T-bet is required for optimal production of IFN-gamma and antigen-specific T cell activation by dendritic cells

Abstract

IFN-gamma is well known as the signature cytokine of CD4+ T helper 1, CD8+, and natural killer cells, but recent studies demonstrate that antigen-presenting cells, in particular dendritic cells (DCs), are another potent source for this proinflammatory cytokine. T-bet, a transcription factor that controls IFN-gamma expression in CD4+ T cells, was reported recently to be expressed in human monocytes and myeloid DCs. In this study we investigate the role of T-bet in this important cell type. The development, differentiation, and activation of bone marrow and splenic DCs were unimpaired in mice lacking T-bet. However, T-bet was essential for the optimal production of IFN-gamma by both CD8alpha+ and CD8alpha- DCs. T-bet-deficient DCs were significantly impaired in their capacity to secrete IFN-gamma after both stimulation with IL-12 alone or in combination with IL-18. Further, T-bet-/- DCs were impaired in their ability to activate the T helper 1 program of adoptively transferred antigen-specific T cells in vivo. The rapid up-regulation of T-bet by IFN-gamma in DCs coupled with a function for DC-derived IFN-gamma in T cell activation may constitute a positive feedback loop to maximize type 1 immunity.

Fig. 1.

Normal development and activation of DCs in T-bet^-/- mice. (A and B) DCs were isolated from wt (Upper) and T-bet^-/- (Lower) mice on a B6 background. DCs derived from BM cultures (A) or spleen-gated on the CD11c⁺ population (B) were stained with the indicated Abs. KO, knockout. (C)wt(Left) and T-bet^-/- (Right) mice were injected i.p. with LPS and spDCs collected at the indicated times. DC maturation was assessed by FACS analysis of CD11c⁺ cells stained with CD86 (B7-2). The data shown are representative of at least four independent experiments.

Fig. 2.

T-bet expression in murine DCs. DCs were sorted by staining with Abs to CD11c and I-A^b and stimulated with IFN-γ as indicated. (A) T-bet mRNA expression was analyzed by real-time PCR in DCs derived from BM cultures (Left) or spleen (Right). (B) spDCs were purified with CD11c magnetic beads (Left) or by three-color FACS with CD8α (Right) and stimulated with IFN-γ (10 ng/ml). Whole extracts were prepared at the indicated time points. T-bet protein levels were detected by immunoblot analysis. The data shown are representative of at least three independent experiments. (C) T-bet mRNA expression analyzed by real-time PCR in spDCs isolated from wt (black) and STAT^-/- (white) mice.

Fig. 3.

Impaired IFN-γ production in T-bet^-/- DCs. B6 wt and T-bet^-/- DCs were seeded at a concentration of 1 × 10⁶ cells per ml and stimulated with IL-12 alone, or in combination with IL-18, at 10 ng/ml. Supernatants were collected after 72 h, and IFN-γ secretion was measured by ELISA. (A) DCs were derived from three independent BM cultures and purified at day 8 by using CD11c magnetic beads. (Left) ELISA results. (Right) IFN-γ mRNA expression measured by real-time PCR from cells collected at 72 h. (B) DCs were isolated from six independent preparations of spleens and sorted (CD11c⁺ and I-A^b). (C) Time-course analysis of IFN-γ secretion from CD11c⁺ I-A^b-sorted DCs from eight pooled spleens per genotype. These results are representative of at least three independent experiments. (D) CD11c⁺CD8α⁺ and CD8α^- subpopulations from wt and T-bet^-/- mice were isolated and stimulated with IL-12 or IL-12 plus IL-18 as described above, and IFN-γ was measured by ELISA. The data are representative of at least three independent experiments. (E) CD11c⁺CD8α⁺ and CD8α^- subpopulations from wt and T-bet^-/- mice were isolated and stimulated with CD40L-transfected NIH 3T3 cells for 72 h, and IFN-γ in supernatants was measured by ELISA. Brackets indicate the fold difference of IFN-γ levels between wt and T-bet^-/- samples. *, not detected; KO, knockout.

Fig. 4.

In vivo function of T-bet in DCs. (A) In vivo proliferation of DO11.10 T cells adoptively transferred into BALB/c recipients. These cells were stained with PE-coupled anticlonotypic KJ126 Ab and propidium iodide. The plots represent levels of CFSE on KJ126-positive and propidium iodide-negative cells. (Upper and Lower) Proliferation of D011.10 T cells primed with peptide-pulsed or unpulsed DCs, respectively. DCs were isolated from wt (Left) and T-bet^-/- (Right) mice. (B) Cytokine secretion from LN cells were harvested from the mice described above and cultured for 96 h with varying numbers of peptide-pulsed wt DCs. Results are expressed as the ratio of IL-2 or IFN-γ produced by DO11.10 T cells from mice primed in vivo with T-bet^-/- as compared with wt peptide-pulsed DCs, with the wt value set arbitrarily at 1.0. KO, knockout.