(A,B) Ex vivo generation of murine tBregs was done in the absence or presence of various TLR ligands (5 µg/ml of ssRNA40; 10 µg/ml of Pam3CSK4 or FSL-1; 5 µg/ml of LPS; 1 µg/ml ODN1826 PS (CpG) and control CpG K) or 10 µg/ml anti-IgM. After 48h, cells were stained for CD20 expression (A) and evaluated for the ability to suppress proliferation of CD3+ T cells stimulated with anti-CD3 Ab (B). FITC- labeled CpG is better uptaken by tBregs when coupled with BLC-arp (continuous line, C), as compared with free CpG (broken line, C). Shaded area is for untreated control cells (C). BLC-arp/CpG (3 µg/ml ODN1826 PS) blocks activity of murine tBregs in vitro, at the same extent as free CpG, as shown by the inability to inhibit proliferation of CFSE-labeled CD3+ T cells (D). Controls were B cells treated with BAFF (B-Mock) and untreated tBregs.