. Proliferation dye-labeled ConA-stimulated responder (Resp) cells were cultured in the bottom well, separated by a semipermeable membrane from indicated Teff cell populations in the top well. Where indicated, CD4 pos CD25 neg cells were added at a ratio of 1:1 to Teff cells (top well). The IL-10-dependency of suppression was assessed by addition of a neutralizing anti-IL-10 antibody in comparison to a non-blocking isotype control antibody (see table). Resp cells were harvested after 72 h and analysed by flow cytometry. Summary bar graphs show the median suppression of proliferation of viable lymphocytes, TCRαβ pos CD4 pos and TCRαβ pos CD8α pos Tresp mediated by the different Teff cell population under each condition (see table). The percentage of suppression was normalized to the negative control (ConA stimulated Resp + CD4 pos CD25 neg Teff) of each condition (+/- CD4 pos CD25 neg , +/- anti IL-10 antibody, +/- isotype control). In case addition of neutralizing IL-10 antibody led to negative values for suppression (i.e. ablation of suppression), values are depicted as 0% suppression. Notably, neutralization of IL-10 completely abrogates the suppression by dnCD25 pos and CD4 pos CD25 pos Teff cells in the Transwell™ system, while it only partially reduces the suppression by dnCD25 neg Teff cells. The results of three independent experiments are shown. Each dot represents one individual dog, bars represent medians. Significance of antibody-dependent effects (anti-IL-10) on suppression was calculated by direct comparison with the corresponding controls (i.e. control without antibody addition and isotype control) using the Mann-Whitney test (two-tailed). (* p < 0.05). " width="100%" height="100%">
Journal: Frontiers in Immunology
Article Title: Distinct characteristics of unique immunoregulatory canine non-conventional TCRαβ pos CD4 neg CD8α neg double-negative T cell subpopulations
doi: 10.3389/fimmu.2024.1439213
Figure Lengend Snippet: Secretion of IL-10 by canine dnCD25 pos and CD4 pos CD25 pos Teff cells is necessary to mediate suppression in the Transwell™ system. Suppression assays were performed in the Transwell™ system as described in Figure 3A . Proliferation dye-labeled ConA-stimulated responder (Resp) cells were cultured in the bottom well, separated by a semipermeable membrane from indicated Teff cell populations in the top well. Where indicated, CD4 pos CD25 neg cells were added at a ratio of 1:1 to Teff cells (top well). The IL-10-dependency of suppression was assessed by addition of a neutralizing anti-IL-10 antibody in comparison to a non-blocking isotype control antibody (see table). Resp cells were harvested after 72 h and analysed by flow cytometry. Summary bar graphs show the median suppression of proliferation of viable lymphocytes, TCRαβ pos CD4 pos and TCRαβ pos CD8α pos Tresp mediated by the different Teff cell population under each condition (see table). The percentage of suppression was normalized to the negative control (ConA stimulated Resp + CD4 pos CD25 neg Teff) of each condition (+/- CD4 pos CD25 neg , +/- anti IL-10 antibody, +/- isotype control). In case addition of neutralizing IL-10 antibody led to negative values for suppression (i.e. ablation of suppression), values are depicted as 0% suppression. Notably, neutralization of IL-10 completely abrogates the suppression by dnCD25 pos and CD4 pos CD25 pos Teff cells in the Transwell™ system, while it only partially reduces the suppression by dnCD25 neg Teff cells. The results of three independent experiments are shown. Each dot represents one individual dog, bars represent medians. Significance of antibody-dependent effects (anti-IL-10) on suppression was calculated by direct comparison with the corresponding controls (i.e. control without antibody addition and isotype control) using the Mann-Whitney test (two-tailed). (* p < 0.05).
Article Snippet: To analyze whether IL-10 is involved in cell-cell contact independent suppression, a neutralizing anti-canine IL-10 antibody (clone 138128, R&D, Biotechne, Wiesbaden, Germany) was used at 10 µg/ml.
Techniques: Labeling, Cell Culture, Membrane, Comparison, Blocking Assay, Control, Flow Cytometry, Negative Control, Neutralization, MANN-WHITNEY, Two Tailed Test
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