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疾病，特应性皮炎（AD）中发挥的作用，与开发这种使人衰弱的疾病的特异性治疗方法高度相关。在健康受试者中，调节性T细胞（Tregs）通常会抑制促炎T细胞。拟议的研究将调查来自AD患者的Tregs是否真的能促进皮肤的炎症反应。主要目的如下：(1)检测Tregs在暴露于过敏性炎症部位表达的因子时转化为分泌Th2细胞因子的致病性T细胞的能力；(2)确定抑制Th2通路的处理是否能增强体外保护性Tregs的诱导。低表达的CD127 （il - 7r1链）将用于从AD患者中分离天然（CD25+CD127lo）和适应性（CD25negCD127lo） treg。数据表明，来自这些患者的天然treg可以获得Th2效应特性，而产生il -10的适应性treg具有保护作用。在Aim 1中，研究将阐明不同CD127lo 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，并确定它们的致病与保护潜力。多色流式细胞术和标准体外T细胞检测将用于分析不同CD127lo Treg型的表型和抑制功能。CD127lo T细胞对AD皮肤中表达的th2促进因子（胸腺基质淋巴生成素（TSLP）、过敏原或细菌超抗原）的反应性将通过流式细胞术成像检测，以识别单细胞水平上进行有丝分裂的IL-4+ T细胞。参与Th2极化和IL-4受体信号传导（STAT5和STAT6）的转录因子在TSLP介导的Treg激活和过敏原对该反应的扩增中的作用将被评估。小干扰rna将用于测试抑制CD25+CD127lo Tregs中Th2信号分子是否可以阻断效应子功能并恢复抑制子活性。在Aim 2中，不同类型的CD127lo Tregs在过敏性炎症部位的存在将首先在特应性斑贴试验（APT）部位的皮肤活检中得到证实。通过分析来自APT位点的角质形成细胞培养的CD25+CD127lo treg的Th2效应特性，将在一种新的皮肤外植体实验中测试treg在这些位点转化为Th2效应物的能力。在Aim 3中，目标是确定可用于增强保护性treg诱导的治疗策略。分泌il -10的Tregs与其他CD127lo Treg类型的关系将使用诱导il -10表达T细胞的过敏原变体（H22-Fel d1）进行分析。过敏原特异性il -10分泌treg将在体外扩增，以确定其保护特性，并比较来自不同CD127lo前体的il -10分泌treg。我们将研究Th2细胞因子在体外抑制诱导产生il -10的Tregs中的作用。在治疗期间，将监测这种Treg类型诱导的变化，已知治疗可改善皮肤状况并减少Th2途径。人们普遍认识到T细胞在炎症中起重要作用，炎症是慢性过敏性皮肤病特应性皮炎（AD）的主要特征。尽管在确定调节性T细胞（Tregs）在过敏性疾病中的作用方面取得了相当大的进展，但我们的知识仍然存在重大差距。研究Tregs如何在AD皮肤中发挥作用的研究与基于T细胞的治疗这种使人衰弱的疾病的发展高度相关。

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. PUBLIC HEALTH RELEVANCE It is widely recognized that T cells play an important role in the inflammation that is a cardinal feature of the chronic allergic skin disease, atopic dermatitis (AD). Although considerable progress has been made in defining the role of regulatory T cells (Tregs) in allergic disease, there are still major gaps in our knowledge. The proposed studies, which investigate how Tregs function in AD skin, are highly relevant to the development of T cell-based treatments for this debilitating disease.