Identification and functional analysis of TNFR2-induced signaling complexes

• Applicant: Professor Dr. Harald Günther Wajant
• Subject Area: Cell Biology ; Biochemistry ; Immunology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 310944718

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.

• DFG Programme: Research Grants