IL-7/IL-7R signaling differentially affects effector CD4⁺ T cell subsets involved in experimental autoimmune encephalomyelitis

Abstract

IL-17-producing CD4⁺ T (Th17) cells, along with IFN-gamma-expressing Th1 cells, represent two major pathogenic T cell subsets in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). The cytokines and transcription factors involved in the development and effector functions of Th1 and Th17 cells have been largely characterized. Among them, IL-23 is essential for the generation of stable and encephalitogenic Th17 cells and for the development of EAE. The IL-7/IL-7R signaling axis participates in cell survival, and perturbation of this pathway has been associated with enhanced susceptibility to MS. A link between IL-23-driven pathogenic T cells and IL-7/IL-7R signaling has previously been proposed but has not been formally addressed. In the present study, we showed that Th17 cells from mice with EAE express high levels of IL-7Ralpha compared to Th1 cells. Using mice that constitutively express IL-7Ralpha on T cells, we determined that sustained IL-7R expression in IL-23R deficient mice could not drive pathogenic T cells and the development of EAE. IL-7 inhibited the differentiation of Th17 cells but promoted IFN-gamma and GM-CSF secretion in vitro. In vivo IL-7/anti-IL-7 mAb complexes selectively expanded and enhanced the proliferation of CXCR3-expressing Th1 cells but did not impact Th17 cells and EAE development in wild-type and IL-23R-deficient mice. Importantly, high IL-7 expression was detected in the CNS during EAE and could drive the plasticity of Th17 cells to IFN-gamma-producing T cells. Together, these data address the contribution of IL-23/IL-23R and IL-7/IL-7R signaling in Th17 and Th1 cell dynamics during CNS autoimmunity.

Introduction

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS), leading to axonal damage and physical impairment. Experimental autoimmune encephalomyelitis (EAE), the mouse model of MS, has been useful in identifying the pathogenic mechanisms at play in MS and in determining that CD4⁺ T helper (Th) cells are essential for the detrimental inflammation characteristic of MS and EAE (1). Historically, Th1 and Th17 cells have been known to drive the inflammatory processes within the CNS by producing IFN-γ and IL-17, respectively (2). Although Th1 or Th17 cells can induce EAE independently, the clinical signs, pathological features, and cells recruited may differ. Th1-polarized cells promote the expression of monocyte attracting chemokines and macrophage-rich infiltrates into the spinal cord, whereas IL-23 polarized Th17 cells activate neutrophil-attracting chemokines, promote neutrophil recruitment, especially in the brain (3), and drive the formation of ectopic lymphoid aggregates (4).

IL-23 is a dimeric cytokine composed of the p40 subunit common with IL-12 and the unique p19 subunit which is essential for the development of EAE, since both IL-23p19 KO and IL-23 receptor-deficient (IL-23R KO) mice are resistant to the development of EAE (5–7). IL-23 maintains and expands Th17 cells (8), induces the production of GM-CSF (9, 10), and promotes the plasticity of Th17 cells into a Th1 cell phenotype (11, 12). Indeed, while Th17 cells differentiated in vitro have a clear and distinct phenotype under strong Th17-polarizing conditions, Th17 cells found in the CNS of mice with EAE modulate their cytokine expression and express IFN-γ (12–14). Few cytokines have been shown to modulate the plasticity of Th17 cells (11, 15) and the identity of the cytokine milieu, which modulates the balance between these effector populations in vivo, remains elusive.

Several cytokines are believed to be important in expanding CD4⁺ T cells. IL-2 is an important survival factor for activated effector T cells, particularly Th1 cells, but limits Th17 cell differentiation and expansion (16). IL-7, besides being crucial for T cell development in the thymus, is important in maintaining the naïve and memory T cell populations in the periphery (17). IL-7/IL-7 receptor (IL-7R) signaling has gained considerable interest in MS because single-nucleotide polymorphisms (SNPs) in the IL-7Rα, which dimerizes with the common γ chain to form the IL-7R, have been associated with increased risk for developing MS (18, 19). The SNP, rs6897932, which is located within the alternatively spliced exon 6 of Il7R, increases the rate of IL7Rα mRNA splicing and results in increased levels of soluble IL7Ra, which competes with membrane-bound IL-7Rα to increase the bioavailability of IL-7 (20). Furthermore, IL-7Rα blockade leads to amelioration of EAE, which is associated with a reduction in CD4⁺ and CD8⁺ T cell numbers (21, 22).

