Presentation_1_Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies.PDF

<p>Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.</p

DataSheet_1_Basic characterization of antibodies targeting receptors of the tumor necrosis factor receptor superfamily.pdf

Many new immunotherapeutic approaches aim on the stimulatory targeting of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) using antibodies with intrinsic or conditional agonism. There is an initial need to characterize corresponding TNFRSF receptor (TNFR)-targeting antibodies with respect to affinity, ligand binding, receptor activation and the epitope recognized. Here, we report a collection of simple and matched protocols enabling the detailed investigation of these aspects by help of Gaussia princeps luciferase (GpL) fusion proteins and analysis of interleukin-8 (IL8) production as an easily measurable readout of TNFR activation. In a first step, the antibodies and antibody variants of interest are transiently expressed in human embryonal kidney 293 cells, either in non-modified form or as fusion proteins with GpL as a reporter domain. The supernatants containing the antibody-GpL fusion proteins can then be used without further purification in cell-free and/or cellular binding studies to determine affinity. Similarly, binding studies with mutated TNFR variants enable the characterization of the antibody binding site within the TNFR ectodomain. Furthermore, in cellular binding studies with GpL fusion proteins of soluble TNFL molecules, the ability of the non-modified antibody variants to interfere with TNFL-TNFR interaction can be analyzed. Last but not least, we describe a protocol to determine the intrinsic and the Fc gamma receptor (FcγR)-dependent agonism of anti-TNFR antibodies which exploits i) the capability of TNFRs to trigger IL8 production in tumor cell lines lacking expression of FcγRs and ii) vector- and FcγR-transfected cells, which produce no or only very low amounts of human IL8. The presented protocols only require standard molecular biological equipment, eukaryotic cell culture and plate readers for the quantification of luminescent and colorimetric signals.</p

Data_Sheet_1_TRAF2 Controls Death Receptor-Induced Caspase-8 Processing and Facilitates Proinflammatory Signaling.pdf

Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity.</p

TNC-scTNF_R2 induces neuroregeneration after H₂O₂-induced oxidative stress.

<p>(<b>A</b>) Differentiated LUHMES cells were incubated with different concentrations of hydrogen peroxide (H₂O₂) for one hour. Then cells were washed with medium und cultivated for additional 24 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations ± SEM). (<b>B–E</b>) LUHMES cells were stimulated with H₂O₂ (100 µM). After one hour the cells were washed with medium und regenerated for the indicated time intervals in medium with or without TNC-scTNF_R2 (100 ng/ml). (<b>B</b>) LUHMES cells were regenerated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). (<b>C</b>) Cells were regenerated for one or three hours, fixed with 4%PFA, permeabilized with 0.1% Triton-X100 and β-III-tubulin was detected with specific antibodies. Cell nuclei were visualized using DAPI. Pictures are projections of eight optical sections (0.4 µm; bar = 50 µm). (<b>D</b>) Number of cells was determined by counting the nuclei (DAPI staining). (<b>E</b>) Cells were regenerated for one hour and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). (D,E) At least 10 different image sections containing a minimum of 500 cells were used to determine the number of total and TUNEL-positive cells. *p values less than 0.05 (** p-value less than 0.001) were considered to be significant (n = 2, shown are the mean values ± SD).</p

TNC-scTNF_R2 induces TNFR2 signaling in R2 MEF.

<p>(<b>A–C</b>) R2 MEF were stimulated with TNC-scTNF_R2 (20 ng/ml) for the indicated times. (<b>A</b>) TNFR2 was immunoprecipitated using MR2-1 antibodies and protein G agarose. The precipitates were analyzed by immunoblot analysis using anti-huTNFR2 (HP9003) and anti-TRAF2 antibodies (NB = non-bound; B = bound). (<b>B</b>) Localization of NFκB p65 was visualized via immunofluorescence microscopy as shown in the upper panel and the number of nuclei showing NFκB translocation was quantified. At least 200 cells per experiment were analyzed (n = 3; shown are the mean values ± SEM of percent NFκB positive nuclei; bar = 20 µm). (<b>C</b>) Phospho-Akt (Ser473) levels in cell lysates were analyzed using immunoblot analysis. Akt was used as a loading control. Representative blot and bar graph show the quantification of the phospho-Akt (Ser473) band. *p values less than 0.05 versus untreated cells were considered to be significant (n = 3; shown are the mean values ± SEM).</p

Genetic engineering of the TNFR2-selective TNF muteins.

