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.

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 Material and Methods. Cells were then analyzed by flow cytometry and the data show frequency of IFN-γ- or IL-10-producing CD4+ T cells.To study cell proliferation, unfractionated spleen cells of NP- or P-mice, were labelled with CFSE as described in Materials and Methods 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 Materials and Methods. The percentage of CD4+CFSElow and CD8+CFSElow indicates the number of replicating CD4+ or CD8+ T cells, respectively.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.#, § 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).nd = not done.