CD4⁺ memory T cells are not the main responders to EPs 7630.

<p>(<b>A</b>) Memory T-helper cells (CD4⁺ CD45RO⁺) were isolated from healthy donor PBMCs by magnetic labeling-based cell sorting. Representative dot plots of the cells (live gate cells with CD4⁺ lymphocytes marked in blue), stained and analyzed by flow-cytometry before and after sorting, are given (<i>left panel</i>). Mean (± SEM) purity of isolated cells was 97.68 ± 0.4%. Isolated cells were then treated with different concentrations of EPs 7630 as indicated for 48 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 5 donors are given (<i>right panel</i>). (<b>B</b>) Memory T-helper cells, isolated as in (A), were treated with different concentrations of EPs 7630 as indicated for 24 h. Subsequently, cells were additionally stimulated with anti-CD3/anti-CD28 antibodies for another 24 h. Quantification of TNF-α, IL-6 and IL-10 in respective culture supernatants was carried out by ELISA. Mean (± SEM) cytokine concentration data from 5 donors are given as percent of 0 μg/ml EPs 7630 group (control). Cytokine concentrations in the absence of EPs 7630 were: 4753±608 pg/ml (TNF-α), 3.4±0.9 pg/ml (IL-6), 737±103 pg/ml (IL-10). (<b>C</b>) Healthy donor PBMCs were stimulated with different concentrations of EPs 7630 as indicated for 4 to 5 h. Brefeldin A was added for the last 3 to 4 h of culture, followed by antibody-based staining of intracellular TNF-α and surface markers and subsequent flow-cytometric analysis. Representative dot plots showing the proportions of TNF-α⁺ cells out of all CD4⁺ cells of one out of 3 independent experiments are given.