T cell factor 1 (TCF-1) is expressed in both developing and mature T cells and has been shown to restrain mature T cell–mediated Th17 responses by inhibiting IL-17 expression. However, it is not clear when TCF-1 is required in vivo to restrain the magnitude of peripheral Th17 responses and what the molecular mechanisms responsible for TCF-1–regulated IL-17 gene expression are. In this study, we showed that conditional deletion of TCF-1 at the early but not later CD4+CD8+ double-positive stage in mice enhanced Th17 differentiation and aggravated experimental autoimmune encephalomyelitis, which correlates with abnormally high IL-17 expression. Expression of TCF-1 in TCF-1–deficient thymocytes but not TCF-1–deficient Th17 cells inhibited IL-17 expression. TCF-1 binds to IL-17 promoter regions, and deletion of two TCF-1 binding sites relieves TCF-1–mediated inhibition of IL-17 promoter activity. Lastly, wild-type TCF-1, but not a TCF-1 mutant that has no intrinsic histone deacetylase activity, was able to inhibit IL-17 expression in TCF-1 deficient mouse thymocytes. Thus, our study demonstrates the requirement of TCF-1 in vivo at stages earlier than double-positive cells to restrain peripheral Th17 immunity by directly binding and inhibiting IL-17 promoter in its intrinsic histone deacetylase–dependent manner.

T cell development in thymus is a process to arm T cells with the capacity to mediate appropriate immune responses in peripheral tissues. Lymphoid progenitors that developed from hematopoietic stem cells in the bone marrow migrate into the thymus to complete sequential maturation stages, including CD4CD8 double-negative (DN), CD4+CD8+ double-positive (DP), and CD4+ or CD8+ single-positive stages (14). Mature single-positive T cells then migrate to the peripheral lymphoid organs to participate adaptive immune responses against pathogens.

When the single CD4+ positive T cells migrate out of thymus, they are naive and are not competent to mediate immune responses. To become effector T cells, they must undergo an activation and differentiation process. This process is initiated upon encountering Ags and eventually differentiates naive T cells into Th cells that include Th1, Th2, Th17, and regulatory T cells (Treg). Th17 cells secrete IL-17 and participate in protective immunity against pathogens, whereas inappropriately exaggerated Th17 responses contribute to pathological immune responses involved in the autoimmunity, such as psoriasis and multiple sclerosis (59).

Besides shaping a T cell repertoire that reacts to foreign but not self-antigens, the thymocyte developmental process also controls the magnitude of T cell responses in the periphery. For example, T cell factor 1 (TCF-1), a transcription factor enriched in hematopoietic cell compartments, regulates T cell development in thymus (1012). Our previous studies have shown that germline deletion of TCF-1 resulted in increased IL-17 expression both in thymus and peripheral T cells and hence led to enhanced Th17 differentiation and more severe experimental autoimmune encephalomyelitis (EAE) (13, 14), indicating the negative role of TCF-1 in the regulation of Th17 immunity. We have some in vitro evidence that TCF-1 loses the ability to regulate IL-17 gene expression in mature T cells. However, because it was a germline deletion, it is not clear when TCF-1 is required in vivo to limit IL-17 gene expression and thus control the scale of Th17 responses in the periphery. By using conditional deletion of TCF-1 at different developmental stages, we demonstrated that CD4-Cre–mediated deletion of TCF-1 at the CD4+CD8+ DP stage did not significantly affect thymic T cell development, peripheral Th17 differentiation, and EAE. In contrast, Vav1-Cre–mediated deletion of TCF-1 at earlier hematopoietic stages disrupts thymic T cell development and potentiates Th17 differentiation and development of EAE. Moreover, expression of TCF-1 in TCF-1−/− thymocytes but not TCF-1−/− Th17 cells was able to downregulate IL-17 expression. We also found that TCF-1–mediated inhibition of IL-17 expression depends on its intrinsic histone deacetylase (HDAC) activity (15). We mapped the TCF-1 binding regions on the IL-17 promoter, and deletion of the DNA fragments containing the TCF-1 binding sites prevented TCF-1 from inhibiting IL-17 promoter. Therefore, we demonstrated the stage-specific requirement of TCF-1 in vivo during early development to control the scale of the peripheral Th17 immune responses via inhibiting IL-17 expression through the intrinsic HDAC activity of TCF-1.

TCF-1fl/fl mice were described previously (16) and obtained from Dr. H.-H. Xue (University of Iowa, Iowa City, IA). Rag1−/−, transgenic CD4-Cre and Vav1-Cre mice were purchased from The Jackson Laboratory. TCF-1fl/fl/CD4-Cre and TCF-1fl/fl/Vav1-Cre were generated by crossing TCF-1fl/fl to CD4-Cre and Vav1-Cre, respectively. For all experiments, mice were 6–10 wk old. All mice were bred and maintained in the specific pathogen-free conditions, and experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at the Beckman Research Institute of City of Hope (IACUC 07023).

Mouse naive CD4+ T cells were isolated from spleens of 6- to 10-wk-old mice by negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). T cells were cultured and differentiated in Iscove’s DMEM (Corning) containing 10% FBS, 50 mM 2-ME, and 100 U/ml penicillin-streptomycin at 37°C with 5% CO2. In brief, 4 × 105 cells/well naive CD4+ T cells were first activated with 0.25 μg/ml anti-CD3 (145-2C11; eBioscience) and 1 μg/ml anti-CD28 (37.51; eBioscience) in goat anti-hamster IgG (0.2 mg/ml; MP Biomedicals, Santa Ana, CA) precoated 24-well plates. Cells were then differentiated in the presence of polarizing cytokine and Ab mixture for 72 h. For Th1, the mixture contained 10 ng/ml recombinant murine (rm) IL-2 (BioLegend), 20 ng/ml rmIL-12 (BioLegend), and 20 μg/ml anti-IL4 (11B11; BioLegend). For Th17, the mixture contained 2 ng/ml rmTGF-β1 (eBioscience) and 25 ng/ml rmIL-6 (eBioscience). For Treg, the mixture contained 5 ng/ml rmTGF-β, 10 μg/ml anti-IFN-γ (XMG1.2; BioLegend), and 10 μg/ml anti–IL-4 (11B11; BioLegend).