Attempts to investigate the effects of IL-7-mediated signaling on pathogenic T cells have yielded conflicting results. It has been proposed that IL-23/IL-23R signaling is essential for the upregulation of IL-7Rα on differentiated Th17 cells and for their expansion (5). Another study further linked Th17 cells with IL-7 by suggesting that IL-7 selectively expands Th17 cells from mice with EAE and MS patients (23). However, the results of this latter study have been questioned, and another laboratory provided evidence that IL-7 does not affect Th17 cell differentiation (21). Although the role of IL-7 has been well studied in development, homeostatic proliferation, and survival of T cells, how the IL-7/IL-7 signaling axis influences disease settings and pathogenic T cell responses, particularly in vivo, has not been fully addressed.

Here, we investigated the effects of IL-7-mediated signaling on the generation of pathogenic Th1 and Th17 cells. We found that IL-7Rα was enriched on Th17 cells in the periphery and CNS of mice with EAE at the peak and onset of the disease. Moreover, we provided evidence that IL-23R-deficient mice, which cannot generate stable Th17 cells, remain resistant to EAE when IL-7Rα was constitutively expressed and when a highly bioactive form of IL-7 complexed to IL-7 antibody (IL-7c) was administered. IL-17 production in IL-23R-deficient mice was not rescued in the presence of constitutively active IL-7Rα or treatment with IL-7c. We further demonstrated that IL-7 selectively promotes Th1 differentiation and the plasticity of Th17 cells. These results are relevant for the development of EAE and MS, since large amounts of IL-7 are produced in the CNS of mice and humans with autoimmunity.

Materials and Methods

Mice

C57BL/6 (B6) mice were purchased from the Jackson Laboratories and bred in the Benaroya Research Institute (BRI) animal facility. Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1triple reporter mice have been described previously (12). These mice were generated by breeding IL-17A-GFP (Biocytogen), IFN-γ knock-in Thy1.1(24), and Foxp3-RFP (25) reporter mice together. IL-23R knock-in (KI) and IL-23R homozygous KI (IL-23R^−/−) GFP reporter mice were previously described (6).IL-7RαTg and ROR-γt-GFP reporter mice were previously described (26, 27). All strains are on the C57BL/6 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 differentiation, naïve CD4⁺CD62L^hiCD44^loCD25− T cells were isolated by FACS sorting (FACSAria, BD Biosciences) and cultured with irradiated (4000 rads) CD4-depleted spleen cells from wild-type (WT) mice and anti-CD3 (2.5 µg/ml, clone 145-2C11) for 5 days in complete RPMI. For Th17 differentiation, 2.5 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) were used. 10 ng/ml rmIL-12 was used for Th1 conditions. For the indicated conditions, 10 ng/ml rmIL-7 (eBioscience) was added to the cultures. For restimulation, T cells were collected and activated with irradiated splenocytes, anti-CD3 and indicated cytokines (rmIL-7 was used at 10 ng/ml, eBioscience).

Antibodies and flow cytometry

For intracellular cytokine staining analysis, cells were incubated 5 hours in complete RPMI containing 50ng/ml phorbol myristate acetate (PMA), 1µg/ml ionomycin (Sigma-Aldrich) and Golgi Stop (BD Biosciences). Cells were then washed with cold PBS and blocked for 10 min with anti-CD16/32 purified antibody (clone 2.4G2, BioXCell). Viability of the cells was assessed by staining with fixable dye eFluor780 (eBioscience). Cells were then stained with anti-CD4 antibody and washed with PBS. Cells were then fixed for 20 min with fixation buffer (BD Biosciences), permeabilized with BD permeabilization/wash buffer (BD Biosciences) and stained with anti-IFN-γ, and anti-IL-17 specific antibodies in permeabilization buffer. Cells were acquired on LSRII (BD Biosciences), and data were analyzed with FlowJo software. The following antibodies were used in our experiments: Antibodies to CD4 (clone L3T4) conjugated to PerCP-Cy5.5 or Alexa700, IL-7Rα (CD127) (clone A7R34) conjugated to PerCP-Cy5.5, IFN-γ (clone XMG1.2) conjugated to PE-Cy7 or PE, were purchased from eBioscience and antibody to IL-17 (clone TC11-18H10.1) conjugated to APC was purchased from Biolegend.

