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: 7292106
• 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/7292106
• 关键词: Non; cytotoxic; functions; lymphoycte; granule

Cell surface proteins control cell-cell interactions between lymphocytes, and the regulated expression of particular surface antigens is widely utilized to delineate lymphocyte differentiation. While most cell surface proteins are expressed on the outer cell membrane directly after biosynthesis, a minority are previously synthesized proteins stored in internal vesicles that undergo exocytosis in response to activating stimuli. This process is mechistically identical to secretion via granule exocytosis, a process we have described as central to cytotoxicity, and more recently, secretion of the chemokine RANTES. In activated T cells, CTLA-4 is found in intracellular vesicles that give rise to functionally active surface CTLA-4 after T cell ligation. Previous work had suggested colocalization of intracellular CTLA-4 and perforin in activated CD8+ T cells. We examined another CTLA-4 containing lymphocyte subset, the CD4+CD25+ "Treg" suppressor cells, to see whether its intracellular storage compartment was lysosomal. When permeabilized human and mouse Tregs were costained for CTLA-4 and lysosomal markers and examined by deconvolution microscopy, no significant costaining was observed in either species. CTLA-4 also costained minimally with the endosomal marker EEA-1 and with intracellular RANTES. In mouse lymph node CD4+CD25+ cells, activation-induced CTLA-4 surface expression is rapid, and resistant to protein synthesis inhibitors for the first hour. Using Rab27a-defective ashen mice that are defective in their ability to exocytose lysosomal/cytotoxic granules after TcR crosslinking, this stimulation nevertheless triggers normal CTLA-4 surface expression in both CD8+ T cell blasts and in the CD4+CD25+ "T regulatory" cells. The Rab27a-independent exocytosis of CTLA-4 indicates that intracellular CTLA-4 in T cells is stored in a third distinct T cell compartment (in addition to the cytotoxic granules and RANTES storage vesicles) that undergoes rapid TcR-induced exocytosis. One model to explain suppression assumes that Tregs conjugated to APC express surface CTLA-4 that sequesters B7 costimulator molecules on the APC. Using confocal microscopy and a TcR transgenic mouse system, we examined antigen-specific conjugates of APC and CD4+T cells with respect to B7-2 expression, comparing the CD4+CD25+ Treg conjugates with CD4+CD25- conjugates. B7-2 was evenly distributed on unconjugated APC and in conjugates with CD4+CD25- T cells, but APC conjugated to Treg had reduced B7-2 staining over the "back" side of the cells, and clearly intensified B7-2 staining in the junctional region of the conjugate. These results suggest that APC costimulator sequestration may be one mechanism for Treg function.

细胞表面蛋白控制淋巴细胞之间的细胞-细胞相互作用，并且特定表面抗原的调节表达被广泛用于描绘淋巴细胞分化。虽然大多数细胞表面蛋白质在生物合成后直接在细胞外膜上表达，但少数是先前合成的蛋白质，储存在内部囊泡中，其响应于激活刺激而进行胞吐。这一过程在机制上与通过颗粒胞吐的分泌相同，这是我们描述为细胞毒性的中心过程，最近，趋化因子RANTES的分泌。在活化的T细胞中，CTLA-4存在于细胞内囊泡中，其在T细胞连接后产生功能活性表面CTLA-4。以前的工作表明，在活化的CD 8 + T细胞中，细胞内CTLA-4和穿孔素共定位。我们检查了另一种含有CTLA-4的淋巴细胞亚群，即CD 4 + CD 25+“Treg”抑制细胞，以观察其细胞内储存区室是否为溶酶体。当透化的人和小鼠胸腺细胞的CTLA-4和溶酶体标志物共染色，并通过去卷积显微镜检查时，在两个物种中均未观察到显著的共染色。CTLA-4还与内体标记物EEA-1和细胞内RANTES最低程度共染色。在小鼠淋巴结CD 4 + CD 25+细胞中，活化诱导的CTLA-4表面表达是快速的，并且在第一个小时内对蛋白质合成抑制剂具有抗性。使用Rab 27 a缺陷型灰白色小鼠（其在TcR交联后外吞溶酶体/细胞毒性颗粒的能力有缺陷），这种刺激仍然触发了CD 8 + T细胞母细胞和CD 4 + CD 25+“T调节”细胞中的正常CTLA-4表面表达。CTLA-4的Rab 27 a非依赖性胞吐表明T细胞中的细胞内CTLA-4储存在经历快速TcR诱导的胞吐的第三个不同的T细胞隔室（除了细胞毒性颗粒和RANTES储存囊泡之外）中。一种解释抑制的模型假设与APC缀合的TdR表达表面CTLA-4，其在APC上螯合B7共刺激分子。使用共聚焦显微镜和TcR转基因小鼠系统，我们检查了APC和CD 4 +T细胞的抗原特异性缀合物对B7-2表达的影响，比较了CD 4 + CD 25 + Treg缀合物和CD 4 + CD 25-缀合物。B7-2均匀地分布在未缀合的APC上和与CD 4 + CD 25- T细胞的缀合物中，但是缀合至Treg的APC在细胞的“背”侧上具有减少的B7-2染色，并且在缀合物的接合区域中明显增强B7-2染色。这些结果表明APC共刺激分子隔离可能是Treg功能的一种机制。