Cultured T cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich, MO) and 1 μg/ml ionomycin (Sigma-Aldrich) in the presence of brefeldin A for 4 h before staining. Both cultured T cells and freshly prepared thymocytes were first stained with fluorescence-conjugated Abs to cell surface markers and then fixed/permeabilized either with the BD Cytofix/Cytoperm kit (BD Biosciences) or Transcription Factor Staining Buffer Set (BD Pharmingen). Cells were then intracellularly strained with fluorescence-conjugated Abs to cytokines or transcription factors. Abs include FITC-CD4 (GK1.5; BioLegend), PE-CD8 (53-6.7; BioLegend), PE-Cy7-CD8 (53-6.7; eBioscience), APC-CD8 (53-6.7; BD Biosciences), PE-Thy1.2 (30-H12; BD Biosciences), PE-TCRβ (H57-597; BD Biosciences), APC-IL-17A (eBio17B7; eBioscience), APC-IFN-γ (XMG1.2; eBioscience), APC-FOXP3 (FJK-16s; eBioscience), and PE-RORγt (Q31-378; BD Biosciences). Flow cytometry was performed by using a FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). DN thymocytes (Thy1.2+CD4CD8) were electronically sorted by using FACSAria II (BD Biosciences).

Total RNA was prepared using the RNeasy Isolation Kit according to the manufacturer’s instructions (Qiagen). cDNA was synthesized using the Tetro cDNA Synthesis Kit (BIO-65043; Bioline). Quantitative real-time PCR (qRT-PCR) was performed using SsoFast EvaGreen Supermix (Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad). Primers are as follows: IL-17A–forward: 5′-TTTAACTCCCTTGGCGCAAAA-3′, IL-17A–reverse: 5′-CTTTCCCTCCGCATTGACAC-3′; IL17F-forward: 5′-TGCTACTGTTGATGTTGGGAC-3′, IL17F-reverse: 5′-AATGCCCTGGTTTTGGTTGAA-3′; CCL20-forward: 5′-GCCTCTCGTACATACAGACGC-3′, CCL20-reverse: 5′-CCAGTTCTGCTTTGGATCAGC-3′; and CCR6-forward: 5′-CCTGGGCAACATTATGGTGGT-3′, CCR6-reverse: 5′-CAGAACGGTAGGGTGAGGACA-3′.

MSCV-IRES-EGFP–based retroviral expression vectors encoding TCF-1 or empty vectors were transfected into Plat-E packaging cells by Lipofectamine 2000 (Invitrogen Life Technologies) mediated transfection. After 48 h, viral supernatants were collected and stored at −80°C until use. For transduction, naive CD4+ T cells were first activated with 0.25 μg/ml anti-CD3 (145-2C11; eBioscience) and 1 μg/ml anti-CD28 (37.51; eBioscience) in goat anti-hamster IgG precoated 24-well plates for 24 h followed by spin-infection with viral supernatants (2500 rpm, 30°C for 2 h) in the presence of 8 μg/ml polybrene (Sigma-Aldrich). After spin-infection, indicated cytokines described above were added to the culture media to induce Th17 differentiation.

Thymocytes were allowed to differentiate on OP9-DL1 cells (gift from Dr. T.P. Bender, University of Virginia Health System, Charlottesville, VA). Briefly, 5 × 105 cells/well electronically sorted Thy1.2+CD4CD8 thymocytes were cultured overnight on an 80% confluent OP9-DL1 monolayer in the flat-bottom 24-well plates in αMEM (Stemcell Technologies) supplemented with 20% FBS, 100 U/ml penicillin-streptomycin, 2 mM l-glutamine (Invitrogen Life Technologies), and 5 ng/ml rmIL-7 (PeproTech). Cocultures were then spin-infected (2500 rpm, 30°C for 2 h) with retroviral supernatants in the presence of 5 μg/ml polybrene. Seventy-two hours posttransduction, cocultures were harvested for flow cytometry.

Active EAE was induced according to the manufacturer’s instructions (Hooke Laboratories, Lawrence, MA). Briefly, mice were immunized with 200 μl of myelin oligodendrocyte glycoprotein 35–55 (MOG35–55) peptide emulsion subcutaneously. On days 0 and 1 after immunization, mice were injected i.p. with 200 ng of Bordetella pertussis toxin (Hooke Laboratories). For Th17- or Th1-induced passive EAE, donor mice were immunized with MOG35–55 subcutaneously. Ten days later, cells were isolated from spleen and lymph node and cultured with 20 μg/ml MOG35–55 for 3 d under either Th17-polarizing conditions (20 ng/ml rmIL23; R&D) or Th1-polarizing conditions (20 ng/ml rmIL-12, R&D; 2 μg/ml α-IL23p19, eBioscience) (17). Rag1−/− recipient mice were then transferred i.p. with 3.0 × 107 MOG35–55–specific Th17 or Th1 cells. The severity of EAE was monitored and evaluated on a scale from 0 to 5 according to Hooke Laboratories’ guideline. In brief, 0 = no disease, 1 = paralyzed tail, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = hind and fore limb paralysis, and 5 = moribund and death. When a mouse was euthanized because of severe paralysis, a score of 5 was entered for that mouse for the rest of the experiment.

Brain and spinal cords were homogenized and single-cell suspensions were prepared using 70-μm cell nylon cell strainers (Fisher Scientific). Cells were collected by centrifugation at 400 relative centrifugal force for 10 min. Cells were then resuspended in 4ml of 30% Percoll (GE Healthcare) and centrifuged onto 5 ml of 70% cushion for 20 min at 2000 rpm. Lymphocytes at 30–70% interface were collected and subjected to cell surface staining with fluorescence-conjugated Abs: FITC-CD45 (30-F11; eBioscience), APC-CD3 (145-2C11; BD Pharmingen), APC-Cy7-Ly6C (HK1.4; eBioscience), PE-Cy7-Ly6G (1A8; BioLegend), and PE-CD11b (M1/70; eBioscience). For intracellular cytokine staining, the CNS infiltrating cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) in the presence of brefeldin A for 4 h and were then stained with PE-Cy7-CD4 followed by intracellular staining of FITC-IFN-γ (XMG1.2; BioLegend), PE-GM-CSF (MP1-22E9; BioLegend), and APC-IL-17A (eBio17B7; eBioscience).

