Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs

The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells. A s ubset of these CpG motifs was previously found to oppose the effects of imm unostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) moth s. Here we show that oligodeoxynucleotides (ODNs) composed of clusters of C pG-N moths could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs. However, non-CpG-co ntaining ODN were also inhibitory, suggesting that neutralization of CpG-S ODNs by CpG-N ODNs in trans was nonspecific. Neutralization of CpG-S motifs by CpG-N motifs in cis was specific, but the degree of inhibition was stro ngly dependent on the particular CpG-S motif being neutralized, with motifs having an A residue 5' to the CG being much more resistant to inhibition t han motifs having a T residue 5' to the CG. The degree of inhibition was de pendent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance. In addition, whereas OD Ns containing extended clusters of CpG-N moths were nonstimulatory, isolate d CpG-N motifs remained stimulatory in most sequence contexts. Finally, CpG -N ODNs were shown to be nonstimulatory when instilled into the lungs of BA LB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in ci s was not observed. These results show that there are precise and fairly co mplex interactions between immunostimulatory and inhibitory sequence moths that govern whether a given DNA is able to activate the vertebrate immune s ystem.