Immature T1 B cells are More Sensitive than Mature Follicular B Cells to anti-IgM Stimulation. (A) Cytoplasmic free calcium levels indicated by indo-1 fluorescence emission ratio at 405nm and 530nm ([Ca²⁺]_i F405/F530) in WT splenic B220⁺ T1 (AA4.1⁺, CD23⁻, IgM⁺⁺), T2 (AA4.1⁺, CD23⁺, IgM⁺⁺), T3 (AA4.1⁺, CD23⁺, IgM⁺)] and mature follicular B cells (AA4.1⁻, CD23⁺, IgM⁺) (Fo) (as described by Allman et. al.(27) and shown in Figure S1) stimulated with 5μg/ml goat anti-mouse IgM F(ab')₂. Arrows indicate time at which cells were stimulated with anti-IgM. Red line at F405/F530 = 0.39 indicates a baseline threshold level, where 90% of unstimulated WT follicular B cells were below this F405/F530 value. (B) Calcium response in WT T1 (green), T2 (blue), T3 (gold) and follicular (red) splenic B cells stimulated with 5μg/ml or 50μg/ml of anti-IgM F(ab')₂. Left panels depict median cytoplasmic calcium level as indicated by indo-1 F405/F530 ratio. Right panels indicate percentage of cells with indo-1 F405/F530 ratio above baseline (0.39). (C) Dose response of WT B cell populations to anti-IgM F(ab')₂ stimulation, as measured by peak median F405/F530 elevation (left panel) or maximal percentage of cells responding above baseline (F405/F530 > 0.39)(right panel). Responses to no stimulation (none) and 16μg/ml ionomycin (iono) are indicated. n=2; similar data obtained from four additional experiments. The sigmoidal dose response curves were generated by non-linear regression; R²>0.96 for each B cell population analyzed (4 parameter logistic equation). (D) Calcium response in WT B220⁺ bone marrow newly formed (NF)(AA4.1⁺, CD23⁻, IgM⁺⁺) (black dashed line) and mature recirculating (AA4.1⁻, CD23⁺, IgM⁺) (purple dashed line) B cells, as well as splenic T1 (green solid line) and follicular (red solid line) B cells stimulated with 5μg/ml anti-IgM F(ab')₂.

Lyn-deficiency has a Major Effect on Calcium Responses in Follicular B Cells but a Lesser Effect on those in T1 B Cells.
(A) Cytoplasmic free calcium levels ([Ca²⁺]_i F405/F530) in splenic T1, T2, T3 and follicular (Fo) B cells from lyn^−/− mice stimulated with 5μg/ml goat anti-mouse IgM F(ab')₂. Red line at indo-1 ratio F405/F530 = 0.39 was the 90^th percentile for the fluorescence ratio of unstimulated WT follicular B cells. (B) Calcium release in WT (blue lines) and lyn^−/− (red lines) T1 and follicular splenic B cells after stimulation with 5μg/ml (dashed lines) and 50μg/ml (solid lines) of anti-IgM F(ab')₂. Left panels depict median [Ca²⁺]_i as measured by fluorescence ratio (F405/F530). Right panels indicate percentage of cells with indo-1 F405/F530 ratio above baseline (0.39). (C) Dose response by WT (solid lines, solid symbols) and lyn^−/− (dashed lines, open symbols) T1 and follicular (Fo) B cells to anti-IgM F(ab')₂ stimulation. Responses to no stimulation (none) and 16μg/ml ionomycin (iono) are indicated. R²>0.95 for each sigmoidal dose response curve (4 parameter logistic equation). n=2 for WT, n=3 for lyn^−/−; similar data obtained in four additional experiments. (D) Calcium response in WT (solid lines) and lyn^−/− (dashed lines) bone marrow newly formed (NF)(black lines) and mature recirculating (purple lines) B cells stimulated with 5μg/ml anti-IgM F(ab')₂. (Note: the serrated line representing [Ca²⁺]_i in lyn^−/− BM mature recirculating B cells reflects the low numbers of cells in this population.)

