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. 2006 Mar 20;203(3):755-66.
doi: 10.1084/jem.20052165. Epub 2006 Mar 6.

T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

Takashi Usui  1 , Jan C Preiss, Yuka Kanno, Zheng Ju Yao, Jay H Bream, John J O'Shea, Warren Strober
Affiliations

T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

Takashi Usui et al. J Exp Med. .

Erratum in

  • J Exp Med. 2006 Apr 17;203(4):1129

Abstract

T helper type 1 (Th1) development is facilitated by interrelated changes in key intracellular factors, particularly signal transducer and activator of transcription (STAT)4, T-bet, and GATA-3. Here we show that CD4+ cells from T-bet-/- mice are skewed toward Th2 differentiation by high endogenous GATA-3 levels but exhibit virtually normal Th1 differentiation provided that GATA-3 levels are regulated at an early stage by anti-interleukin (IL)-4 blockade of IL-4 receptor (R) signaling. In addition, under these conditions, Th1 cells from T-bet-/- mice manifest IFNG promotor accessibility as detected by histone acetylation and deoxyribonuclease I hypersensitivity. In related studies, we show that the negative effect of GATA-3 on Th1 differentiation in T-bet-/- cells arises from its ability to suppress STAT4 levels, because if this is prevented by a STAT4-expressing retrovirus, normal Th1 differentiation is observed. Finally, we show that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels. These findings lead to a model of T cell differentiation that holds that naive T cells tend toward Th2 differentiation through induction of GATA-3 and subsequent down-regulation of STAT4/IL-12Rbeta2 chain unless GATA-3 levels or function is regulated by T-bet. Thus, the principal function of T-bet in developing Th1 cells is to negatively regulate GATA-3 rather than to positively regulate the IFNG gene.

