Non-cytotoxic functions of lymphoycte granule exocytosis

T淋巴细胞通过分泌影响其他细胞的介质发挥功能。分泌通过两条途径发生：“构成”途径，其中新合成的蛋白质通过高尔基体并立即通过小囊泡的胞吐释放；以及“受调节”的途径，在这种途径中，介质储存在较大的颗粒中，直到TcR参与发出它们的胞吐信号。虽然在淋巴细胞中，受调节的途径只与细胞毒性和穿孔素和颗粒酶的分泌有关，但我们已经研究了是否通过该途径分泌其他介质，以及它是否在CD4+和CD8+ T细胞中都起作用。我们已经评估了颗粒胞吐对CD8+和CD4+ T细胞母细胞分泌趋化因子的重要性。纯化后的CD4+和CD8+人T细胞原细胞亚群均能快速分泌颗粒酶b-己糖氨基酶，以响应板结合抗cd3，这表明CD4+ T细胞具有功能性颗粒样隔室。在这些条件下，我们测量了趋化因子和g-干扰素的分泌，使用对环己亚胺（阻断蛋白质合成）和Brefeldin A（阻断高尔基体输出）的抗性来评估颗粒的贡献。在CD4+和CD8+ T细胞中，tcr诱导的RANTES和MIP-1a在2-4小时时对这些药物产生耐药性，而tcr诱导的g-干扰素被消除。后来，构成途径主导趋化因子的分泌。显微镜和流式细胞术显示，RANTES存在于穿孔素阴性CD8+和CD4+ T细胞母细胞的颗粒中。这些结果表明，颗粒在CD8+和CD4+效应T细胞中快速递送非细胞毒性介质。流式细胞术实验进一步证明了CD4+ T细胞中调控通路的运作，表明溶酶体膜标记物LAMP-1和LAMP-2在静止T细胞表面检测不到，但在TcR作用1小时内，CD4+和CD8+细胞表面均表达。

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. Although in lymphocytes the regulated pathway has been exclusively associated with cytotoxicity and the secretion of perforin and granzymes, we have investigated whether other mediators are secreted via this pathway, and if it operates in both CD4+ and CD8+ T cells. We have evaluated the importance of granule exocytosis to chemokine secretion by both CD8+ and CD4+ T cell blasts. Purified subpopulations of both CD4+ and CD8+ human T cell blasts were found to rapidly secrete the granule enzyme b-hexosaminidase in response to plate-bound anti-CD3, suggesting that CD4+ T cells have a functional granule-like compartment. We measured the secretion of chemokines and g-interferon under these conditions, using resistance to cycloheximide (which blocks protein synthesis) and Brefeldin A (which blocks Golgi export) to assess the contribution of granules. In both CD4+ and CD8+ T cells, TcR-induced secretion of RANTES and MIP-1a at 2-4 hours was resistant to these drugs, while TcR-induced g-interferon was abolished. At later times, the constitutive pathway dominates chemokine secretion. Microscopy and flow cytometry show that RANTES is present in granules in perforin-negative CD8+ and CD4+ T cell blasts. These results demonstrate that granules rapidly deliver non-cytotoxic mediators in both CD8+ and CD4+ effector T cells. Further evidence for operation of the regulated pathway in CD4+ T cells comes from flow cytometry experiments showing that the lysosomal membrane markers LAMP-1 and LAMP-2 are undetectable on the surface of resting T blasts, but are expressed on the surface of both CD4+ and CD8+ blasts within an hour of TcR engagement.