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; 5, moribund.

Isolation of CNS mononuclear cells

Mice were sacrificed at the peak of disease and perfused with cold PBS. Brain and spinal cords were isolated and digested for 30 min at 37°C with Collagenase D at a concentration of 2.5mg/ml (Roche). Mononuclear cells were isolated over a 37% / 70% Percoll gradient (VWR), washed twice with complete medium and collected in medium for further analysis.

IL-7/M25 complex treatment

Recombinant mouse IL-7 was purchased from eBioscience (San Diego, CA). M25 anti-IL-7 antibody was purchased from Bio X Cell (West Lebanon, NH). IL-7/M25 complexes (IL-7c) were generated as previously described (28). Briefly, each mouse received complexes generated from a 30-minute incubation at 37°C of 1.5µg IL-7 with 15µg mAb M25. WT mice immunized with MOG_35–55 in CFA received 3 injections of IL-7c every other day starting at day 1 after immunization. ROR-γt-GFP mice were sacrificed six days after the first injection.

Statistical analysis

Statistical analyses were 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

IL-7 receptor is highly expressed on Th17 cells

Th17 and Th1 cells can induce EAE with different pathological phenotypes and distinct clinical signs (3, 4). Because high levels of IL-7 receptor have been proposed to render cells more responsive to IL-7-mediated signaling (17), we examined IL-7 receptor (IL-7Rα) expression on these subsets in vivo during the course of EAE. We took advantage of a triple reporter mouse (Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1) in which cells expressing Foxp3, IL-17, and IFN-γ can be detected based on RFP, GFP, and Thy1.1 expression, respectively, to identify the proportion of Foxp3⁺ T and cytokine-producing effector Th17 and Th1 cells ex vivo. This strategy allowed the detection of cytokine-producing cells without permeabilization and activation with PMA/ionomycin, which can impact IL-7Rα expression. First, we analyzed at the peak of EAE IL-7Rα expression on Foxp3⁻ and Foxp3⁺ CD4⁺ T cells, which contain effector and regulatory T cells respectively (Figure 1A). Consistent with published data, in the spleen and lymph nodes (LNs) of mice at the peak and onset of EAE, CD4⁺ Foxp3⁻ effector T cells expressed higher levels of IL-7Rα, while Foxp3⁺ T cells showed diminished expression of the receptor (Figure 1B–C and not shown). Next, we examined the patterns of IL-7Rα expression on effector Th1 and Th17 cells after immunization with MOG_35–55 and CFA to induce EAE development. Unexpectedly, Th17 cells expressed high amounts of IL-7Rα in both the spleen and LNs at the onset of the disease, while the expression of IL-7Rα on Th1 cells was significantly lower (Figure 1D–F). The enrichment of IL-7Rα on Th17 cells was also observed at the peak of the disease (Figure 1G–I). Therefore the differential expression of IL-7Rα on Th1 and Th17 cells remained constant at all measured stages of the disease, including at the peak of EAE.

Figure 1. Th17 cells express high levels of IL-7Rα.

(A–I) Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1 triple reporter mice were immunized for EAE with MOG_35–55 emulsified in CFA. (A–C) Surface expression of IL-7Rα (B) was analyzed on CD4+ Foxp3⁻ cells (solid line gate and histogram) and CD4+ Foxp3⁺ cells (dashed line gate and histogram) from the spleen/lymph nodes of immunized mice (as gated in A). (C) Summary of IL-7Rα MFI on these populations. (D–I) Expression of GFP (IL-17A), indicative of Th17 cells (bold line), and Thy1.1 (IFN-γ), representing Th1 cells (thin line), on CD4⁺CD44⁺ T cells from the spleen (top) and lymph nodes (bottom) and IL-7Rα expression on these T cell populations were determined at the onset (day 9–12, D–F) and peak (day 14–18, G–I) of the disease. (E and H) Surface expression of IL-7Rα on IL-17A⁺ Th17 cells (bold line histogram) and IFN-γ⁺ Th1 cells (thin line histogram) is compared to isotype control (shaded grey histogram). (F and I) Summary of IL-7Rα expression quantified by mean fluorescent intensity (MFI) on gated subsets. (J) Identification of IL-23R⁺ (bold line gate) and IL-23R⁻ (thin line gate) effector CD4⁺CD44⁺ T cells in the draining lymph nodes of IL-23R-GFP mice 8–10 days after immunization with MOG_35–55/CFA. (K) Staining of IL-7Rα on IL-23R⁻ (thin line histogram) and IL-23R⁺ (bold line histogram) CD4⁺ T cells as gated in (J). Statistics were performed with Student’s t test (^*p < 0.05, ^**p < 0.01). Data are representative of at least 2 independent experiments with at least 3 mice each experiment.

