SYK is required for TLR-induced B cell activation. (A and B) Number of live control (Syk^fl/+MCM) and mutant (Syk^fl/−MCM) B cells after 48-h (A) or 72-h (B) culture with the indicated stimuli, normalized to the number in control B cells stimulated with anti-IgM, which was set to 100. Medium only (med), lipid A from S. minnesota (LAS) or E. coli (LAE), synthetic lipid A (SLA), and CD40L and IL4 (C+I). A, n = 6 (Syk^fl/+MCM) and 8 (Syk^fl/−MCM); B, n = 10 (Syk^fl/+MCM) and 14 (Syk^fl/−MCM). (C) Cell surface expression of CD86 (as measured by mean fluorescence intensity [MFI]) on control or SYK-deficient B cells cultured for 24 h with the indicated stimuli; one of three independent experiments. n = 8 (Syk^fl/+MCM) and 6 (Syk^fl/−MCM). (D) Fraction of B cells that had divided after 72 h of culture with the indicated stimuli. n = 10 (Syk^fl/+MCM) and 14 (Syk^fl/−MCM). (E) Histogram of expression of intracellular SYK measured by flow cytometry in control B cells or mutant B cells that had divided or remained undivided in cultures with LPS; representative of experiments with 11 Syk^fl/+MCM and 12 Syk^fl/−MCM mice. (F) B cells were isolated from radiation chimeras reconstituted with BM cells of the indicated genotypes infected with a retroviral vector expressing GFP and Bcl-x_L. Histograms on the left show overlay of GFP expression in B cells taken straight out of mice (ex vivo) or after culture for 72 h with LPS or in medium only (med), indicating an increase in GFP expression in cultured Syk^fl/−MCM B cells. 2D dot plots on the right show dilution of the CPD plotted against GFP expression, showing LPS-induced proliferation in Syk^fl/+MCM but not Syk^fl/−MCM B cells; numbers indicate percentage of cells in each quadrant; representative of three experiments (total n = 10 of each genotype). (G) Control or SYK-deficient B cells labeled with CFSE or CPD, respectively, were injected into TLR4-deficient mice that were treated with LPS. Flow cytometric analysis of CFSE⁺ or CPD⁺Syk⁻ B cells isolated from the spleen of recipient mice 3 d later shows that some B cells had undergone division as seen by dilution of dye (boxes). Percentages of cells that had divided are indicated; representative of two experiments. (H) Number of live B cells remaining after 48-h culture of wild-type B cells with a range of concentrations of three SYK inhibitors, BAY 61-3606 (BAY), PRT062607 (PRT), or R406, or with concentrations of vehicle (DMSO) equivalent to those used for the inhibitors. Cells were cultured in medium only or with LPS or CD40L + IL4, and cell numbers were normalized to cells treated with no inhibitor, which was set to 100. Representative of three independent experiments. Graph shows mean ± SEM of triplicates. For comparison, the numbers of control (Syk^fl/+MCM, n = 6) and mutant (Syk^fl/−MCM, n = 8) B cells remaining after 48 h of culture are also shown, normalized to the number of control cells, which was set to 100% (data taken from experiment in A). Graphs show mean ± SEM. Statistical analysis was performed using a Mann–Whitney test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.