ABSTRACT
Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-gamma-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-alpha2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-alpha in cells that express the IFN-alpha receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.
Subject(s)
Adjuvants, Immunologic/pharmacology , B-Lymphocytes/drug effects , Chemokines, CXC/biosynthesis , CpG Islands/immunology , Oligodeoxyribonucleotides/pharmacology , B-Lymphocytes/immunology , Cells, Cultured , Chemokine CXCL10 , DNA Primers/chemistry , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dose-Response Relationship, Drug , Humans , Interferon alpha-2 , Interferon-alpha/pharmacology , Interferon-gamma/pharmacology , Kidney/drug effects , Kidney/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Membrane Glycoproteins/metabolism , Monocytes/drug effects , Monocytes/immunology , Oligodeoxyribonucleotides/classification , Receptor, Interferon alpha-beta , Receptors, Cell Surface/metabolism , Receptors, Interferon/metabolism , Recombinant Proteins , Toll-Like Receptor 9 , Toll-Like Receptors , TransfectionABSTRACT
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.
Subject(s)
B-Lymphocytes/drug effects , CpG Islands/immunology , Inosine/analogs & derivatives , Lymphocyte Activation/drug effects , Membrane Glycoproteins/drug effects , Oligodeoxyribonucleotides/pharmacology , Receptors, Cell Surface/drug effects , T-Lymphocytes/drug effects , Amino Acid Motifs/immunology , Animals , B-Lymphocytes/immunology , Cell Line , Female , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/immunology , Heterocyclic Compounds/pharmacology , Humans , Inosine/chemistry , Inosine/immunology , Inosine/pharmacology , Interferon-alpha/immunology , Lymphocyte Activation/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Structure , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/immunology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes/immunology , Th1 Cells/drug effects , Th1 Cells/immunology , Toll-Like Receptor 9 , Toll-Like ReceptorsABSTRACT
Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have been shown to possess immune modulatory capacities. We investigated the effects of LNA substitutions on immune stimulation mediated by antisense ODN G3139 or CpG ODN 2006. LNA ODNs were tested for their ability to stimulate cytokine secretion from human immune cells or TLR9-dependent signaling. Phosphorothioate chimeric LNA/DNA antisense ODNs with phosphodiester-linked LNA nucleobases at both ends showed a marked decrease of immune modulation with an increasing number of 3' and 5' LNA bases. In addition, guanosine-LNA and cytosine-LNA or simply cytosine-LNA substitutions in the CpG dinucleotides of ODN 2006 led to strong decrease or near complete loss of immune modulation. TLR9-mediated signaling was similarly affected. These data indicate that increasing amounts of LNA residues in the flanks or substitutions of CpG nucleobases with LNA reduce or eliminate the immune stimulatory effects of CpG-containing phosphorothioate ODN.
