Fig. 1
High-efficiency antigens but not low-efficiency antigens elicited CD40L in 2D2-FIG splenocytes. 2D2-FIG splenocytes were cultured with 3.2 μM MOG35–55 (MOG), NFM13–37 (NFM), GMCSF-MOG (G-MOG), GMCSF-NFM (G-NFM), or GM-CSF in duplicate. After 4 h of culture, 10 μg/ ml PE-conjugated anti-mouse CD40L mAb (Armenian Hamster IgG, MR-1) or control PE-conjugated anti-trinitrophenol-KLH mAb (Armenian Hamster IgG, HTK888) were added to cultures and incubated for an additional 2 h. Cells were then stained for CD3, CD4, and CD25. Shown are representative histograms of CD40L (a) and CD25 (b) expression (x-axis) of CD3+ CD4+ T cells activated with designated antigens or cytokines. Shown are percentages of CD40L+ (c) or CD25+ T cells (d) of the total CD3+ CD4+ T cell population following culture with designated treatments. C57BL/6 DCs (20,000/well) were cultured for 2 days with designated concentrations of MOG, G-MOG, or GM-CSF + MOG (e), or NFM, G-NFM, or GM-CSF + NFM (f). DCs were washed extensively and then were parked for 4 days in a second culture without antigen. DCs were then irradiated and cultured in the absence of antigen with naïve responder 2D2-FIG T cells (25,000/well). Thus, antigen acquired during the first culture that persisted over the second culture was assayed by responder T cells in the third culture. g 2D2-FIG splenocytes were cultured with 1 μM MOG or 100 nM NFM in triplicate. At indicated time points (x-axis), culture supernatants were harvested and assayed for IL-2 bioactivity by use of a transformed, IL-2-dependent mouse T cell line (SJL-PLP.1, 20,000 cells/well). e–g Cultures were pulsed with 1 μCi of [3H]thymidine during the last 24 h of a 3-day culture. These data are representative of three independent experiments. c, e, f Statistical significance was analyzed by use of a one-way ANOVA. d, g Statistical significance was analyzed by use of a one-tailed t test. (*p < 0.05, **p < 0.01, ***p < 0.001). e (a) G-MOG vs GM-CSF + MOG (p < 0.05) and (b) G-MOG vs MOG (p < 0.05). f (a) G-NFM vs GM-CSF + NFM (p < 0.05) and (b) G-NFM vs NFM and (c) GM-CSF + NFM vs NFM (p < 0.05). Error bars represent standard deviations