recombinant soluble il 21 receptor  (R&D Systems)

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Follicular Helper–like T Cells in the Lung Highlight a Novel Role of B Cells in Sarcoidosis

doi: 10.1164/rccm.202012-4423OC

Figure Lengend Snippet: Sarcoidosis-affected BAL T cells lack classical T follicular helper (Tfh) cell markers but express high levels of CD40L and IL-21. Antigen-experienced CD4 + T cells (CD45RO + ; for complete gating, see Figure E1 in the online supplement) from BAL of a patient with sarcoidosis, tonsillar tissue, and peripheral blood of healthy donors (CD45RO-negative [naive] cells are shown as additional control) were analyzed by flow cytometry. ( A and C ) Frequency of T cells with a CXCR5 + PD-1 + Tfh and CXCR5 ++ PD-1 ++ germinal center Tfh cell phenotype. ( B and D ) Analysis of CD40L expression (no restimulation; geometric mean fluorescence intensity [MFI]) and IL-21 (after short-term restimulation). Representative flow cytometry data and statistical analysis are shown. Individual samples are depicted by symbols with bars showing the mean. Data are from 10 sarcoidosis-affected BAL (11 for IL-21), 5 tonsil, and 16 healthy donor blood samples. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Kruskal-Wallis test). ns = not significant ( P ⩾ 0.05, Kruskal-Wallis test); Sarc = sarcoidosis.

Article Snippet: To block T-cell help, an antibody against CD40L (clone TRAP1; 20 μg/ml) and/or recombinant soluble IL-21 receptor (R&D Systems; 10 μg/ml) were added.

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Follicular Helper–like T Cells in the Lung Highlight a Novel Role of B Cells in Sarcoidosis

doi: 10.1164/rccm.202012-4423OC

Figure Lengend Snippet: Lung-infiltrating T cells are polyfunctional cytokine producers and have a tissue-resident phenotype. T cells from BAL of a patient with sarcoidosis, tonsillar tissue, and peripheral blood of healthy donors were analyzed by flow cytometry. Plots are gated on antigen-experienced (CD45RO + ) CD4 + T cells except for the “naive” panel, which was gated on CD45RO − cells. ( A ) Analysis of IL-21, IFN-γ, IL-2, and IL-17 production by intracellular staining after short-term restimulation in vitro . ( B ) Cytokine coexpression analysis indicating the percentage of cells producing neither IL-21, IFN-γ, IL-2, nor IL-17 (0), a single one of these four cytokines (1+), IL-21 in combination with one of the other cytokines (2+), IL-21 together with two of the other cytokines (3+), and all four cytokines together (4+). ( C ) Analysis of CD69 and CXCR3 expression. Representative flow cytometry data and statistical analysis are shown. Individual samples are depicted by symbols with bars showing the mean. Data are from 11 sarcoidosis-affected BAL (7 for IFN-γ and 10 for CD69 and CXCR3), 5 tonsil, and 16 healthy donor blood samples. * P < 0.05, ** P < 0.005, *** P < 0.001, and **** P < 0.0001 (Kruskal-Wallis test). ns = not significant ( P ⩾ 0.05, Kruskal-Wallis test); Sarc = sarcoidosis.

Article Snippet: To block T-cell help, an antibody against CD40L (clone TRAP1; 20 μg/ml) and/or recombinant soluble IL-21 receptor (R&D Systems; 10 μg/ml) were added.

Techniques: Flow Cytometry, Staining, In Vitro, Expressing

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Follicular Helper–like T Cells in the Lung Highlight a Novel Role of B Cells in Sarcoidosis

doi: 10.1164/rccm.202012-4423OC

Figure Lengend Snippet: BAL T cells provide potent help for the differentiation of B cells into plasmablasts. ( A ) Heterologous memory B cells were cocultured with no T cells (Ctr), naive CD4 + T cells from blood, sarcoidosis-affected BAL CD4 + T cells, non–T follicular helper and germinal center T follicular helper cells from tonsils for 7 days in the presence of superantigen (staphylococcal enterotoxin B). T-cell help was blocked by addition of an antibody against CD40L and neutralization of IL-21 with recombinant soluble IL-21 receptor. Percentage of B cells with a plasmablast phenotype (CD27 high CD38 + ) was determined by flow cytometry. Representative flow cytometry data and statistical analysis of two to seven independent experiments are shown. Each experiment is depicted by a different symbol and consisted of one to three individual cell culture wells. Only statistically significant differences are indicated. ( B ) Analysis of the culture supernatants from the experiment above for IgM, IgA, and IgG by ELISA. ( C ) BAL supernatants from 17 patients with sarcoidosis (10 patients from the study cohort and seven additional samples) and 7 control subjects (idiopathic cough, lung cancer, pulmonary metastases, or patients without apparent lung disease receiving BAL for exclusion of pulmonary tuberculosis) were analyzed for immunoglobulin subclasses by ELISA. Capture and biotinylated detection antibodies for ELISA are listed in Table E3. Human Immunoglobulin Calibrator (The Binding Site) was used as standard. Bars in all panels display mean with SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Statistical tests: Kruskal-Wallis test ( A ) and Mann-Whitney U test ( B and C ). Sarc = sarcoidosis; Tfh = T follicular helper cell.

Article Snippet: To block T-cell help, an antibody against CD40L (clone TRAP1; 20 μg/ml) and/or recombinant soluble IL-21 receptor (R&D Systems; 10 μg/ml) were added.

Techniques: Neutralization, Recombinant, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Binding Assay, MANN-WHITNEY