T-bet is essential for encephalitogenicity of both Th1 and Th17 cells

Abstract

The extent to which myelin-specific Th1 and Th17 cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is controversial. Combinations of interleukin (IL)-1beta, IL-6, and IL-23 with transforming growth factor beta were used to differentiate myelin-specific T cell receptor transgenic T cells into Th17 cells, none of which could induce EAE, whereas Th1 cells consistently transferred disease. However, IL-6 was found to promote the differentiation of encephalitogenic Th17 cells. Further analysis of myelin-specific T cells that were encephalitogenic in spontaneous EAE and actively induced EAE demonstrated that T-bet expression was critical for pathogenicity, regardless of cytokine expression by the encephalitogenic T cells. These data suggest that encephalitogenicity of myelin-specific T cells appears to be mediated by a pathway dependent on T-bet and not necessarily pathway-specific end products, such as interferon gamma and IL-17.

Figure 1.

Th17 cells differentiated in vitro with TGF-β and IL-6 do not transfer EAE. Naive Vα2.3/Vβ8.2 transgenic splenocytes were differentiated in vitro with MBP Ac1-11 plus TGF-β + IL-6 or IL-12 for 3 d. (A) Cells were harvested and analyzed by flow cytometry. Cells were gated on CD4⁺ T cells, and intracellular IL-17 and IFN-γ were analyzed. Data are representative of three independent experiments (percentages are shown). (B) Cells harvested from A were adoptively transferred into wild-type B10.PL recipient mice. Data are representative of six independent experiments (means ± SEM). (C) Naive Vα2.3/Vβ8.2 transgenic splenocytes were differentiated in vitro with MBP Ac1-11 and TGF-β + IL-6 for 3 d. The Th17 cells were rested for 4 d and restimulated with MBP Ac1-11 in the absence of exogenous cytokines for 2 d. The cells were analyzed for intracellular cytokines by flow cytometry (gated on CD4⁺ T cells; top) or transferred into naive B10.PL mice (Table I). CNS mononuclear cells were isolated from recipient B10.PL mice on day 7 after adoptive transfer (bottom). Flow cytometry was used to evaluate IFN-γ– or IL-17–producing T cells. Cells were gated on CD45 and Vβ8 TCR-positive cells. Data are representative of two independent experiments (percentages are shown).

Figure 2.

Encephalitogenicity correlates with IFN-γ production and T-bet expression. (A) Naive splenocytes from wild-type B10.PL mice were activated in vitro with anti-CD3/CD28 plus different combinations of cytokines for 72–96 h. ELISA was performed to detect IL-17 and IFN-γ secretion. Data are representative of three independent experiments (means ± SEM). (B) Naive Vα2.3/Vβ8.2 transgenic splenocytes were differentiated in vitro with MBP Ac1-11 plus different combinations of cytokines for 3 d and transferred into naive B10.PL mice. Data are representative of two independent of experiments (means ± SEM). (C) Cells from B were harvested and flow cytometry was used to evaluate IFN-γ, IL-17, IL-10, and T-bet (white, T-bet; gray, isotype control) expression in CD4⁺ T cells before transfer. Data are representative of two independent experiments (percentages are shown).

Figure 3.

T-bet expression correlates with encephalitogenicity. (A) Splenocytes from a Vα2.3/Vβ8.2 transgenic mouse with spontaneous EAE were activated in vitro with MBP Ac1-11, MBP Ac1-11 plus TGF-β, or MBP Ac1-11 plus TGF-β/IL-6 for 3 d. Flow cytometry was used to evaluate cytokine production and T-bet expression on CD4⁺ T cells. Data are representative of two independent experiments (percentages are shown). (B) B6 wild-type mice were injected with siRNA-Tbet or siRNA-NS at the time of immunization with MOG35-55/CFA. The lymph nodes were removed on day 11 after immunization and stimulated with MOG 35–55 for 3 d. Flow cytometry was used to evaluate cytokine production on activated CD4⁺ T cells and to confirm T-bet suppression that was >95% (not depicted). Data are representative of two independent experiments (percentages are shown). (C) Naive Vα2.3/Vβ8.2 transgenic splenocytes were differentiated into Th17 cells in vitro with MBP Ac1-11 plus TGF-β/IL-6 for 3 d. Cells were rested and restimulated with MBP Ac1-11 in the presence of irradiated feeders or with1 µg/ml each of plate-bound anti-CD3/CD28. After 48 h, cells were analyzed by flow cytometry. Cells were gated on CD4 and T-bet expression was analyzed in each quadrant based on IL-17 and IFN-γ expression (gray, isotype control; white, T-bet). Data are representative of two independent experiments (percentages are shown). (D) Naive Vα2.3/Vβ8.2 transgenic splenocytes were differentiated into Th17 cells in vitro with MBP Ac1-11 plus TGF-β/IL-6 for 3 d. Cells were rested for 4 d and transfected with siRNA-Tbet or siRNA-NS, followed by a 2-d restimulation with MBP Ac1-11 with nontransfected irradiated feeder cells. 5 × 10⁶ cells per mouse were transferred into naive B10.PL mice. Data are representative of three independent experiments (means ± SEM). (E) T-bet suppression was verified by flow cytometry. Data are representative of two independent experiments (percentages are shown). (F) ELISA was performed to detect IL-17 and IFN-γ secretion of the cells before transfer. Data are representative of two independent experiments (means ± SEM).

