Functional modulation of regulatory T cells and analyses of the underlying mechanisms

调节性T细胞的功能调节及潜在机制分析

• 批准号: 445886458
• 负责人: Professor Dr. Alexander Steinkasserer
• 金额: --
• 依托单位: Immunmodulatorische Abteilung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/445886458?language=en
• 关键词: Functional; modulation; regulatory; cells; analyses

The overall aim of this project is the identification of new pathways to specifically modulate Treg differentiation and stability in the context of autoimmunity. We have shown that the specific loss of CD83 expression by Tregs, surprisingly leads to the downregulation of Treg-specific differentiation markers and the induction of an inflammatory profile. In addition, these Treg-specific conditional knockout mice showed aggravated autoimmunity and an impaired resolution of inflammation. Within this proposal we will focus on the following major aims:Aim #1: Are Treg specific TLR pathways modified by CD83 expression? Previously we showed that soluble CD83 modulates the TLR4 pathway by binding to MD2 and TLR4. Noteworthy, TLRs are expressed at much higher levels on Tregs when compared to e.g. with effector T cells. It was also shown that LPS binding to TLR2 induces Treg proliferation with a temporal loss of the suppressive function. It is still obscure how these effects are mechanistically regulated. Thus, using our CD83 cKO Treg mice we will investigate if CD83 expression modulates Treg function and stability via the MD2-TLR pathways by destabilizing factors such as IRAK1. Aim #2: Decipher new signaling pathways involved in CD83-dependent Treg modulation. With the help of gene arrays we identified several pathways potentially involved in CD83 dependent modulation of Treg cells. However, for a detailed mechanistic analyses signaling events must be investigated also on protein levels, including phosphorylation events. Thus, within aim 2 we will compare unstimulated and stimulated Treg cells derived from CD83 cKO mice by phospho-proteomics. This approach will enable us not only to confirm data derived from aim 1 but also to identify new pathways and involved networks. Aim #3: Identify and characterize newly elucidated CD83 modulated pathways in human Tregs. Two different approaches will be used: first, CD83 expression will be knocked down in human Tregs using siRNAs, and re-stimulated cells will be investigated regarding specific target genes which have been identified within aim 1 and 2, on RNA and protein levels. In addition we will knockdown CD83 in naïve human T cells, differentiate them into iTregs and analyze them again, in comparison to murine Tregs, on RNA and protein levels.Aim #4: Characterize CD83 impact on the establishment of different tissue resident Tregs. Very recently, it has been shown that non-lymphoid tissue (NLT) Tregs show different expression profiles and tissue adaptations. Thus, within this aim we will examine if CD83 is differentially expressed in different NLT Tregs compared to lymphoid Tregs. Interestingly, a recent study reported a higher CD83 expression in colonic Tregs when compared to skin Tregs. With the main focus on colonic Tregs and IBD we will investigate how NLT Tregs, derived from CD83 cKO mice, are influenced by the loss of CD83 expression.

该项目的总体目标是鉴定在自身免疫背景下特异性调节Treg分化和稳定性的新途径。我们已经证明，Treg表达的CD 83特异性丧失令人惊讶地导致Treg特异性分化标志物的下调并诱导炎症特征。此外，这些Treg特异性条件性敲除小鼠显示出加重的自身免疫和炎症消退受损。在这个提议中，我们将集中在以下主要目标：目标#1：Treg特异性TLR通路是否被CD 83表达修饰？以前我们发现可溶性CD 83通过与MD 2和TLR 4结合来调节TLR 4通路。值得注意的是，当与例如效应T细胞相比时，TLR以高得多的水平在T细胞上表达。还显示LPS与TLR 2结合诱导Treg增殖，同时暂时丧失抑制功能。这些影响是如何被机械地调节的仍然是模糊的。因此，使用我们的CD 83 cKO Treg小鼠，我们将研究CD 83表达是否通过不稳定因子如IRAK 1经由MD 2-TLR途径调节Treg功能和稳定性。目标#2：破译参与CD 83依赖性Treg调节的新信号通路。在基因阵列的帮助下，我们鉴定了可能参与Treg细胞的CD 83依赖性调节的几种途径。然而，为了进行详细的机理分析，还必须在蛋白质水平上研究信号传导事件，包括磷酸化事件。因此，在目标2中，我们将通过磷酸化蛋白质组学比较来源于CD 83 cKO小鼠的未刺激和刺激的Treg细胞。这种方法将使我们不仅能够确认来自aim 1的数据，而且能够识别新的途径和相关网络。目的#3：鉴定和表征人TCFs中新阐明的CD 83调节途径。将使用两种不同的方法：首先，将使用siRNA敲低人T细胞中的CD 83表达，并将在RNA和蛋白质水平上研究再刺激的细胞中已在目标1和2中鉴定的特定靶基因。此外，我们将敲低幼稚人T细胞中的CD 83，将其分化为iTclad，并在RNA和蛋白质水平上与小鼠Tclad进行比较，再次分析它们。最近，已经表明，非淋巴组织（NLT）THBG显示不同的表达谱和组织适应。因此，在这个目标内，我们将检查与淋巴样T细胞相比，CD 83是否在不同的NLT T细胞中差异表达。有趣的是，最近的一项研究报告了结肠Tumor中的CD 83表达高于皮肤Tumor。主要集中在结肠TCLs和IBD，我们将研究来自CD 83 cKO小鼠的NLT TCLs如何受到CD 83表达缺失的影响。