T-bet is a critical determinant in the instability of the IL-17-secreting T-helper phenotype

Abstract

IL-23, an IL-12-related cytokine, induces an IL-17-secreting T-helper phenotype that is involved in autoimmune diseases and host defense against certain pathogens. Although the transcription factors required for development of IL-23-stimulated cells are unknown, we show that T-bet is a critical negative regulator of the IL-23-primed T-cell phenotype, which we term Th1beta. Th1 or Th1beta Tbx21-/- cultures secrete higher than WT levels of IL-17 in response to T-cell receptor (TCR) or IL-23 + IL-18 stimulation. Ectopic T-bet expression in Th1beta cells promotes IFN-gamma secretion but decreases IL-17 production. Although antigen-receptor stimulation of Th1beta cells stimulates IL-17 production, it also induces the IFN-gamma-independent expression of T-bet and progression to a Th1 cytokine secretion pattern. T-bet is required for the progression to the Th1 phenotype, because Tbx21-/- Th1beta cultures maintain the IL-17-secreting phenotype after 2 weeks of culture. Addition of IFN-gamma to Tbx21-/- Th1beta cultures cannot recover the progression to the Th1 phenotype, suggesting T-bet, rather than IFN-gamma, mediates Th1beta to Th1 progression. The transient nature of the Th1beta phenotype suggests that these cells are a component of type I immunity and that T-bet expression is a critical determinant of Th1 versus Th1beta cell fate.

Figure 1.

IL-23-cultured CD4⁺ T cells transiently secrete IL-17 and progress to a Th1 cytokine-secreting profile. (A) CD4⁺ T cells from C57BL/6 mice were activated with α-CD3 and α-CD28 and cultured with α-IL-4. IL-12 or IL-23 was added to the culture to generate Th1 or Th1β cells, respectively. Cells were cultured for 3 days and expanded in the presence of IL-12 or IL-23 for 2 additional days. After 5 days, cells were left unstimulated or were restimulated with α-CD3 for 24 hours, and cell-free supernatants were analyzed for IL-17 and IFN-γ secretion using ELISA. (B) Th1β cells were cultured for 5 days as in panel A. Cells were replated with media alone or restimulated with α-CD3 and α-CD28 for 5 hours in the presence of Brefeldin A. Cells were fixed, permeabilized, and stained with PE-α-IL-17 and FITC-α-IFN-γ antibodies and analyzed for intracellular cytokine staining by flow cytometry. Numbers indicate percentage of cells within each region. (C) Th1 or Th1β cells differentiated for 5 days (week 1) in panel A were cultured for 5 additional days (week 2) with or without plate-bound α-CD3. Th1 and Th1β cultures were maintained in media containing IL-12 or IL-23, respectively. At day 10, live cells were cultured in the presence or absence of plate-bound α-CD3 for 24 hours, and supernatants were analyzed for IFN-γ and IL-17 production using ELISA. Unstimulated cultures at day 10 did not produce detectable levels of cytokine. (D) Five-day Th1β cultures differentiated in the presence or absence of α-IFN-γ (10 μg/mL) were restimulated as in panel A, and cell-free supernatants were measured for IL-17 quantities using ELISA (week 1). In parallel, 5-day differentiated cells were cultured for 5 more days (week 2) in the presence of plate-bound α-CD3 and maintained in media containing IL-23 with or without α-IFN-γ as indicated in the figure. Two-week cultures were restimulated as in panel C, and IL-17 levels from cell-free supernatants were measured using ELISA. ELISA data are represented as mean ± SE of replicate samples.

Figure 2.

