Non-cytotoxic functions of lymphoycte granule exocytosis

在抗原识别后，效应性T淋巴细胞分泌各种影响因子，形成反应细胞网络。这些介质中的大多数是新合成的蛋白质，可以立即从T细胞输出。然而，细胞毒性淋巴细胞有一个标志性的“调节”分泌途径，在这个途径中，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。另一家研究克隆细胞毒性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。当我们检测从血液和短期培养的人T细胞中纯化的记忆和效应亚群的激活诱导的RANTES分泌时，我们发现预先形成的RANTES分泌早期爆发，比溶酶体/细胞毒颗粒的胞吞作用更快。去卷积荧光显微镜显示，细胞内RANTES存在于不与溶酶体标记物、穿孔素或颗粒酶共定位的囊泡中，这一结果得到了超薄EM切片的免疫金标染色的证实。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胞浆囊泡中含有预先形成的RANTES，提供快速的局部趋化因子释放，在抗原识别后可以招募其他炎症细胞。作为后续项目，我们研究了负调控因子CTLA-4在T细胞母细胞和CD4CD25“T调节”细胞中的细胞内定位和表面表达。这种蛋白与抗原呈递细胞上的共刺激分子强烈结合，并向T细胞发出负面信号，从而终止了抗原诱导的激活。在这两种类型的T细胞中，去卷积显微镜显示CTLA-4存在于与溶酶体或内体标记不显著共定位的囊泡中。在小鼠淋巴结CD4 CD25细胞中，激活诱导的CTLA-4表面表达迅速，并对蛋白质合成抑制剂耐受1小时。使用TCR交联后不能排出溶酶体/细胞毒颗粒的Rab27a缺陷灰白小鼠，这种刺激在CD8T细胞母细胞和CD4CD25“T调节”细胞中触发正常的CTLA-4表面表达。CTLA-4的Rab27a非依赖性胞吐作用表明，T细胞内的CTLA-4储存在经历TCR诱导的快速胞吐的另一个不同的T细胞隔室中。

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.