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Figures

Figure 1.
Figure 1.
Elevated IL-4 and GATA-3 and committed Th2 cells in T-bet−/− mice. (A) Naive CD4+ T cells and whole CD4+ T cells from T-bet−/−, T-bet+/+, and wild-type mice were stimulated with anti-CD3/anti-CD28 and maintained under Th1 conditions (IL-12 only) and expanded with IL-2. On day 6, the cells were subjected to intracellular cytokine staining after restimulation with PMA/ionomycin. These data are representative of results obtained from three independent experiments. (B) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under Th1 conditions (IL-12 only) or neutral (no cytokines or antibodies added) conditions for 48 h, and then culture supernatants were harvested for IFN-γ and IL-4 determination by ELISA. The data shown are averages ± SE derived from three independent experiments. (C) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with anti-CD3/ anti-CD28 under Th1 conditions (IL-12 only) or neutral conditions and maintained for the time periods shown in the figure. At each time point, cells were harvested and total RNAs were isolated and subjected to real-time PCR analysis for GATA-3 mRNA quantification. Independently, total RNAs from freshly isolated naive and memory CD4+ T cells were prepared for day 9 samples. The data shown are averages ± SE derived from three independent experiments.
Figure 2.
Figure 2.
T-bet–independent Th1 development and IL-12Rβ2 chain expression. (A) Naive CD4+ T cells from T-bet−/− and wild-type mice (purified by flow cytometric sorting) were stimulated with Con A/APC (30 Gy-irradiated splenocytes from wild-type mice) under optimal Th1 conditions (IL-12 + anti–IL-4), suboptimal Th1 conditions (IL-12 only or anti–IL-4 only), neutral conditions, or suboptimal Th2 (IL-4 only) conditions. The cells were then expanded with IL-2 as well as cytokines and antibodies on days 2 and 4. On day 8, the cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4 and IFN-γ. See Materials and methods for further details. (B) Experimental design was the same as in A, except that cells subject to two kinds of stimulation (Con A/APC and anti-CD3/anti-CD28) were compared. These data are representative of results obtained from three independent experiments. (C) Naive CD4+ T cells from T-bet−/− and wild-type mice (purified by flow cytometric sorting) were stimulated with anti-CD3/anti-CD28 in the presence of IL-12 and 20 μg/ml anti–IL-4 (for optimal Th1 conditions) or with 5 ng/ml IL-12 (for suboptimal Th1 conditions). The cells were expanded by the addition of IL-2 on day 4 of culture and then washed and restimulated with PMA/ionomycin or plate-bound (pb) anti-CD3 on day 6, and culture supernatants were collected after 6 and 24 h, respectively, for assay of IFN-γ by ELISA (See Materials and methods).
Figure 3.
Figure 3.
T-bet−/− cells cultured under optimal Th1 conditions (in the presence of anti–IL-4) display normal IFNG promoter accessibility (histone acetylation). (A) Naive CD4+ T cells from T-bet+/+ and T-bet−/− mice were stimulated with anti-CD3/anti-CD28 under suboptimal (IL-12) or optimal (IL-12 plus anti–IL-4) Th1 conditions. After 3 d, the cells were subjected to ChIP assay as described in Materials and methods. Ig, isotype control H3, α-acetyl-histone 3. (B) DNase I hypersensitivity pattern of the IFNG locus of wild-type naive T cells stimulated with anti-CD3/anti-CD28 and cultured under optimal, suboptimal, or neutral conditions for 6 d. Hypersensitivity sites I and II are indicated by arrows. (C) Comparison of DNase I hypersensitivity patterns of the IFNG locus in wild-type and T-bet−/− naive CD4+ T cells cultured under optimal or optimal conditions for 6 d. Hypersensitivity sites I and II are indicated by arrows.
Figure 4.
Figure 4.
STAT4 signaling is impaired in T-bet−/− CD4+ T cells. (A) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under either suboptimal Th1 conditions (IL-12 only) or neutral conditions. After maintenance for the time periods shown in the figure, whole cell lysates were subjected to Western blot analysis. Independently, lysates from freshly isolated naive CD4+ T cells were prepared for day 0 samples. Similar results were obtained in three other independent experiments. (B) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under either optimal Th1 conditions (IL-12 + anti–IL-4) or suboptimal Th1 conditions (IL-12 only), and then expanded with IL-12 as well as cytokines and antibodies on days 2 and 4. On day 7, the cells were restimulated with fresh IL-12 for 30 min and lysed for Western blot analysis. Recombinant GATA-3 protein was prepared from GATA-3–expressing retrovirus-transfected Phoenix-Eco packaging cell line. Similar results were obtained in three other independent experiments. (C) Naive CD4+ T cells from T-bet−/− mice were stimulated and maintained as described in B. On day 2, the cells were infected with either control retrovirus (GFP-RV) or STAT4-expressing retrovirus (STAT4-RV). On day 7, the cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4 and IFN-γ. (D) The cells used in C were sorted by a flow cytometer according to GFP expression on day 6 and then lysed for Western blot analysis. The lysate of the Th2 line from T-bet−/− mice was also prepared as a positive control for GATA-3 protein. These data are representative of those obtained in three independent experiments.