Greater Effect of CD22 on Follicular B Cells Than on T1 B Cells.
(A) Surface levels of CD22, CD16/CD32 (which on B cells is primarily FcγRIIb), CD5, and CD72 on WT splenic T1 (dashed lines) and follicular B cells (solid lines). Isotype control shown as filled-in gray histograms. See table 1 for mean fluorescent intensity quantification. (B) Cytoplasmic free calcium levels ([Ca²⁺]_i F405/F530) in splenic T1 and follicular B cells from cd22^−/− (green lines), WT (blue lines) and lyn^−/− mice (red lines) stimulated with 2, 5 and 50μg/ml goat anti-mouse IgM F(ab')₂. Arrows indicate time at which cells were stimulated with anti-IgM. Similar data were obtained in multiple experiments. The delayed calcium response in lyn^−/− B cells compared to cd22^−/− B cells likely reflects the positive signaling function of Lyn in phosphorylating BCR ITAMs. (C) Cytoplasmic free calcium levels ([Ca²⁺]_i F405/F530) in bone marrow Newly Formed B cells (NF) from WT (blue lines), cd22^−/− (green lines), lyn^−/− mice (red lines) and motheaten-viable mice that have a loss of function mutation in the SHP-1 gene, ptpn6 (shp-1^me-v/me-v) (orange lines), that were stimulated with 2, 5 and 50μg/ml goat anti-mouse IgM F(ab')₂. Arrows indicate time at which cells were stimulated with anti-IgM. Similar data were obtained in multiple experiments. Analysis of mature B cell responses was not possible because of the extremely limited size of this population in motheaten-viable mice. (D) Surface levels of CD22 in splenic follicular (Fo) and T1 B cells from cd22^+/− and WT mice. (E) Cytoplasmic free calcium levels in splenic T1 (solid lines) and follicular B cells (dashed lines) from cd22^+/− (green lines) and WT mice (blue lines).

The Effect of Lyn-Deficiency on Receptor Editing and Proliferative Responses.
(A) Relative RAG1 and RAG2 mRNA levels in sorted populations of WT and lyn^−/− immature BM B cells. RAG transcripts were normalized to GAPDH transcript levels in each sample. Pre-B represents cells from the pro-pre B cell BM gate (Figure S1) with small lymphocyte size on forward scatter. Each data point represents data from 1 mouse; results from 2 experiments are shown. (B) Frequency of Igλ⁺ cells in B cells populations from the BM and spleen of WT (gray bars) and lyn^−/− (black bars) mice. n=11 mice for WT, n=10 for lyn^−/−; bars represent mean ± std dev; * indicates P=0.01, ** P=0.04 (unpaired t-test). (C) Frequency of RS recombination events in genomic DNA of B cell subpopulations. The amount of RS recombinations in sorted Igλ⁻ B cell populations was determined by quantitative PCR of genomic DNA and standardized to the number of actin genes. Shown is the frequency of RS recombination normalized to the RS recombination frequency in WT follicular B cells. Each data point represents data from 1 mouse; results from 2 experiments are shown. (D) Proliferative responses of T1, combined T2-T3, or follicular (Fo) B cells sorted from spleens of WT (gray bars) or lyn−/− (black bars) mice and then stimulated for 48 hours with 15 μg/ml anti-IgM F(ab')₂. (E) Proliferative responses of WT and lyn−/− follicular B cells to stimulation with various doses of goat anti-mouse IgM F(ab')₂, as measured by 3H-thymidine incorporation during the last 4 hours of stimulation. Bars represents data from 2 sets of pooled spleen cells per genotype; each set of pooled cells from 2–3 mice; each sample tested in duplicate or triplicate; bars indicate means, error bars indicate standard error of the mean.

