(A) Shown are the numbers of bone marrow IgM⁻ pro and pre B cells (B220⁺, AA4⁺, CD23⁻, IgM⁻) and newly formed (NF) B cells (B220⁺, AA4⁺, CD23⁻, IgM⁺⁺) per mouse tibia (left panel), and transitional (T1, T2 and T3) and mature follicular B cells in the spleen (right panel) of wild type and Lyn-deficient young adult mice, as determined by flow cytometry using AA4, CD23 and IgM levels to distinguish these populations. WT (gray bars) and Lyn^−/− (black bars) mice. n=4 per group; similar results obtained from multiple other experiments; bars represent mean ± std dev; * indicates P<0.01 and *** indicates P<0.0001 (unpaired t-test). (B) Percentages of B cells that are Lyn^−/− in different bone marrow (n=13) and splenic (n=18) populations from mice reconstituted with a mixture of 80% Lyn^−/− CD45.1 bone marrow cells and 20% wild type (CD45.2) bone marrow cells. The two genotypes were distinguished with the allelic difference at CD45. CD45.1 was detected with biotinylated monoclonal antibody A20 and CD45.2 was detected with pacific blue-labeled monoclonal antibody 104. Data from individual mice are shown; the means are shown by horizontal bars; B cell populations described above from bone marrow (solid gray symbols) and spleen (open symbols); *indicates P<0.01, and ** indicates P<0.001 (paired t-test). This experiment was repeated once with similar results.

(A)Representative intracellular staining of Bim in total splenic B cells of a wild type (solid gray line) and a Lyn^−/− (dashed black line) mouse, as well as staining with an isotype control (solid gray histogram) is shown. Geometric mean fluorescence of intracellular Bim (B–D), Bcl-2 (B) and Bcl-x (B) in different splenic B cell subpopulations from wild type (n=5) and Lyn^−/− (n=5) mice (B), in wild type and Lyn^−/− splenic B cell populations from mice reconstituted with a mixture of 75% Lyn^−/− bone marrow cells and 25% wild type bone marrow cells (n=7 or 8 per group) (C) and in splenic B cell populations from Ig^HEL transgenic (n =5) and Ig^HEL transgenic Lyn^−/− (n=4) mice (D). WT (gray bars) and Lyn^−/− (black bars); bars represent mean ± std dev; * indicates P<0.01, ** indicates P<0.001, *** indicates P<0.0001 (unpaired t-test) The data in panels C and D were from single experiments with the indicated numbers of mice. The data in A and B were representative of several independent experiments. Note, data represented in panels B, C and D were obtained on different days and therefore MFI values from cannot be compared between different panels.

(A) Survival in vitro of cultured unstimulated splenic B cell populations. Splenic B cell populations from wild type or Lyn-deficient mice were isolated by cell sorting and were cultured for 24 hr in 10% FCS-containing medium, after which the numbers of viable cells were counted by flow cytometry. WT (gray bars) and Lyn^−/− (black bars); bars represent mean ± std dev of 3 or 4 replicates per group except for Lyn^−/− T1 cells which had only 2 replicates; * P<0.01, *** P<0.0001 (unpaired t-test). (B) Numbers of T1, T2, T3 and follicular B cells from the spleens of wild type (solid gray bars, n=3), Lyn^−/−(solid black bars, n=5), Bim^−/− (hatched gray bars, n=5), and Bim^−/− Lyn^−/− (hatched black bars, n=6) mice. (C) Numbers of T1, T2, T3 and follicular B cells from the spleens of wild type (solid gray bars, n=4), Lyn^−/− (solid black bars, n=5), Bcl-2 transgenic (hatched gray bars, n=3), and Bcl-2 transgenic Lyn^−/− (hatched black bars, n=4) mice. Each experiment was repeated once with similar results. (D) Ratio of the numbers of CD45.2 Lyn^−/− Bcl-2 transgenic and CD45.1/.2 Lyn^+/+ Bcl-2 transgenic cells from splenic T1, T2, T3 and follicular (FO) B cells from 7 mixed bone marrow chimeric mice generated from a 50%/50% mixture of bone marrow cells from the two donor genotypes into lethally irradiated CD45.1 recipients. In this experiment, the recipient mice were CD45.1, the Lyn-expressing cells were CD45.1/D45.2 heterozygotes, and Lyn-deficient cells were CD45.2. CD45.1 and CD45.2 were distinguished as described in the legend to Figure 1B, except that the anti-CD45.2 reagent was labeled with flourescein. Shown are data from all of the chimeric mice generated with this mixture, all of which were generated at one time and analyzed subsequently at one time. For panels B, C, and D, the values among the four genotypes were statistically different (ANOVA) at the following levels: + <0.05, * <0.01, **<0.01, ***<0.001. For panel D, ANOVA indicated that the groups were statistically different and posthoc analysis indicated that T1 was different from FO at the level shown.

(A) fraction of B cells from wild type (grey bars, n=4) or Lyn^−/− (black bars, n=5) mice with elevated levels of CD69; ** P<0.001, *** P<0.0001 (unpaired t-test). (B) mean expression level of CXCR5 on different bone marrow and splenic B cell subpopulations from wild type (n=3) and Lyn^−/− (n=3) mice. (C) median expression level of the MHC-II molecule I-A^b in wild type (n=4) and Lyn^−/− (n=5) mice. Experiments were repeated at least once with similar results.

(A) Representative example of intracellular free calcium measurement of wild type B cells responding to anti-IgM stimulation at the time indicated by the arrow. The dashed line represents the level of fluorescence below which were 90% of wild type B cells of that subpopulation prior to stimulation. (B) Comparison of the fraction of B cells with spontaneous ex vivo intracellular calcium levels above the 90% threshold line as described in panel A (note: the wild type values are all 10% by definition). (C) Mean levels of phospho-Erk intracellular staining of wild type or Lyn-deficient B cells of different subsets (geometric mean fluorescence intensity). Experiments were repeated at least once with similar results. Statistical analysis: the values between the two genotypes were statistically different (unpaired t-test) at the following levels: + <0.05, * <0.01.

Levels of phospho-S473 Akt (A) and of phospho-S6 (B) were determined in T1 and follicular B cells from wild type (upper panels) and Lyn^−/− (lower panels) mice. Shown are levels of signaling-related phosphorylations for unstimulated B cells, B cells stimulated with 5 µg/ml anti-IgM for 30min (red curves) or 120min (orange curves) or with 50µg/ml anti-IgM for 30min (blue curves) or 120 min (green curves). Results are representative of 2–3 mice per genotype analyzed on 2 different occasions.