Figure. 5. TCR signal-driven IL-12Rb2 upregulation is cell division-dependent. Purified C7 and C24 CD4+ Tg cells were cultured with CD4-depleted splenocytes from naïve B6 mice in the presence or absence of E6 peptide. (A) Flow cytometry plots showing IL-12Rβ2 and T-bet expression in Tg cells 48 h after stimulation. In some E6-stimulated cultures, mAbs to IL-12p70, IL-4 and IFN-g were added (TCRdriven) while in others, mAbs to IL-4 and IFN-g, together with recombinant IL-12p70, were included (IL-12driven). (B) Kinetic analysis of the percentage and absolute number of IL-12Rβ2-expressing Tg cells by flow cytometry. Data shown are the mean ± sd of triplicate cultures. Statistical differences between C7 and C24 cultures were determined by Students t-test (*p<0.05, **p < 0.01, ***p<0.001, ns., not statistically significant). The difference in the proportion of IL-12Rb2+ C7 and C24 cells was statistically significant (at least p<0.05) for all time points, unless indicated otherwise. (C-E) CTV-labelled C7 and C24 T cells were stimulated with E6 peptide and IL-12 in the presence of anti-IL-4 and anti-IFN-γ mAbs. Where indicated, paclitaxel (200 nM) was added at the beginning of the cultures. (C) Flow cytometric analysis of T-bet and IL-12Rβ2 expression on CTVlabelled C7 and C24 cells 66 h after peptide stimulation. The percentage of (D) Tbethi and (E) T-betint populations in C7 and C24 cultures with or without paclitaxel examined by flow cytometry at 52 h after peptide stimulation. Bars that are black or red represent C7 and C24 populations, respectively. Data shown are mean ± sd of triplicate cultures. Statistical differences between untreated and paclitaxel-treated cultures were determined by Students t-test (*p<0.05, **p < 0.01). (F) Flow cytometric analysis of the effect of paclitaxel treatment on IL-12 versus IFN-g augmented T-bet expression. As specified, cells were exposed to TCR-driven (anti-IL-12p70, anti-IL-4 and anti-IFN-g mAbs), IL-12-driven (5 ng/mL IL-12p70 and anti-IFN-g and anti-IL-4 mAbs) or IFN-g-driven (anti-IL-12p70, anti-IL-4 mAbs) culture conditions and analyzed at 66 h. All data are representative of at least two independent experiments with similar results.
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