Chromatin immunoprecipitation (ChIP) on qRT-PCR was performed with the ChIP-IT High Sensitivity Kit (53040; Active Motif). Briefly, 1.5 × 107 cells per ChIP were obtained from thymus or differentiated Th17 cells. Cells were fixed and sheared as described in the ChIP-IT High Sensitivity Kit manual. ChIP reactions were then performed on 20 μg of the prepared chromatin using 4 μg of anti–TCF-1 Ab (C63D9; Cell Signaling) or anti–acetyl-histone H3 (06-599; Millipore). Four micrograms of IgG was used as negative control. Input DNA and DNA pull-down after immunoprecipitation were analyzed by qRT-PCR as described above. Primers that cover ∼2000 bp of the IL-17A promoter region were designed as follows: P1-forward: 5′-AAACCCTATGCAGTTGGTACA-3′, P1-reverse: 5′-CATCTCTCCAGCTCCATGGA-3′; P2-forward: 5′-AGCCCATAAAGAAGCCAATGT-3′, P2-reverse: 5′-TCCTGGCTTTCTACGTGTCA-3′; P3-forward: 5′-GGGTGAAAGAGGACATTGCC-3′, P3-reverse: 5′-TGCCAGGTCTTTTCCCATTC-3′; P4-forward: 5′-TGACACGTAGAAAGCCAGGA-3′, P4-reverse: 5′-GATTGCCAGGTCTTTTCCCA-3′; P5-forward: 5′-GGCAATCAGAGGTGTGTGTG-3′, P5-reverse: 5′-CTCAGAAGTTGCAGCACCTC-3′; P6-forward: 5′-CTATCGGTCCACCTCATGCT-3′, P6-reverse: 5′-GGAGATGAGGGATGAGAAGGG-3′; P7-forward: 5′-GTTAGTAGTCTCCACCCGGC-3′, P7-reverse: 5′-TGAGGTTCGGTATCAAGCCT-3′; P8-forward: 5′-GAGTGGGTTTCTTTGGGCAA-3′, P8-reverse: 5′-AGCATGACTTCTTGGGAGCT-3′; and P9-forward: 5′-AGCTCCCAAGAAGTCATGCT-3′, P9-reverse: 5′-TACGTCAAGAGTGGGTTGGG-3′.

Previous studies of TCF-1 function mostly depend on using a strain of germline knockout mice that cannot determine the tissue- and time-specific function of TCF-1 (10). To determine the critical stages in which TCF-1 is required to restrain peripheral Th17 responses, we crossed conditional TCF-1 (TCF-1fl/fl) mice (16) to CD4-Cre and Vav1-Cre mice, respectively. CD4-Cre induces gene deletion at the CD4+CD8+ DP stage (18), whereas Vav1-Cre induces deletion at earlier hematopoietic stages, including the CD4CD8 DN stage (19). We performed PCR analysis of the TCF-1 locus to detect the deletion of the TCF-1 gene (Fig. 1A). As expected, there was no deletion at all in the absence of Cre, as shown in TCF-1fl/fl mice. However, TCF-1 deletion was detected in CD4+CD8+ thymocytes and peripheral T cells but not in CD4CD8 thymocytes of the TCF-1fl/fl/CD4-Cre mice. In contrast, TCF-1fl/fl/Vav1-Cre mice deleted TCF-1 in CD4CD8, CD4+CD8+, and peripheral T cells, confirming that Vav1-Cre induced TCF-1 deletion at the stage earlier than CD4CD8 DN cells. To determine the effects of deletion of TCF-1 at different developmental stages on peripheral Th17 responses, EAE was induced (Fig. 1B). Interestingly, no obvious differences of EAE development in terms of disease onset and severity were observed between TCF-1fl/fl and TCF-1fl/fl/CD4-Cre mice, with a comparable mean peak disease score of ∼3. However, the clinical score of TCF-1fl/fl/Vav1-Cre mice reached over 3.5, indicating more severe EAE than that observed in TCF-1fl/fl/CD4-Cre mice. These results demonstrated that deletion of TCF-1 at earlier stages, but not at later DP stages, potentiated Th17 immunity responsible for EAE. Moreover, TCF-1fl/fl/Vav1-Cre mice had a significantly reduced number of peripheral T cells compared with TCF-1fl/fl and TCF-1fl/fl/CD4-Cre mice (Fig. 1C), suggesting fewer T cells, but with higher potency in the induction of EAE when TCF-1 was deleted by Vav1-Cre.

FIGURE 1.

Vav1-Cre– but not CD4-Cre–induced deletion of TCF-1 potentiates EAE and Th17 differentiation. (A) Floxed and deleted TCF-1 loci in indicated population of thymocytes and peripheral T cells from indicated genotypes of mice, as determined by PCR analysis. Data shown are representative of three independent experiments. (B) Mean clinical score of EAE in indicated genotypes of mice on different days after MOG35–55 immunization. (TCF-1fl/fl n = 6, TCF-1fl/fl/CD4-Cre n = 6, and TCF-1fl/fl/Vav1-Cre n = 6). (C) Total number of CD4+ T cells in the spleens of indicated genotypes of mice (n = 3). (D) Percentage of IL-17+ cells among indicated genotypes of naive CD4+ T cells stimulated under Th0 or Th17 priming conditions, as determined by flow cytometric analysis. Right panels are the quantification of three biological replicas. (E) Expression levels of indicated critical Th17 genes in differentiated Th17 cells of indicated genotypes, as determined by qRT-PCR. *p < 0.05, **p < 0.01. ns, not significant.

FIGURE 1.

Vav1-Cre– but not CD4-Cre–induced deletion of TCF-1 potentiates EAE and Th17 differentiation. (A) Floxed and deleted TCF-1 loci in indicated population of thymocytes and peripheral T cells from indicated genotypes of mice, as determined by PCR analysis. Data shown are representative of three independent experiments. (B) Mean clinical score of EAE in indicated genotypes of mice on different days after MOG35–55 immunization. (TCF-1fl/fl n = 6, TCF-1fl/fl/CD4-Cre n = 6, and TCF-1fl/fl/Vav1-Cre n = 6). (C) Total number of CD4+ T cells in the spleens of indicated genotypes of mice (n = 3). (D) Percentage of IL-17+ cells among indicated genotypes of naive CD4+ T cells stimulated under Th0 or Th17 priming conditions, as determined by flow cytometric analysis. Right panels are the quantification of three biological replicas. (E) Expression levels of indicated critical Th17 genes in differentiated Th17 cells of indicated genotypes, as determined by qRT-PCR. *p < 0.05, **p < 0.01. ns, not significant.

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Because Th17 cells contribute to EAE development (20), we wondered how stage-specific deletion of TCF-1 affects Th17 differentiation. Even under Th0 conditions, we have already observed an increased percentage of IL-17+ cells in naive CD4+ T cells from TCF-1fl/fl/Vav1-Cre mice, whereas IL-17 expression was hardly detectable in TCF-1fl/fl and TCF-1fl/fl/CD4-Cre T cells. Under Th17 polarization conditions, T cells from TCF-1fl/fl and TCF-1fl/fl/CD4-Cre mice were equivalent in the generation of IL-17+ cells, whereas ∼100% more IL-17+ cells were developed from TCF-1fl/fl/Vav1-Cre T cells (Fig. 1D). Increased IL-17+TCF-1fl/fl/Vav1-Cre T cells correlated with the significant increased expression of Th17 signature genes, including IL-17A, IL-17F, CCR6, and CCL20 (Fig. 1E). In contrast, naive CD4+ T from TCF-1fl/fl/Vav1-Cre mice had no defects in Th1 and Treg differentiation (Supplemental Fig. 1), suggesting the selective role of TCF-1 in Th17 differentiation. These results demonstrate that TCF-1 is required at early but not later CD4+CD8+ stages during T cell development to restrain peripheral Th17 immunity by selectively controlling the potential of Th17 differentiation.

