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Figure S1. FCRL3 enhances CpG-induced proliferation in a concentration-dependent manner. Blood CD19+ B cells purified by negative selection were cultured with or without biotinylated F(ab’)2 fragments of FCRL3 or IgG1 isotype control mAbs at the various indicated concentrations plus SA (20 μg/ml) in the presence or absence of CpG 2006 (2.5 μg/ml). After 48 hours of culture, MTS dye was added for 1 hour before recording the absorbance at 490 nm with a plate reader. The data presented are mean ± SD of experiments from three different donors.

Figure S2. FCRL3 expression is induced in transitional cord blood (CB) B cells following CPG exposure, but is downregulated by newly differentiated PCs. CB B cells isolated by negative selection were cultured (2.5 × 105 per well) with or without CpG (2.5 μg/ml). The cells were harvested on days 0, 2, and 4, and stained for CD24 and CD38, as well as with a biotinylated FCRL3-specific mAb (histogram-black line) or an isotype control (histogram-gray shade) followed by SA-PE before analysis by flow cytometry. Contour plots (column 1) indicate CD24 versus CD38 expression and the numbers represent the percentages of gated CD24lowCD38++ PC relative to the total live events. The numbers within the histograms specify the MFIR (upper values) and the percentage of FCRL3 positive events relative to the isotype control (lower values). One representative experiment of three is shown.

Figure S3. FCRL3 differentially enhances CpG-induced whole-cell tyrosine phosphorylation among blood B cell subsets. CD19+ blood B cells purified by negative selection were cultured overnight in the presence or absence of CpG (2.5 μg/ml). Cells harvested the next day were surface stained for CD19, IgD, and CD27 as well as biotinylated F(ab’)2 fragments of FCRL3 or IgG1 isotype control mAbs (3 μg/ml) before ligation with SA (20 μg/ml) and stimulation for 20 minutes at 37°C. Cells were immediately fixed, permeabilized, and intracellularly stained with Abs specific for phospho-tyrosine (pTyr) or conjugated isotype control Abs. The dot plot shows gating for the four respective populations from which staining in the histograms is derived. MFIR values resulting from the indicated stimulatory conditions are shown in the histograms.

Figure S4. FCRL3 isexpressed by the human SUDHL5 B celllineand is upregulated by CpGstimulation.SUDHL5 cells were cultured (2.5 × 105 per well) with or without CpG (2.5 μg/ml). Cells were harvested after 48 hours and stained with biotinylated anti-FCRL3 (3D2) (black line) or mouse IgG1 isotype control (gray histogram) mAbs and counterstained with SA-PE before analysis by flow cytometry. The numbers within the histograms specify the MFIR (upper values) and the percentage of FCRL3 positive events relative to the isotype control (lower values).

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