Identification and functional analysis of TNFR2-induced signaling complexes

在到期的项目中，我们取得了以下发现和进展：i)我们在caspase抑制的巨噬细胞中证明了TNFR2通过激活细胞对内源性TNF/TNFR1的敏感性而诱导坏死性下垂，通过耗尽TRAF2与cIAP1或cIAP2的复合体来触发坏死性下垂。Ii)我们进一步确定了一条新的合作途径，在该途径中，TNFR2和TNFR1协同刺激RIPK1激酶活性依赖的基因诱导，该途径从RIPK3和MLKL上游的坏死性下垂信号中分叉出来。值得注意的是，TRAF1和A20是TNFR2和TNFR1信号复合体的两个组成部分，是这一途径的靶标。Iii)我们发现夏平拮抗肿瘤坏死因子受体2与肿瘤坏死因子受体1的协同作用。Iv)我们开发了多种新的TNFR2激动剂，并在他们的帮助下证明了仅刺激TNFR2就足以在体外和体内扩大Tregs并促进其抑制活性。值得注意的是，尽管TNFR2刺激巨噬细胞的深刻影响主要依赖于TNFR2调节TNFR1信号质量的能力，但到目前为止，TNFR2刺激对Tregs的影响被发现是独立于内源性TNF/TNFR1轴的。在这些成果的基础上，目前的项目提案追求以下目标：i)对新发现的TNFR2和caspase限制性的TNFR1诱导的RIPK1活性依赖的基因诱导信号通路进行后续分析。我们将在分子水平上阐明如何控制TNFR2这一途径。我们将进一步鉴定该通路的靶基因，并评估其在表达病毒或细菌caspase-8抑制剂的巨噬细胞中对肿瘤坏死因子和/或肿瘤坏死因子诱导的PAMPs的炎症反应的相关性。Ii)鉴定和鉴定巨噬细胞中独立于内源性肿瘤坏死因子-肿瘤坏死因子受体1轴的TNFR2活性和功能。因此，我们将分析缺乏肿瘤坏死因子和肿瘤坏死因子受体1的巨噬细胞与肿瘤坏死因子受体2诱导的信号事件及其与巨噬细胞存活和活性的相关性。Iii)评估Tregs和CD8+T细胞中的TNFR1-TNFR2串扰。我们将通过在存在和不存在caspase抑制的情况下进行全面的TNFR1/2共刺激实验来解决这个问题。4)肿瘤坏死因子的中长期活性是两种肿瘤坏死因子受体触发的多种反馈机制作用的综合结果。我们将评估这种作用于肿瘤坏死因子受体信号复合体水平的串扰机制。为此，我们将分析TNFR1和TNFR2启动对随后刺激的肿瘤坏死因子受体形成信号复合体的影响。特别是，我们将调查TRAF1、A20或未知因素是否与此类情景相关。

In the expired project, we made among others the following findings and progresses: i) We demonstrated in caspase-inhibited macrophages that TNFR2 enables necroptosis induction by sensitizing the cells for endogenous TNF/TNFR1-triggred necroptosis by depletion of complexes of TRAF2 with cIAP1 or cIAP2. ii) We identified furthermore a novel cooperative pathway in which TNFR2 and TNFR1 cooperate to stimulate RIPK1 kinase activity-dependent gene induction and which bifurcates from necroptosis signaling upstream of RIPK3 and MLKL. Notably, TRAF1 and A20, two components of the TNFR2 and TNFR1 signaling complexes, are targets of this pathway. iii) We found that sharpin antagonizes necroptotic TNFR2-TNFR1 cooperation. iv) We developed various novel TNFR2 agonists and demonstrated by their help that stimulation of TNFR2 alone is sufficient to expand Tregs in vitro and in vivo and to promote their suppressive activity. Notably, while the profound effects of selective TNFR2 stimulation in macrophages were mainly dependent on the ability of TNFR2 to modulate the quality of TNFR1 signaling, the effects of TNFR2 stimulation on Tregs were found to be so far independent from the endogenous TNF/TNFR1 axis. Based on these achievements the current project proposal pursues now the following objectives: i) Follow up analysis of the newly identified TNFR2- and caspase-restricted TNFR1-induced RIPK1 kinase activity-dependent gene inductive signaling pathway. We will clarify how controls TNFR2 this pathway at the molecular level. We will further identify target genes of this pathway and evaluate its relevance for the inflammatory response against TNF and/or TNF-inducing PAMPs in macrophages expressing viral or bacterial caspase-8 inhibitors. ii) Identification and characterization of TNFR2 activities and functions in macrophages which are independent from the endogenous TNF-TNFR1 axis. We will therefore analyze TNF- and TNFR1-deficient macrophages with respect to TNFR2-induced signaling events and their relevance for survival and activity of macrophages. iii) Evaluation of the TNFR1-TNFR2 crosstalk in Tregs and CD8+ T-cells. We will address this issue by comprehensive TNFR1/2 costimulation experiments in the presence and absence of caspase inhibition. iv) The mid- and long-term activities of TNF are the integrated result of the effects of various feed-back mechanisms triggered by the two TNF receptors. We will evaluate such crosstalk mechanisms which act on the level of the TNF receptor signaling complexes. For this purpose, we will analyze the effect of TNFR1 and TNFR2 priming on signaling complex formation by subsequently stimulated TNF receptors. Especially, we will investigate whether TRAF1, A20 or yet unknown factors are of relevance in such scenarios.