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。被普遍接受的细胞毒介质是穿孔素和丝氨酸蛋白酶的颗粒酶亚家族。组织蛋白酶W是硫醇蛋白水解酶家族中的一个新成员，其编码基因在NK和细胞毒性T淋巴细胞中均有表达，提示其在细胞毒作用中具有重要作用，尽管最近的一篇论文报道了组织蛋白酶W基因敲除的淋巴细胞具有正常的细胞毒活性。虽然对组织蛋白酶W的活性一无所知，但另一个研究小组已报道该蛋白定位于内质网。我们推测，组织蛋白酶W是一种颗粒细胞毒介质，具有与颗粒酶相似的功能冗余的蛋白酶活性。为了研究组织蛋白酶W，我们在大肠杆菌中表达了人组织蛋白酶原W，并制备了一系列抗组织蛋白酶W的单抗。NK细胞提取物和较小程度的CTL提取液的Western blotting显示mAb与单一的25-30kD条带反应，在那里加工，催化活性的组织蛋白酶W是预期的。当人NK92细胞被PMA和离子霉素刺激以触发颗粒吐出时，组织蛋白酶W在3小时内从细胞中排出并释放到培养液中，与抗CD3抗体处理人CD8+CTL的结果相似。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单抗对NK细胞的荧光显微镜观察显示，成熟的组织蛋白酶W在细胞质内呈泡状/颗粒状表达，与穿孔素和颗粒酶A染色共存，而原组织蛋白酶W以更弥漫的方式表达，与内质网一致。目前，我们正在使用抗组织蛋白酶W单抗从NK92细胞中纯化成熟的酶，并对其酶学性质进行研究。我们检测了由表面标志物定义的人类血液T淋巴细胞亚群中颗粒胞吐的功能状态。当直接从血液中纯化并立即进行重定向细胞毒活性测定时，只有CD8+效应T细胞具有细胞毒活性。经抗CD3/抗CD28交联后，48h后记忆性T细胞呈细胞毒作用，而NAVE和记忆性T细胞在72h后均呈细胞毒作用。用流式细胞仪分析固定和通透的外周血淋巴细胞时，CD8+T细胞的效应亚群表达颗粒标志物颗粒酶A、B和穿孔素的作用最强。CD8+效应记忆细胞仅有少量颗粒标记表达，其他CD8+T细胞亚群和所有CD4+亚群均为阴性。CD_4~+和CD_8~+细胞对所有颗粒标志均呈阳性，两种细胞均表现为活化诱导的溶酶体膜标志CD107a的表面表达。

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 subfamily of serine proteases. mRNA encoding a novel member of the thiol protease family, cathepsin W, has been shown to be expressed exclusively in NK and cytotoxic T lymphocytes, suggesting a functional role in cytotoxicity, although a recent paper reported that cathepsin W knockout lymphocytes have normal cytotoxic activity. While nothing is known about cathepsin W protease activity, the protein has been reported by another group to be localized in the endoplasmic reticulum,. We hypothesize that cathepsin W is a granule cytotoxic mediator with functionally redundant protease activity similar to granzymes. In order to study cathepsin W, we expressed human procathepsin W in E. coli and made a series of monoclonal antibodies against it. Western blots of extracts of NK cells and to a lesser extent CTL show mAb reactivity 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. When human NK92 cells were stimulated with PMA and ionomycin to trigger granule exocytosis, cathepsin W was depleted from the cells and released into the medium within 3 hours and 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 on NK cells with anti-cathepsin W mAbs show that mature cathepsin W is expressed within the cytoplasm in a vesicular/granular pattern colocalizing with stains for perforin and granzyme A, while procathepsin W is seen in a more diffuse pattern consistent with endoplasmic reticulum. Currently we are using anti-cathepsin W mAb to purify the mature enzyme from NK92 cells and allow study of its enzymatic properties.We have examined the functional status of granule exocytosis in nave, memory, and effector subpopulations of human blood T lymphocytes phenotypically defined by surface markers. When purified directly from blood and immediately assayed by redirected cytotoxicity, only CD8+ effector T cells showed cytotoxic activity. After anti-CD3/anti-CD28 crosslinking, memory T cells became cytotoxic after 48 hours of culture, while both nave and memory cells were cytotoxic after 72 hours. When fixed and permeabilized blood lymphocytes were analyzed by flow cytometry, the effector subset of CD8+ T cells expressed the granule markers granzyme A and B and perforin most strongly. There was limited granule marker expression in CD8+ effector memory cells, while other CD8+ T cell subsets and all CD4+ subsets were negative for all granule markers tested. Both CD4+ and CD8+ blasts were positive for all granule markers, and both cells showed activation-induced surface expression of the lysosomal membrane marker CD107a.