ENDOTHELIAL CELL INJURY BY CYTOLYTIC LYMPHOCYTES

溶细胞淋巴细胞对内皮细胞的损伤

• 批准号: 2625720
• 负责人: Mitzi Nagarkatti
• 金额: $ 15.35万
• 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/2625720
• 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 在各种感染过程中诱导的炎症， 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）的癌症免疫疗法，显著的内皮细胞 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。 这个实验室的研究 证明了各种活化的细胞毒性淋巴细胞[细胞毒性T细胞] 淋巴细胞（CTL），淋巴因子激活的杀伤（LAK）细胞，和双 阴性（DN）T细胞]可以介导有效的MHC-不受限制的裂解， 靶细胞，包括内皮细胞，当通过CD 44激活时。 同时，CTL+IL-2转染受照小鼠，可诱导受照小鼠血管内皮细胞增殖， 细胞损伤和血管渗漏综合征（VLS）。 此外，他们还 显示穿孔素敲除（KO）和FasL缺陷小鼠表现出降低 IL-2诱导的VLS。 目前的研究是阐明机制， 用IL-2诱导的VLS作为模型，观察内皮细胞损伤。 它将测试 激活的CTL之间相互作用的失调 或表达CD 44的LAK细胞和携带适当的CD 44的内皮细胞。 配体如透明质酸（HA）可导致内皮细胞溶解 和诱导VLS。 CD 44在VLS诱导中的作用将在下文中详细描述。 使用CD 44 KO小鼠研究。 这些老鼠是否 对IL-2给药后VLS诱导的抗性以及IL-2是否 来自这些小鼠的激活的CTL或LAK细胞可以触发VLS， 处理。 CD 44变体亚型在粘附和细胞增殖中的作用 内皮细胞的细胞毒性将使用CD 44外显子特异性 KO小鼠和同种型特异性构建体在CD 44缺陷型小鼠中的转染 溶细胞淋巴细胞 为了进一步检验VLS由以下原因引起的假设， 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，基于上述结果，将尝试 使用可溶性CD 44 Rg融合蛋白在体内阻断VLS诱导， 针对CD 44变体同种型的mAb。 CD 44透明质酸的作用 内皮细胞损伤中的相互作用将使用突变体融合蛋白进行测试。 蛋白（CD 44 MutRg）或抗CD 44 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， 细胞损伤和可能的治疗方法，以防止这一点，在一个 各种疾病状态，包括癌症的免疫治疗。