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: 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

Journal: Cell reports. Medicine

Article Title: Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

doi: 10.1016/j.xcrm.2023.101210

Figure Lengend Snippet: Figure 2. Characterization of anti-Pseudomonas responses by surface binding and membrane protein immunoassays (A) Summary of the positive percentage of antibody surface binding against PA46 at the indicated dilution. Representative flow results and comparisons of CF versus non-CF are in Figures S1A and S1B. (B) Overview of anti-OMP46 level in patients with CF with different lung function at the indicated dilutions. (C) Workflow of screening specific antigens from OMP46 interacting with CF sera using LC-MS. Two samples each from ppFEV1 >90% CF and ppFEV1 <70% CF groups were used. IgG antibodies in CF sera were bound with protein A beads via its Fc region and specific antigens from OMP46 via its Fab region. The im- munobinding proteins were purified and analyzed through liquid chromatography (LC)-MS, in which OprI was identified as the top candidate; see STAR Methods for detailed information. (D) Summary of all ant-OprI IgG isotype levels in patients with CF and non-CF control subjects. (E) Serial dilution in 2-fold of determining anti-OprI IgG titer shown in diluted curve (left) and area under curve (right) using ELISA. (F) The anti-OprI IgG1 (left) and IgG4 (right) titers in three groups. All summaries were visualized as polar plots, n = 10 in each group; the asterisk represents significant differences in the isotype between CF with reduced and healthy lung function cohorts using Mann-Whitney test, *p < 0.05. Area under curve (AUC) comparison was using Kruskal-Wallis test, ****p < 0.0001.

Article Snippet: All used antibodies were listed: Goat antihuman IgG BIOT (1:50 dilution, SouthernBiotech#2040-08); Mouse anti-human IgG1 Hinge BIOT (1:50 dilution, SouthernBiotech#9052-08); Mouse anti-human IgG2 Fc BIOT (1:50 dilution, SouthernBiotech#9070-08); Mouse anti-human IgG3 Hinge BIOT (1:50 dilution, SouthernBiotech#9210-08); Mouse anti-human IgG4 Fc BIOT (1:50 dilution, SouthernBiotech#9052-08); Streptavidin-PE (1:250 dilution, eBioscience #12-4317-87)

Techniques: Binding Assay, Membrane, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Control, Serial Dilution, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Comparison

Journal: Cell reports. Medicine

Article Title: Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

doi: 10.1016/j.xcrm.2023.101210

Figure Lengend Snippet: Figure 4. Key antibody biophysical and func- tional profiling replication using an indepen- dent validation cohort (A) Serial dilution in 2-fold of determining anti-OprI IgG titer shown in diluted curve (left) and AUC (right) using ELISA. (B) Anti-OprI IgG1 (left) and IgG4 (right) in the sera of CF validation cohorts. (C) ADCD analysis mediated by CF sera from vali- dation cohorts (n = 10 each). All statistical compar- isons were conducted using Mann-Whitney test, *p < 0.05, ****p < 0.0001.

Article Snippet: All used antibodies were listed: Goat antihuman IgG BIOT (1:50 dilution, SouthernBiotech#2040-08); Mouse anti-human IgG1 Hinge BIOT (1:50 dilution, SouthernBiotech#9052-08); Mouse anti-human IgG2 Fc BIOT (1:50 dilution, SouthernBiotech#9070-08); Mouse anti-human IgG3 Hinge BIOT (1:50 dilution, SouthernBiotech#9210-08); Mouse anti-human IgG4 Fc BIOT (1:50 dilution, SouthernBiotech#9052-08); Streptavidin-PE (1:250 dilution, eBioscience #12-4317-87)

Techniques: Biomarker Discovery, Serial Dilution, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Cell reports. Medicine

Article Title: Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

doi: 10.1016/j.xcrm.2023.101210

Figure Lengend Snippet: Figure 6. Change in anti-Oprl serologies following modulator treatment (A) Paired comparison of anti-Oprl IgG1 in plasma acquired from a cohort of adult subjects with CF immediately prior to and on average 5 months post-ETI treatment initiation (n = 20). Plasma dilution 1:256. Comparison was performed using paired t test, **p < 0.01. (B) Plasma serial dilution in 2-fold of anti-OprI IgG1 titer shown in diluted curve (left) and AUC (right). (C) Change in Pseudomonas abundance (log10 CFU/mL) in the same cohort as measured by quantitative cultures prior to and >1 month after ETI treatment initiation. Comparisons were made using unpaired t test, ***p < 0.001. (D) Regression analysis of the anti-OprI IgG1 predictor of clinical outcomes after ETI. Statistical comparisons were analyzed by a linear mixed model (via the lmerTest R package) using study visit as the only fixed effect and a random intercept for each subject.

Article Snippet: All used antibodies were listed: Goat antihuman IgG BIOT (1:50 dilution, SouthernBiotech#2040-08); Mouse anti-human IgG1 Hinge BIOT (1:50 dilution, SouthernBiotech#9052-08); Mouse anti-human IgG2 Fc BIOT (1:50 dilution, SouthernBiotech#9070-08); Mouse anti-human IgG3 Hinge BIOT (1:50 dilution, SouthernBiotech#9210-08); Mouse anti-human IgG4 Fc BIOT (1:50 dilution, SouthernBiotech#9052-08); Streptavidin-PE (1:250 dilution, eBioscience #12-4317-87)

Techniques: Comparison, Clinical Proteomics, Serial Dilution