TCF-1 deficiency skews Th2 fate to Th1 fate (21). Besides Th17 cells, Th1 cells can also contribute to EAE induction, but these two forms of EAE display distinct lesion sites and immune profiles in CNS (22, 23), indicating different mechanisms are involved in the development of Th17- and Th1-mediated EAE. Although our in vitro experiment did not show significant difference in Th1 differentiation between TCF-1fl/fl/Vav1-Cre mice and TCF-1fl/fl and TCF-1fl/fl/CD4-Cre control (Supplemental Fig. 1), it is not clear whether deletion of TCF-1 affects Th1 function in term of induction of EAE in vivo. We thus compared the function of differentiated Th1 and Th17 cells by means of induction of passive EAE. Interestingly, Rag1−/− recipient mice (which have a congenital deficiency in mature B and T cells) reconstituted with cells from spleen and lymph node of TCF-1fl/fl, TCF-1fl/fl/CD4-Cre, or TCF-1fl/fl/Vav1-Cre mice stimulated under Th1 priming conditions developed equivalent EAE (Fig. 2A, 2B). However, cells from TCF-1fl/fl/Vav1-Cre mice stimulated under Th17 priming conditions induced more severe EAE in Rag1−/− recipient mice (Fig. 2C), which was also indicated by a significantly increased absolute number of CNS-infiltrating lymphocytes, including T cells, neutrophils, and monocytes (Fig. 2D, 2E, gate strategy for different cells shown in Supplemental Fig. 2). In addition, intracellular staining of CNS-infiltrating cells showed a greatly increased percentage of CD4+IL-17+IFN-γ+ and CD4+IL-17+GM-CSF+ cells, which are believed to be pathogenic for EAE (57), in the CNS of Rag1−/− mice adoptively transferred with Th17-priming conditions–treated cells from TCF-1fl/fl/Vav1-Cre mice (Fig. 2F, 2G). Taken together, these findings supported that the exaggerated EAE resulted from deletion of TCF-1 at an early T cell developmental stage was because of enhanced Th17 but not Th1 responses.

FIGURE 2.

Deletion of TCF-1 by Vav1-Cre potentiates Th17- but not Th1-mediated EAE. (A and C) Mean clinical scores of EAE in Rag1−/− mice on different days after adoptively transferred with indicated genotypes of MOG33–35-expanded Th1 (A) or Th17 (C) cells. (B and D) Number of lymphocytes infiltrated into the CNS of Rag1−/− mice adoptively transferred with indicated Th1 (B) or Th17 (D) cells at the peak of disease as shown in (A) or (C). (E) Quantification of cells expressing surface markers characteristic of various types of lymphocytes as determined by flow cytometric analysis at the peak of disease from Rag1−/− mice receiving Th17 cells. (F) Flow cytometric analysis of intracellular IL-17, GM-CSF, and IFN-γ in CD4+ T cells that infiltrated CNS of Rag1−/− mice receiving Th17 cells at the peak of disease. (G) Percentage of cells positive for indicated cytokines among CD4+ T cells infiltrated into CNS of Rag1−/− mice. Data are pooled from three experiments. *p < 0.05, **p < 0.01. ns, not significant.

FIGURE 2.

Deletion of TCF-1 by Vav1-Cre potentiates Th17- but not Th1-mediated EAE. (A and C) Mean clinical scores of EAE in Rag1−/− mice on different days after adoptively transferred with indicated genotypes of MOG33–35-expanded Th1 (A) or Th17 (C) cells. (B and D) Number of lymphocytes infiltrated into the CNS of Rag1−/− mice adoptively transferred with indicated Th1 (B) or Th17 (D) cells at the peak of disease as shown in (A) or (C). (E) Quantification of cells expressing surface markers characteristic of various types of lymphocytes as determined by flow cytometric analysis at the peak of disease from Rag1−/− mice receiving Th17 cells. (F) Flow cytometric analysis of intracellular IL-17, GM-CSF, and IFN-γ in CD4+ T cells that infiltrated CNS of Rag1−/− mice receiving Th17 cells at the peak of disease. (G) Percentage of cells positive for indicated cytokines among CD4+ T cells infiltrated into CNS of Rag1−/− mice. Data are pooled from three experiments. *p < 0.05, **p < 0.01. ns, not significant.

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Because we observed increased IL-17 expression in mature CD4+ T cells from TCF-1fl/fl/Vav1-Cre mice (Fig. 1D), we next wondered whether these increased IL-17–producing cells originated from thymus. Vav1-Cre– but not CD4-Cre–induced deletion of TCF-1 affected thymocyte development indicated by thymic weight (Fig. 3A, left panel) and cellularity (Fig. 3A, right panel) as well as developmental stages defined by CD4 and CD8 surface markers (Fig. 3B), suggesting that TCF-1 plays a major role at rather earlier developmental stages to ensure normal thymocyte development. Intracellular IL-17 staining indicated that more mature CD4+ TCRhi thymocytes from TCF-1fl/fl/Vav1-Cre mice produce IL-17 (3.10%) than those from TCF-1fl/fl (0.03%) and TCF-1fl/fl/CD4-Cre (0.2%) mice (Fig. 3B). This was consistent with our observation that peripheral CD4+ cells from TCF-1fl/fl/Vav1-Cre mice generated more IL-17–producing cells, even in Th0-neutral conditions (Fig. 1D). When gated on an earlier CD4CD8 TCR stage, we still found that more CD4CD8 thymocytes from TCF-1fl/fl/Vav1-Cre mice produced IL-17 (4.51%) compared with those from TCF-1fl/fl (0.42%) and TCF-1fl/fl/CD4-Cre (0.48%) mice (Fig. 3B). Therefore, Vav1-Cre–induced deletion of TCF-1 resulted in heightened IL-17 expression at as early as the CD4CD8 DN stage, and such heightened IL-17 expression was maintained at the mature CD4+ T cell stage, even after migrating out of thymus to the periphery. Because RORγt is required to stimulate IL-17 expression (24), RORγt expression was assessed at different thymocyte developmental stages in all three groups (Fig. 3C). Consistent with previous observations (25), RORγt was upregulated at the CD4+CD8+ DP stage but downregulated at the mature CD4+ stage in control TCF-1fl/fl mice. RORγt expression levels overlapped well between TCF-1fl/fl and TCF-1fl/fl/CD4-Cre thymocytes at CD4CD8, CD4+CD8+, and CD4+ stages. In contrast, a portion of CD4CD8 DN and CD4+ thymocytes expressed higher levels of RORγt in TCF-1fl/fl/Vav1-Cre mice (Fig. 3C, arrows), which correlated with the observed increased IL-17 expression. Taken together, these data indicate that TCF-1 is required at early but not late developmental stages to inhibit IL-17 expression during T cell development.