Figure 4.

Myelin-specific T cells in the CNS of IFN-γ–deficient mice express IL-17 and T-bet. EAE was induced in B6/IFN-γ^−/− mice by immunization with MOG35-55 emulsified in CFA. The CNS-infiltrating mononuclear cells were isolated from the brains and spinal cords of 14 mice 5 d after the mice began to develop signs of EAE. The cells were cultured overnight with or without 2 µg/ml MOG35-55. PMA/ionomycin was added to all cells during the last 5 h of culture. Cells were gated on CD45 and CD4. (A) Intracellular IL-17 expression. (B) Intracellular T-bet expression (white, T-bet; gray, isotype control). (C) T-bet expression was analyzed in the CD4⁺IL-17⁺ and CD4⁺IL-17⁻ T cells in the absence or presence of MOG35-55 (white, T-bet; gray, isotype control). (D) Naive TCR transgenic splenocytes were differentiated with IL-6 + TGF-β, rested, restimulated with antigen, and transferred into naive B10.PL mice. Inflammatory cells were isolated in the CNS and gated on CD45 and CD4, and T-bet expression in the IL-17⁺ and IFN-γ⁺ T cells was determined (white, T-bet; gray, isotype control). Data in A–D are representative of two independent experiments (percentages are shown).

Figure 5.

IFN-γ^−/− and T-bet^−/− mice generate myelin-specific Th17 cells, although EAE susceptibility differs. B6, B6/IFN-γ^−/−, and B6/T-bet^−/− mice were immunized with MOG35-55 emulsified in CFA. The draining lymph nodes of three mice per group were removed on day 12 and cultured with MOG35-55 for 3 d. (A) Cells were gated on CD4 and IL-17 expression was analyzed in the activated (CD44^hi) T cells (percentages are shown). (B) T-bet expression in the CD4⁺ T cells was analyzed (white, T-bet; gray, isotype control; percentages are shown). (C) The percentage of T-bet⁺ cells was determined by gating on the CD4⁺IL-17⁺ T cells. (D) Splenocytes from B6, B6/IFN-γ^−/−, and B6/T-bet^−/− mice immunized with MOG35-55 were stimulated in vitro with MOG35-55 and transferred into wild-type B6 mice and monitored for EAE development. Data are representative of two independent experiments (means ± SEM).

Figure 6.

IL-6, in the absence of exogenous TGF-β, promotes the differentiation of encephalitogenic Th17 cells. (A) Naive TCR transgenic splenocytes were differentiated with MBPAc1-11 and IL-6, IL-1β, IL-23, or IL-1β + IL-23. Cells were gated on CD4⁺ T cells and T-bet expression was analyzed. IFN-γ and IL-17 expression on CD4⁺ T-bet⁺ cells was then analyzed (percentages are shown). (B) Naive TCR transgenic splenocytes were differentiated with IL-6, TGF-β, or both and cytokine production was determined by ELISA from the supernatants (means ± SEM). (C) Naive TCR transgenic splenocytes were differentiated with IL-12 or IL-6 (±TGF-β) in the presence of neutralizing antibodies for IFN-γ, IL-12, and IL-4. Flow cytometry analyzed IL-17, IFN-γ, and T-bet expression in these cells (percentages are shown). (D) ELISA was used to measure cytokine expression from supernatants of cells in C (means ± SEM). (E) The cells in D were transferred into naive B10.PL mice (5 × 10⁶ cells per mouse) and monitored for development of EAE. Data are representative of three independent experiments (means ± SEM).

Figure 7.

TGF-β contributes to IL-17 expression but not the encephalitogenic capacity of T cells differentiated with IL-6. Naive TCR transgenic splenocytes were differentiated with IL-12 (Th1), IL-6 + TGF-β + anti–IL-12/IFN-γ/IL-4, IL-6 + anti–IL-12/IFN-γ/IL-4, or IL-6 + anti–IL-12/IFN-γ/IL-4/TGF-β. (A) T-bet (white histogram; gray, isotype control) expression in total CD4⁺ T cells was determined by flow cytometry (top). IL-17 and IFN-γ expression in CD4⁺ T cells was determined (middle). IFN-γ and IL-17 were gated on and T-bet levels were determined in each population. Because the number of IL-17⁺ T cells was low with IL-6 + anti–IL-12/IFN-γ/IL-4/TGF-β differentiation, T-bet levels could not accurately be determined. (B) IL-17 levels from the supernatants from A were determined by ELISA. IFN-γ levels were undetectable in all IL-6 cultures (not depicted; means ± SEM). (C) The T cell populations differentiated with IL-6 were transferred in to B10.PL (5 × 10⁶ cells per mouse) and monitored for EAE development (means ± SEM). (D) The CNS-infiltrating cells were isolated from the mice receiving the IL-6-differentiated (neutralizing other cytokines) T cells on day 12 after transfer. The cells were stimulated in vitro overnight with MBPAc1-11, and intracellular IFN-γ and IL-17 were analyzed by flow cytometry. Data in A–D are representative of two independent experiments.