Impaired survival, proliferation, and persistence of Rictor-deficient B cells. (A-B) Spleen cells were isolated from Cre-ER^T2Rictor^fl/fl (cKO) or control (WT) mice 2 weeks after starting tamoxifen treatment, and were cultured for indicated times (A) or for 1 day (B) in the presence or absence of BAFF. Viable cells were counted and analyzed by flow cytometry, and B-cell survival was determined by calculating the percentages of live B-cell (7-AAD^- B220⁺) numbers relative to input. Shown are mean (± SEM) for the 3 mice of each genotype within 1 representative analysis of 3 independent replicate experiments yielding the same results. (C) Increased apoptosis of Rictor-deficient B cells. Rictor was acutely depleted in the Cre-ER^T2Rictor^fl/fl mice as in Figure 3, and annexin-V staining was performed using splenic B cells. Shown are representative histograms in the transitional (B220⁺ AA4.1⁺) and mature (B220⁺ AA4.1^-) B-cell gates (left), and mean (± SEM) frequencies of annexin-V⁺ B cells (right): n = 5 vs 5. (D) Competitive homeostasis of mature B cells. LN cells of Cre-ER^T2Rictor^fl/fl mice (CD45.2) or WT CD45.2 controls were mixed with equal numbers of WT CD45.1 LN cells, and transferred into Rag2^−/− mice. Mice were then treated with tamoxifen and were analyzed by flow cytometry 4 weeks after the adoptive transfer. Shown are mean (± SEM) ratios of CD45.2 B cells to CD45.1 competitors in the spleen and LN (n = 6 WT vs 6 cKO). (E) Homeostatic proliferation of Rictor-deficient B cells. LN cells of donors [Cre-ER^T2Rictor^fl/fl (cKO) mice or controls] were labeled with CFSE and then were transferred into Rag2^−/− mice, after which mice were treated with tamoxifen. Shown are representative histograms of CSFE fluorescence measured in the CD19⁺ B220⁺ viable lymphoid gate 4 weeks after transfer and initiation of tamoxifen (n = 4 WT vs 4 cKO). (F) Spleen cells were harvested from Cre-ER^T2Rictor^fl/fl (cKO) or control (WT) mice 2 weeks after starting tamoxifen treatment and then were cultured (2 days) in the presence or absence of anti-IgM or LPS, followed by addition of BrdU and further culture (18 hours). Percentages of BrdU⁺ B cells were measured by flow analyses in the viable cell gate. Shown is 1 result representative of those in 3 independent experiments, each consisting of 4 mice of each genotype.