FIGURE 3.

Vav1-Cre–induced deletion of TCF-1 leads to abnormal IL-17 expression in thymus. (A) Weight (left panel) and number of total thymocytes (right panel) of the thymus (n = 3) from indicated genotypes of mice. (B) Thymocytes of different genotypes of mice were either gated on CD4CD8 or CD4+ and TCRβhi (top panels). Percentage of IL-17+ cells in CD4CD8 TCRβlo and CD4+ TCRβhi population of indicated genotype of mice was determined (middle panels). Bottom panels are the quantification of percentage of IL-17+ cells shown on middle panels from three mice of each genotype. (C) Expression of RORγt in indicated population of cells from indicated genotypes of mice, detected by intracellular staining. Arrow indicates the very high levels of RORγt population in CD4CD8 and CD4+ thymocytes of TCF-1fl/fl/Vav1-Cre mice. *p < 0.05, **p < 0.01. ns, not significant.

FIGURE 3.

Vav1-Cre–induced deletion of TCF-1 leads to abnormal IL-17 expression in thymus. (A) Weight (left panel) and number of total thymocytes (right panel) of the thymus (n = 3) from indicated genotypes of mice. (B) Thymocytes of different genotypes of mice were either gated on CD4CD8 or CD4+ and TCRβhi (top panels). Percentage of IL-17+ cells in CD4CD8 TCRβlo and CD4+ TCRβhi population of indicated genotype of mice was determined (middle panels). Bottom panels are the quantification of percentage of IL-17+ cells shown on middle panels from three mice of each genotype. (C) Expression of RORγt in indicated population of cells from indicated genotypes of mice, detected by intracellular staining. Arrow indicates the very high levels of RORγt population in CD4CD8 and CD4+ thymocytes of TCF-1fl/fl/Vav1-Cre mice. *p < 0.05, **p < 0.01. ns, not significant.

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We next determined whether forced expression of TCF-1 could inhibit IL-17 expression in developing and mature T cells from TCF-1fl/fl/Vav1-Cre mice. Just like in mature T cells with germline deletion of TCF-1 (13), expression of TCF-1 by retrovirus-mediated gene transduction in mature T cells from TCF-1fl/fl/Vav1-Cre mice was unable to reduce IL-17 expression to wild-type (WT) levels under Th17 priming conditions (Fig. 4A). Next, we determined the effects of TCF-1 on thymocytes cocultured with OP9-DL1 stroma cells (26). TCF-1fl/fl CD4CD8 DN thymocytes could develop to CD4+CD8+ DP thymocytes with or without expression of exogenous TCF-1 from retrovirus (Fig. 4B). TCF-1fl/fl/CD4-Cre CD4CD8 DN thymocytes were able to develop to CD4+CD8+ DP thymocytes, however, with reduced efficiency indicated by a reduced percentage of CD4+CD8+ cells. Exogenous TCF-1 could restore the percentage of CD4+CD8+ cells in TCF-1fl/fl/CD4-Cre thymocytes to WT level (Fig. 4B), indicating the intact function of exogenous TCF-1. In contrast to TCF-1fl/f and TCF-1fl/fl/CD4-Cre thymocytes, the majority of TCF-1fl/fl/Vav1-Cre CD4CD8 DN thymocytes stayed at the CD4CD8 DN stage, even in the presence of exogenous TCF-1 (Fig. 4B). We next determined the effects of exogenous TCF-1 on IL-17 expression. IL-17 was hardly detectable in thymocytes (including DN and developed DP) from TCF-1fl/fl and TCF-1fl/fl/CD4-Cre mice with or without exogenous TCF-1 (Fig. 4C), whereas CD4CD8 DN thymocytes from TCF-1fl/fl/Vav1-Cre mice had abundant IL-17 expression (Fig. 4C), consistent with what we observed in vivo (Fig. 3B). Indeed, exogenous TCF-1 significantly suppressed IL-17 expression in TCF-1fl/fl/Vav1-Cre thymocytes (Fig. 4C). Taken together, these data further demonstrated that TCF-1 is required to suppress IL-17 expression at early T cell developmental stages.

FIGURE 4.

Forced expression of TCF-1 in TCF-1–deficient CD4CD8 thymocytes but not mature CD4+ T cells from TCF-1fl/fl/Vav1-Cre mice inhibits IL-17 expression. (A) Percentage of IL-17+/GFP+ cells among indicated genotypes of CD4+ T cells transduced with retrovirus expressing GFP along or together with TCF-1 and differentiated under Th17-priming conditions for 3 d, as determined by flow cytometry analysis. Bottom panel is the quantification of three biological replicas. (B) Flow cytometric analysis of CD4 and CD8 among thymocytes ex vivo differentiated from CD4CD8 thymocytes sorted from indicated genotypes of mice, transduced with retrovirus expressing GFP along or together with TCF-1 and cocultured for 3 d with OP9-DL1 stroma cells and IL-7. The number in each quadrant indicated the percentage of cells in gated area among GFP+ cells. Bottom panel is the quantification the percentage of CD4+CD8+ cells shown on top panels. (C) Percentage of IL-17+/GFP+ cells among ex vivo differentiated thymocytes described in (B). Bottom panel is the quantification of the results. **p < 0.01. ns, not significant.

FIGURE 4.

Forced expression of TCF-1 in TCF-1–deficient CD4CD8 thymocytes but not mature CD4+ T cells from TCF-1fl/fl/Vav1-Cre mice inhibits IL-17 expression. (A) Percentage of IL-17+/GFP+ cells among indicated genotypes of CD4+ T cells transduced with retrovirus expressing GFP along or together with TCF-1 and differentiated under Th17-priming conditions for 3 d, as determined by flow cytometry analysis. Bottom panel is the quantification of three biological replicas. (B) Flow cytometric analysis of CD4 and CD8 among thymocytes ex vivo differentiated from CD4CD8 thymocytes sorted from indicated genotypes of mice, transduced with retrovirus expressing GFP along or together with TCF-1 and cocultured for 3 d with OP9-DL1 stroma cells and IL-7. The number in each quadrant indicated the percentage of cells in gated area among GFP+ cells. Bottom panel is the quantification the percentage of CD4+CD8+ cells shown on top panels. (C) Percentage of IL-17+/GFP+ cells among ex vivo differentiated thymocytes described in (B). Bottom panel is the quantification of the results. **p < 0.01. ns, not significant.

