Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: TNF-α does not impact the levels of TNFR1 and TNFR2 on Th1 and Th17 cells. Purified CD4 + T cell subsets were stimulated with 1 µg/mL of TNF-α for 24 h and stained, for flow cytometry, with anti-human TNFR1 or TNFR2 mAbs. ( A ) Representative histograms showing the expression levels of TNFR1 and TNFR2 on Th1 and Th17 cells at baseline conditions and upon TNF-α stimulus. Solid black line histograms, isotype control; black histograms, Th1 cells; grey histograms, Th17 cells; dashed line histograms, TNF-α-treated Th1 cells; long dashed line histograms, TNF-α-treated Th17 cells. The levels of TNFR1 ( B ) and TNFR2 ( C ) were measured on purified CD4 + T cell subpopulations obtained from two to four healthy controls. Bars represent the mean values ± SD. Statistical analyses were carried out with Kruskal–Wallis followed by Dunn’s multiple comparison tests. ** p < 0.01.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Staining, Flow Cytometry, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Effect of TNFR1 or TNFR2 blockade on the production of IFN-γ and IL-17 by Th1 and Th17 cells. Purified CD4 + T lymphocyte subpopulations were incubated with neutralizing antibodies to TNFR1 or TNFR2 for 1 h prior to a 4-day stimulus with TNF-α (1 µg/mL). Intracellular staining of IFN-γ and IL-17 was assessed by flow cytometry. ( A ) Representative dot plots of Th1 and Th17 cells treated with anti-TNFR1 or anti-TNFR2 in the presence or absence of TNF-α. An isotype control was used to discard non-specific effects of the neutralizing antibodies. The fold increase in the percentages of IFN-γ ( B , D ) or IL-17 ( C , E ) producers was measured on Th1 and Th17 cells obtained from two to six healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive treatment with TNF-α or TNF-α plus TNFRs blocking mAbs. For statistical analysis, Kruskal–Wallis and Dunn’s multiple comparison tests were performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Incubation, Staining, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Expression of TNFR1 and TNFR2 on Th1 and Th17 cells present in the peripheral blood of rheumatoid arthritis (RA) patients treated with adalimumab. Cell staining for flow cytometry analysis was performed on PBMC samples from healthy controls ( n = 9) and RA patients ( n = 10) before (PRE) and after (POST) treatment with adalimumab. The levels of TNFR1 ( A ) and TNFR2 ( C ) are expressed in MFI values. The frequency of TNFR1 ( B ) and TNFR2 ( D )-expressing lymphocytes are also shown. Each symbol represents data for one individual. Mean values ± SD are indicated. Significance was assessed with non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test (for MFI data) or parametric one-way ANOVA plus Tukey’s post-test (for lymphocyte frequencies data). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Expressing, Staining, Flow Cytometry, Comparison

Journal: iScience

Article Title: Functional and structural modifications of influenza antibodies during pregnancy

doi: 10.1016/j.isci.2022.104088

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human IgG2-Fc PE , Southern Biotech , CAT # 9060-09; RRID: AB_2796635.

Techniques: Virus, Produced, Recombinant, Luciferase, Plasmid Preparation, Software, Luminex, Magnetic Beads

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: (A, B) Determination of IC 50 values in a competition ELISA for compound binding to human and cyno IL-7Rα and γc. The assay format is IL-7Rα-(His) 6 -tagged ECD or Fc-γc ECD immobilized on 96-well plates. Tracers are C-terminal biotinylated forms of the reference IL-7Rα and γc peptide ligands, each pre-complexed with Neutravidin-HRP (NA-HRP). (C, D) Label-free measurement of the equilibrium dissociation constant (K D ) for peptide binding to IL-7Rα and γc extracellular domains with peptides was performed by biolayer interferometry as detailed in methods. Red lines depict 1:1 binding fit from Gator™ software. Blue lines represent individual BLI sensogram data taken every 0.1 seconds. Inset table displays comparison of ELISA data and BLI kinetic data.