Immature B cells are More Sensitive than Mature Follicular B Cells to Stimulation with Antigen.
(A) Cytoplasmic free calcium levels, indicated by indo-1 fluorescence emission ratio as in figure 1A, of Ig_hel transgenic B cells stimulated with 100ng/ml Hen Egg Lysozyme (HEL). Arrows indicate time at which cells were stimulated with HEL. Red line at F405/F530 = 0.88 indicates a baseline threshold level, where 90% of unstimulated WT follicular B cells were below this F405/F530 value. Note, T2 and T3 B cells cannot be distinguished in these mice because transgenic expression of Ig causes uniformly high expression levels of surface IgM. Difference in Y-axis scale between data shown in figure 2 and figure 1 was due to change in flow cytometer equiptment used. (B) Antigen stimulation of WT B cells transgenic for Ig_hel. Calcium release by T1, T2–3 and mature follicular B cells stimulated with various concentrations of hen egg lysozyme (HEL). (C) Dose response of WT Ig_hel B cell populations to HEL stimulation, as measured by the peak calcium response seen in (B). n=2; similar data obtained from three additional experiments.

Lyn Deficiency Strongly Enhances Activation of Erk in Follicular B Cells but has a Lesser Effect on T1 B Cells.
(A) Erk activation, as measured by flow cytometry analysis of intracellular staining with a monoclonal antibody recognizing the activating phosphorylations of Erk1 and Erk2, in B cell populations identified by B220, IgM, CD24 and CD23 expression (see Figure S3). Phospho-Erk histograms of unstimulated WT splenic B220⁺ T1 (CD24⁺⁺, CD23⁻, IgM⁺⁺) or follicular (CD24⁺, CD23⁺, IgM⁺) B cells (filled-in gray histograms) or of these cells stimulated with 1μg/ml (light blue) or 50μg/ml (purple) goat anti-mouse IgM F(ab')₂ or with PMA 1μg/ml (orange dashed) for 2.5 minutes as shown. (B) Erk activation dose response of WT B cell populations after 2.5 minutes of anti-IgM F(ab')₂ stimulation. Response to 1μg/ml PMA is indicated. n=4; data points represent the mean ± std dev; R²>0.98 for each sigmoidal dose response curve (4 parameter logistic equation); similar data were obtained in two additional experiments. (C) Comparison of Erk activation in BM and spleen B cell populations. n=4; bars represent means ± std. dev.; one of three experiments with similar findings. (D) Erk activation in WT (blue lines) and lyn^−/− (red lines) T1 (left panel) and follicular B cells (right panel). Phospho-Erk geometric mean fluorescent intensity (MFI) levels of unstimulated B cell populations (filled-in gray histogram for WT, dashed red histogram for lyn^−/−) and B cells stimulated with 50μg/ml anti-IgM F(ab')₂ for 2.5 minutes. (E) Comparison of Erk activation dose response of WT (solid lines, solid symbols) and lyn^−/− (dashed lines, open symbols) splenic T1 (green lines) and follicular B cells (red lines). n=4 per genotype; data points represent the mean ± std dev; R²>0.97 for each sigmoidal dose response curve (4 parameter logistic equation); similar data were obtained in 3 additional experiments.

Lyn Tyrosine-Phosphorylation Status in B Cells of Different Developmental Stages.
(A) Sorted WT splenic B cell populations were stimulated with 50μg/ml anti-IgM F(ab')₂ for 2 minutes (S) or with media alone (U) and were lysed in SDS-PAGE buffer. Lysates were analyzed by simultaneously immunoblotting with antibody to total Lyn protein (53 and 56kd isoforms) and site-specific antibodies against either C-terminal inhibitory phosphotyrosine of Lyn (Lyn pY507) or the phosphorylated activation loop of Src-family kinases (SFK pY416) which identifies Lyn pY397 (this antibody cross reacts with multiple other SFKs including Fyn [59kd] and Blk [55kd]). BAP31 served as a protein loading control. Unstimulated lyn^−/− splenic B cells are also shown (Lyn). Similar results were obtained in three additional experiments. The ratio of phosphorylated Lyn to total Lyn, quantified by densitometry, is provided. Ratios are normalized to unstimulated T1 B cells for the pY397:Lyn blot and to unstimulated T3 B cells for the pY507:Lyn blot. (B) purified lymph node B cells (which are primarily follicular B cells) from WT and cd22^−/− mice, were stimulated, lysed, immunoblotted and probed with specific antibodies, as in (A). Similar results were obtained in one additional experiment. As in (A), the ratio of phosphorylated Lyn to total Lyn, normalized to unstimulated WT B cells, is provided.