Close modal

Histone deacetylation is an evolutionarily conserved epigenetic modification mechanism responsible for gene repression (27). Recent studies have shown that TCF-1 had intrinsic HDAC activity (15). Considering the dual capacity of DNA binding and intrinsic HDAC activity in TCF-1, we wondered whether TCF-1 binds to IL-17 locus and silences IL-17 expression via its intrinsic HDAC activity. To answer this question, we first determined whether TCF-1 binds to IL-17 promoter by ChIP assays with nine primer pairs covering the IL-17A promoter regions (P1 to P9) ∼2000 bp upstream of the transcriptional starting site (Fig. 5A). The enrichment of TCF-1 binding to IL-17 promoter was observed in the region from P4 to P8 in TCF-1fl/fl thymus versus differentiated Th17 cells (Fig. 5B). To determine which TCF-1 binding regions are functional, each individual region from P4 to P8 was deleted to assess the effects on IL-17 reporter activity. Consistent with previous results (28, 29), RORγt stimulated IL-17 reporter. Whereas TCF-1 inhibited RORγt-dependent IL-17 reporter, deletion of the P4 or P7 regions, but not the P5, P6, or P8 regions, of IL-17 promoter significantly relieved TCF-1–mediated inhibition of IL-17 reporter (Fig. 5C), suggesting that P4 and P7 are two functional TCF-1 binding sites responsible for the inhibition of IL-17 promoter activity. To determine whether TCF-1 is required for epigenetic modification critical for IL-17 expression, we compared histone H3 acetylation levels in the regions from P4 to P8. Because of the deletion of TCF-1 in TCF-1fl/fl/Vav1-Cre thymus, P5 and P8 had higher H3 acetylation levels (Fig. 5D), indicating that these two regions underwent TCF-1–dependent histone deacetylation in the WT thymus. These data indicate the possibility that TCF-1 mediates histone deacetylation in the regions around these binding sites. We further observed that, in contrast to WT TCF-1, TCF-1-5aa mutant, which diminished HDAC activity (15), was unable to inhibit IL-17 expression in DN thymocytes obtained from TCF-1fl/fl/Vav1-Cre mice, which is comparable to virus expression of GFP alone (Fig. 5E). Taken together, these results demonstrated that TCF-1 binds to IL-17 promoter and silences IL-17 expression through its intrinsic HDAC activity.

FIGURE 5.

TCF-1 binds to IL-17 gene locus and inhibits IL-17 expression through its intrinsic HDAC activity. (A) Schematic representation of the location of nine regions, P1–P9, on IL-17 promoter. (B) Enrichment of TCF-1 binding to nine regions on IL-17 promoter, detected by ChIP assay using anti–TCF-1 Ab or IgG control in WT thymus or differentiated Th17 cells. (C) IL-17 promoter–luciferase reporter activity in HEK293T cells transfected with the indicated expression plasmids. Δ4, deletion of P4; Δ5, deletion of P5; Δ6, deletion of P6; Δ7, deletion of P7; Δ8, deletion of P8 region shown in (A). Luciferase data are normalized to Renilla luciferase activity. (D) DNA was precipitated with anti–acetyl-histone H3 Abs. qRT-PCR–based ChIP analysis was used to measure histone H3 acetylation levels in indicated regions. (E) Percentage of IL-17+/GFP+ cells among thymocytes ex vivo differentiated from CD4CD8 cells sorted from TCF-1fl/fl/Vav1-Cre mice, transduced with retrovirus expressing GFP along or together with TCF-1 or TCF1-Mut5aa and cocultured with OP9-DL1 stromal cells for 3 d, as determined by flow cytometry. Right panel is the quantification of three biological replicas. *p < 0.05, **p < 0.01. ns, not significant.

FIGURE 5.

TCF-1 binds to IL-17 gene locus and inhibits IL-17 expression through its intrinsic HDAC activity. (A) Schematic representation of the location of nine regions, P1–P9, on IL-17 promoter. (B) Enrichment of TCF-1 binding to nine regions on IL-17 promoter, detected by ChIP assay using anti–TCF-1 Ab or IgG control in WT thymus or differentiated Th17 cells. (C) IL-17 promoter–luciferase reporter activity in HEK293T cells transfected with the indicated expression plasmids. Δ4, deletion of P4; Δ5, deletion of P5; Δ6, deletion of P6; Δ7, deletion of P7; Δ8, deletion of P8 region shown in (A). Luciferase data are normalized to Renilla luciferase activity. (D) DNA was precipitated with anti–acetyl-histone H3 Abs. qRT-PCR–based ChIP analysis was used to measure histone H3 acetylation levels in indicated regions. (E) Percentage of IL-17+/GFP+ cells among thymocytes ex vivo differentiated from CD4CD8 cells sorted from TCF-1fl/fl/Vav1-Cre mice, transduced with retrovirus expressing GFP along or together with TCF-1 or TCF1-Mut5aa and cocultured with OP9-DL1 stromal cells for 3 d, as determined by flow cytometry. Right panel is the quantification of three biological replicas. *p < 0.05, **p < 0.01. ns, not significant.

Close modal

Previous studies have shown TCF-1 was able to restrain peripheral Th17 responses, and in vitro experiments show that TCF-1 negatively regulates peripheral Th17 responses at T cell developmental stages in thymus rather than at the mature stage of T cells in the periphery (13, 14). However, there was no in vivo evidence to support stage-specific requirement of TCF-1 for limiting peripheral Th17 immunity. Furthermore, the mechanisms responsible for TCF-1–mediated inhibition of Th17 responses remain unknown. In this study, we answered the critical question of when TCF-1 is required to control the peripheral Th17 responses, given that T cell development is a sequential multistep process. We found that deletion of TCF-1 early in the hematopoietic stage, but not in later DP stages, resulted in exaggerated Th17 differentiation and EAE development. In addition, deletion of TCF-1 at early but not later DP stages leads to significant disruption of thymocyte development, demonstrating that TCF-1 plays a major role during early thymocyte development. Mechanically, we show that TCF-1 binds to and inhibits IL-17 promoter to prevent IL-17 expression via its intrinsic HDAC activity.

The IL-17 gene locus must be kept inactive by TCF-1 prior to developing thymocytes entering the DP stage. This is indicated by the fact that deletion of TCF-1 at the DP stage does not cause enhanced IL-17 expression in thymus, which means that the suppressive function of TCF-1 for the IL-17 gene is not required at the DP stage or later. Once TCF-1–mediated inhibition is imprinted on the IL-17 gene locus, which we believe occurred no later than the DN stage, it can last even after T cell migration out of the thymus, resulting in prevention of exaggerated Th17 responses in the periphery. Furthermore, our results highlight the specific requirement of HDAC activity of TCF-1 for suppression of IL-17 gene activity during T cell development. The HDAC family consists of a large number of family members, many of which play indispensable roles in regulating T cell development, including DN to DP transition, cell survival, and TCR signaling in DP thymocytes (3033). Although several HDAC proteins are expressed in thymus, it seems they are unable to compensate for the HDAC activity of TCF-1, as indicated by abnormal high levels of IL-17 expression (or natural Th17 cells) in thymus of TCF-1fl/fl/Vav1-Cre mice. This phenomenon highlights the specific requirement of HDAC activity of TCF-1 for keeping the IL-17 gene inactive early during T cell development.

