Journal: Nature Communications

Article Title: Modeling heterogeneous signaling dynamics of macrophages reveals principles of information transmission in stimulus responses

doi: 10.1038/s41467-025-60901-3

Figure Lengend Snippet: A Schematic of the NFκB signaling network. Extrinsic noise is represented by parameter variation in the Receptor, Adaptor, and Core modules. B Workflow for identifying information loss by denoising individual signaling modules. This involves collapsing the distribution of the respective parameters to a single value. C Maximum mutual information (Maximum MI) between the ligands and NFκB signaling codons for the original network and after denoising the Receptor (rcp), Adaptor (adp), or Core (core) modules (labeled at the bottom). MI is calculated five times per condition, shown as individual data points. Data are presented as mean values ±SD. D Maximum MI of the network under no noise, noise added to the Receptor module (rcp), Adaptor module (adp), or core NFκB-IκBα module (core) (labeled at the bottom). The no-noise condition is based on calculations from 1000 identical cells. MI is calculated five times per condition, shown as individual data points and as mean values ±SD. E Illustration of the successive information loss along the pathway due to noise in indicated modules. Blue shade represents information flow, gray shade represents information loss. F Bar plots of average coefficient of variation (CV) of Receptor (TNF, LPS, CpG, pIC, and Pam), Adaptor (adp), and Core (core) module parameters, calculated from 10 permutation-sampled datasets, shown as data points and as mean values ±SD. ‘r-syn’ is the receptor synthesis rates (k54 for TNFR, k68 for TLR1/2, k85 for TLR9, k35 for TLR4, k77 for TLR3); ‘C-deg’ is the signaling complex degradation rate (k58, k61, and k64 for TNF module, k75 for Pam module, k44 for LPS module, k83 for pIC module); ‘endo’ is the endosomal import rate (k88 for CpG module, k36 and k40 for LPS module, k79 for pIC module); ‘TAK-ac is the TAK1 activation rate (k52 and k65); ‘time-d1’ and ‘time-d2’ is the time delay parameters in NFκB mediated transcription of IκBα (k99 and k101); NFκBtot is the total initial NFκB abundance within single cell. Colored bars indicate receptor modules, gray bars indicate adaptor and core modules.

Article Snippet: To block paracrine feed-forward TNF signaling during PAMP responses, we co-treated the cells with 5 μg/mL of soluble TNF receptor II (Recombinant Mouse sTNFRII/TNFRSF1B, 426-R2-050, R&D Systems).

Techniques: Labeling, Activation Assay

Journal: Immunity

Article Title: Six distinct NFκB signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses

doi: 10.1016/j.immuni.2021.04.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant Mouse sTNFRII/TNFRSF1B , R & D Systems , 426-R2–050.

Techniques: Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Software, Cell Tracking Assay, Flow Cytometry

Journal: Journal of immunological methods

Article Title: Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

doi: 10.1016/j.jim.2021.113173

Figure Lengend Snippet: Fig. 1. Characterization of anti-mouse TNFR2 antibodies in vitro. (A and B) mTNFR2 stable transfected CHO-K1 cells were incubated with 3-fold increasing con centrations of each rat IgG2a mAbs, and binding was detected by flow cytometry assessing TNFR2 + population percentage (A) and gMFI (B). (C) TNFα ligand competition with generated antibodies assessed by FACS. Data represented as a three-parameter gMFI dose-response curve fit of the blocker antibodies with appropriate controls incubations with 3-fold increasing concentrations. Two benchmark hamster-anti-mTNFR2 antibodies with known blocking activity were added as controls. All data based on mean and SEM is representative of three independent experiments.

Article Snippet: First, a single estimation screening of KD value was performed with an expected saturating concentration of 100 nM His tagged mTNFR2 (R&D Systems, 426-R2/CF) 100 nM diluted in 10× Kinetics Buffer (KB) followed by a dissociation step.

