Regulation of Th1 responses by IL-2 receptor blockade

IL-2 受体阻断调节 Th1 反应

• 批准号: 6597808
• 负责人: JOHN F MCDYER
• 金额: $ 13.15万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-08 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6597808
• 关键词: CD28 molecule; CD40 molecule; SDS polyacrylamide gel electrophoresis; binding sites; biological signal transduction; clinical research; cytokine receptors; enzyme linked immunosorbent assay; flow cytometry; gel mobility shift assay; gene expression; helper T lymphocyte; human subject; immunotherapy; interferon gamma; interleukin 12; interleukin 2; monoclonal antibody; polymerase chain reaction; receptor binding; site directed mutagenesis

DESCRIPTION (provided by applicant): Monoclonal antibody therapy directed at the alpha chain (TAC/CD25) of the high affinity IL-2 receptor (IL-2R) is a growing therapy in transplantation and autoimmune disease. However, the mechanisms by which this therapy may limit immune responses have not been fully elucidated. Using a humanized anti-TAC antibody in vitro, we investigated the role of IL-2R blockade in the regulation of Thl immune responses. Our preliminary data indicate HAT inhibits production of the Thl effector cytokine, IFN-y, and the central regulatory Thl cytokine, IL-12, from activated peripheral blood mononuclear cell (PBMC) cultures. We hypothesize CD40 ligand (CD40L)/CD40 interactions play a major role in HAT-mediated inhibition of IL-12- dependent Thl responses and show CD40L expression on activated T cells is biphasic, with HAT inhibiting late phase CD40L. In SA#1, we will extend this studies to determine whether differential regulation of CD40L on naive and memory T cells by CD28 costimulation and/or IL-2R signaling accounts for biphasic expression. We will also determine the role of IL-2R signaling in regulating IL-12R expression and IL-12 responsiveness in T cells, since IL-12 cannot restore IFN-gamma in the presence of HAT. In SA#2, we will focus on the molecular mechanisms by which IL-2R blockade and/or CD28 costimulation regulates CD40L gene expression. Using a 1.3Kb CD40L promoter reporter construct and transiently transfecting a Thl cell clone, we will test putative Stat5 binding elements to determine whether IL-2R blockade regulates CD40L expression at these sites. In SA#3, we hypothesize that HAT directly inhibits IFN-gamma production from T cells in an IL-12-independent, cell cycle-dependent mechanism. To test this, we will assess the effects of HAT on cell cycle progression, using carboxyfluorescin diacetate succinimidyl ester (CFSE)-Iabeling and co-staining for IFN-y in activated T cells subjected to cell cycle arrest at various stages. The PI, Dr. John McDyer is a junior faculty member and committed to a career in academic medicine and understanding the mechanisms through which anti-TAC .regulates human immune responses; this knowledge will improve our understanding of the role of IL-2R blockade in human T cell responses and differentiation and may expand the role for HAT antibody therapy in the treatment of immune-mediated diseases. This K08 will provide the foundation for a research career dedicated to translational studies in immunology.

描述(由申请人提供)： 针对高亲和力白介素2受体(IL-2R)α链(TAC/CD25)的单抗治疗在移植和自身免疫性疾病中是一种日益成熟的治疗方法。然而，这种疗法限制免疫反应的机制尚未完全阐明。使用体外人源化的抗TAC抗体，我们研究了IL-2R阻断在调节Th1免疫应答中的作用。我们的初步数据表明，HAT抑制激活的外周血单个核细胞(PBMC)培养中Th1效应细胞因子干扰素-γ和中央调节Th1细胞因子IL-12的产生。我们假设CD40配体(CD40L)/CD40相互作用在HAT介导的抑制IL-12依赖的THL反应中起主要作用，并表明活化的T细胞上CD40L的表达是双相的，HAT抑制晚期CD40L。在SA#1中，我们将扩展这项研究，以确定CD28共刺激和/或IL-2R信号对CD40L在初始T细胞和记忆T细胞上的差异调节是否解释了双相表达。我们还将确定IL-2R信号在调节T细胞中IL-12R表达和IL-12反应性中的作用，因为IL-12不能在HAT存在的情况下恢复干扰素-γ。在SA#2中，我们将重点介绍IL-2R阻断和/或CD28共刺激调节CD40L基因表达的分子机制。通过构建一个1.3Kb的CD40L启动子报告基因并瞬时转染THL细胞克隆，我们将测试可能的Stat5结合元件，以确定IL-2R阻断是否调节这些位点的CD40L表达。在SA#3中，我们假设HAT以一种IL-12非依赖的、细胞周期依赖的机制直接抑制T细胞产生干扰素-γ。为了验证这一点，我们将评估HAT对细胞周期进程的影响，使用羧基荧光素二乙酸酯琥珀酰亚胺酯(CFSE)标记和联合染色在不同阶段细胞周期停滞的激活T细胞中检测干扰素-γ。约翰·麦克代尔博士是一名初级教员，致力于学术医学和了解抗TAC调节人类免疫反应的机制；这一知识将提高我们的理解 IL-2R阻断在人类T细胞反应和分化中的作用，并可能扩大HAT抗体治疗在治疗免疫介导性疾病中的作用。这个K08将为致力于免疫学翻译研究的研究生涯奠定基础。