Figure 7.

Active Ras restores the differentiation of 3-83Igi-low immature B cells in vitro and in vivo. (A) Representative analysis of CD21 and CD23 expression in either gfp control (top) or N-rasD12 (bottom) transduced 3-83Igi-low immature B cells at days 0 and 2 of BAFF culture as described in Fig. 5 A, and in the presence or absence of MAPK pharmacological inhibitors as described in Fig. 6 B. 0.3% DMSO was included as solvent control. Numbers indicate frequencies of live transduced B220⁺ B cells in quadrant gates. (B) Mean frequency (±SD; n = 2 pooled mice in each of three individual experiments) of CD23⁺CD21⁺ transduced B cells at day 2 of BAFF culture as described in A. Graphs represent data from treatment with 10 µM U0126 in 0.04% DMSO (top) or 10 µM SB203580 in 0.3% DMSO (bottom) and appropriate DMSO-only controls. (C) Schematic for the generation of N-rasD12 and gfp control retrogenic mice. (D) Representative flow cytometric analysis of spleen cells from N-rasD12 and gfp control retrogenic mice described in C. Expression of the indicated markers on CB17 (from gfp retrogenic mice; top), N-rasD12 transduced 3-83Igi-low (middle), and gfp transduced 3-83Igi-low (bottom) cells is shown. Donor cells were gated as B220⁺IgM^b+ for CB17 and B220⁺IgM^a+GFP⁺ for 3-83Igi-low. Numbers are frequencies of cells in the indicated gates, and data are representative of six retrogenic mice from one experiment. Similar results were obtained in an additional independent experiment with three to four retrogenic mice per group. (E) Mean frequency of CD23^–/lowCD24^high (T1), CD23^highCD24^high (T2), CD21⁺CD23⁺ (T2/mature), and IgD^highIgM^low (T2/mature) donor spleen cells in the retrogenic mice described in C and D. CB17 (from gfp retrogenic mice), N-rasD12 transduced 3-83Igi-low, and gfp transduced 3-83Igi-low cells are shown. Data represent arithmetic means ± SD of six retrogenic mice from one experiment.