The adjuvant activity of CpG DNA requires T-bet expression in dendritic cells

Abstract

Treatment with synthetic oligodeoxynucleotides containing CpG motifs (CpG ODNs) is remarkably protective against otherwise lethal infection. Here, we describe an essential role for the transcription factor T-bet in mediating the protective function of CpG ODNs. Loss of T-bet in conventional CD11c(hi) dendritic cells (DCs) and in plasmacytoid DCs impaired production of IFNs. Strikingly, in contrast to Rag2-/- mice, Rag2-/- mice that also lacked T-bet (DKO) could not be rescued from lethal Listeria monocytogenes infection by prior treatment with CpG ODN. Rescue was achieved by adoptive transfer of CD11c(hi) DCs from WT, but not T-bet-/-, CpG ODN-treated donor mice. We conclude that T-bet in DCs is required for the adjuvant activity of CpG ODN in infection, revealing its vital role in innate immunity.

Fig. 1.

Rag2^-/- mice deficient in T-bet (double-knockout, DKO) succumb to infection with L. monocytogenes despite treatment with CpG ODN. (A) Survival curves of B6 WT and Rag2^-/- mice. (B) Survival curves of BALB/c WT, T-bet^-/-, and Rag2^-/-T-bet^-/- (DKO) mice. All mice were treated (i.p.) with 50 μg of CpG ODN, control ODN, or PBS only. Three days later, mice were challenged with 1,000 LD₅₀ of Listeria (i.p.). Cumulative survival rates were monitored on a daily basis for 14 days. All immunizations and bacterial challenge experiments were performed on a minimum of 10 mice per group. Statistical significance was evaluated by using a Kaplan–Meier survival analysis.

Fig. 2.

T-bet expression in DCs is dependent on IFNγ. (A and B) Classical DCs (DX5^neg/CD11c^hi/MHC-II^hi) (A) and pDCs (DX5^neg/CD11c^Int/B220^hi) (B) were FACS-sorted (purity >95%) from the spleens of B6 and 129S/v WT, 129S/v Stat1^-/-, and B6 IFNγ^-/- mice. (C) B cells were positively selected with anti-CD19-coated magnetic beads from the spleens of WT and IFNγ^-/- mice and stimulated with CpG ODN (1 μg/ml) alone or in combination with exogenous IL-12 (10 ng/ml) and IFNγ (10 ng/ml). At the indicated times, mRNA was prepared from each sample for real-time PCR analysis. T-bet mRNA is expressed as copies per 10,000 mRNA copies of β-actin.

Fig. 3.

T-bet is required for optimal production of type I/II IFNs. (A) IFNγ production from classical DCs (DX5^neg/CD11c^hi/MHC-II^hi) and pDCs (DX5^neg/CD11c^Int/B220^hi). (B) IL-12p40 production from classical and pDCs. (C) IFNα production from pDCs. DCs were FACS-sorted (purity >95%) from WT and T-bet^-/- spleens and stimulated with CpG ODN (1 μg/ml) alone or in combination with exogenous IL-12 (10 ng/ml) and IFNγ (10 ng/ml). At the indicated times, supernatants were harvested for ELISA to measure IFNγ, IL-12p40, and IFNα. These results represent at least three independent experiments, except for pDC production of IFNγ and IL-12, which represents two independent experiments. *, not detected.

Fig. 4.

Adoptive transfer of DCs from CpG ODN-treated WT but not T-bet^-/- mice restores resistance of Rag2^-/-T-bet^-/- DKO mice to lethal L. monocytogenes. WT and T-bet^-/- donor mice were treated with CpG ODN for 72 h. Classical DCs (DX5^neg/CD11c^hi/MHC-II^hi) were FACS-sorted (purity >95%) from donor mice and adoptively transferred to Rag2^-/- and DKO mice (7 × 10⁵ per mouse) that were challenged with L. monocytogenes (250 LD₅₀) a day later. Survival was monitored for 21 days. Statistical significance between groups ranged from P < 0.001 to P < 0.0001.