗粒中。当我们检查激活诱导的RANTES分泌从纯化的记忆和效应器亚群的CD 8+人T细胞从血液以及短期培养的母细胞，我们发现了一个更快的动力学比溶酶体/细胞毒性颗粒的胞吐作用的预先形成的RANTES分泌的早期爆发。去卷积荧光显微镜显示，细胞内RANTES存在于囊泡中，不与溶酶体标志物，穿孔素或颗粒酶共定位，结果证实了免疫金染色的ESPREEM切片。与这些CD 8+淋巴细胞一样，CD 4+母细胞和NK细胞也在高度可动员的非溶酶体胞质囊泡中含有预先形成的RANTES，提供了局部趋化因子释放的快速爆发，可以在抗原识别后招募其他炎性细胞。作为一个后续项目，我们已经检查了细胞内的定位和表面表达的负调节CTLA-4在T细胞母细胞和在CD 4 + CD 25+“T调节”细胞。这种蛋白质与抗原呈递细胞上的共刺激分子结合，并向T细胞发出负信号，从而终止抗原诱导的活化。在两种类型的T细胞中，去卷积显微镜显示CTLA-4存在于不与溶酶体或内体标记物显著共定位的囊泡中。在小鼠淋巴结CD 4 + CD 25+细胞中，活化诱导的CTLA-4表面表达是快速的，并且在第一个小时内对蛋白质合成抑制剂具有抗性。使用在TcR交联后不能胞吐溶酶体/细胞毒性颗粒的Rab 27 a缺陷型灰白色小鼠，这种刺激触发了CD 8 + T细胞母细胞和CD 4 + CD 25+“T调节”细胞中的正常CTLA-4表面表达。CTLA-4的Rab 27 a非依赖性胞吐表明T细胞中的细胞内CTLA-4储存在经历快速TcR诱导的胞吐的另一个不同的T细胞区室中。