Pathogenicity of IFN-γ–producing Th17 cells is independent of T-bet

Abstract

During the development of experimental autoimmune encephalomyelitis (EAE), the proportion of pathogenic and myelin-specific cells within CNS-infiltrating cytokine producing T helper (Th) cells is unknown. Using an IL-17A-IFN-γ double reporter mouse and I-A^b/MOG_38–49 tetramer, we show here that IL-17+ IFN-γ+ Th cells, which are expanded in the CNS during EAE, are highly enriched in MOG-specific T cells. We further demonstrate that IL-23 is essential for the generation and expansion of IFN-γ producing Th17 cells independently of the Th1-associated transcription factors T-bet, STAT1 and STAT4. Furthermore, Th17 and IL-17+ IFN-γ+ Th cells can induce CNS autoimmunity independently of T-bet. While T-bet is crucial for Th1 mediated EAE T-bet is dispensable for Th17 cell-mediated autoimmunity. Our results suggest the existence of different epigenetic programs that regulate IFN-γ expression in Th1 and Th17 cells.

Introduction

Several subsets of cytokine-producing Th cells infiltrate the CNS during the course of EAE. However, the breakdown of the blood brain barrier during EAE facilitates T cell entry into the CNS independent of antigenic specificity. Yet, it is unclear which proportion of cytokine-producing T cells is directed against myelin antigens and contributes to tissue damage.

Th17 cells have been characterized as one of the major pathogenic Th cell populations underlying the development of many autoimmune diseases (1). IL-23 enhances and stabilizes Th17 cells (2–4) and is critical for the development of autoimmune diseases such as EAE (4).

However, several observations have recently challenged the pathogenic role of Th17 cells: First, IL-17A- and IL-17F-deficient mice are only partially resistant to the development of EAE (5, 6). Second, Th17 cells are plastic, and have been described to lose IL-17 and acquire IFN-γ expression in a T-bet and STAT4-dependent manner, questioning whether EAE pathogenicity could be attributed to Th17 or Th1 cells (7–9). Indeed, in several autoimmune diseases, including multiple sclerosis (MS), and rheumatoid arthritis (RA), CD4+ effector T cells produce both IL-17 and IFN-γ (10–12), but little is known about their generation. Third, recent studies showed that T-bet is required for the pathogenicity of Th17 cells (7, 13).

Prior to the identification of Th17 cells, several studies investigated the requirement of T-bet for the development of EAE. We and others have demonstrated an essential role for T-bet in EAE disease development (14–16). However, T-bet is also expressed by dendritic cells (DCs) and B cells, which may directly and/or indirectly affect disease development (17, 18).

Here we show that during EAE, Th17 cells expressing GM-CSF and/or IFN-γ are highly enriched in MOG-specific T cells in the CNS and can induce disease independently of Th1 cells. IL-23 signaling is crucial for the development of IL-17+ IFN-γ+ T cells and expression of the IL-23R promotes their expansion and maintains their pathogenic profile. We further demonstrate that, although this subset can express IFN-γ and T-bet, their development and pathogenicity is independent of T-bet. Together, our data suggest that while the modulation of T-bet might be important to control Th1-mediated autoimmunity, it is ineffective at controlling Th17-mediated autoimmune manifestations.

Materials and Methods

Mice

C57BL/6J (B6), IL-12p40^−/−, Tbx21^−/−, Stat1^−/− and Stat4^−/− mice were purchased from the Jackson Laboratories and Taconic. IL-17A GFP mice are from Biocytogen. IFN-γ KI Thy1.1, T-bet^fl/fl CD4^Cre and Tbx21^−/− Eomes^fl/fl CD4^Cre mice mice were generously provided by Drs. Casey T. Weaver, Steven Reiner and Binfeng Lu, respectively. IL-23R GFP reporter mice were previously described (2). All strains are on the C57BL/6J background. All animals were bred and maintained under specific pathogen-free conditions at the Benaroya Research Institute (Seattle, WA) and all experiments were performed in accordance with the guidelines of the Benaroya Research Institute Animal Care and Use Committee.

