Figure 7.

CD40L synergizes with TI-1 antigens to activate teleost IgM⁺ B cells. (A) Spleen leukocytes were incubated with CD40L (5 μg/ml), TNP-KLH (5 μg/ml), TNP-LPS (5 μg/ml), combinations of any of the TNP-bound antigens with CD40L, or left unstimulated (control) for 72 h at 20°C. After this time, cells were labeled with anti-IgM and anti-MHC II mAbs and analyzed by flow cytometry. Representative dot plots showing MHC II expression levels on IgM⁺ B cells from one representative fish are shown, in which IgM⁺ B cells were gated in control cells (gray), CD40L-treated cells (blue), TNP-antigen (red), and combination of CD40L plus TNP-antigen (purple). Histograms showing MHC II mean fluorescence intensity (MFI) on IgM⁺ gated B cells are also shown. (B) MFI values for surface MHC II were calculated on IgM⁺ B cells under the different treatments, and average values plotted as mean + SD (n = 12). (C) Spleen leukocytes were incubated as described above and after 72 h, an ELISPOT was conducted. Representative wells and quantification of spot forming cells are shown as mean + SD (n = 12). In all cases, statistical differences were evaluated by a one-way ANOVA followed by a two-tailed Student's t-test, where ^**p ≤ 0.01 and ^***p ≤ 0.005.