Article Snippet: Biotinylated mouse anti-human IgG2 Fc (Southern Biotech Cat# 9070–08) was used as the capture antibody, and the rabbit polyclonal antibody against MDK1188, followed by a mouse anti-rabbit IgG Fc HRP (Genscript Cat# A01856-200) as the detection antibody.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Software, Comparison

Journal: Immunity

Article Title: Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets

doi: 10.1016/j.immuni.2021.05.001

Figure Lengend Snippet:

Article Snippet: Mouse anti-human IgG2 Fc-HRP , Southern Biotech , Cat# 9080-05; RRID: AB_2796633.

Techniques: Virus, Clinical Proteomics, Infection, Recombinant, RNA Binding Assay, Transfection, Enzyme-linked Immunosorbent Assay, Bioprocessing, Multiplex Assay, Isolation, Gene Expression, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Divergent trajectories of antiviral memory after SARS-CoV-2 infection

doi: 10.1038/s41467-022-28898-1

Figure Lengend Snippet: Humoral immune responses were assessed in acute and convalescent by binding antibody ELISA for total IgG specific to the a Spike glycoprotein and b Nucleocapsid, quantification of c IgG memory B cells specific to the spike glycoprotein, and d pseudoneutralisation antibody titres. A two-tailed Wilcoxon rank-sum test was used to compare between study time points. The boxplots all display the median values with the first and third quartile, and the whiskers represent the highest and lowest values no more than 1.5 times the interquartile range from the corresponding hinge. A generalised additive mixed model (GAMM) by restricted maximum likelihood—right-hand plots—was used to fit the immunological measures (log10 transformed) taken at multiple study time points, using Gaussian process smooth term. The GAMM plots the ribbon represents the 95% confidence interval around the fitted value. Disease severity group was included in the GAMM as a linear predictor and a participant identifier was included as a random effect. See Table for number of individuals evaluated per assay.

Article Snippet: For detection of anti-spike IgG2 and IgG4 steps modified as follows: (1) Plates were additionally coated with commercially available human immunoglobulin control (recombinant human IgG2 lambda or recombinant human IgG4 lambda (Bio-Rad)) to serve as internal controls, (2) Mouse anti-human IgG2 Fd-AP or mouse anti-human IgG4 Fc-AP (Southern Biotech) were used, and (3) Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the immunoglobulin control reached a specified OD405.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transformation Assay

Journal: Nature Communications

Article Title: Divergent trajectories of antiviral memory after SARS-CoV-2 infection

doi: 10.1038/s41467-022-28898-1

Figure Lengend Snippet: SARS-CoV-2 spike-specific antibody isotype and subclasses measured post-infection: a IgM, b IgA, c IgG1 and d IgG3. Antibody function measure post-SARS-CoV-2 infection: e antibody-dependent NK cell activation (ADNKA), f antibody-dependent neutrophil phagocytosis (ADNP), g antibody-dependent monocyte phagocytosis (ADMP) and h antibody-dependent complement deposition (ADCD). i Polar plot of various antibody isotype, subclass and function data, minimum-maximum normalised. The boxplots all display the median values with the first and third quartile, and the whiskers represent the highest and lowest values no more than 1.5 times the interquartile range from the corresponding hinge. A two-tailed Wilcoxon rank-sum test was used to compare between study time points. A generalised additive mixed model (GAMM) by restricted maximum likelihood—right-hand plots—was used to fit the immunological measures (log10 transformed) taken at multiple study time points, using Gaussian process smooth term. The GAMM plots the ribbon represents the 95% confidence interval around the fitted value. Disease severity group was included in the GAMM as a linear predictor and a participant identifier was included as a random effect. See Table for number of individuals evaluated per assay.

Article Snippet: For detection of anti-spike IgG2 and IgG4 steps modified as follows: (1) Plates were additionally coated with commercially available human immunoglobulin control (recombinant human IgG2 lambda or recombinant human IgG4 lambda (Bio-Rad)) to serve as internal controls, (2) Mouse anti-human IgG2 Fd-AP or mouse anti-human IgG4 Fc-AP (Southern Biotech) were used, and (3) Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the immunoglobulin control reached a specified OD405.

Techniques: Infection, Activation Assay, Two Tailed Test, Transformation Assay