CD4⁺ T cell preparation and T cell differentiation

For T cell differentiations, naïve CD4+CD62L^hi CD25− T cells were isolated by FACS sorting (FACSAria, BD Biosciences) and cultured with irradiated spleen cells from IL-12p40^−/− mice for 7 days in complete RPMI, 2.5 µg/ml of anti-CD3 in the presence of 5 ng/ml rhTGF-β (R&D Systems), 30 ng/ml rmIL-6 (Peprotech), 10 µg/ml anti-IFN-γ and 10 µg/ml anti-IL-4 (NIH/NCI BRB Preclinical Repository). For restimulation, T cells were recovered, activated with fresh splenocytes, anti-CD3 with or without IL-23 (20 ng/ml, R&D Systems).

Antibodies and Flow cytometry

Intracellular cytokine and intranuclear stainings from CNS-infiltrating cells or in vitro differentiated cells were performed according to the manufacturer’s instructions (Biolegend/eBioscience). A viability dye (ebioscience) was used to exclude dead cells. For surface cytokine staining, cells were stimulated with PMA/ionomycin and stained with anti-IL-17 and anti-IFN-γ antibodies. I-A^b/MOG_38–49 tetramer was obtained through the NIH tetramer facility and used according to their guidelines. All samples were analyzed on an LSRII flow cytometer (BD Biosciences), and data were analyzed with the FlowJo software (Tree Star).

EAE induction

EAE was induced by subcutaneous immunization of mice into the flanks with an emulsion of MOG_35–55 peptide (100 µg) emulsified in complete Freund adjuvant supplemented with 4mg/ml of M. tuberculosis extract H37Ra (Difco). In addition, the animals received 200 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2. Clinical signs of EAE were assessed according to the following score: 0, no signs of disease; 1, loss of tail tonicity; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and forelimb paralysis.

Statistical analysis

Statistical analysis was conducted with GraphPad Prism software. P values were calculated with Student’s paired t-test. P values of less than 0.05 were considered significant, *≤ 0.05, **≤0.01, ***≤0.001. Error bars denote ± SEM as indicated.

Results and Discussion

CNS-infiltrating IL-17+ IFN-γ+ T cells are highly enriched in MOG-specific cells during EAE

We used an IL-17A-IFN-γ double reporter mouse in which cells expressing IFN-γ and IL-17 can be detected and isolated based on Thy1.1 and GFP expression, respectively, in conjunction with MOG_38–49 /I-Ab tetramer to identify the proportion of antigen-specific and cytokine-producing T cells during EAE. After EAE induction, IFN-γ+ IL-17- and IL-17+ IFN-γ- T cells represent the majority of CD4+ T cells present in the LN, with limited expression of GM-CSF and few IL-17+ IFN-γ+ T cells (Fig. 1A, Supplemental Fig. 1A). Remarkably, CNS cytokine secretion increases by 77 fold for IL-17+ IFN-γ+ T cells (Fig. 1A). CNS-infiltrating T cells are further characterized by higher T-bet, Rorγt, IL-23R and GM-CSF expression and lower TGF-β3 expression in comparison to peripheral T cells (Supplemental Fig. 1B). The phenotype of CNS-infiltrating T cells is in sharp contrast to newly in vitro differentiated Th17 cells (Supplemental Fig. 1C), which do not express T-bet or GM-CSF (Supplemental Fig. 1D).

Figure 1. Signature of pathogenic IL-17+ IFN-γ+ Th17 cells.

