Non-cytotoxic functions of lymphoycte granule exocytosis

淋巴细胞颗粒胞吐作用的非细胞毒性功能

• 批准号: 6557495
• 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/6557495
• 关键词: beta N acetylhexosaminidase; cell population study; chemokine; cytotoxic T lymphocyte; exocytosis; flow cytometry; granule; helper T lymphocyte; interferons; lymphocyte; secretion

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.

T 淋巴细胞通过分泌影响其他细胞的介质发挥作用。分泌通过两种途径发生：“组成型”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；以及“调节”途径，其中介质被储存在较大的颗粒中，直到 TcR 参与信号其胞吐作用。尽管在淋巴细胞中，受调节的途径仅与细胞毒性以及穿孔素和颗粒酶的分泌相关，但我们研究了其他介质是否通过该途径分泌，以及它是否在 CD4+ 和 CD8+ T 细胞中起作用。我们评估了颗粒胞吐作用对 CD8+ 和 CD4+ T 细胞母细胞分泌趋化因子的重要性。发现纯化的 CD4+ 和 CD8+ 人类 T 细胞母细胞亚群响应板结合的抗 CD3 快速分泌颗粒酶 b-己糖胺酶，表明 CD4+ T 细胞具有功能性颗粒样区室。我们测量了这些条件下趋化因子和 g-干扰素的分泌，利用对放线菌酮（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 小时的分泌对这些药物具有耐药性，而 TcR 诱导的 g-干扰素则被废除。在后来的时间里，组成型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。 CD4+ T 细胞中调节途径运作的进一步证据来自流式细胞术实验，表明溶酶体膜标记物 LAMP-1 和 LAMP-2 在静息 T 母细胞表面检测不到，但在 TcR 参与一小时内在 CD4+ 和 CD8+ 母细胞表面表达。