IL-23R is required for the maintenance of Th17 cells and is expressed on effector T cells as they differentiate into Th17 cells (5). Thus, we analyzed the expression of IL-7Rα on IL-23R⁺ cells from the draining LNs of MOG-immunized IL-23R-GFP reporter mice before disease onset. We found that IL-7Rα was more highly expressed on IL-23R⁺ CD4⁺ T cells than IL-23R⁻ CD4⁺ T cells (Figure 1J–K), consistent with our results that IL-7R expression is upregulated on Th17 cells at the onset and peak of EAE development. Thus, we observed that Th17 cells constantly express higher levels of IL-7Rα during all stages of the disease course. The elevated levels of IL-7Rα on Th17 cells compared to Th1 and Foxp3⁺ T cells suggest that Th17 cells might be more sensitive and responsive to IL-7 and expand in response to this cytokine. Alternatively, the higher expression of IL-7Rα on Th17 cells could indicate that these cells did not respond to IL-7 and therefore did not downregulate IL-7Rα.

Sustained IL-7R signaling does not replace IL-23R signaling for the generation of pathogenic T cells

To address this question, we first tested the sensitivity to and requirement of IL-7/IL-7R signaling in Th17 cells by providing sustained IL-7R expression on T cells. T cells from IL-23R deficient (IL-23R ^−/−) mice immunized with MOG_35–55 have impaired Th17 cell expansion and effector function, making them resistant to the development of EAE (5, 6). It was proposed that IL-23/IL-23R interactions drive IL-7Rα expression, which is essential for Th17 cell proliferation and expansion (5), further suggesting that forced expression of IL-7R on IL-23R^−/− T cells could rescue the expansion of Th17 cells. To test this hypothesis, we took advantage of mice that constitutively express the IL-7Rα on CD4⁺ and CD8⁺ T cells (IL-7RαTg) (26). Since IL-7Rα is quickly downregulated upon TCR ligation, IL-7/IL-7R signaling, and other cytokine signals, we first tested the ability of IL-23R^−/−/IL-7RαTg CD4⁺ T cells to maintain high levels of IL-7Rα expression. Eight days after immunization with MOG_35–55/CFA, CD4⁺ T cells isolated from the draining LNs of IL-7RαTg and IL-23R^−/−/IL-7RαTg mice maintained high levels of IL-7Rα expression, whereas IL-23R^−/− CD4⁺ T cells downregulated the receptor (Figure 2A–B). Furthermore, IL-7/IL-7R signaling was highly functional, since cells from IL-23R^−/−/IL-7RαTg mice were also capable of phosphorylating STAT5 at higher levels in response to IL-7 compared to cells from IL-23R^−/− mice (Figure 2C). Thus, constitutive IL-7Rα expression and sustained STAT5 signaling in IL-23R^−/−/IL-7RαTg mice bypass the possible requirement of IL-23 for IL-7Rα expression and make the mice suitable to address whether IL-7/IL-7R signaling could compensate for the lack of IL-23 in the development of EAE.

Figure 2. IL-7- and IL-23-mediated signaling events act independently on T cells.