(A) Expression of IFN-γ and IL-17 in CD4+ T cells from the CNS or LN of IFN-γ Thy1.1/IL-17 GFP reporter mice during EAE. (B) Representative proportion of I-Ab/MOG_38–49 tetramer+ CD4+ T cells in the CNS during EAE progression. (C) Percentage of MOG-specific CD4+ T cells within cytokine-producing subsets in the CNS of mice with EAE. Data are from two independent experiments with 6 mice total (mean ± SEM). (D) Percentage of GM-CSF+ cells among CNS-infiltrating, cytokine-producing Th cell subsets. Data are from at least 12 mice per group (mean ± SEM) and are pooled from three independent experiments. (E) Percentage of IL-23R GFP+ cells in CNS-infiltrating CD4+ T cell subsets from IL-23R GFP reporter mice with EAE. Data are from 5 mice per group (mean ± SEM). (F) CNS-infiltrating CD4+ IL-17+ IFN-γ- and IL-17+ IFN-γ+ T cells from mice with EAE were sorted and activated 2 days later with irradiated IL-12b^−/− splenic feeder cells, anti-CD3 in the presence or absence of IL-23. IL-17 and IFN-γ intracellular staining was performed seven days later. One representative experiment out of two is shown.

Numerous studies have investigated the capacity of distinct T cell subsets to induce EAE upon adoptive transfer and after their differentiation or expansion in the presence of appropriate cytokines for a short period in vitro. However, these conditions may not fully recapitulate the phenotype of effector T cells infiltrating the CNS in vivo during EAE.

To determine which T cell subset might play the most important role in EAE pathogenicity, we examined the antigenic specificity of CNS-infiltrating cytokine-producing T cells using MOG_38–49 /I-Ab tetramer (Fig. 1A, 1B). Among double negative T cells, very few cells (6.2% ± 2.1) were MOG specific. Among IFN-γ+ and IL-17+ T cells, 19.4% ± 5.7 and 24.8% ± 6.3 were MOG_38–49 /I-Ab+, respectively. However, there was a significant enrichment (42% ± 5.6) of MOG-specific T cells in CNS-infiltrating IL-17+ IFN-γ+ T cells suggesting an enhanced pathogenic role of these cells in EAE (Fig. 1C). Furthermore, CNS-infiltrating IL-17+ IFN-γ+ T cells contained the highest percentage of GM-CSF+ cells (Fig. 1D) and expressed high levels of GM-CSF, Rorγt and T-bet (Supplemental Fig. 2A). These data are consistent with recent findings showing the importance of GM-CSF for pathogenic T cells (21, 22). Similarly to IL-17+, IL-17+ IFN-γ+ T cells express CXC13 which might attract neutrophils cells in the CNS. In addition these cells express, GranzymeB and Perforin suggesting that they could exhibit a direct cytolytic activity on cells from the CNS (Supplemental Fig. 2B).

Similarly to IL-17+ IFN-γ- cells, IL-17+ IFN-γ+ T cells express very high levels of the IL-23R protein (Fig. 1E). To address the effect of IL-23 on those cells, we isolated these CNS-infiltrating T cells and analyzed their fate after in vitro stimulation. The isolation of both T cell subsets from the CNS of mice with EAE and their activation in absence of IL-23 led to the partial loss of either IL-17 or IFN-γ expression (Fig. 1F). In contrast, stimulation of IL-17+ IFN-γ- T cells in the presence of IL-23 resulted in the appearance of IL-17+ IFN-γ+ cells. It also significantly increased the percentage of and stabilized IL-17+ IFN-γ+ T cells (Figure 1F). Therefore IL-23 likely maintains IL-17+ IFN-γ+ T cells present in target tissues under inflammatory conditions.

