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。分泌通过两种途径发生：“组成性”途径，其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;和“调节性”途径，其中介质储存在较大颗粒中，直到TcR接合信号它们的胞吐作用。尽管在淋巴细胞中，调节途径仅与细胞毒性和穿孔素和颗粒酶的分泌相关，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CD 4+和CD 8 + T细胞中起作用。我们已经评估了颗粒胞吐对CD 8+和CD 4 + T细胞母细胞分泌趋化因子的重要性。发现纯化的CD 4+和CD 8+人T细胞母细胞亚群响应于板结合的抗CD 3而快速分泌颗粒酶b-己糖胺酶，表明CD 4 + T细胞具有功能性颗粒样隔室。我们测量了在这些条件下的趋化因子和g-干扰素的分泌，使用放线菌酮（其阻断蛋白质合成）和布雷菲德菌素A（其阻断高尔基体出口）的抗性来评估颗粒的贡献。在CD 4+和CD 8 + T细胞中，TcR诱导的RANTES和MIP-1a在2-4小时的分泌对这些药物具有抗性，而TcR诱导的g-干扰素被消除。在以后的时间，组成性途径主导趋化因子分泌。显微镜和流式细胞术显示RANTES存在于穿孔素阴性的CD 8+和CD 4 + T细胞母细胞的颗粒中。这些结果表明，颗粒在CD 8+和CD 4+效应T细胞中快速递送非细胞毒性介质。CD 4 + T细胞中调节途径的操作的进一步证据来自流式细胞术实验，其显示溶酶体膜标记物LAMP-1和LAMP-2在静息T母细胞的表面上不可检测，但在TcR接合的一小时内在CD 4+和CD 8+母细胞的表面上表达。

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.