Late developmental plasticity in the T helper 17 lineage

Abstract

Development of T helper (Th) 17 cells requires transforming growth factor (TGF)-beta and interleukin (IL)-6 and is independent of the Th1 pathway. Although T cells that produce interferon (IFN)-gamma are a recognized feature of Th17 cell responses, mice deficient for STAT4 and T-bet-two prototypical Th1 transcription factors-are protected from autoimmunity associated with Th17 pathogenesis. To examine the fate and pathogenic potential of Th17 cells and origin of IFN-gamma-producing T cells that emerge during Th17 immunity, we developed IL-17F reporter mice that identify cells committed to expression of IL-17F and IL-17A. Th17 cells required TGF-beta for sustained expression of IL-17F and IL-17A. In the absence of TGF-beta, both IL-23 and IL-12 acted to suppress IL-17 and enhance IFN-gamma production in a STAT4- and T-bet-dependent manner, albeit with distinct efficiencies. These results support a model of late Th17 developmental plasticity with implications for autoimmunity and host defense.

Figure 1. Requirement for TGF-β in the Maintenance of IL-17 Expression by Th17-Polarized Cells

(A) FACS-sorted naive CD4⁺ T cells from OT-II TCR transgenic mice were cultured with irradiated Il12b^−/− (IL-12p40-deficient) splenic feeder cells and 5 μg/ml OVAp for 7 days under Th17-polarizing conditions (TGF-β, 5 ng/ml; IL-6, 20 ng/ml; anti-IFN-γ, 10 μg/ml; anti-IL-4, 10 μg/ml), then stained intracellularly for IL-17A and IFN-γ after PMA/ionomycin activation for 5 hr in the presence of monensin (1° culture; left panel). Cells were harvested and restimulated with irradiated Il12b^−/− splenic feeder cells in the presence of indicated cytokine(s) at the same doses or, in the case of IL-23 and IL-12, at 1 ng/ml, with anti-IFN-γ, anti-IL-4, and OVAp for an additional two rounds (7 days each) and stained intracelluarly for IL-17A and IFN-γ after PMA-ionomycin activation (post 3° culture; right panels). (B) Ifng^{Thy1.1/Thy1.1} OT-II naive CD4⁺ T cells were cultured with irradiated Il12b^−/− splenic feeder cells under Th17-polarizing conditions for 6 days and a fraction of the recovered cells were stained intracellularly for IL-17A and IFN-γ after PMA-ionomycin activation (left panel). A second fraction of recovered CD4⁺ T cells were activated for 5 hr with OVAp and Il12b^−/− feeder cells, and Thy1.1⁺ (IFN-γ⁺) cells were depleted by magnetic sorting. Thy1.1 depletion was confirmed by surface staining for Thy1.1 (middle panel), and the isolated Thy1.1- cells were cultured with Il12b^−/− feeder cells and OVAp and either TGF-β plus IL-6 or IL-23 in the presence of anti-IFNγ plus anti-IL-4 for three additional rounds (right panels), then stained intracellulary for IL-17A and IFN-γ after PMA-ionomycin activation. Numbers in each quadrant indicate percentages of total CD4⁺ T cells. All data are representative of at least three independent experiments.

Figure 2. Th17 Cells Respond to IL-12 to Rapidly Upregulate IFN-γ and Extinguish IL-17

OT-II CD4⁺ T cells were cultured with irradiated Il12b^−/− splenic feeder cells under Th17-polarizing condition for 7 days (1° culture, top panel), and a fraction of recovered T cells were restimulated and analyzed for intracellular IL-17A and IFN-γ as in Figure 1. The remaining T cells were cultured again with irradiated Il12b^−/− splenic feeder cells in the presence of indicated cytokine(s), anti-IFNγ, anti-IL-4, and OVAp as in Figure 1 for 7 days (2° culture, middle panel), and a fraction was reanalyzed for intracellular cytokine expression. The remaining recovered cells from secondary cultures were recultured under identical cytokine conditions for a third and fourth round (3° and 4° cultures, bottom panel), then analyzed for intracellular IL-17A and IFN-γ after PMA-ionomycin activation as before. Numbers in quadrants indicate percentage of total CD4⁺ cells in each. Data are representative of at least three experiments.

Figure 3. IFN-γ-Producing Effectors Arise from IL-17-Producing Precursors

(A) FACS-sorted naive CD4⁺ T cells from Il17f^{Thy1.1/Thy1.1} reporter mice were cultured with irradiated Il12b^−/− splenic feeder cells under Th17-polarizing conditions for 6 days as in Figure 1. A fraction of recovered cells were analyzed for expression of CD4, Thy1.1 (IL-17F), and intracellular cytokine IL-17A and IFN-γ after PMA-ionomycin stimulation for 5 hr in the presence of monensin. The flow-cytometric plots are gated on CD4⁺ cells. (B) Cells from (A) were collected and Thy1.1⁺ (IL-17F⁺) cells isolated by magnetic sorting. The purity of sorted Thy1.1⁺ cells was confirmed by surface staining of Thy1.1. Numbers in the histograms are the fraction of CD4⁺ T cells in the indicated gates. (C and D) Thy1.1⁺ cells were restimulated with irradiated Il12b^−/− splenic feeder cells in the presence of indicated cytokine(s), anti-IFNγ, anti-IL-4, and anti-CD3 as in Figure 1 for a second (C) and fourth round (D) and were stained for CD4, Thy1.1 (IL-17F), and intracellular IL-17A and IFN-γ after PMA-ionomycin activation for 5 hr. Numbers in each quadrant indicate the percentage of total CD4⁺ T cells. Data are representative of at least three independent experiments.

