Mechanism of Lymphocyte-Mediated Cytotoxicity

淋巴细胞介导的细胞毒性的颗粒胞吐模型假设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细胞之间的突触样空间。普遍接受的细胞毒性介质是穿孔蛋白和丝氨酸蛋白酶的颗粒酶亚家族。编码巯基蛋白酶家族新成员组织蛋白酶W的mRNA已被证明仅在NK和细胞毒性T淋巴细胞中表达，这表明其在细胞毒性中起功能作用，尽管最近的一篇论文报道，敲除组织蛋白酶W的淋巴细胞具有正常的细胞毒性活性。虽然组织蛋白酶W的活性尚不清楚，但另一组研究人员已报道该蛋白定位于内质网。我们假设组织蛋白酶W是一种颗粒细胞毒性介质，具有类似于颗粒酶的功能冗余蛋白酶活性。为了研究组织蛋白酶W，我们在大肠杆菌中表达了人原组织蛋白酶W，并制备了一系列针对它的单克隆抗体。NK细胞提取物和CTL在较小程度上显示单个25-30kD带的单抗反应性，其中加工的，催化活性的组织蛋白酶W有望。当PMA和离子霉素刺激人NK92细胞引发颗粒胞吐时，组织蛋白酶W在3小时内从细胞中被消耗并释放到培养基中，当用抗cd3处理人CD8+ CTL时也看到类似的结果。在这两种情况下，cat W释放与颗粒酶A释放平行。用抗组织蛋白酶W单克隆抗体对NK细胞进行荧光显微镜观察显示，成熟的组织蛋白酶W在细胞质内以囊泡/颗粒状表达，与穿孔素和颗粒酶a的染色共定位，而原组织蛋白酶W则以更弥散的形式表达，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细胞中纯化成熟酶，并对其酶学性质进行研究。我们研究了人类血液T淋巴细胞的中性、记忆和效应亚群中颗粒胞吐的功能状态，这些亚群的表型由表面标记物定义。当直接从血液中纯化并立即进行重定向细胞毒性检测时，只有CD8+效应T细胞表现出细胞毒性活性。经抗cd3 /抗cd28交联后，记忆T细胞在培养48小时后具有细胞毒性，而记忆T细胞在培养72小时后均具有细胞毒性。流式细胞术分析固定淋巴细胞和渗透淋巴细胞时，CD8+ T细胞的效应亚群表达颗粒标记物颗粒酶A、B和穿孔蛋白最强烈。CD8+效应记忆细胞中颗粒标志物表达有限，而其他CD8+ T细胞亚群和所有CD4+亚群对所有颗粒标志物检测均为阴性。CD4+和CD8+细胞对所有颗粒标记物均呈阳性，两种细胞均表现出活化诱导的溶酶体膜标记物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.