Figure 1. Antigen-specific and polyclonal BMEM cells are responsive to TLR agonists in vitro and display an enhanced capacity to differenti- ate into ASCs compared with NF B cells. NP-BMEM and PE-BMEM were sorted by FACS, along with NF B cells. NP-BMEM and NF B cells were stained with DDAO and PE-BMEM, and NF B cells were stained with CFSE. Cells were cultured for 3 d with 10 µg/ml LPS, whereupon the cell surface phenotype was analyzed for the expression of B220 and CD138 (A) or DDAO or CFSE (B), with the percentage of B220+CD138+ cells depicted (A). (B) Filled histograms, B cells + IL-4; gray lines, BMEM; black lines, NF B cells. To analyze the reactivity of BMEM to TLR agonists in the absence of BCR cross-linking, poly-BMEM were used. Spleens from NP-KLH–immunized C1-cre/ROSA-YFP mice were enriched for BMEM, with poly-BMEM present in the remaining population sorted by the phenotype B220+IgG1/YFP+CD38+DumpCD80+. Dump gates, CD43+IgD+IgM+ cells (C). Both poly-BMEM and NF B cells were labeled with DDAO and cultured for 3 d in the presence of either 10 µg/ml LPS or 5 µM CpG, whereupon the cell surface phenotype was analyzed for the expression of B220 and CD138 (D) and DDAO dye dilution (E). (E) Gray lines, B cells + IL-4; black lines, NF or poly-BMEM as indicated. Proliferation as a function of CD138 expres- sion is shown, with the percentage of cells that have diluted DDAO and are expressing CD138 shown (F). The ability of cells derived from either NF B cells (open bars) or BMEM (filled bars) after 3 d of LPS culture to secrete antibody of the IgM, IgG1, IgG2a, IgG2b, IgG3, IgE, and IgA isotypes was analyzed by ELISpot analysis (F). The response profiles of poly-BMEM and NF B cells to a panel of TLR agonists was examined after 3 d of culture with the indicated agonist. The agonists used, their abbreviations, the receptors for which they are specific for, and the concentrations used are as follows: Pam3CSK4 (P3C), TLR1/2, 1 µg/ml; LPS (LPS), TLR4, 10 µg/ml; FSL-1 (FSL1), TLR6/2, 1 µg/ml; S-27609 (609), TLR7, 1 µg/ml; CpG ODN 1826 (CpG), TLR9, 5 µM, CD40 2016 IN VIVO ACTIVATION OF MEMORY B CELLS | Benson et al.