Regulation of Th1 responses by IL-2 receptor blockade

描述（由申请人提供）： 针对高亲和力IL-2受体（IL-2 R）的α链（TAC/CD 25）的单克隆抗体疗法是移植和自身免疫性疾病中的日益增长的疗法。 然而，这种疗法可能限制免疫反应的机制尚未完全阐明。 在体外使用人源化抗TAC抗体，我们研究了IL-2 R阻断在调节Thl免疫应答中的作用。 我们的初步数据表明，HAT抑制从活化的外周血单核细胞（PBMC）培养物产生Thl效应细胞因子IFN-γ和中枢调节性Thl细胞因子IL-12。 我们假设CD 40配体（CD 40 L）/CD 40相互作用在HAT介导的IL-12依赖性Thl应答的抑制中起主要作用，并且显示活化的T细胞上的CD 40 L表达是双相的，其中HAT抑制晚期CD 40 L。 在SA#1中，我们将扩展这项研究，以确定CD 28共刺激和/或IL-2 R信号传导对幼稚和记忆T细胞上的CD 40 L的差异调节是否导致双相表达。 我们还将确定IL-2 R信号转导在调节IL-12 R表达和IL-12在T细胞中的反应性中的作用，因为IL-12在HAT存在下不能恢复IFN-γ。 在SA#2中，我们将关注IL-2 R阻断和/或CD 28共刺激调节CD 40 L基因表达的分子机制。 使用1.3Kb CD 40 L启动子报告基因构建体并瞬时转染Th 1细胞克隆，我们将测试推定的Stat 5结合元件以确定IL-2 R阻断是否调节这些位点的CD 40 L表达。 在SA#3中，我们假设HAT以IL-12非依赖性、细胞周期依赖性机制直接抑制T细胞产生IFN-γ。 为了测试这一点，我们将使用羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）标记和在经历细胞周期停滞的活化T细胞中的IFN-γ共染色来评估HAT对细胞周期进程的影响。 PI，John McDyer博士是一名初级教员，致力于学术医学的职业生涯，并了解抗TAC调节人类免疫反应的机制;这些知识将提高我们的理解 IL-2 R阻断在人T细胞应答和分化中的作用，并可能扩大HAT抗体疗法在治疗免疫介导的疾病中的作用。 该K 08将为致力于免疫学转化研究的研究生涯提供基础。

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.