IL-23 enhances Th17 cell plasticity and the generation of IL-17+ IFN-γ+ T cells

IL-12 and IFN-γ are negative regulators of RORγt, IL-17 and GM-CSF; it was therefore striking to observe the existence of an antigen-specific population expressing all three cytokines concomitantly. Our data would suggest that IL-23 might be important for the generation of Th cells co-expressing IL-17, IFN-γ and GM-CSF. To address this question, we chronically stimulated differentiated Th17 cells in the presence of IL-23. This led to the emergence of Th17 cells, which produced IFN-γ and concomitantly expressed T-bet and Rorγt (Fig. 2A, 2B). Of note, GM-CSF expression was markedly increased in IL-17+ IFN-γ+ T cells compared to IL-17-single producing cells (Fig. 2C). This phenomenon was specific to IL-23 since IL-17+ IFN-γ+ T cells were absent from IL-23R-deficient mice (Fig. 2A, 2B, Supplemental Fig. 2C). Next, we analyzed the requirement of IL-23 for the generation of these cells in vivo. Because IL-23RKO mice are protected from EAE, we analyzed the percentage of cytokine-producing cells in the peripheral immune organs ten days after immunization. There was no difference in the number of IFN-γ+ T cells between WT and IL-23RKO mice (Fig. 2D). However, IL-23RKO mice displayed a significant decrease in the percentage of IL-17+ IFN-γ− and IL-17+ IFN-γ+ T cells (Fig. 2D). Thus, chronic exposure of Th17 cells to IL-23 leads to the induction of IL-17+ IFN-γ+ (GM-CSF+) T cells in vitro and in vivo. It is tempting to speculate that IL-17+ IFN-γ+ T cells emerge to a higher extent in the target tissues during autoimmune disease development where they are constantly exposed to their autoantigen in an IL-23-rich cytokine environment.

Figure 2. IL-23 induces IL-17+ IFN-γ+ Th17 cells in vitro.

(A) Intracellular cytokine staining for IL-17 and IFN-γ in Th17 cells from WT and IL-23R^−/− mice stimulated for two rounds in the presence or absence of IL-23. (B) Frequency of RORγt+ IL-17+ and T-bet+ IFN-γ+ cells under the indicated cytokine conditions. Data are representative of at least three independent experiments (mean ± SEM). (C) Expression of GM-CSF among WT IL-17+ IFN-γ- and IL-17+ IFN-γ+ Th17 cells propagated with IL-23 for three rounds of stimulation. Data are from 4 independent experiments (mean ± SEM). (D) WT and IL-23R^−/− mice were immunized with MOG_35–55 emulsified in CFA. At day 10, the proportion of IL-17+, IFN-γ+ and GM-CSF+ cells among splenic CD4+ T cells was determined by intracellular cytokine staining. The graph represents cumulative data of 5–7 mice per group, from two independent experiments (mean ±SEM).

IFN-γ production by IL-17+ T cells is independent of bona fide Th1-specific transcriptions factors

The ontogeny and factors controlling IL-17+ IFN-γ+ T cells are not known. Because these cells express T-bet and IFN-γ (Fig. 1A, 2A, 2B), and T-bet has been described as a critical factor for Th17 cell pathogenicity (7, 16), we next determined if Th1-specific transcription factors were required for their differentiation in response to IL-23 in vitro. We differentiated naïve T cells lacking IFN-γ-driving transcription factors into Th17 cells and expanded these cells with IL-23. Importantly, IL-17+ IFN-γ+ cells were present whether T cells originated from WT, STAT1KO, STAT4KO or T-betKO mice (Fig. 3A, 3B, 3C). Eomesodermin (Eomes) did not compensate for T-bet deficiency since the loss of both transcription factors did not hamper the generation of IL-17+ IFN-γ+ T cells (Fig. 3D), Neither did STAT4 nor STAT1 in the T-betKO, which was assessed by phosphorylation of STAT4 or STAT1 in WT vs T-bet-deficient Th17 cells cultured with IL-23 (Supplemental Fig 2D, E). This indicates that Th17 cells can express IFN-γ after chronic stimulation with IL-23 in the absence of transcriptional factors that regulate IFN-γ production by Th1 cells such as T-bet, Eomes, STAT1 and STAT4 (Fig. 3E). These data argue against the model that IL-23 is upstream of T-bet as previously described (13).

Figure 3. Th1-associated transcription factors are not required for the IL-23-dependent induction of IL-17A+ IFN-γ+ CD4+ T cells.

Naïve CD4+ T cells from STAT1^−/− (A), STAT4^−/− (B), Tbx21^−/− (C) and Tbx21^−/−/Eomes^fl/fl CD4^Cre (D) and WT control mice were primed as Th17 cells, restimulated with IL-23 two more times and analyzed. In A – D, numbers in each quadrant indicate the percentage of total, viable CD4+ T cells. (E) Summary (mean) of all priming experiments. Error bars denote ± SEM. Data are from two or three experiments per group.