(A–C) IL-7RαTg, IL-23R^−/−/IL-7RαTg, and IL-23R^−/− mice were immunized with MOG_35–55 in CFA. Eight to ten days after immunization, CD4⁺ T cells from the draining lymph nodes were analyzed for IL-7Rα expression (A) and quantified in (B). (C) STAT5 phosphorylation from CD4⁺ T cells isolated from the draining lymph nodes of MOG-immunized mice, in response to IL-7 or no stimulation. (D) Frequency of IL-23R^−/− (CD45.1) and IL-23R^−/−/IL-7RαTg (CD45.2) TCR-β⁺CD4⁺ cells recovered in the spleen 7 days after transfer into TCR-βδ^−/− mice, assessed by flow cytometry. Numbers indicate percentage of each cell population among CD4⁺ TCR-β⁺ T cells. Data are representative of at least 2 independent experiments with n≥2 mice in each experiment. (E–H) Wild-type (WT), IL-7RαTg, IL-23R^−/−, and IL-23R^−/−/IL-7RαTg mice were immunized with MOG/CFA. (E–H) The percentage of IL-17⁺ and IFN-γ⁺ CD4⁺ T cells (representative dot plot, E and summary, F) from the lymph nodes of these animals 10 days after immunization was determined by intracellular cytokine staining. (G) Clinical course of EAE over time in these mice. Results are shown as mean clinical score ± SEM (n ≥ 5 mice per group/experiment). Data from 3 combined experiments are shown. (H) Percentages of IL-17⁺, IFN-γ⁺, IL-17⁺IFN-γ⁺, and GM-CSF⁺ CD4⁺ T cells from the CNS of WT and IL-7RαTg mice at the peak of the disease are shown. Results from one representative experiment of two independent experiments are shown.

To determine how constitutive expression of IL-7Rα affects the proliferation and expansion of CD4⁺ T cells, we sorted naïve (CD62L^hiCD44^lo) CD4⁺ T cells derived from IL-23R^−/− and IL-23R^−/−/IL-7RαTg mice and allowed them to expand under lymphopenic conditions in TCRβδ-deficient mice. In this system, transferred cells have access to an abundant source of IL-7, which allows them to survive and expand very rapidly in an IL-7-dependent manner. Seven days after transfer, we recovered fewer IL-23R^−/− cells, compared to IL-23R^−/−/IL-7RαTg cells (Figure 2D), showing that increased expression and signaling through the IL-7R induces preferential expansion and survival of IL-23R-deficient T cells.

To test whether constitutive expression of IL-7Rα on IL-23R-deficient cells can affect cytokine production, we immunized WT, IL-7RαTg, IL-23R^−/−, and IL-23R^−/−/IL-7RαTg mice with MOG_35–55/CFA and determined cytokine production of CD4⁺ T cells by intracellular cytokine staining. IL-7RαTg mice had similar frequencies and percentages of IL-17⁺ and IFN-γ⁺ CD4⁺ T cells to WT mice (Figure 2E–F). Consistent with a role for IL-23 in the maintenance and expansion of Th17 cells, IL-23R-deficient mice had fewer IL-17-producing T cells and similar proportions of IFN-γ⁺ cells. Importantly, cytokine production by Th cells in IL-23R^−/−/IL-7RαTg mice phenocopied that of IL-23R deficiency (Figure 2E–F). Although we did not see changes in cytokine production from CD4⁺ T cells isolated from IL-23R^−/− and IL-23R^−/−/IL-7RαTg mice, we tested whether constant IL-7Rα expression on T cells can bypass the requirement for IL-23R in EAE development. To this end, we immunized WT, IL-23R^−/−, IL-23R^+/−/IL-7RαTg, and IL-7RαTg mice for EAE development with MOG_35–55/CFA. Whereas IL-7RαTg mice developed classical signs of EAE to the same extent as WT mice (Figure 2G), IL-23R^−/− and IL-23R^−/−/IL-7RαTg mice did not develop any disease (Figure 2G). As a result, T cells failed to infiltrate the CNS of IL-23R^−/− and IL-23R^−/−/IL-7RαTg mice (data not shown). Constitutive expression of IL-7Rα on T cells did not impact the balance of Th1, Th17, and IFN-γ-producing Th17 (Th1/17) cells in the CNS of mice that developed EAE (Figure 2I). However, we found an increase in GM-CSF-producing CD4⁺ T cells in the CNS of IL-7RTg mice afflicted with EAE (Figure 2H). Thus, providing constitutive expression of IL-7Rα on IL-23R^−/− T cells does not rescue disease susceptibility or significantly impact the percentage of IL-17⁺ CD4⁺ T cells, showing that sustained IL-7R expression does not compensate for the absence of IL-23/IL-23R signaling in the generation of pathogenic Th17 cells.

