title: Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs.
abstract: The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells. A subset of these CpG motifs was previously found to oppose the effects of immunostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) motifs. Here we show that oligodeoxynucleotides (ODNs) composed of clusters of CpG-N motifs could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs. However, non-CpG-containing 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 strongly 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 than motifs having a T residue 5' to the CG. The degree of inhibition was dependent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance. In addition, whereas ODNs containing extended clusters of CpG-N motifs were nonstimulatory, isolated CpG-N motifs remained stimulatory in most sequence contexts. Finally, CpG-N ODNs were shown to be nonstimulatory when instilled into the lungs of BALB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in cis was not observed. These results show that there are precise and fairly complex interactions between immunostimulatory and inhibitory sequence motifs that govern whether a given DNA is able to activate the vertebrate immune system.
authors.affiliation: Genzyme Corporation, Framingham, MA 01701-9322, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zhao
authors.forename: S H
authors.initials: SH
authors.lastname: Cheng
authors.forename: N S
authors.initials: NS
authors.lastname: Yew
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics*; D000280:Administration, Inhalation; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018899:CpG Islands / Q000235:genetics* / Q000276:immunology*; D005260:Female; D015316:Genetic Therapy; D018664:Interleukin-12 / Q000096:biosynthesis / Q000276:immunology; D008168:Lung / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D013154:Spleen / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D009838:Oligodeoxyribonucleotides; D018664:Interleukin-12