Figure 5.
Figure 5.
T-bet overexpression suppresses GATA-3 and Th2 cytokines. (A) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under strict Th2 conditions (IL-4 + anti–IFN-γ + anti–IL-12). On day 2, the cells were infected with control (mock) or T-bet–expressing retrovirus (T-bet-RV). Finally, on day 7, GFP+ cells were sorted by flow cytometry and lysed for Western blot studies. These data are representative of those obtained in three independent experiments. (B) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under optimal Th1 conditions. On day 2, the cells were infected with control (mock) or STAT4-, IL-12Rβ2 chain–, or T-bet–expressing retroviruses. Finally, on day 7, cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4. The values shown were the percentages of IL-4+ cells in the GFP+ population. These data are representative of those obtained from two independent experiments. (C) An established murine Th2 cell line (D10 cells) was stimulated with conalbumin/APCs and then infected with a T-bet–expressing retrovirus on days 2, 3, 4, and 5. On day 14, GFP+ cells were enriched by flow cytometric sorting and restimulated with antigen/APC. Finally, on day 21, GFP+ and GFP− cells were separated by sorting (purities were >92%) and lysed for Western blot analysis. (D) T-bet–expressing and nonexpressing D10 cells were obtained and cultured as described in C. On day 21, the cells were restimulated with either high (2 μg/ml) or low (0.2 μg/ml) concentrations of plate-bound anti-CD3 and/or conalbumin/APCs. 48 h after such restimulation, culture supernatants were collected and IL-4, IL-5, and IFN-γ were measured by ELISA. Similar results were obtained in three independent experiments. (E) T-bet–expressing and nonexpressing D10 cells were obtained and cultured as described in C. On day 21, 2 μg/ml actinomycin D was added to the cultures, and cells were harvested every 30 min for extraction of total RNA and subsequent real-time PCR analysis for GATA-3 mRNA. The left panel depicts the relative amount of GATA-3 mRNA normalized by 18S rRNA, and the right panel depicts the percent decreases from the nontreated sample. Similar results were obtained in two independent experiments.
Figure 6.
Figure 6.
T-bet is not a direct inducer of IL-12Rβ2 chain. (A) T-bet–expressing and nonexpressing D10 cells were obtained and cultured as described in Fig. 4 C and harvested for IL-12Rβ2 chain staining on day 21. The values shown in each panel depict the Δ mean fluorescence intensities (MFI)s for the IL-12Rβ2 chain stain after subtraction of the MFIs of a control stain. Open histograms depict isotype controls, and filled histograms depict IL-12Rβ2 chain–specific staining. (B) Naive CD4+ T cells from T-bet−/− and wild-type mice were stimulated with Con A/APCs under optimal Th1 (IL-12 + anti–IL-4) or Th2 conditions. On day 2, the cells were infected with control (mock), STAT4-, or T-bet–expressing retroviruses, and on day 5, surface IL-12Rβ2 chain staining was performed. The values shown in each panel represent the ΔMFIs of IL-12Rβ2 chain expression in either GFP− or GFP+ populations after subtraction of the MFIs of control stainings.
Figure 7.
Figure 7.
STAT4 signal induces T-bet in the absence of STAT1. (A) Naive CD4+ T cells from BALB/c and STAT1−/− mice were stimulated with anti-CD3/anti-CD28 under Th2 conditions for 4 d. 24 h after stimulation, the cells were infected with mock-RV or STAT4-RV. On day 4, the cells were washed extensively and maintained under Th1 conditions (IL-12 and anti–IL-4 antibody) for another 3 d. Finally, the cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4 and IFN-γ. Similar results were obtained in three independent experiments. (B) The same cells used in A were initially cultured under Th2 conditions for 4 d and then either cultured under Th1 conditions or continued under Th2 conditions for 3 d. The cells were then sorted on day 7 according to GFP expression to obtain cells expressing STAT4-RV and then lysed for Western blot analysis. Similar results were obtained in three independent experiments. (C) The same cells used in A were initially cultured under Th2 conditions for 4 d and then either cultured under Th1 conditions with two concentrations of IL-12 or continued under Th2 conditions for 3 d. The cells were then sorted on day 7 according to GFP expression to obtain cells expressing STAT4-RV or GFP-RV (mock) and then lysed for Western blot analysis. Similar results were obtained in three independent experiments. (D) Naive CD4+ T cells from C57BL/6, STAT1−/−, IFN-γ−/−, and IFN-γR−/− mice were stimulated with anti-CD3/anti-CD28 either under strict Th1 conditions (IL-12 + anti–IL-4 antibody) or suboptimal Th1 conditions (IL-12 only) and expanded by hu-IL-2. On day 7, these cell lines were lysed and subjected to Western blot analysis. Similar results were obtained in two independent experiments.
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