Neuroregeneration by a TNFR2 Agonist Figure 2. Bioactivity and receptor selectivity of the TNF muteins. Mouse embryonic fibroblasts (MEF) from TNFR12/2/ TNFR22/2 mice stably transfected with the chimeric receptors TNFR1- Fas or TNFR2-Fas (A) or Kym-1 cells (B) were stimulated with wildtype human TNF (huTNF; N), scTNFR2 (&) or TNC-scTNFR2 (m). Where indicated, MEF TNFR2-Fas were pretreated with the ligand/receptor stabilizing monoclonal antibody 80M2 (open symbols; 1 mg/ml; 30 min) before TNF treatment. Cell viability was determined by crystal violet staining after 24 hours (n = 3, shown are the mean values 6 SEM). doi:10.1371/journal.pone.0027621.g002 rhabdomyosarcoma cell line Kym-1 (Fig. 2B), which endogenously expresses both TNF receptors and is highly sensitive to TNF- induced cytotoxicity [22]. Using near to saturating concentrations (10 ng/ml), where both scTNFR2 and TNC-scTNFR2 showed maximum bioactivity in a long term bioassay (induction of cell death in Kym-1 cells as shown in 2B), we further investigated the kinetics of NFkB p65 nuclear translocation (Fig. 3). Whereas scTNFR2 stimulation only resulted in a significant translocation after 60 minutes with 32% of the cells showing nuclear NFkB, TNC-scTNFR2 stimulation resulted in a rapid NFkB translocation with an apparent peak at 30 minutes (85% NFkB+ cells), which was maintained at a high level for at least one hour (60% NFkB+ cells), further demonstrating the superior bioactivity of TNC-scTNFR2. Figure 3. Comparison of the NFkB translocation after stimula- tion with scTNFR2 or TNC-scTNFR2. Kym-1 cells were stimulated with scTNFR2 or TNC-scTNFR2 (10 ng/ml) for 10, 30 or 60 minutes. Cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100 and stained with anti-NFkB p65 antibodies and Alexa-Fluor546-labeled secondary antibodies. Cell nuclei were visualized using DAPI. Shown are representative images at point of time 30 minutes (upper panel) and the quantification of the number of NFkB p65-positive nuclei. At least 200 cells were analyzed per condition. *p values less than 0.01 versus untreated controls were considered to be significant (n = 2; shown are the mean values 6 SD; bar = 20 mm). doi:10.1371/journal.pone.0027621.g003 TNC-scTNFR2 induces clustering of TNFR2 To further analyze the bioactivity of TNC-scTNFR2 we used the human TNFR2 (huTNFR2) expressing cell line R2 MEF [23]. We recently demonstrated in these cells that upon activation of TNFR2 by soluble TNF in combination with 80M2, the adaptor protein TRAF2 is recruited to the receptor and the classical NFkB pathway is activated [23]. Here we demonstrate that stimulation of R2 MEF with TNC-scTNFR2 results in co-immunoprecipitation of TRAF2 with TNFR2 (Fig. 4A) and activation of the classical NFkB pathway (Fig. 4B). In addition, the PI3K-PKB/Akt pathway was activated, as evident by the increased phosphoryla- tion level of PKB/Akt after TNC-scTNFR2 stimulation (Fig. 4C). The enhanced activation of NFkB (Fig. 3) suggests a more efficient and/or faster formation of TNFR2 signaling complexes by TNC-scTNFR2. To confirm this, we investigated the ligand- induced recruitment of TRAF2, the essential cytosolic signal transducer of TNFR2, to the membrane using immunofluores- cence analysis of a TRAF2-eGFP reporter (Fig. 5). Unstimulated PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27621