Mechanism of Lymphocyte-Mediated Cytotoxicity

淋巴细胞介导的细胞毒性的颗粒胞吐模型假定抗原触发预先形成的细胞毒性介质快速分泌到细胞毒性淋巴细胞与其结合靶之间的突触样空间中。普遍接受的细胞毒性介质是穿孔素和丝氨酸蛋白酶的颗粒酶亚家族。编码巯基蛋白酶家族的新成员组织蛋白酶W的mRNA已显示仅在NK和细胞毒性T淋巴细胞中表达，表明在细胞毒性中的功能作用，尽管最近的一篇论文报道组织蛋白酶W敲除的淋巴细胞具有正常的细胞毒性活性。虽然对组织蛋白酶W的蛋白酶活性一无所知，但另一个研究小组已经报道该蛋白质定位于内质网。我们假设，组织蛋白酶W是一种颗粒细胞毒性介质与功能冗余的蛋白酶活性类似颗粒酶。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。用免疫印迹法检测NK细胞提取物和CTL细胞提取物，结果显示mAb与25- 30 kD的单一条带反应，其中预期有经处理的催化活性的组织蛋白酶W。其他mAb与45 kD条带反应，与组织蛋白原W一致。当用PMA和离子霉素刺激人NK 92细胞以触发颗粒胞吐时，组织蛋白酶W从细胞中耗尽并在3小时内释放到培养基中，并且当用抗CD 3处理人CD 8 + CTL时观察到类似的结果。在这两种情况下，如通过颗粒酶A释放所测量的，猫W释放脱颗粒。用抗组织蛋白酶W单克隆抗体对NK细胞进行荧光显微镜检查显示，成熟组织蛋白酶W在细胞质内以与穿孔素和颗粒酶A染色共定位的囊泡/颗粒模式表达，而组织蛋白酶原W以与内质网一致的更弥散的模式表达。目前，我们正在使用抗组织蛋白酶W单克隆抗体纯化成熟的酶从NK 92细胞，并允许其酶学properties的研究，我们已经研究了功能状态的颗粒胞吐作用的幼稚，记忆，和效应子亚群的人血T淋巴细胞表型定义的表面标记。当直接从血液中纯化并立即通过重定向细胞毒性测定时，仅CD 8+效应T细胞显示细胞毒性活性。在抗CD 3/抗CD 28交联后，记忆T细胞在培养48小时后变得具有细胞毒性，而原始细胞和记忆细胞在72小时后都具有细胞毒性。当通过流式细胞术分析固定和透化的血液淋巴细胞时，CD 8 + T细胞的效应子亚群表达颗粒标志物颗粒酶A和B以及穿孔素最强烈。在CD 8+效应记忆细胞中有有限的颗粒标志物表达，而其他CD 8 + T细胞亚群和所有CD 4+亚群对所有测试的颗粒标志物均为阴性。CD 4+和CD 8+原始细胞对所有颗粒标志物均呈阳性，两种细胞均显示活化诱导的溶酶体膜标志物CD 107 a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.