Non-cytotoxic functions of lymphocyte granule exocytosis

淋巴细胞颗粒胞吐作用的非细胞毒性功能

• 批准号: 6948361
• 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/6948361
• 关键词: T cell receptor; cell population study; chemokine; cytotoxic T lymphocyte; enzyme linked immunosorbent assay; exocytosis; flow cytometry; granule; helper T lymphocyte; histology; intracellular transport; lymphocyte; protein localization; protein transport; secretion; vesicle /vacuole

Effector T lymphocytes function by secretion of mediators which influence other cells, and most of these mediators such as cytokines are secreted via the "constitutive" pathway, in which newly synthesized proteins are immediately released by exocytosis of small vesicles. In the "regulated" secretory pathway mediators are stored in cytoplasmic granules until activation signals their exocytosis, and in lymphocytes this pathway has been exclusively associated with cytotoxicity. In cloned CTL, the chemokine RANTES had been reported to use the latter pathway and to be stored in cytotoxic granules. We examined activation-induced RANTES secretion from purified subpopulations of CD8+ human T cells from blood as well as short term cultured blasts, measuring supernatant RANTES by ELISA assay. CD8+ memory and effector blood cells as well as blasts secreted RANTES within 30 minutes, while other mediators such as interferon-g required hours to be detectable. The regulated pathway was implicated for RANTES secretion by the lack of inhibition by cycloheximide and Brefeldin A. 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 cytoplasmic vesicles in the memory and effector subpopulations of CD8+ blood T cells as well as blasts, but is undetectable in the nave subpopulation. By confocal microscopy intracellular RANTES appears as vesicles that do not colocalize with proteins such as perforin or granzymes, nor with endosomal or ER markers. 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. When stimulated by TcR crosslinking, the RANTES positive vesicles were depleted with a half-time of 20 minutes, while at this time less than 10% of the granzyme-positive granules were depleted. Thus we have described a novel regulated secretory pathway in CD8+ T lymphocytes, capable of rapid delivery of preformed RANTES after antigen recognition. We are currently assessing the role of the regulated secretory in CD4+ T cells.

效应T淋巴细胞通过分泌影响其他细胞的介质发挥功能，并且这些介质中的大多数如细胞因子通过“组成型”途径分泌，其中新合成的蛋白质通过小囊泡的胞吐作用立即释放。在“调节的”分泌途径中，介质储存在细胞质颗粒中，直到激活信号发出它们的胞吐作用，并且在淋巴细胞中，该途径仅与细胞毒性相关。在克隆的CTL中，已报道趋化因子RANTES使用后一种途径并储存在细胞毒性颗粒中。我们研究了活化诱导的RANTES分泌从纯化的亚群的CD 8+人T细胞从血液以及短期培养的母细胞，测量上清液RANTES的ELISA测定。CD 8+记忆和效应血细胞以及原始细胞在30分钟内分泌RANTES，而其他介质如干扰素-g需要数小时才能检测到。由于放线菌酮和布雷菲德菌素A缺乏抑制作用，因此调节途径与RANTES分泌有关。在TcR触发后超过3小时的时间，这些药物阻断分泌，表明向组成性途径的转换。透化细胞的流式细胞术显示，RANTES存在于CD 8+血液T细胞的记忆和效应子亚群以及原始细胞的细胞质囊泡中，但在幼稚细胞亚群中检测不到。通过共聚焦显微镜，细胞内RANTES表现为囊泡，其不与蛋白质如穿孔素或颗粒酶共定位，也不与内体或ER标记物共定位。这种缺乏共定位证实了更高的分辨率技术的去卷积显微镜，无论是视觉和数字像素强度的统计分析。免疫胶体金染色法证实RANTES存在于囊泡中，囊泡在形态上与颗粒不同。当通过TcR交联刺激时，RANTES阳性囊泡在20分钟的半衰期内耗尽，而此时少于10%的颗粒酶阳性颗粒被耗尽。因此，我们已经描述了一种新的调节分泌途径，在CD 8 + T淋巴细胞，能够快速交付预先形成的RANTES抗原识别后。我们目前正在评估CD 4 + T细胞中调节分泌的作用。