Non-cytotoxic functions of lymphoycte granule exocytosis

效应性T淋巴细胞通过分泌影响其他细胞的介质来发挥作用。分泌通过两种途径发生：一种是“构成”途径，在这种途径中，新合成的蛋白质通过高尔基体，并通过小囊泡的胞吐作用立即释放；另一种是“调节”途径，即介质储存在较大的颗粒中，直到TCR参与释放它们的胞吐作用。在淋巴细胞中，调节途径仅与细胞毒性和穿孔素和颗粒酶的分泌(颗粒胞吐途径)有关。我们研究了用于分泌趋化因子的T细胞途径，特别是主要的T细胞产物RANTES。纯化的CD4+和CD8+T细胞在平板结合的抗CD3抗体刺激TCR后2小时内分泌RANTES。环己亚胺(阻止蛋白质合成)和Brefeldin A(阻止高尔基体出口)没有抑制作用，这与调控途径有关。在TCR触发这些药物后超过3小时的时候，这些药物会阻止分泌，表明转换到构成途径。对通透性细胞的流式细胞术分析表明，RANTES存在于CD8+和CD4+T细胞母细胞中，而胞浆RANTES在TCR交联后迅速丢失(比颗粒标记物快约6倍)。在共聚焦显微镜下，细胞内的RANTES以小泡的形式出现，不与溶酶体颗粒中的穿孔素和颗粒酶等蛋白质共存。去卷积显微镜的高分辨率技术在视觉上和对数字像素强度的统计分析中都证实了这种缺乏共定位的情况。超薄EM切片的免疫金染色法证实，RANTES存在于与颗粒形态不同的小泡中。因此，我们描述了一种新的调节T淋巴细胞分泌途径，能够在抗原识别后快速递送预先形成的趋化因子。

Effector T lymphocytes function by secretion of mediators which influence other cells. Secretion occurs via two pathways the "constitutive" pathway, in which newly synthesized proteins pass through the Golgi and are immediately released by exocytosis of small vesicles; and the "regulated" pathway in which mediators are stored in larger granules until TcR engagement signals their exocytosis. In lymphocytes the regulated pathway has been exclusively associated with cytotoxicity and the secretion of perforin and granzymes (granule exocytosis pathway). We investigated the T cell pathway used for chemokine secretion, particularly the major T cell product RANTES. Purified populations of both CD4+ and CD8+ human T cell blasts were found to secrete RANTES within two hours after TcR stimulation by plate-bound anti-CD3. The regulated pathway was implicated by the lack of inhibition by cycloheximide (which blocks protein synthesis) and Brefeldin A (which blocks Golgi export). At times greater than 3 hours after TcR triggering these drugs block secretion, indicating a switch to the constitutive pathway. Flow cytometry of permeabilized cells shows that RANTES is present in both CD8+ and CD4+ T cell blasts and that cytoplasmic RANTES is lost rapidly after TcR crosslinking (~ 6x faster than granule markers). By confocal microscopy intracellular RANTES appears as vesicles that do not colocalize with proteins such as perforin and granzymes in the lysosomal granules. This lack of colocalization was confirmed by the higher resolution technique of deconvolution microscopy, both visually and by statistical analysis of digital pixel intensities. Immunogold staining of ultrathin EM sections confirmed that RANTES is present in vesicles that are morphologically distinct from the granules. Thus we have described a novel regulated secretory pathway in T lymphocytes, capable of rapid delivery of preformed chemokines after antigen recognition.