IL-7 differentially affects the differentiation of CD4 T cells in vitro

While the IL-7/IL-7R axis cannot rescue the pathogenic T cell program driven by IL-23, it is possible that it works in concert with or sequentially after IL-23. To address this possibility, we investigated the effect of IL-7 on T helper cell differentiation of Th1 and Th17 cells. We activated WT naive CD4⁺ T cells with anti-CD3 and irradiated antigen presenting cells (APCs) under neutral (Th0), Th1, or Th17 conditions in the presence or absence of IL-7 and further analyzed the frequencies of cytokine-producing T cells in each condition by intracellular cytokine staining. We found that IL-7 significantly induced IFN-γ under neutral and Th1 conditions (Figure 3A). Because GM-CSF is expressed by encephalitogenic Th1 and Th17 cells (9, 10), we analyzed its expression by in vitro polarized cells in response to IL-7. We found that addition of IL-7 to the Th0, Th1, and Th17 cell cultures increased the frequency of GM-CSF⁺ cells (Figure 3A–B). In contrast, IL-7 did not promote but rather impaired Th17 cell differentiation in vitro (Figure 3A–B). Together these data demonstrate the capacity of IL-7 to selectively expand Th1 cells, while inhibiting Th17 cells, and to drive GM-CSF production.

Figure 3. IL-7 enhances Th1 but inhibits Th17 differentiation in vitro.

(A–B) Sorted naïve (CD44^loCD62L^hiCD25) CD4⁺ T cells were stimulated with antigen-presenting cells (APCs) and αCD3 (Th0), plus IL-12 (Th1), or IL-6 and TGF-β (Th17) in the presence or absence of IL-7. Five days after stimulation, cells were stimulated with PMA/ionomycin and intracellular cytokine staining was performed for IL-17, IFN-γ, and GM-CSF (A) on live CD4⁺ T cells. (B) Summary of the mean percentage of IFN-γ-, GM-CSF-, IL-17-expressing CD4⁺ T cells is shown relative to the condition with medium alone. The percentages of cytokine-producing T cells are represented as mean ± SEM in each differentiation condition in the presence or absence of IL-7. Data are representative of at least 4 independent experiments. Statistics were performed with Student’s t test (^*p < 0.05, ^**p < 0.01, ns, not significant).

IL-7/M25 complexes promote Th1 differentiation after immunization with MOG_35–55/CFA

Although IL-7RαTg provided sustained expression of IL-7Rα on T cells, we considered the possibility that IL-7 availability in vivo might be scarce. Therefore, to test the importance of IL-7 on T cell expansion in vivo, and to ensure that a constant supply of IL-7 was available, we generated and administered IL-7 complexes (IL-7/M25 complexes: IL-7c) to WT mice. Treatment with IL-7c increases the potency of IL-7 50- to 100-fold, compared to IL-7 alone, and boosts lymphocyte numbers, particularly T cells, 2- to 3-fold (28). As a result, this strategy enabled us to test the effects of IL-7 on CD4⁺ T cell expansion and survival in vivo. We treated ROR-γt-reporter mice (27) with IL-7c or PBS at days 0, 2, and 4, and harvested cells from the spleen at day 6. Total numbers of CD4⁺ T cells were elevated 2 to 2.5 times in IL-7c treated mice compared to PBS treated mice (Figure 4A). We examined how IL-7 affects the pool of Foxp3⁺ T cells upon treatment with IL-7c. IL-7c mildly decreased the frequency of Foxp3⁺ CD4⁺ T cells but due to the increase in total T cell numbers, the absolute number of these cells was significantly increased compared to PBS-treated groups (Figure 4B), an effect that may be reflective of the low levels of expression by Foxp3⁺ cells. These results are in agreement with the reported effects of IL-7 on Foxp3⁺ T cells (29).

Figure 4. Expansion of IFN-γ⁺ T cells by IL-7c in vivo after immunization with MOG_35–55/CFA.

