Mechanism of Lymphocyte-Mediated Cytotoxicity

淋巴细胞介导的细胞毒作用的颗粒吐出模型假定抗原引发的预先形成的细胞毒介质迅速分泌到细胞毒淋巴细胞与其结合靶细胞之间的突触样间隙中。公认的细胞毒介质是穿孔素和颗粒酶。然而，编码一种新的组织蛋白酶W的mRNA仅在NK和细胞毒性T淋巴细胞中表达，目前还没有关于其功能的有意义的现有信息。我们推测它可能是颗粒状细胞毒介质，如颗粒酶，或者是穿孔素加工酶。为了验证这些模型，我们在大肠杆菌中表达了人Proathepsin W，并制备了一系列针对它的单抗。在NK细胞和CTL提取物的Western blotting中，这些单抗中的一些与单一的25-30kD条带反应，在那里加工后的组织蛋白酶W具有催化活性。另一些单抗与原蛋白W反应，与45kD条带一致。使用这些单抗跟踪组织蛋白W激活后的命运，用PMA和离子霉素刺激人NK细胞株NK92以触发颗粒胞吐，在两种情况下都会导致组织蛋白W从细胞中耗尽并在3小时内释放到培养液中。当用抗CD3抗体处理人CD8+CTL时，也看到了类似的结果。在这两种情况下，CAT W释放与颗粒酶A释放相平行的脱颗粒。抗组织蛋白酶W单抗的荧光显微镜显示，加工蛋白在细胞质内以囊泡/颗粒的方式表达，而原组织蛋白酶W以更弥漫的方式表达，与内质网一致。 我们还在检测由表面标志物定义的人类血液T淋巴细胞的原始、记忆和效应亚群中颗粒胞吐的功能状态。这些研究表明，CD4+和CD8+T细胞的效应亚群表达颗粒标记最强，记忆亚群也表达大多数颗粒标记，但初治T细胞几乎不表达这些标记。当通过激活后CD107a的表面表达来评估颗粒的胞吐作用时，似乎只有效应亚群具有胞吐的能力。

The granule exocytosis model of lymphocyte-mediated cytotoxicity postulates an antigen-triggered rapid secretion of preformed cytotoxic mediators into the synapse-like space between the cytotoxic lymphocyte and its bound target. The generally accepted cytotoxic mediators are perforin and the granzyme proteases. However, mRNA encoding a novel cathepsin protease, cathepsin W, has been shown to be expressed exclusively in NK and cytotoxic T lymphocytes with no meaningful current information as to its function. We hypothesize that it is either a granule cytotoxic mediator like granzymes or that is a perforin processing enzyme. In order to test these models, we expressed human procathepsin W in E. coli and made a series of monoclonal antibodies against it. In Western blots of extracts of NK cells and CTL, some of these mAb react with a single 25-30kD band, where processed, catalytically active cathepsin W is expected. Other mAb react with a 45kD band, consistent with procathepsin W. Using these mAbs to follow the fate of cathepsin W after activation, stimulation of the human NK cell line NK92 with PMA and ionomycin to trigger granule exocytosis causes depletion of cathepsin W from the cells and its release into the medium within 3 hours in both cases. Similar results were seen when human CD8+ CTL were treated with anti-CD3. In both cases cat W release paralleled degranulation as measured by granzyme A release. Fluorescence microscopy with anti-cathepsin W mAbs show that the processed protein is expressed in a vesicular/granular pattern within the cytoplasm, while procathepsin W is seen in a more diffuse pattern consistent with endoplasmic reticulum. We are also examining the functional status of granule exocytosis in nave, memory, and effector subpopulations of human blood T lymphocytes phenotypically defined by surface markers. These studies show that effector subsets of both CD4+ and CD8+ T cells express granule markers most strongly, memory subsets are also positive for most granule markers, but nave T cells show negligible expression of these markers. When granule exocytosis is assessed by surface expression of CD107a after activation, only effector subsets appear to have the ability to exocytose.