Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity.
Synthetic phosphorothioate oligodeoxynucleotides (ODN) bearing unmethylated CpG motifs can mimic the immune-stimulatory effects of bacterial DNA and are recognized by Toll-like receptor 9 (TLR9). Past studies have demonstrated that nucleotide modifications at positions at or near the CpG dinucleotides can severely affect immune modulation. However, the effect of nucleotide modifications to stimulate human leukocytes and the mechanism by which chemically modified CpG ODN induce this stimulation are not well understood. We investigated the effects of CpG deoxyguanosine substitutions on the signaling mediated by human TLR9 transfected into nonresponsive cells. ODN incorporating most of these substitutions stimulated detectable TLR9-dependent signaling, but this was markedly weaker than that induced by an unmodified CpG ODN. One of the most active ODN tested contained deoxyinosine for deoxyguanosine substitutions (CpI ODN), but its relative activity to induce cytokine secretion on mouse cells was much weaker than on human cells. The activity was dependent on TLR9, as splenocytes from mice genetically deficient in TLR9 did not respond to CpI ODN stimulation. It is surprising that CpI ODN were nearly as strong as CpG ODN for induction of human B cell stimulation but were inferior to CpG ODN in their ability to induce T helper cell type 1 effects. These data indicate that certain deoxyguanosine substitutions in CpG dinucleotides are tolerated to stimulate a TLR9-mediated immune response, but this response is insufficient to induce optimal interferon-alpha-mediated effects, which depend on the presence of an unmodified CpG dinucleotide. These studies provide a structure-activity relationship for TLR9 agonist compounds with diverse immune effects.
Vollmer
Jörg
J
Coley Pharmaceutical, Elisabeth-Selbert-Str. 9, 40764 Langenfeld, Germany. jvollmer@coleypharma.com
Weeratna
Risini D
RD
Jurk
Marion
M
Davis
Heather L
HL
Schetter
Christian
C
Wüllner
Meike
M
Wader
Tanja
T
Liu
Ming
M
Kritzler
Andrea
A
Krieg
Arthur M
AM
D020816:Amino Acid Motifs / Q000276:immunology; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D002460:Cell Line; D018899:CpG Islands / Q000276:immunology*; D005260:Female; D006571:Heterocyclic Compounds / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D007288:Inosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D016898:Interferon-alpha / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects* / Q000276:immunology; D008562:Membrane Glycoproteins / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D018417:Th1 Cells / Q000187:drug effects / Q000276:immunology; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors
D006571:Heterocyclic Compounds; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D007288:Inosine; C012271:deoxyinosine