A GpC-Rich Oligonucleotide Acts on Plasmacytoid Dendritic Cells To Promote Immune Suppression

Abstract

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-β–dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3+ T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.

High doses of CpG oligodeoxynucleotides stimulate a tolerogenic TLR9–TRIF pathway

“…Moreover our data indicate that (d) immunostimulant activity by lower-dose CpG requires, instead, the TLR9-MyD88 pathway as well as production of the proinflammatory cytokine IL-23; (e) genetic deficiency of MyD88 negates functional effects by lower-dose CpG, implying that direction of response depends upon ligand ARTICLE concentration, which likely affects the affinity with which TLR9 recruits downstream adaptor and signalling molecules, including TRAF3 and TRAF6; (f) the protective effects of CpG in experimental allergy in vivo require a TLR9-TRIF circuit but not MyD88-dependent events. Not secondarily, we also confirmed that (g) a non-CpG ODN with two GpC motifs is exclusively immunosuppressive in the same setting, an effect we have recently demonstrated not to require TLR9 and be, in fact, contingent on TLR7-and TRIF-dependent signalling events 10 . As a corollary, a role, per se, of the phosphorothioate backbone in the TLR7 (GpC-dependent) and TLR9 (higher-dose CpG-dependent) pathways of tolerogenesis is ruled out by the stimulatory effect of lower-dose CpG on the same backbone, which is in line with the dual effects both of CpG on a phosphodiester backbone and of bacterial DNA.…”

“…4b). (As expected 10 , under the same experimental conditions, the tolerogenic effects of GpC required neither TLR9 nor MyD88, Fig. 4b.)…”

“…S4). As expected 10 , the GpC-ODN manifested IDO1-dependent tolerogenic activity in a dose-independent fashion (that is, at both 1 and 10 mg ml À 1 ) (Fig. 1c).…”

“…2d). On the other hand, pDCs stimulation by GpC resulted in measurable TGF-b in culture supernatants at as early as 24 h, further underlining the qualitatively different mechanism of action of CpG and GpC in TLR signalling 10 .…”

“…Human pDCs stimulated with TLR7 or TLR9 ligands induce CD8 þ LAG-3 þ Foxp3 þ CTLA-4 þ regulatory (Treg) cells that suppress alloreactive memory T cells via an IDO1-dependent mechanism 9 . Unlike TLR9, TLR7 is targeted by synthetic GpCODNs, yet its univocally tolerogenic signalling exploits IDO1 as a downstream effector mechanism 10 .…”