ENDOTHELIAL CELL INJURY BY CYTOLYTIC LYMPHOCYTES

溶细胞淋巴细胞对内皮细胞的损伤

• 批准号: 2901325
• 负责人: Mitzi Nagarkatti
• 金额: $ 19.44万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2901325
• 关键词: CD44 molecule; cell adhesion; cell cell interaction; chimeric proteins; cytolysins; cytotoxic T lymphocyte; cytotoxicity; gene expression; genetically modified animals; hyaluronate; interleukin 2; laboratory mouse; lymphokine activated killer cell; monoclonal antibody; pore forming protein; protein isoforms; transfection; tumor necrosis factor alpha; vascular endothelium

DESCRIPTION (Adapted from Investigator's Abstract): At sites of chronic inflammation induced during the course of a variety of infections, autoimmune diseases, allograft rejection, graft-versus-host disease and immunotherapy of cancer using interleukin-2 (IL-2), significant endothelial cell injury is known to occur, leading to pathogenesis of severe toxicity, the underlying reason for which remains unclear. Studies from this lab demonstrated that a variety of activated cytotoxic lymphocytes [cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, and double negative (DN) T cells] can mediate efficient MHC-unrestricted lysis of target cells, including the endothelial cells, when activated through CD44. Also transfer of CTL+IL-2 into irradiated mice could trigger endothelial cell injury and vascular leak syndrome (VLS). Moreover, they have also shown that perforin knockout (KO) and FasL-defective mice exhibit decrease IL-2-induced VLS. The current study is to delineate the mechanism of endothelial cell injury by using IL-2-induced VLS as a model. It will test the hypothesis that dysregulation of the interaction between activated CTL or LAK cells expressing CD44 and endothelial cells bearing the appropriate ligand such as hyaluronic acid (HA), could lead to endothelial cell lysis and induction of VLS. The role of CD44 in the induction of VLS will be investigated using CD44 KO mice. The question of whether such mice are resistant to VLS induction upon IL-2 administration and whether IL-2 activated CTL or LAK cells from these mice can trigger VLS will be addressed. The role played by CD44 variant isoforms in adhesion and cytotoxicity of endothelial cells will be studies using CD44-exon specific KO mice and transfection of isoform-specific constructs in CD44-deficient cytolytic lymphocytes. To further test the hypothesis that VLS results from direct endothelial cell lysis, the role of FasL, perforin and TNF in VLS induction will be studied using double-KO (FasL and perforin-deficient) and TNFR-KO mice. Lastly, based on the above results, attempts will be made to block the VLS induction in vivo using soluble CD44Rg fusion proteins and mAbs against CD44 variant isoforms. The role of CD44-hyaluraonate interactions in endothelial cell injury will be tested using a mutant fusion protein (CD44MutRg) or Fab fragments of anti-CD44 mAbs. Together, the studies should shed new lights on the basic mechanisms involved in cytotoxic-endothelial cell interactions leading to endothelial cell injury and possible therapeutic approaches to prevent this, in a variety of disease states, including immunotherapy of cancer.

描述（改编自研究者摘要）：在慢性病部位 各种感染过程中引起的炎症， 自身免疫性疾病、同种异体移植排斥、移植物抗宿主病和 使用白细胞介素 2 (IL-2) 进行癌症免疫治疗，显着改善内皮细胞 已知会发生细胞损伤，导致严重毒性的发病机制， 其根本原因尚不清楚。 该实验室的研究 证明多种活化的细胞毒性淋巴细胞[细胞毒性T 淋巴细胞 (CTL)、淋巴因子激活的杀伤细胞 (LAK) 和双 阴性（DN）T细胞]可以介导有效的MHC非限制性裂解 当通过 CD44 激活时，靶细胞，包括内皮细胞。 将 CTL+IL-2 转移到受辐射的小鼠体内也可以触发内皮细胞 细胞损伤和血管渗漏综合征（VLS）。 此外，他们还 表明穿孔素敲除 (KO) 和 FasL 缺陷小鼠表现出减少 IL-2 诱导的 VLS。 目前的研究旨在阐明其机制 使用IL-2诱导的VLS作为模型研究内皮细胞损伤。 它将测试 假设激活的 CTL 之间的相互作用失调 或表达CD44的LAK细胞和带有适当的内皮细胞 透明质酸（HA）等配体可能导致内皮细胞裂解 和VLS的诱导。 CD44 在 VLS 诱导中的作用是 使用 CD44 KO 小鼠进行研究。 问题是这样的老鼠是否是 IL-2 给药后对 VLS 诱导产生抗性以及 IL-2 是否 来自这些小鼠的激活的 CTL 或 LAK 细胞可以触发 VLS 已解决。 CD44 变体亚型在粘附和粘附中发挥的作用 将使用 CD44 外显子特异性研究内皮细胞的细胞毒性 CD44 缺陷型 KO 小鼠和异构体特异性构建体的转染 溶细胞淋巴细胞。 为了进一步检验 VLS 产生的假设 直接内皮细胞裂解，FasL、穿孔素和 TNF 在 VLS 中的作用 将使用双 KO（FasL 和穿孔素缺陷）研究诱导 TNFR-KO 小鼠。 最后，根据上述结果，将尝试 使用可溶性 CD44Rg 融合蛋白阻断体内 VLS 诱导， 针对 CD44 变体亚型的单克隆抗体。 CD44-透明质酸的作用 将使用突变融合来测试内皮细胞损伤中的相互作用 蛋白 (CD44MutRg) 或抗 CD44 mAb 的 Fab 片段。 总之，这些研究应该为基本机制提供新的线索 参与细胞毒性-内皮细胞相互作用，导致内皮细胞 细胞损伤和可能的治疗方法来预防这种情况 各种疾病状态，包括癌症的免疫疗法。