Identification and functional analysis of TNFR2-induced signaling complexes

在已到期的项目中，我们取得了以下发现和进展：i）我们在半胱天冬酶抑制的巨噬细胞中证明，TNFR 2通过耗尽TRAF 2与cIAP 1或cIAP 2的复合物而使细胞对内源性TNF/TNFR 1介导的坏死性凋亡敏感，从而能够诱导坏死性凋亡。ii）我们还鉴定了一种新的协同途径，其中TNFR 2和TNFR 1协同刺激RIPK 1激酶活性依赖性基因诱导，并且其从RIPK 3和MLKL上游的坏死性凋亡信号传导分叉。值得注意的是，TRAF 1和A20，TNFR 2和TNFR 1信号复合物的两个组分，是该途径的靶点。 iii）我们发现sharpin拮抗坏死性肿瘤坏死因子受体2-肿瘤坏死因子受体1的协同作用。iv）我们开发了各种新的TNFR 2激动剂，并通过它们的帮助证明了单独刺激TNFR 2足以在体外和体内扩增TcR并促进它们的抑制活性。值得注意的是，虽然选择性TNFR 2刺激在巨噬细胞中的深远影响主要取决于TNFR 2调节TNFR 1信号传导质量的能力，但TNFR 2刺激对TNF的影响迄今为止被发现与内源性TNF/TNFR 1轴无关。基于这些成就，本项目提案现在追求以下目标：i）对新鉴定的TNFR 2和半胱天冬酶限制性TNFR 1诱导的RIPK 1激酶活性依赖性基因诱导信号通路进行后续分析。我们将阐明如何在分子水平上控制TNFR 2这一途径。我们将进一步确定该途径的靶基因，并评估其与表达病毒或细菌caspase-8抑制剂的巨噬细胞中TNF和/或TNF诱导PAMP的炎症反应的相关性。ii）鉴定和表征巨噬细胞中独立于内源性TNF-TNFR 1轴的TNFR 2活性和功能。因此，我们将分析TNF-和TNFR 1缺陷的巨噬细胞的TNFR 2诱导的信号事件和它们的相关性的生存和活动的巨噬细胞。iii）评估T细胞和CD 8 + T细胞中的TNFR 1-TNFR 2串扰。我们将通过全面的TNFR 1/2共刺激实验在存在和不存在半胱天冬酶抑制剂的情况下解决这个问题。iv）TNF的中期和长期活性是由两种TNF受体触发的各种反馈机制作用的综合结果。我们将评估这样的串扰机制，作用于TNF受体信号复合物的水平。为此，我们将分析TNFR 1和TNFR 2引发对随后刺激的TNF受体形成信号复合物的影响。特别是，我们将调查TRAF 1，A20或未知因素是否与这种情况有关。

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.