T-bet negatively regulates Th1β development. (A) WT and Tbx21^-/- CD4⁺ T cells were cultured under Th1 (IL-12 + α-IL-4) or Th1β (IL-23 + αx-IL-4) conditions for 5 days and restimulated with α-CD3 for 18 hours. Cell-free supernatants were analyzed for IFN-γ or IL-17 using ELISA. Unstimulated populations did not produce detectable levels of cytokine. RNA was extracted from T cells activated for 18 hours, and duplicate samples were quantified by real-time PCR for IL-17A mRNA. Cycle number was normalized to β2-microglobulin mRNA and is represented as fold induction relative to unstimulated wild-type Th1 cells. ND indicates not detected. (B) WT CD4⁺ T cells from C57BL/6 mice were cultured with α-IL-4 alone (Unsk.) or under Th1 or Th1β conditions for 5 days as described in Figure 1A. Protein lysates from freshly isolated CD4⁺ T cells or differentiated cells were assayed for T-bet expression by immunoblot. (C) WT Th1β cells were cultured for 2 days and infected with a bicistronic retrovirus expressing EGFP alone (vector control) or T-bet and EGFP (T-bet RV). Cells were cultured for 3 more days, and EGFP-positive cells were sorted and restimulated with α-CD3 or left unstimulated for 18 hours. Cell-free supernatants were analyzed for IL-17 and IFN-γ production. Unstimulated cell populations did not produce detectable cytokine levels. ELISA data are represented as mean ± SE of replicate samples.

Figure 3.

T-bet negatively regulates cytokine-induced IL-17 production but does not augment cytokine receptor expression on Th1 or Th1β cells. (A) WT or Tbx21^-/- Th1 or Th1β cells were cultured for 5 days as in Figure 1A (1° culture). Cells were restimulated with media alone or the indicated cytokines (2° culture) for 18 hours and analyzed for IFN-γ or IL-17 production using ELISA. ND indicates not detected. (B) WT or Tbx21^-/- Th1 or Th1β cells were cultured as in Figure 1A. IL-12Rβ2, IL-23R, or IL-18Rα expression was assessed using flow cytometry. Numbers within each histogram represent mean fluorescent intensities averaged from 2 independent experiments. Black line indicates receptor staining; gray fill, control staining. ELISA data are represented as mean ± SE of replicate samples.

Figure 4.

Antigen-receptor stimulation induces T-bet expression in Th1β cells, which is required for its progression to an IFN-γ secreting state. (A) WT C57BL/6 Th1 and Th1β cells were cultured for 5 days and left unstimulated or restimulated with α-CD3 with or without 15 μg/mL α-IFN-γ for 4 hours. Protein lysates were analyzed for T-bet expression by immunoblot (B) WT and Tbx21^-/- Th1β cells were cultured for 5 days and left unstimulated or were restimulated with α-CD3 for 24 hours. Cell-free supernatants were collected on day 6 and measured for IFN-γ or IL-17 secretion using ELISA. In parallel, 5-day differentiated cells were replated on α-CD3-coated plates and IL-23 for 5 more days as in Figure 1C. After the culture period, live cells were washed and left unstimulated or restimulated with α-CD3 for 24 hours. Cell-free supernatants were collected on day 11. Supernatants from both time points were analyzed for IL-17 and IFN-γ production using ELISA. (C) Tbx21^-/- Th1β cells were cultured as in panel B with or without the indicated doses of IFN-γ during the second week of culture. Cells were left unstimulated or were restimulated with α-CD3 for 24 hours, and cell-free supernatants on days 6 and 11 were analyzed for IL-17 production using ELISA. Unstimulated cell populations did not produce detectable cytokine levels (B-C). ELISA data are represented as mean ± SE of replicate samples.

Figure 5.

Proposed model for Th1 and Th1β development. CD4⁺ T-helper cells (Th) in the presence of IL-12 or IL-23 develop into Th1 or Th1β phenotypes, respectively. Although T-bet induces Th1 development, it negatively regulates IL-23-mediated T-cell development. Antigen-receptor engagement of differentiated IL-17-secreting Th1β cells induces T-bet expression and promotes a switch to a Th1 cytokine-secreting phenotype.