Interestingly, if the IL-17 gene locus does not undergo TCF-1–mediated inhibition like TCF-1 deficiency at the early development stage, it will lead to an elevated Th17 differentiation and Th17 immunity. This consequence is irreversible, as reconstitution of TCF-1 in mature T cells was unable to reduce IL-17 expression to normal levels. The mechanisms of how TCF-1 closes the IL-17 gene locus in early developing thymocytes rather than in mature T cells remains largely unknown. Our results suggest that the IL-17 gene locus in mature T cells was relatively inaccessible for TCF-1 compared with that in the early T cell developmental stage. This may result from chromatin remolding during later developmental stages and/or lack of cofactors that present in early thymocytes but not in mature T cells, which could be an interesting topic for future investigation.

Exaggerated Th17 responses are associated with many types of autoimmune diseases (8, 34). To develop effective treatment for autoimmune diseases, many studies have focused on understanding the mechanisms of regulating Th17 differentiation and Th17–Treg balance in the peripheral immune system. Our study suggests that peripheral Th17 responses can be greatly influenced by specific developmental events in the central immune system, thus providing an additional layer of control to prevent autoimmune diseases.

We thank Dr. Hai-Hui Xue for generously sharing TCF-1fl/fl mice. We appreciate the help by City of Hope–supported cores, including animal, genomic, and flow cytometer cores. In addition, we thank Chris Gandhi and Keely Walker for their careful proofing/editing work on the manuscript.

This work was supported by National Institutes of Health Grants R01-AI053147 and R01-AI109644 and institutional pilot funding. Research reported in this publication was also supported by the National Cancer Institute of the National Institutes of Health under Award P30CA33572, which includes work performed in the animal, genomic, flow cytometer, and mass spectrometric cores supported by this grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • ChIP

    chromatin immunoprecipitation

  •  
  • DN

    double-negative

  •  
  • DP

    double-positive

  •  
  • EAE

    experimental autoimmune encephalomyelitis

  •  
  • HDAC

    histone deacetylase

  •  
  • MOG35–55

    myelin oligodendrocyte glycoprotein 35–55

  •  
  • qRT-PCR

    quantitative real-time PCR

  •  
  • rm

    recombinant murine

  •  
  • TCF-1

    T cell factor 1

  •  
  • Treg

    regulatory T cell

  •  
  • WT

    wild-type.

