Therefore, it was possible that PL2-3 IC elicited a strong TLR9 signal not easily regulated by FcγRIIB. Although TLR9-expressing AM14 cells respond more robustly to DNA fragments enriched for CG dinucleotides than to CG-poor DNA fragments 14, 25, CG-poor DNA fragments can still be bound by TLR9 26. To extend our analysis

to weak TLR9 ligands, normally incapable of promoting AM14 selleck cell cycle entry, we decided to use IC that contained defined dsDNA fragments derived from CG-poor portions of the genome. CG-poor dsDNA is the prevalent class of DNA found in the mammalian genome, and representative sequences such as sentrin-specific peptidase 1 (SenP1), a 557 fragment containing only four CG dinucleotides routinely induce minimal activation of AM14 B cells 14. CGneg, a sequence completely devoid of CG dinucleotides, was constructed to examine TLR9 specificity, and also fails to promote AM14 B-cell proliferation 11. By contrast, Clone 11 is a 573 bp long dsDNA fragment corresponding to a CG-rich unmethylated sequence found in the promoter region of the murine preribosomal RNA gene complex. Such CG-rich regions, denoted CpG islands, comprise about 2% of the mammalian genome 27. IgG2a IC incorporating Clone 11 are potent activators of AM14 B cells 14. To determine whether IC containing CG-poor

dsDNA fragments could Trametinib in vivo activate R2− AM14 B cells, we used IC consisting of 1D4 bound to Bio-SenP1 or Bio-CGneg. As a control for CG-rich DNA, we used 1D4 bound to Bio-Clone 11 IC. Similar to the results obtained with PL2-3, Clone 11 IC-activated R2+ and R2− AM14 B cells had almost identical dose–response curves. However, the R2− AM14 B cells proliferated significantly better than the R2+ AM14 B cells when stimulated with SenP1 or CGneg IC (Fig. 3A). These results indicate that FcRIIB does indeed regulate B-cell responses to endogenous TLR9 ligands; however, its regulatory capacity is only revealed with weak TLR9 ligands. To verify that the enhanced R2− AM14 B-cell response to SenP1 IC was still TLR9-dependent,

we tested the effect of the TLR9 inhibitor, oligodeoxynucleotide (ODN) INH-18, and the control (non-inhibitory) ODN, INH-48 28. The R2− AM14 B-cell responses to SenP1 IC were blocked by INH-18 but not by INH-48 (Fig. 3B). These PAK5 results demonstrate that in the absence of FcγRIIB-mediated inhibition, AM14 B cells respond to otherwise nonstimulatory DNA through a TLR9-dependent mechanism. AM14 B cells respond to RNA-containing IC through coengagement of the BCR and TLR7. TLR7-dependent AM14 B-cell responses to RNA IC are modest when compared with TLR9-dependent responses to CG-rich DNA IC, but can be significantly enhanced by addition of IFNα 18. To determine whether the absence of FcγRIIB promoted AM14 B-cell responses to RNA IC, we stimulated R2+ and R2− AM14 cells with increasing concentrations of the RNA-specific IgG2a mAb BWR4 29.