μMT mice as a tool for examining NKT-enhanced Ab production. (A) Thymocytes (THY) and splenocytes (SPN) were assessed for TCRβ⁺, CD1d tetramer⁺ cells (dot plots) and CD1d⁺ splenocytes (histograms). Data are representative of several (> 6) determinations. (B) Mice were immunized as described in “Immunization and sera collection,” and sera were collected at times indicated before Bio-Plex analysis. Data show serum IL-4 and IFNγ concentration (mean ± SEM, n = 2 for vehicle, n = 5 for C57Bl/6 and n = 2 for μMT). Samples were analyzed in duplicate. No response was elicited with α-GC in CD1d^−/− mice (not shown). Data are representative of 2 independent experiments. (C) Complement-mediated lysis of Thy1.2- and CD4-labeled cells depletes T and NKT cells resulting in a B cell–enriched preparation. Data are representative of several (> 6) determinations. (D) CFSE-labeled C57Bl/6 B cells were adoptively transferred to μMT mice. Cells were detected in recipient spleen after 18 hours (top panel) and 2 weeks after immunization with NP-KLH/Alum (bottom panel). (E) B cells were transferred into μMT recipients, which were then untreated (naive) or immunized 24 hours later with NP-KLH/Alum by the intraperitoneal or subcutaneous route. Sera were obtained after 2 weeks and anti-NP IgG1 detected by enzyme-linked immunosorbent assay. Data show A405 at a 1 of 1000 dilution of serum. (F) After T/NKT-depletion, CD19⁺ cells and CD19⁻ (non-B/non-T cells) were resolved by magnetic sorting. C57Bl/6 mice were adoptively transferred with 2 × 10⁷ CD19⁺ cells or 10⁶ CD19⁻ cells before immunization with NP-KLH/α-GC. The graph shows that the resulting Ab response was the result of the CD19⁺ fraction. The CD19⁻ cellular fraction was determined by flow cytometry to be approximately 50% granulocytes and 50% DCs (not shown). Data in panels D-F show the results from single experiments.