Non-cytotoxic functions of lymphoycte granule exocytosis

在抗原识别后，效应T淋巴细胞分泌多种影响的介质，形成应答细胞网络。这些介质大多数是新合成的蛋白质，立即从T细胞中输出。然而，细胞毒性淋巴细胞有一个标志性的“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，直到抗原信号触发它们的快速胞吐。另一个研究克隆细胞毒性T细胞的实验室报告称，趋化因子RANTES也储存在细胞毒性颗粒中。当我们从血液中纯化的CD8+人T细胞的记忆亚群和效应亚群以及短期培养的母细胞中检测激活诱导的RANTES分泌时，我们发现预先形成的RANTES分泌的早期爆发具有比溶酶体/细胞毒性颗粒的胞吐更快的动力学。反褶积荧光显微镜显示细胞内RANTES存在于不与溶酶体标记物、穿孔蛋白或颗粒酶共定位的囊泡中，这一结果通过超薄电镜切片的免疫金染色证实。与这些CD8+淋巴细胞一样，CD4+母细胞和NK细胞也在高度可动员的非溶酶体细胞质囊泡中含有预先形成的RANTES，提供快速爆发的局部趋化因子释放，可以在抗原识别后招募其他炎症细胞。作为后续项目，我们研究了负调节因子CTLA-4在T细胞母细胞和CD4+CD25+“T调节”细胞中的细胞内定位和表面表达。该蛋白与抗原呈递细胞上的共刺激分子紧密结合，并向T细胞发出负信号，从而终止抗原诱导的激活。在这两种类型的T细胞中，反褶积显微镜显示CTLA-4存在于与溶酶体或内体标记物不明显共定位的囊泡中。在小鼠淋巴结CD4+CD25+细胞中，激活诱导的CTLA-4表面表达迅速，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交联后不能排出细胞溶酶体/细胞毒性颗粒的rab27a缺陷灰鼠，这种刺激在CD8+ T细胞母细胞和CD4+CD25+“T调节性”细胞中触发正常的CTLA-4表面表达。不依赖rab27a的CTLA-4胞吐表明，T细胞内的CTLA-4储存在另一个不同的T细胞室中，经历tcr诱导的快速胞吐。

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.