Immune Response to Cat: Regulatory and Effector T Cells

描述（由申请人提供）：了解T细胞在慢性过敏性炎症性皮肤病（特应性皮炎（AD））中的作用，与开发这种使人衰弱的疾病的特异性治疗方法高度相关。调节性T细胞（Tcells）通常抑制健康受试者中的促炎性T细胞。拟议的研究将调查AD患者的TdR是否真的有助于皮肤的炎症反应。主要目的如下：（1）检测Tcl 4转化为致病性T细胞的能力，所述致病性T细胞在暴露于在变应性炎症部位表达的因子时分泌Th 2细胞因子;（2）确定减少Th 2途径的治疗是否可以在体外增强保护性Tcl 4的诱导。低表达的CD 127（IL-7 R1链）将用于从AD患者中分离天然（CD 25 + CD 127 lo）和适应性（CD 25 + CD 127 lo）T细胞。数据表明，这些患者的天然Treg可以获得Th 2效应器特性，而产生IL-10的适应性Treg具有保护作用。在目标1中，研究将阐明不同CD 127 lo Treg类型的特性，比较它们对AD皮肤中表达的T细胞活化因子的易感性，并确定它们的致病性与保护性潜力。多色流式细胞术和标准体外T细胞测定将用于分析不同CD 127 lo Treg类型的表型和抑制功能。将使用流式细胞术成像检查CD 127 lo T细胞对AD皮肤中表达的Th 2促进因子（胸腺基质淋巴细胞生成素（TSLP）、过敏原或细菌超抗原）的反应性，以鉴定在单细胞水平经历有丝分裂的IL-4+ T细胞。参与Th 2极化和IL-4受体信号传导的转录因子（STAT 5和STAT 6）的作用将在TSLP介导的Treg活化和过敏原扩增该应答中进行评估。小干扰RNA将用于测试抑制CD 25 + CD 127 lo TcR中的Th 2信号传导分子是否可以阻断效应子功能并恢复抑制子活性。在目的2中，在过敏性炎症部位存在不同类型的CD 127 lo T细胞将首先在来自特应性斑贴试验（APT）部位的皮肤活检中确认。将在新的皮肤外植体测定中通过分析与来自APT部位的角质形成细胞一起培养的CD 25 + CD 127 lo TdR的Th 2效应物特性来测试TdR在这些部位转化为Th 2效应物的能力。在目的3中，目标是确定可用于增强诱导保护性T细胞的治疗策略。将使用诱导表达IL-10的T细胞的过敏原变体（H22-Fel d 1）来分析分泌IL-10的T细胞与其他CD 127 lo Treg类型的关系。将在体外扩增变应原特异性IL-10分泌型TCLs，以确定其保护特性并比较源自不同CD 127 lo前体的IL-10分泌型TCLs。将检测Th 2细胞因子在体外抑制产生IL-10的T细胞因子的诱导中的作用。在已知改善皮肤状况和减少Th 2途径的治疗期间，将离体监测这种Treg类型的诱导的变化。

DESCRIPTION (provided by applicant): Understanding the role that T cells play in the chronic allergic inflammatory skin disorder, atopic dermatitis (AD), is highly relevant to the development of specific treatments for this debilitating disease. Regulatory T cells (Tregs) normally suppress pro-inflammatory T cells in healthy subjects. The proposed studies will investigate whether Tregs from AD patients can actually contribute to inflammatory responses in the skin. The primary objectives are as follows: (1) To examine the capacity for Tregs to convert to pathogenic T cells which secrete Th2 cytokines upon exposure to factors expressed at the site of allergic inflammation; (2) To determine whether treatments which diminish Th2 pathways can enhance the induction of protective Tregs in vitro. Low expression of CD127 (IL-7R 1 chain) will be used to isolate natural (CD25+CD127lo) and adaptive (CD25negCD127lo) Tregs from AD patients. Data suggests that natural Tregs from these patients can acquire Th2 effector properties, while IL-10-producing adaptive Tregs are protective. In Aim 1, studies will elucidate the properties of distinct CD127lo Treg types, compare their susceptibility to T cell-activating factors expressed in AD skin, and determine their pathogenic versus protective potential. Multi-color flow cytometry and standard in vitro T cell assays will be used to analyze the phenotype and suppressive function of distinct CD127lo Treg types. Responsiveness of CD127lo T cells to Th2-promoting factors expressed in AD skin (thymic stromal lymphopoietin (TSLP), allergen, or bacterial superantigen) will be examined using flow cytometry imaging to identify IL-4+ T cells undergoing mitosis at the single-cell level. The role of transcription factors involved in Th2 polarization and IL-4 receptor signaling (STAT5 and STAT6) will be assessed in Treg activation mediated by TSLP and in amplification of this response by allergen. Small interfering RNAs will be used to test whether inhibiting Th2 signaling molecules in CD25+CD127lo Tregs can block effector function and restore suppressor activity. In Aim 2, the presence of distinct types of CD127lo Tregs at the site of allergic inflammation will first be confirmed in skin biopsies from atopy patch test (APT) sites. The capacity for Tregs to convert to Th2 effectors at these sites will be tested in a novel skin explant assay by analyzing the Th2 effector properties of CD25+CD127lo Tregs cultured with keratinocytes from APT sites. In Aim 3, the goal is to identify treatment strategies which could be used to enhance induction of protective Tregs. The relationship of IL-10-secreting Tregs to other CD127lo Treg types will be analyzed using an allergen variant (H22-Fel d 1) which induces IL-10-expressing T cells. Allergen-specific IL-10-secreting Tregs will be expanded in vitro in order to determine their protective properties and to compare IL-10-secreting Tregs derived from distinct CD127lo precursors. The role of Th2 cytokines in inhibiting induction of IL-10-producing Tregs in vitro will be examined. Changes in induction of this Treg type will be monitored ex vivo during treatments which are known to improve skin condition and to diminish Th2 pathways.