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

该项目的总体目标是确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特异性CD83表达的缺失，令人惊讶地导致treg特异性分化标志物的下调和炎症谱的诱导。此外，这些treg特异性条件敲除小鼠表现出加重的自身免疫和炎症消退受损。在本提案中，我们将重点关注以下主要目标：目标#1:Treg特异性TLR通路是否被CD83表达修饰？之前我们发现可溶性CD83通过与MD2和TLR4结合来调节TLR4通路。值得注意的是，与效应T细胞相比，tlr在treg上的表达水平要高得多。研究还表明，LPS与TLR2结合可诱导Treg增殖，但抑制功能暂时丧失。目前尚不清楚这些效应是如何被机械调节的。因此，使用我们的CD83 cKO Treg小鼠，我们将研究CD83表达是否通过MD2-TLR途径通过IRAK1等不稳定因子调节Treg的功能和稳定性。目标2：破译cd83依赖性Treg调制中涉及的新信号通路。在基因阵列的帮助下，我们确定了几种可能参与CD83依赖性Treg细胞调节的途径。然而，为了进行详细的机制分析，还必须在蛋白质水平上研究信号事件，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自CD83 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从目标1中得到的数据，而且还能确定新的途径和相关网络。目标#3：鉴定和表征人类treg中新阐明的CD83调节通路。将使用两种不同的方法：首先，使用sirna敲除人类treg中的CD83表达，然后在目标1和目标2中确定的特定靶基因的RNA和蛋白质水平上对重新刺激的细胞进行研究。此外，我们将敲除naïve人类T细胞中的CD83，将其分化为iTregs，并再次分析它们，与小鼠treg相比，在RNA和蛋白质水平上。目的4：表征CD83对不同组织驻留treg建立的影响。最近，非淋巴组织（NLT） Tregs表现出不同的表达谱和组织适应性。因此，为了达到这个目的，我们将研究CD83在不同的NLT treg和淋巴样treg中的表达是否存在差异。有趣的是，最近的一项研究报告称，与皮肤treg相比，结肠treg中CD83的表达更高。主要关注结肠treg和IBD，我们将研究来自CD83 cKO小鼠的NLT treg如何受到CD83表达缺失的影响。

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.