Figure 4. Divergence of Transcriptomes of Th17 Progeny Restimulated under Different Cytokine Conditions

FACS-sorted naive CD4⁺ T cells from Il17f^{Thy1.1/Thy1.1} mice were cultured with irradiated Il12b^−/− splenic feeder cells under Th17-polarizing conditions for 6 days. Cells were collected and Thy1.1⁺ (IL-17F⁺) cells were isolated by magnetic sorting as in Figure 3. Isolated Thy1.1⁺ cells were restimulated with anti-CD3 and anti-CD28 in the presence of indicated cytokine(s) and processed for mRNA quantification of the indicated genes at 6 hr (Fasl and Gzma) or 18 hr after stimulation by real-time PCR. All data were normalized to 18S RNA and are expressed as fold differences (log₂) relative to anti-CD3- and anti-CD28-stimulated cells. Data are representative of two or three experiments.

Figure 5. Transition of Th17 Precursors into IFN-γ-Producing Cells Is Stat4 and T-bet Dependent

(A) CD4⁺ T cells from WT (BALB/c), Stat4^−/−, or Tbx21^−/− mice were cultured with either WT or Il12b^−/− splenic feeder cells and anti-CD3 under Th17-polarizing conditions for 7 days, and then a fraction was analyzed for CD4 and intracellular IL-17A and IFN-γ after PMA-ionomycin stimulation for 5 hr. (B) Recovered CD4⁺ T cells were restimulated with either irradiated WT or Il12b^−/− splenic feeder cells in the presence of indicated cytokine(s), anti-IFNγ, anti-IL-4, and anti-CD3 for an additional 5 days and analyzed as in (A). (C and D) Naive CD4⁺ T cells from BALB or B6 WT, BALB.Stat4^−/−, or B6.Tbx21^−/− mice were cultured as in (A) for one round, a fraction was analyzed for CD4 and intracellular cytokines (IL-17A and IFN-γ) as in (A), and the remainder was recultured for three additional rounds under the indicated cytokine conditions as in (B). After the third round of culture, recovered BALB.WT and BALB.Stat4^−/− CD4⁺ T cells ([C], left and right panels) or B6.WT and B6.Tbx21^−/− ([D], left and right panels) were activated with PMA-ionomycin and analyzed for CD4 and intracellular IL-17A and IFN-γ. Numbers in quadrants indicate percentages of total CD4⁺ T cells in each. Data are representative of at least two independent experiments.

Figure 6. A Subset of Th17 Cells Extinguish IL-17 and Transition to Single-Positive IFN-γ Cells in Transfer Model of Colitis

(A and B) FACS-sorted CD4⁺CD45RB^hi T cells (4 ×10⁵) or CD4⁺CD45RB^lo (2 ×10⁵) T cells isolated from pooled spleen and LNs of Il17f^{Thy1.1/Thy1.1} mice were transferred into congenic Rag1^−/− mice as indicated. Th17-polarized T cells were generated from naive Il17f^{Thy1.1/Thy1.1} CD4⁺ T cells with Il12b^−/− APCs and anti-CD3 (2.5 μg/ml) as in Figure 1, in the absence (Th17 − IL-23) or presence (Th17 + IL-23) of 10 ng/ml IL-23. Thy1.1⁻ (IL-17F⁻ ) and Thy1.1⁺ (IL-17F⁺) cells were isolated by FACS sorting (see Figure S8), and 3 ×10⁵ cells were transferred as indicated. As shown in (A), 8 weeks after transfer, mice were sacrificed and intestinal tissues were collected for histological processing and analysis. Shown are representative sections of colon from indicated experimental groups; all magnifications represent 40×. (B) shows the incidence and severity of colitis 4 (upper panel) or 8 (lower panel) weeks after transfer of the indicated cell populations. n represents the number of recipients in each group. (C and D) IL-17A, Thy1.1 (IL-17F), and IFN-γ expression of CD4⁺ T cells isolated from MLNs of indicated recipient mice 4 weeks (upper two panels) or 8 weeks (lower panel) after cell transfer. Recovered cells were stimulated ex vivo with PMA-ionomycin and analyzed for CD4, Thy1.1, and intracellular IL-17A and IFN-γ. Numbers in each quadrant indicate percentages of total CD4⁺ T cells (C). Cumulative data for frequencies of cytokine-positive CD4⁺ T cells, as analyzed as in (C), are shown in (D). Data are the means ± SEM for tissues and groups indicated. All data are representative of results from five similar experiments.

Figure 7. Effects of IL-23 and IL-12 Blockade on Th17 Precursors in Transfer Model of Colitis

(A) Th17-polarized T cells were generated from naive Il17f^{Thy1.1/Thy1.1} CD4⁺ T cells with Il12b^−/− APCs and anti-CD3 (5 μg/ml) as in Figure 1, in the absence (Th17 -IL-23) or presence (Th17 +IL-23) of 5 ng/ml IL-23. Thy1.1⁺ (IL-17F⁺) cells were isolated by FACS sorting and 3 × 10⁵ cells were transferred into congenic Rag1^−/− mice as indicated. Recipients were administered 100 μg of anti-IL-23R, anti-IL-12p40, or isotype control via i.p. injection in saline 1 day prior and 1 day after T cell transfers and once weekly thereafter (five total doses). Mice were sacrificed 4 weeks after transfer and analyzed histologically and scored for development of colitis. (B) Mice that received Th17 -IL-23 (IL-17F⁺) T cells and treated as in (A) were sacrificed 4 weeks after transfer, and cells recovered from MLNs were stimulated ex vivo with PMA ionomycin and analyzed for CD4 and intracellular IL-17A and IFN-γ by FACS. Data are the means from five mice in each treatment group. *p < 0.05 and **p < 0.01 versus control treatment group. Data are representative of two similar experiments.