Techniques: In Vitro, Transfection, Incubation, Binding Assay, Flow Cytometry, Generated, Blocking Assay, Activity Assay

Journal: Journal of immunological methods

Article Title: Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

doi: 10.1016/j.jim.2021.113173

Figure Lengend Snippet: Fig. 2. Characterization of anti-mTNFR2 mAbs targeting CRDs 1–4. (A) Schematic representation of the 6 mouse-human TNF2 chimeras CRD1-CRD4 (Cystein Rich Domain). (B) The targeting CRD of each mAb were determined by cell ELISA with mouse-human TNFR2 domain swap mutants. Data represented as a three-parameter OD450–620 detection based on mean and SD of three independent experiments. (C) The domain epitopes of the 13 mAbs are indicated on a hTNFR2-hTNFα trimer structure (PDB: 3ALQ), 74% similar to mouse TNFR2. The CRDs for one TNFR2 receptor are shown in indicated colors.

Article Snippet: First, a single estimation screening of KD value was performed with an expected saturating concentration of 100 nM His tagged mTNFR2 (R&D Systems, 426-R2/CF) 100 nM diluted in 10× Kinetics Buffer (KB) followed by a dissociation step.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunological methods

Article Title: Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

doi: 10.1016/j.jim.2021.113173

Figure Lengend Snippet: Fig. 3. Characterization of anti-mouse TNFR2 antibodies with ex vivo material. (A) TNFR2 expression upon binding of anti-mTNFR2 antibodies to Treg cell pop ulation. Detection by commercial hamster anti-TNFR2 direct labeled with PE with the respective hamster-isotype control labeled with PE (left). Generated rat anti- mTNFR2 antibodies and a rat isotype control were detected by a secondary antibody anti-rat AF647 label (right). Gating strategy shown in (Sup. Fig. 2 A) The isotype control has been overlaid in each anti-mTNFR2 antibody histogram represented with % of max. Data representative of two independent experiments. (B) TNFR2 expression upon binding of anti-mTNFR2 antibodies to activated CD8+ cells. Detection by commercial hamster anti-TNFR2 direct labeled with PE with the respective hamster-isotype control labeled with PE (left). Generated rat anti-mTNFR2 antibodies and a rat isotype control were detected by a secondary antibody anti-rat PE label (right). Gating strategy not shown. Gating strategy for CD8+ population was done on unstained OT1 activated cells. First, OT1 cells were gated based on FSC-A / SSC-A properties. Next, single cells were gated based FSC-A / FSC-H. CD8+ population were gated as CD8-PerCP-Vio700 positive. Next to the CD8+ population, a mouse TNFR2+ gate was set with a rat isotype control via histogram. The isotype control has been overlaid in each anti-mTNFR2 antibody histogram represented with % of max. Data representative of single experiment out of two independent experiments. (C)TNFR2 expressing Treg cells co-staining, representation of can didates 18 and 25 with a benchmark antibody, clone TR75–89. Data representative of two independent experiments. (D) Costimulation of CD8+ T-cells with anti- TNFR2 antibodies. Assessment of in vitro CD8+ T-cell costimulation for different anti-TNFR2 antibodies (plate bound anti-CD3 at 0.5 μg/mL). Anti-TNFR2 antibodies were plate bound in 2-fold decreasing dilution starting at 50 μg/mL. Data representative of three independent experiments with n = 3 biological replicates on the read out of IFNγ in supernatant at 50 μg/mL per each candidate and mean of independent experiment. Blank was subtracted, IFNγ was calculated based on the standard curve and normalized based on single incubation of anti-CD3 antibodies as 0% costimulation and double incubation of anti-CD3 + anti-CD28 antibodies as a 100% costimulation.

Article Snippet: First, a single estimation screening of KD value was performed with an expected saturating concentration of 100 nM His tagged mTNFR2 (R&D Systems, 426-R2/CF) 100 nM diluted in 10× Kinetics Buffer (KB) followed by a dissociation step.

Techniques: Ex Vivo, Expressing, Binding Assay, Labeling, Control, Generated, Staining, In Vitro, Incubation