Role of SOCS in Regulation of Transplantation Tolerance

SOCS 在移植耐受调节中的作用

• 批准号: 6727883
• 负责人: Stanislaw M Stepkowski
• 金额: $ 29.7万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2005-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727883
• 关键词: CD40 molecule; apoptosis; biological signal transduction; cytokine; helper T lymphocyte; homologous transplantation; immune tolerance /unresponsiveness; immunoregulation; laboratory mouse; laboratory rat; mitogen activated protein kinase; protein protein interaction; transcription factor; transplant rejection

DESCRIPTION (provided by applicant): Although current immunosuppression has dramatically improved organ allograft survival, the majority of patients develop impaired graft function and chronic rejection. Therefore, the ultimate goal--to radically improve graft survival--is to induce transplantation tolerance of allografts without the need for continuous drug therapy. It became obvious that tolerance induction requires two phases, namely, initial deletion (apoptosis) of alloreactive T cells and generation of regulatory T (Treg) cells. We have induced tolerance by promoting apoptosis by anti-CD40 Ligand antibody alone or in combination with CTLA4Ig or apoptosis-inducing agent, WP744. In addition, donor cells or donor/recipient histocompatibility proteins may be added to the protocols to promote generation of interleukin (IL)-4- producing T helper (Th2)reg cells to test our hypothesis that tolerance depends upon the expansion of Th2reg cells with IL-4-driven Jak3/Stat5/Stat6 feedback loops. Function of Th2reg cells is positively controlled by Jak/Stat molecules and negatively controlled by SOCS family proteins, including suppressor of cytokine signaling (SOCS)1, SOCS2, SOCS3, SOCS5, cytokine inducible SH-2 containing protein (CIS)-1, and tyrosine phosphatase SH2 containing protein (Shp)-1. We have developed quantitative real-time PCR (QRT-PCR) method to measure SOCS mRNA expression. Our preliminary experiments showed that tolerance correlated with increased expression of SOCS3 mRNA. Therefore, we plan to examine the kinetics of SOCS expression during induction and maintenance of tolerance by QRT-PCR and Western blot (WB) methods. These results will be correlated with tyrosine phosphorylation (by immunoprecipitation [IP]/WB) and DNA binding (by electrophoretic mobility shift assay [EMSA]) of Stat5 and Stat6 during tolerance induction. We postulate that mice over-expressing SOCS3 should be easier to induce tolerance in comparison to normal or SOCS3-deficient mice. We plan to directly link IL-4-induced Stat5 and Stat6 activation with SOCS3 expression: the IL-4-responsive D10 T cell line will be co-transfected with Stat5- or Stat6-driven luciferase reporter gene and different SOCS. Such an approach will directly evaluate whether Stat5 or Stat6 activation is regulated by SOCS3 and/or other SOCS. These experiments will provide the first evidence for the role of SOCS in induction of transplantation tolerance.

描述（由申请人提供）： 尽管目前的免疫抑制已显着提高了器官同种异体移植物的存活率，但大多数患者会出现移植物功能受损和慢性排斥反应。因此，从根本上提高移植物存活率的最终目标是诱导同种异体移植物的移植耐受性，而不需要连续的药物治疗。很明显，耐受诱导需要两个阶段，即同种异体反应性 T 细胞的初始删除（凋亡）和调节性 T (Treg) 细胞的生成。我们通过单独使用抗 CD40 配体抗体或与 CTLA4Ig 或细胞凋亡诱导剂 WP744 组合促进细胞凋亡来诱导耐受。此外，可以将供体细胞或供体/受体组织相容性蛋白添加到方案中，以促进产生白细胞介素 (IL)-4 的 T 辅助 (Th2)reg 细胞的产生，以检验我们的假设，即耐受性取决于 Th2reg 细胞的扩增IL-4 驱动的 Jak3/Stat5/Stat6 反馈回路。 Th2reg 细胞的功能受 Jak/Stat 分子正向控制，并受 SOCS 家族蛋白负向控制，包括细胞因子信号传导抑制蛋白 (SOCS)1、SOCS2、SOCS3、SOCS5、含有细胞因子诱导性 SH-2 的蛋白 (CIS)-1 和酪氨酸磷酸酶SH2含有蛋白(Shp)-1。我们开发了实时定量PCR (QRT-PCR) 方法来测量SOCS mRNA 表达。我们的初步实验表明耐受性与 SOCS3 mRNA 表达增加相关。因此，我们计划通过 QRT-PCR 和蛋白质印迹 (WB) 方法检查在诱导和维持耐受过程中 SOCS 表达的动力学。这些结果将与耐受诱导期间 Stat5 和 Stat6 的酪氨酸磷酸化（通过免疫沉淀 [IP]/WB）和 DNA 结合（通过电泳迁移率变动测定 [EMSA]）相关。我们假设与正常或 SOCS3 缺陷的小鼠相比，过度表达 SOCS3 的小鼠应该更容易诱导耐受。我们计划将 IL-4 诱导的 Stat5 和 Stat6 激活与 SOCS3 表达直接联系起来：IL-4 响应的 D10 T 细胞系将​​与 Stat5 或 Stat6 驱动的荧光素酶报告基因和不同的 SOCS 共转染。这种方法将直接评估 Stat5 或 Stat6 激活是否受 SOCS3 和/或其他 SOCS 调节。这些实验将为 SOCS 在诱导移植耐受中的作用提供第一个证据。