Selective deficiency of T-bet in T cells does not prevent EAE development and CNS-infiltrating IFN-γ+ IL-17+ T cells

Our data raises the following questions: Can the emergence of IL-17+ IFN-γ+ T cells in response to IL-23 be recapitulated in vivo in the absence of T-bet? Furthermore, can T-bet-deficient Th17 cells induce EAE? To address these questions, we immunized mice in which T-bet was specifically deleted in T cells (T-bet^fl/fl CD4^Cre). In contrast to T-betKO mice (14), T-bet^fl/fl CD4^Cre mice were not resistant to the development of EAE. However, these mice developed EAE with delayed onset (16.75±4.1 vs 12.7±4.1) and lower incidence (66.0% vs 91.7%) compared to control mice (Fig. 4A, B). Consistent with the role of T-bet in the differentiation of Th1 cells (14, 17), T-bet^fl/fl CD4^Cre mice lacked most IFN-γ+ Th1 cells in the CNS (Fig. 4B), indicating that Th17 cells induced EAE in the absence of Th1 cells. Furthermore, T-bet^fl/fl CD4^Cre mice displayed an increase in the percentage of IL-17+ IFN-γ− T cells infiltrating the CNS. Interestingly, the frequency of IL-17+ IFN-γ+ T cells was similar between both groups as well as their level of GM-CSF production (Fig. 4B, C). Thus, while the delayed onset and lower incidence of T-bet^fl/fl CD4^Cre mice compared to control mice may indicate a role for T-bet-expressing B cells (16), DCs (17) and Th1(14, 17) cells in EAE development, it also demonstrates that IL-17+ and IL-17+ IFN-γ+ Th17 cells can induce EAE independently of Th1 cells and in a T-bet-independent manner. Our data are in agreement with previous data showing that encephalitogenicity of Th17 cells correlates with high T-bet and IFN-γ expression (7, 13, 15) since we see enhanced IFN-γ and T-bet expression in IL-17+ IFN-γ+ T cells compared to IL-17+ T cells but our data which address the selective defect of T-bet in T cells, argue against a direct role of T-bet in the pathogenicity of Th17 cells and its absolute requirement for the development of EAE. Our results further suggest that IFN-γ expression is regulated differently in Th1 versus Th17 cells. Interestingly, T-bet was recently shown to interact with the transcription factor Runx1, and to inhibit Runx1-mediated transactivation of Rorc (23). It is therefore possible that the association between T-bet and Runx1 does not occur in IFN-γ+ T-bet+ Th17 cells which allows for co-expression of T-bet and Rorγt and continuation of the Th17 differentiation program. Future experiments are needed to determine the mechanisms by which T-bet affects the function of Rorγt in T cells that produce both IL-17 and IFN-γ.

Figure 4. T-bet^fl/fl CD4^Cre mice develop EAE and have CNS-infiltrating IL-17+ IFN-γ+ CD4+ T cells.

(A) Clinical scores of T-bet^fl/fl and T-bet^fl/fl CD4^Cre mice over time (n = 6 mice per group). (B) Intracellular cytokine staining of IL-17 and IFN-γ in CNS-infiltrating CD4+ T cells. Numbers in each quadrant indicate the percentage of total, viable CD4+ T cells. (C) Quantification of cytokine producing cells in the CNS of T-bet^fl/fl and T-bet^fl/fl CD4^Cre mice. Data are representative of two independent experiments with at least six mice per group (mean ± SEM).

In summary, our study underlines the plasticity of pathogenic Th17 cells present in the CNS during EAE and the importance of IL-23 in this process. It further establishes the pathogenic potential of IL-17+ IFN-γ+ T cells in EAE by demonstrating that MOG-specific, GM-CSF+ T cells are selectively enriched in this population. Finally it demonstrates that Th17 cells can induce CNS autoimmunity independently of T-bet. Together, these results have important implications for the development of drugs to target Th17 cells, their effector cytokines and their functions.