Optimal TLR9 signal converts tolerogenic CD4-8- DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses.

Abstract TLR ligands have been reported to promote DC maturation and enhance CD8+ CTL responses. We have demonstrated previously that CD4-8- DCs secreting TGF-beta stimulate CD4+ Tr1 cell responses. Here, we have assessed whether TLR4 and TLR9 signaling through LPS and CpG stimulation can convert CD4-8- DC-induced tolerance. We demonstrate that immature OVA-pulsed CD4-8- DCs cultured in medium with LPS (2 microg/ml) and CpG (5 microg/ml) for 8 h became mature DCs (DCOVA) with no TGF-beta secretion. CpG-treated, CD4-8- DCOVA-secreting IL-6/IL-15 induced IFN-gamma/IL-17-secreting/T-bet- and ROR-gammat-expressing CD4+ Th1/Th17, whereas LPS-treated CD4-8- DCOVA stimulated IFN-gamma-secreting/T-bet-expressing CD4+ Th1 responses. The former also significantly stimulated more efficient OVA-specific CD8+ T cell responses and antitumor immunity against OVA-expressing BL6-10OVA tumor cells than the latter (P<0.05). CpG-treated, CD4-8- DCOVA-stimulated CD4+ Th1/Th17 cell responses and antitumor immunity were found to be reduced by using neutralizing anti-IL-6, IL-15, and NK1.1 antibodies in wild-type C57BL/6 mice, IL-15R-/- mice for immunization, or CD4-8- (IL-6-/-) DCOVA for immunization in C57BL/6 mice. Interestingly, in vitro-generated CD4+ Th17 cells significantly enhanced LPS-treated, CD4-8- DCOVA-induced in vivo antitumor immunity via increasing CD8+ CTL responses (P<0.05), although they did not show any direct killing activity against tumor cells in vitro. In addition, prolonged 48 h CpG-treated CD4-8- DCOVA dramatically diminished its cytokine secretion, stimulatory effect, and antitumor immunity. Taken together, our data demonstrate an effect of conversion of tolerogenic DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses by optimal CpG signaling, which may advance current understanding of the importance of TLR9 signaling in a DC-based cancer vaccine.