(A–D) ROR-γt-GFP reporter mice were treated with IL-7/M25 complexes (IL-7c) or PBS three times at 2-day intervals. (A) On day 6, cells from the spleen were quantified for total CD4⁺ T cells. The frequencies and numbers of Foxp3⁺, ROR-γt⁺ Th17, and CXCR3⁺ Th1 cells were determined by intranuclear and surface staining (GFP and CXCR3) respectively on CD4⁺ T cells from the spleen. (B) Summary of frequency (top) and number (bottom) of Foxp3-, ROR-γt-, CXCR3-expressing CD4⁺ T cells. (C–E) WT and IL-23R^−/− mice were immunized with MOG_35–55/CFA and treated with (IL-7c) or PBS on days 1, 3, and 5 after immunization. (C) Eight days after immunization, CD4⁺ T cells from the draining lymph nodes were counted. (F) CD4⁺ T cells were stimulated with PMA/ionomycin for intracellular cytokine staining to assess the frequencies and numbers of Th1 and Th17 cells in each strain (D, representative dot plot and E, summary). (F) Proliferation of Th17 (top) and Th1 cells (bottom) from immunized PBS- and IL-7c-treated WT mice was compared by Ki67 staining in IL-17⁺ and IFN-γ⁺ CD4⁺ T cells respectively. The graphs represent cumulative data of 2–5 mice per group, from at least 2 independent experiments (mean ±SEM). Statistics were performed with Student’s t test (^*p < 0.05, ^**p < 0.01, ns, not significant). (G) Clinical scores of WT and IL-23R^−/− mice immunized with MOG_35–55/CFA and treated with PBS or IL-7c at days 1, 3, and 5 after immunization. Results are shown as mean ± SEM over time (n ≥ 6 mice per group). Data from 2 combined experiments are shown.

Because Th17 cells have been shown to display a large degree of plasticity in vivo by expressing IFN-γ, we investigated the presence of Th17 cells via the expression of their hallmark transcription factor ROR-γt. We analyzed the frequency and absolute number of ROR-γt⁺ Th17 and CXCR3⁺ Th1 cells 6 days after the first injection of IL-7c. Flow cytometric analysis of T cells from the spleen of treated mice revealed a reduced frequency of ROR-γt-expressing CD4⁺ T cells in IL-7c-treated mice, while no change in the absolute number of ROR-γt⁺ T cells was observed between both groups (Figure 4B). However, we found that IL-7c boosted the frequency and number of CXCR3⁺ Th1 cells compared to PBS-treated mice (Figure 4B). Thus, IL-7 enhanced IFN-γ production and the number of Th1 cells but did not expand Th17 or Treg cells in vivo, supporting our in vitro data.

Next, we assess the effects of IL-7 complexes in the generation and expansion of Th1 and Th17 cells in wild type control and IL-23R-deficient mice immunized with MOG35–55 and treated with IL-7c as described above. Total numbers of CD4⁺ T cells from the LNs increased 2.5-fold in IL-7c treated WT mice compared to PBS treated mice (Figure 4C). In accordance with data from the ROR-γt-GFP reporter mice (Figure 4A–B), IL-7c treatment significantly enhanced the absolute number of Th1 cells in WT and IL-23R^−/− mice compared to PBS treatment (Figure 4D–E). However, we did not observe a change in the number and frequency of Th17 cells generated after in vivo treatment with IL-7c upon immunization (Figure 4D–E). The generation of IFN-γ⁺IL-17⁺ T cells remained unchanged between PBS- and IL-7C–treated B6 and IL-23R^−/− mice (Figure 4D). These results, together with the results observed in IL-7RαTg mice (Figure 2), demonstrate that increased IL-7/IL-7R signaling through administration of IL-7c does not rescue Th17 differentiation but acts to promote Th1 differentiation, reflective of what we see in vitro.

Together our data point to a role for IL-7 in promoting the differentiation of Th1 cells (Figure 3A–B and Figure 4D) and increasing the numbers of Th1 cells (Figure 4B and 4E). However, it is unclear whether IL-7 also played a role in the proliferation of Th1 cells. To determine whether this was the case, we analyzed Ki67 expression in Th1 and Th17 cells from PBS- or IL-7c-treated mice. Interestingly, Th17 cells proliferated more rapidly than Th1 cells in immunized mice that received PBS and their proliferation was not affected by IL-7c (Figure 4F). The increased proliferation of Th17 cells, compared to Th1 cells that we observed, is in accordance with previous reports (14). In contrast, Th1 cells proliferated to a much larger extent in the presence of IL-7c (Figure 4F). Collectively, these results demonstrate that IL-7c selectively promotes the proliferation of Th1 but not Th17 cells.