1
Jameson
,
S. C.
,
K. A.
Hogquist
,
M. J.
Bevan
.
1995
.
Positive selection of thymocytes.
Annu. Rev. Immunol.
13
:
93
126
.
2
Ma
,
J.
,
R.
Wang
,
X.
Fang
,
Z.
Sun
.
2012
.
β-catenin/TCF-1 pathway in T cell development and differentiation.
J. Neuroimmune Pharmacol.
7
:
750
762
.
3
Klein
,
L.
,
B.
Kyewski
,
P. M.
Allen
,
K. A.
Hogquist
.
2014
.
Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see).
Nat. Rev. Immunol.
14
:
377
391
.
4
Starr
,
T. K.
,
S. C.
Jameson
,
K. A.
Hogquist
.
2003
.
Positive and negative selection of T cells.
Annu. Rev. Immunol.
21
:
139
176
.
5
Lee
,
Y.
,
A.
Awasthi
,
N.
Yosef
,
F. J.
Quintana
,
S.
Xiao
,
A.
Peters
,
C.
Wu
,
M.
Kleinewietfeld
,
S.
Kunder
,
D. A.
Hafler
, et al
.
2012
.
Induction and molecular signature of pathogenic TH17 cells.
Nat. Immunol.
13
:
991
999
.
6
Codarri
,
L.
,
G.
Gyülvészi
,
V.
Tosevski
,
L.
Hesske
,
A.
Fontana
,
L.
Magnenat
,
T.
Suter
,
B.
Becher
.
2011
.
RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.
Nat. Immunol.
12
:
560
567
.
7
El-Behi
,
M.
,
B.
Ciric
,
H.
Dai
,
Y.
Yan
,
M.
Cullimore
,
F.
Safavi
,
G. X.
Zhang
,
B. N.
Dittel
,
A.
Rostami
.
2011
.
The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.
Nat. Immunol.
12
:
568
575
.
8
Elloso
,
M. M.
,
M.
Gomez-Angelats
,
A. M.
Fourie
.
2012
.
Targeting the Th17 pathway in psoriasis.
J. Leukoc. Biol.
92
:
1187
1197
.
9
Skepner
,
J.
,
R.
Ramesh
,
M.
Trocha
,
D.
Schmidt
,
E.
Baloglu
,
M.
Lobera
,
T.
Carlson
,
J.
Hill
,
L. A.
Orband-Miller
,
A.
Barnes
, et al
.
2014
.
Pharmacologic inhibition of RORγt regulates Th17 signature gene expression and suppresses cutaneous inflammation in vivo.
J. Immunol.
192
:
2564
2575
.
10
Verbeek
,
S.
,
D.
Izon
,
F.
Hofhuis
,
E.
Robanus-Maandag
,
H.
te Riele
,
M.
van de Wetering
,
M.
Oosterwegel
,
A.
Wilson
,
H. R.
MacDonald
,
H.
Clevers
.
1995
.
An HMG-box-containing T-cell factor required for thymocyte differentiation.
Nature
374
:
70
74
.
11
Schilham
,
M. W.
,
A.
Wilson
,
P.
Moerer
,
B. J.
Benaissa-Trouw
,
A.
Cumano
,
H. C.
Clevers
.
1998
.
Critical involvement of Tcf-1 in expansion of thymocytes.
J. Immunol.
161
:
3984
3991
.
12
Okamura
,
R. M.
,
M.
Sigvardsson
,
J.
Galceran
,
S.
Verbeek
,
H.
Clevers
,
R.
Grosschedl
.
1998
.
Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1.
Immunity
8
:
11
20
.
13
Ma
,
J.
,
R.
Wang
,
X.
Fang
,
Y.
Ding
,
Z.
Sun
.
2011
.
Critical role of TCF-1 in repression of the IL-17 gene.
PLoS One
6
:
e24768
.
14
Yu
,
Q.
,
A.
Sharma
,
A.
Ghosh
,
J. M.
Sen
.
2011
.
T cell factor-1 negatively regulates expression of IL-17 family of cytokines and protects mice from experimental autoimmune encephalomyelitis.
J. Immunol.
186
:
3946
3952
.
15
Xing
,
S.
,
F.
Li
,
Z.
Zeng
,
Y.
Zhao
,
S.
Yu
,
Q.
Shan
,
Y.
Li
,
F. C.
Phillips
,
P. K.
Maina
,
H. H.
Qi
, et al
.
2016
.
Tcf1 and Lef1 transcription factors establish CD8(+) T cell identity through intrinsic HDAC activity.
Nat. Immunol.
17
:
695
703
.
16
Steinke
,
F. C.
,
S.
Yu
,
X.
Zhou
,
B.
He
,
W.
Yang
,
B.
Zhou
,
H.
Kawamoto
,
J.
Zhu
,
K.
Tan
,
H. H.
Xue
.
2014
.
TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4(+) T cell fate and interact with Runx3 to silence Cd4 in CD8(+) T cells.
Nat. Immunol.
15
:
646
656
.
17
Kang
,
Z.
,
C. Z.
Altuntas
,
M. F.
Gulen
,
C.
Liu
,
N.
Giltiay
,
H.
Qin
,
L.
Liu
,
W.
Qian
,
R. M.
Ransohoff
,
C.
Bergmann
, et al
.
2010
.
Astrocyte-restricted ablation of interleukin-17-induced Act1-mediated signaling ameliorates autoimmune encephalomyelitis.
Immunity
32
:
414
425
.
18
Lee
,
P. P.
,
D. R.
Fitzpatrick
,
C.
Beard
,
H. K.
Jessup
,
S.
Lehar
,
K. W.
Makar
,
M.
Pérez-Melgosa
,
M. T.
Sweetser
,
M. S.
Schlissel
,
S.
Nguyen
, et al
.
2001
.
A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival.
Immunity
15
:
763
774
.
19
de Boer
,
J.
,
A.
Williams
,
G.
Skavdis
,
N.
Harker
,
M.
Coles
,
M.
Tolaini
,
T.
Norton
,
K.
Williams
,
K.
Roderick
,
A. J.
Potocnik
,
D.
Kioussis
.
2003
.
Transgenic mice with hematopoietic and lymphoid specific expression of Cre.
Eur. J. Immunol.
33
:
314
325
.
20
Cua
,
D. J.
,
J.
Sherlock
,
Y.
Chen
,
C. A.
Murphy
,
B.
Joyce
,
B.
Seymour
,
L.
Lucian
,
W.
To
,
S.
Kwan
,
T.
Churakova
, et al
.
2003
.
Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.
Nature
421
:
744
748
.
21
Yu
,
Q.
,
A.
Sharma
,
S. Y.
Oh
,
H. G.
Moon
,
M. Z.
Hossain
,
T. M.
Salay
,
K. E.
Leeds
,
H.
Du
,
B.
Wu
,
M. L.
Waterman
, et al
.
2009
.
T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-gamma.
Nat. Immunol.
10
:
992
999
.
22
Kroenke
,
M. A.
,
T. J.
Carlson
,
A. V.
Andjelkovic
,
B. M.
Segal
.
2008
.
IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition.
J. Exp. Med.
205
:
1535
1541
.
23
Stromnes
,
I. M.
,
L. M.
Cerretti
,
D.
Liggitt
,
R. A.
Harris
,
J. M.
Goverman
.
2008
.
Differential regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells.
Nat. Med.
14
:
337
342
.
24
Ivanov
,
I. I.
,
B. S.
McKenzie
,
L.
Zhou
,
C. E.
Tadokoro
,
A.
Lepelley
,
J. J.
Lafaille
,
D. J.
Cua
,
D. R.
Littman
.
2006
.
The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.
Cell
126
:
1121
1133
.
25
Sun
,
Z.
,
D.
Unutmaz
,
Y. R.
Zou
,
M. J.
Sunshine
,
A.
Pierani
,
S.
Brenner-Morton
,
R. E.
Mebius
,
D. R.
Littman
.
2000
.
Requirement for RORgamma in thymocyte survival and lymphoid organ development.
Science
288
:
2369
2373
.
26
Holmes
,
R.
,
J. C.
Zuniga-Pflucker
.
2009
.
The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.
Cold Spring Harb. Protoc.
2009
:
pdb prot5156
.
27
Rusche
,
L. N.
,
A. L.
Kirchmaier
,
J.
Rine
.
2003
.
The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae.
Annu. Rev. Biochem.
72
:
481
516
.
28
Wang
,
X.
,
Y.
Zhang
,
X. O.
Yang
,
R. I.
Nurieva
,
S. H.
Chang
,
S. S.
Ojeda
,
H. S.
Kang
,
K. S.
Schluns
,
J.
Gui
,
A. M.
Jetten
,
C.
Dong
.
2012
.
Transcription of Il17 and Il17f is controlled by conserved noncoding sequence 2.
Immunity
36
:
23
31
.
29
Sen
,
S.
,
F.
Wang
,
J.
Zhang
,
Z.
He
,
J.
Ma
,
Y.
Gwack
,
J.
Xu
,
Z.
Sun
.
2018
.
SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate RORγt activity in a PKC-θ-dependent manner.
Proc. Natl. Acad. Sci. USA.
115
:
E458
E467
.
30
Haery
,
L.
,
R. C.
Thompson
,
T. D.
Gilmore
.
2015
.
Histone acetyltransferases and histone deacetylases in B- and T-cell development, physiology and malignancy.
Genes Cancer
6
:
184
213
.
31
Dovey
,
O. M.
,
C. T.
Foster
,
N.
Conte
,
S. A.
Edwards
,
J. M.
Edwards
,
R.
Singh
,
G.
Vassiliou
,
A.
Bradley
,
S. M.
Cowley
.
2013
.
Histone deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice.
Blood
121
:
1335
1344
.
32
Cabrero
,
J. R.
,
J. M.
Serrador
,
O.
Barreiro
,
M.
Mittelbrunn
,
S.
Naranjo-Suárez
,
N.
Martín-Cófreces
,
M.
Vicente-Manzanares
,
R.
Mazitschek
,
J. E.
Bradner
,
J.
Avila
, et al
.
2006
.
Lymphocyte chemotaxis is regulated by histone deacetylase 6, independently of its deacetylase activity.
Mol. Biol. Cell
17
:
3435
3445
.
33
Kasler
,
H. G.
,
B. D.
Young
,
D.
Mottet
,
H. W.
Lim
,
A. M.
Collins
,
E. N.
Olson
,
E.
Verdin
.
2011
.
Histone deacetylase 7 regulates cell survival and TCR signaling in CD4/CD8 double-positive thymocytes.
J. Immunol.
186
:
4782
4793
.
34
Gaffen
,
S. L.
,
R.
Jain
,
A. V.
Garg
,
D. J.
Cua
.
2014
.
The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing.
Nat. Rev. Immunol.
14
:
585
600
.

The authors have no financial conflicts of interest.

Supplementary data