Effects of 4-1BB ligation in spleen cells of NP- and P-mice stimulated with Ag85B protein on IFN-γ-producing CD4⁺ T cells and on proliferation of CD4⁺ and CD8⁺ T cells.

<p>To analyze IFN-γ- or IL-10-producing CD4⁺ T cells, unfractionated spleen cells of P- or NP-mice were cultured with Ag85B protein (5 µg/ml) in the presence of IgG2a control Ab or agonist anti-4-1BB mAb (5 µg/ml). After 2 days (for IL-10 analysis) or 4 days (for IFN-γ analysis) of culture and an overnight incubation with brefeldin A cells were stained with CD4-PE-Cy5 and then intracellular stained with PE anti-IL-10 or FITC anti-IFN-γ Ab as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Material and Methods</a>. Cells were then analyzed by flow cytometry and the data show frequency of IFN-γ- or IL-10-producing CD4⁺ T cells.</p><p>To study cell proliferation, unfractionated spleen cells of NP- or P-mice, were labelled with CFSE as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a> and then stimulated with Ag85B protein (5 µg/ml) in the presence of agonistic anti-4-1BB mAb (5 µg/ml) or rat IgG2a control Ab (5 µg/ml) for 4 days. At this time point cells were labelled with CD4-PE and CD8-PerCP and analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>. The percentage of CD4⁺CFSE^low and CD8⁺CFSE^low indicates the number of replicating CD4⁺ or CD8⁺ T cells, respectively.</p><p>The results are presented as mean ± SEM of the percentage of CD4⁺ T cells or CD8⁺ T cells staining for the indicated markers. Pooled data of 3 independent experiments are shown.</p><p>#, § statistical significance for differences between groups determined by ANOVA and Bonferroni-type multiple t-test (# Ag85B/control IgG vs unstimulated; § Ag85B/control IgG vs Ag85B/anti-4-1BB mAb).</p><p>nd = not done.