Figure 4.

Proliferative responses of B cell subsets after stimulation with toll-like receptor (TLR) ligands (A) Gating strategy defining CD21⁺IgM⁺, CD21⁺IgM⁺, and IgG⁺ B cells, exemplified for unstimulated and Pam3Cys-SK4 stimulated peripheral blood mononuclear cells (PBMCs) after 5 days of culture. (B) CellTrace Violet staining of the three B-cell subsets defined in (A) using multicolor flow cytometry. PBMCs were cultured for 5d. A representative animal out of three is shown. (C) Percentage of proliferating B cell subsets, defined as shown in (A,B), after stimulation of PBMCs with the indicated TLR ligands. Triplicates cultures from three different animals are shown. (D) Proliferation of FACS-sorted B cell subsets after stimulation for 3 days was determined using ³H-thymidine incorporation. P1 was defined and sorted as CD21⁺IgM⁺ subset, P2 as IgG⁺ subset, P3 as CD21⁻IgM⁺CD11R1⁺ subset, and P4 as CD21⁻IgM⁺CD11R1⁻ subset (for gate definition see Figure 2). (C,D) Mean values are indicated by horizontal bars. Error bars show SDs. Statistical significance was calculated using ANOVA followed by Tukey’s test.