(A) C57Bl/6 mice were immunized with NP-KLH or NP-KLH plus α-GC. After 28 days, all mice received a booster vaccine (NP-KLH). Sera were collected on day 35 and endpoint IgM, IgG1, IgG2b, IgG2c and IgG3 titers determine by ELISA. (B) Thymocytes and splenocytes were obtained from CD40L−/− and C57Bl/6 mice and then analyzed by flow cytometry. Dot plots (left) show CD1d tetramer+/TCRβ+ cells. Histograms (right) show expression of CD40L by gated CD1d tetramer+/TCRβ+ cells. Data show representative analyses from two CD40L−/− mice and numerous (>50) C57Bl/6 mice. (C) C57Bl/6 and CD40L−/− mice were immunized with NP-KLH plus α-GC on d 0 and boosted with NP-KLH on day 28 before bleeding on day 35. Endpoint IgM, IgG1, IgG2b, IgG2c and IgG3 titers in the sera collected on day 35 were then determined by ELISA. Each data point in (A) and (C) represents an individual mouse and line indicates geometric mean titer. Statistically significant differences between groups were determined using Mann Whitney U test.