T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells

Abstract

Differentiation of naive CD4(+) T cells into IFN-gamma-producing T helper 1 (T(H)1) cells is pivotal for protective immune responses against intracellular pathogens. T-bet, a recently discovered member of the T-box transcription factor family, has been reported to play a critical role in this process, promoting IFN-gamma production. Although terminal T(H)1 differentiation occurs over days, we now show that challenge of mice with a prototypical T(H)1-inducing stimulus, Toxoplasma gondii soluble extract, rapidly induced IFN-gamma and T-bet; T-bet induction was substantially lower in IFN-gamma-deficient mice. Naive T cells expressed little T-bet, but this transcription factor was induced markedly by the combination of IFN-gamma and cognate antigen. Human myeloid antigen-presenting cells showed T-bet induction after IFN-gamma stimulation alone, and this induction was antagonized by IL-4 and granulocyte/macrophage colony-stimulating factor. Although T-bet was induced rapidly and directly by IFN-gamma, it was not induced by IFN-alpha, lipopolysaccharide, or IL-1, indicating that this action of IFN-gamma was specific. Moreover, T-bet induction was dependent on Stat1 but not Stat4. These data argue for a model in which IFN-gamma gene regulation involves an autocrine loop, whereby the cytokine regulates a transcription factor that promotes its own production. These findings substantially alter the current view of T-bet in IFN-gamma regulation and promotion of cell-mediated immune responses.

Figure 1

IFN-γ signaling regulates T-bet expression. Murine splenocytes were collected from four wild-type or four IFN-γ^−/− mice 6 h after T. gondii antigen challenge as well as from four untreated mice from each strain. IFN-γ (A) and T-bet (B) mRNA levels were measured by quantitative real-time reverse transcription–PCR (RT-PCR). (C) Splenocytes obtained from BALB/c/Stat4^+/+ (wild type) and BALB/c/Stat4^−/− (Stat4 KO) mice were treated with IFN-γ for 3 h. T-bet mRNA levels were assayed by quantitative real-time RT-PCR. (D) Splenocytes obtained from 129/SvEv/Stat1^+/+ (wild type) and 129/SvEv/Stat1^−/− (Stat1 KO) mice were treated with IFN-γ for 3 h in vitro. T-bet mRNA levels were assayed by quantitative real-time RT-PCR, using mRNA from Con-A-activated murine splenocytes for normalization. This level is arbitrarily designated as 1.0.

Figure 2

IFN-γ in conjunction with cognate antigen (Ag) induces T-bet and T_H1 differentiation in naive CD4⁺ T cells. Naive CD4⁺ T cells were isolated from lymph nodes of 5C.C7 T cell receptor transgenic/Rag2-deficient mice, incubated with APCs and anti-IL-12 (10 μg/ml) in the presence (solid lines) or absence (dashed lines) of peptide antigen (100 μM) and various amounts of exogenous IFN-γ as indicated. Neutralizing anti-IFN-γ antibody (10 μg/ml) was added to selected cultures stimulated with peptide without exogenous IFN-γ (□). At 24 h, T-bet mRNA (A) and IFN-γ mRNA (B) levels were determined by quantitative RT-PCR. (C) In similar experiments, exogenous IFN-γ (5 units/ml) and anti-IFN-γ antibody blockade were analyzed for their effects on IFN-γ and IL-4 production, as detected by staining fixed, permeabilized cells for cytosolic cytokine content, at 72 h. Numbers in the upper right quadrant of each histogram indicate the percentage of IFN-γ-positive cells.

Figure 3

T-bet is induced rapidly in monocytes and DC by IFN-γ. Purified human monocytes were treated as indicated with IFN-γ and/or LPS, and T-bet mRNA levels were measured by Northern blotting (A Upper) and by real-time PCR (B). The experiment shown in A represents 3 h of stimulation; a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to confirm equal loading (A Lower). (C) Cultured human myeloid DC were either removed (shaded) or maintained (unshaded) in IL-4- and GM-CSF-containing medium before treatment with IFN-γ or LPS as indicated. T-bet levels were assayed by quantitative RT-PCR.

Figure 4

T-bet mRNA is induced specifically by IFN-γ but not IFN-α. Human monocytes were stimulated for 3 h with various concentrations of IFN-γ and IFN-α as indicated. T-bet mRNA levels were measured by real-time RT-PCR.