Figure 2. NKT-derived CD40L is dispensable for Ab production.

(A) Outlines the strategy used for generating mixed bone marrow chimeras. Jα18^−/− mice have a gene deletion in the TCR locus and do not rearrange the Vα14/Jα18 invariant TCR found on Type I NKT cells. (B) Spleens were obtained from immunized Jα18^−/−/C57Bl/6 and Jα18^−/−/CD40L^−/− chimeric mice and analyzed by flow cytometry for CD45.2^+/+ donor cells and residual CD45.1^+/+ recipient cells. (C) Graph shows expression of B cell, T cell and dendritic cell markers for each of the chimeras. (D) Dot-plots in upper panels show CD1d tetramer⁺/TCRβ⁺ NKT cells. Histograms in middle panels show CD1d expression. Anti-CD1d and isotype control mAb binding are represented by filled and empty histograms respectively. Dot-plots in lower panels show donor-derived (CD45.2^+/+) versus recipient-derived (CD45.2^−/−) tetramer-binding cells. Data in B-D are representative of three of each chimera. (E) CD45.2^+/+ (C57Bl/6) mice were lethally irradiated and engrafted with a 50/50 mixture of donor bone marrow cells from Jα18^−/− mice (CD45.2^+/+) and CD45.1^+/+ mice. After 12 weeks peripheral blood leukocytes were analyzed by flow cytometry for CD45.2 and CD45.1 expression. Graph shows relative percentages of CD45.1 and CD45.2 cells in the periphery of 5 re-constituted recipients. (F-H) Jα18^−/−/C57Bl/6 and Jα18^−/−/CD40L^−/− mixed chimeras were immunized as indicated. Sera were collected on d 28 (primary) and 35 (secondary) and ELISA assays were performed to analyze NP-specific (F) IgG1, (G) IgG2b and (H) IgG2c titers. Each data point represents an individual mouse and the lines indicate geometric mean titer. Two outliers were removed from the NP-KLH, primary sera group in (F). Data shown are representative of 2 independent experiments. Statistically significant differences between control and experimental mice were determined using Mann Whitney U test.