Non-cytotoxic functions of lymphoycte granule exocytosis

细胞表面蛋白控制淋巴细胞之间的细胞间相互作用，而特定表面抗原的调节表达被广泛用于描述淋巴细胞的分化。虽然大多数细胞表面蛋白在生物合成后直接表达在细胞外膜上，但少数是先前合成的蛋白质，储存在内部小泡中，在激活刺激下经历胞吐。这个过程在机械上与通过颗粒胞吐分泌的过程相同，我们已经将这个过程描述为细胞毒性的中心过程，最近，趋化因子RANTES的分泌。在激活的T细胞中，CTLA-4存在于细胞内的小泡中，这些小泡在T细胞连接后产生具有功能活性的表面CTLA-4。以前的工作表明，在激活的CD8+T细胞中，CTLA-4和穿孔素在细胞内共存。我们检查了另一个含有CTLA-4的淋巴细胞亚群，即CD4+CD25+“Treg”抑制细胞，以确定其细胞内储存室是否为溶酶体。用CTLA-4和溶酶体标记对渗透性人和小鼠Tregs进行共染色，并在去卷积显微镜下观察，在这两个物种中都没有观察到显著的共染色。CTLA-4与内体标记EEA-1和细胞内RANTES也有极低程度的共染色。在小鼠淋巴结CD4+CD25+细胞中，活化诱导的CTLA-4表面表达迅速，且对蛋白质合成抑制剂耐受1小时。使用TCR交联物后排出溶酶体/细胞毒颗粒能力有缺陷的Rab27a缺陷的灰质小鼠，这种刺激仍能在CD8+T细胞母细胞和CD4+CD25+“T调节”细胞中触发正常的CTLA-4表面表达。CTLA-4的Rab27a非依赖性胞吐表明，T细胞内的CTLA-4储存在第三个不同的T细胞室(除了细胞毒颗粒和RANTES储存囊之外)，该T细胞经历了TCR诱导的快速胞吐。一种解释抑制的模型假设Tregs与APC结合，表达表面CTLA-4，它将B7共刺激分子隔离在APC上。利用共聚焦显微镜和TCR转基因小鼠系统，我们检测了APC和CD4+T细胞的抗原特异性结合物对B7-2的表达，比较了CD4+CD25+Treg结合物和CD4+CD25-结合物。B7-2均匀分布在未结合的APC和与CD4+CD25-T细胞结合的APC上，但与Treg结合的APC减少了细胞背面的B7-2染色，并明显增强了结合体交界区的B7-2染色。这些结果提示，APC共刺激分子的隔离可能是Treg功能的一种机制。

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.