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）： 虽然目前的免疫抑制已显着提高器官移植存活率，大多数患者发展受损的移植功能和慢性排斥反应。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，而不需要持续的药物治疗。很明显，耐受诱导需要两个阶段，即同种异体反应性T细胞的初始缺失（凋亡）和调节性T（Treg）细胞的产生。我们已经通过单独的抗CD 40配体抗体或与CTLA 4 Ig或凋亡诱导剂WP 744组合促进凋亡来诱导耐受。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介素（IL）-4的辅助性T（Th 2）reg细胞的产生，以测试我们的假设，即耐受性取决于具有IL-4驱动的Jak 3/Stat 5/Stat 6反馈环的Th 2 reg细胞的扩增。Th 2 reg细胞的功能受Jak/Stat分子的正控制，并受SOCS家族蛋白的负控制，包括细胞因子信号传导抑制因子（SOCS）1、SOCS 2、SOCS 3、SOCS 5、含细胞因子诱导SH-2的蛋白（CIS）-1和含酪氨酸磷酸酶SH 2的蛋白（Shp）-1。我们建立了实时荧光定量PCR（QRT-PCR）方法来测量SOCS mRNA的表达。我们的初步实验表明，耐受性与SOCS 3 mRNA表达的增加相关。因此，我们计划通过QRT-PCR和Western blot（WB）方法检测SOCS表达在诱导和维持耐受过程中的动力学。这些结果将与耐受诱导期间Stat 5和Stat 6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。我们推测，与正常或SOCS 3缺陷小鼠相比，过表达SOCS 3的小鼠应该更容易诱导耐受。我们计划将IL-4诱导的Stat 5和Stat 6活化与SOCS 3表达直接联系起来：IL-4应答性D10 T细胞系将与Stat 5或Stat 6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Stat 5或Stat 6激活是否受SOCS 3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。