<p>(<b>A,B</b>) Schematic representation of the TNF variants used in this study. CT: cys-tag, HT: his-tag; TNF: huTNFR2-specific (D143N/A145R) TNF module (aa 80–233); L: GGGGS-linker; TNC: trimerization domain of human tenascin C (aa 110–139). (<b>C</b>) Coomassie staining and (<b>D</b>) immunoblot analysis of scTNF_R2 (1) and TNC-scTNF_R2 (2). Purified TNF variants were analyzed by 8% SDS-PAGE under reducing (r.) or non-reducing (n.r.) conditions and either stained with Coomassie or immunoblotted with anti-his-tag antibodies. (<b>E</b>) TNF muteins were analyzed by HPLC size exclusion chromatography using a BioSep-Sec-2000 column. Peak positions of relevant standard proteins are indicated (200 kDa; 67 kDa and 29 kDa).</p

TNC-scTNF_R2 induces formation of TNFR2 signaling complexes.

<p>(<b>A,B</b>) R2 MEF were transfected with pTRAF2-eGFP. After 24 hours, cells were incubated with or without 80M2 (1 µg/ml) for 5 minutes on ice and subsequently incubated with scTNF_R2 or TNC-scTNF_R2 (10 ng/ml) for 10 minutes at 37°C. Then cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and localization of huTNFR2 (<b>A</b>) or huTNF (<b>B</b>) was detected with specific antibodies and Alexa-Fluor546-labeled secondary antibodies. Cell nuclei were visualized using DAPI. Pictures are optical sections obtained by confocal fluorescence microscopy (bar = 10 µm). White arrows indicate areas of colocalization.</p

Bioactivity and receptor selectivity of the TNF muteins.

<p>Mouse embryonic fibroblasts (MEF) from TNFR1^−/−/TNFR2^−/− mice stably transfected with the chimeric receptors TNFR1-Fas or TNFR2-Fas (<b>A</b>) or Kym-1 cells (<b>B</b>) were stimulated with wildtype human TNF (huTNF; •), scTNF_R2 (▪) or TNC-scTNF_R2 (▴). Where indicated, MEF TNFR2-Fas were pretreated with the ligand/receptor stabilizing monoclonal antibody 80M2 (open symbols; 1 µg/ml; 30 min) before TNF treatment. Cell viability was determined by crystal violet staining after 24 hours (n = 3, shown are the mean values ± SEM).</p

TNC-scTNF_R2-mediated neuroregeneration is dependent on PI3K-PKB/Akt signaling.

<p>(<b>A</b>) LUHMES cells were stimulated with LY294002 (25 µM) for either 24 hours or for 1 hour. If stimulated for 1 hour, medium was exchanged to differentiation medium and cells were cultivated for further 23 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values ± SEM). (<b>B,C</b>) LUHMES cells were stimulated with hydrogen peroxide (H₂O₂; 100 µM) with or without LY294002 (25 µM). After one hour the cells were washed with medium und regenerated in medium with or without TNC-scTNF_R2 (100 ng/ml). (<b>B</b>) Cells were incubated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). (<b>C</b>) Cells were regenerated for one hour, fixed with 4% PFA and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). At least 5 different image sections containing a minimum of 250 cells were used to determine the number of total and TUNEL-positive cells (n = 3, shown are the mean values ± SEM). **p values less than 0.001 were considered to be significant.</p

TNC-scTNF_R2 protects neurons against catecholaminergic cell death.

<p>(<b>A</b>) Differentiated LUHMES cells were incubated with different concentrations of 6-hydroxydopamine (6-OHDA) for 20 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations ± SEM). (<b>B</b>) LUHMES cells were stimulated with 6-OHDA (32 µM). Cells were stimulated with TNC-scTNF_R2 (100 ng/ml) one, two or four hours after 6-OHDA addition and incubated for a total time period of 20 hours. Cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). *p values less than 0.05 (** p-value less than 0.001) were considered to be significant.</p