To test how increased levels of IL-7 by exogenous administration affects EAE development, we immunized WT and IL-23R-deficient mice with MOG_35–55 to induce disease and treated them with IL-7c. We observed a slight increase, though not statistically significant, in the severity of the disease of IL-7c-treated compared to PBS-treated WT mice (Figure 4G). This was associated with an increase number of IFN-γ⁺ CD4⁺ T cells generated in the spleen of WT mice treated with IL-7c (data not shown). On the other hand, IL-23R-deficient mice, whether or not treated with IL-7c, were resistant to the development of EAE (Figure 4G). These results show that IL-7 complexes selectively expand Th1 cells but not Th17 cells in the periphery. As a result, IL-7c treatment, similarly to IL-7Rα overexpression, is incapable of restoring disease susceptibility in IL-23R-deficient mice.

IL-7 promotes the conversion of Th17 cells to a Th1 phenotype

CD4⁺ T cells infiltrating the target organ in various autoimmune diseases are phenotypically very different from CD4⁺ T cells present in peripheral immune tissues because they become plastic and modulate their cytokine production. Therefore, we next examined whether CNS infiltrating CD4⁺ T cells during EAE could have a specific IL-7Rα expression profile and IL-7 responsiveness compared to CD4⁺ T cells from peripheral immune tissues. We analyzed the expression of IL-7 in the CNS of naive mice and mice with EAE. IL-7 mRNA was very highly expressed in the CNS of both naive mice and mice with EAE signs (Figure 5A), suggesting that it could play an important role in modulating the balance between IFN-γ⁺ and IL-17⁺ T cells.

Figure 5. Role of IL-7-IL-7R signaling in Th17 plasticity.

(A) IL-7 mRNA expression in the spleen (Spl) and CNS of naive and mice with EAE at the peak of the disease, was measured by real time PCR. (B–D) CNS-infiltrating Th1, Th1/17, and Th17 cells were detected by Thy1.1 and GFP surface expression in triple reporter (Foxp3-RFP/IFN-γ-Thy1.1/IL-17-GFP) mice with EAE. (C) IL-7Rα expression on the different effector CD4⁺ T cells gated in (B). (D) Summary of IL-7Rα MFI on Th1, Th1/17, and Th17 cells. (E) Naïve CD4⁺ T cells were differentiated into Th17 cells for the primary stimulation (1°). Seven days later the cells were restimulated with APCs and anti-CD3 in the absence or presence of IL-7 (2°). Five days after each stimulation, the cells were stained intracellularly for IFN-γ and IL-17 after PMA/ionomycin stimulation. Results are from at least 2 independent experiments of n≥2 mice/group.

To determine how the substantial presence of IL-7 in the CNS correlates with IL-7R positivity on Th1, Th17, and Th1/17 cells, we immunized the Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1 triple reporter mice for EAE and analyzed the cytokine expression by CD4⁺ T cells in the CNS at the peak of the disease. Consistent with peripheral T cells (Figure 1A–K), a hierarchy of IL-7Rα expression was apparent, with IL-7Rα being more expressed on Th17 cells, followed by Th1/17 cells, and then Th1 cells (Figure 5B–D). Similar results were found when the CNS was separated into the brain and spinal cord or when the CNS was examined at the onset of the disease (data not shown). Because low IL-7Rα levels indicate a recent IL-7-mediated signaling event, this finding suggests that Th17 cells that maintain their phenotype (IL-17 production) do not respond to IL-7, while those that respond downregulate the IL-7Rα and express IFN-γ.

To address this hypothesis, we differentiated naïve T cells into Th17 cells and chronically stimulated Th17 cells in the presence of IL-7. Whereas restimulation alone did not impact Th17 cells, the presence of IL-7 induced IFN-γ production by Th17 cells (Figure 5E). These data not only confirm the inherent plastic nature of Th17 cells but they also suggest that restimulation of Th17 cells in the presence of IL-7 induces Th1/17 or ex-Th17 cells. Thus, high levels of IL-7 in the CNS of mice, during EAE, induce the production of IFN-γ from Th17 cells and drive the development of Th1/17 and Th1 cells.

Discussion

Th1 and Th17 cells play a central pathogenic role in both EAE and MS (1, 3). IL-23 is essential for the generation of pathogenic CD4⁺ T cells and for the induction of EAE (5, 6, 30). On the other hand, IL-7 is required for lymphopoiesis and in many aspects of naïve and memory T cell survival and maintenance in the periphery (31). A connection between IL-7 and IL-23-induced pathogenic Th17 cells has emerged based on previous studies showing that IL-7 promoted Th17 expansion and that IL-23 induced the expression of IL-7Rα on Th17 cells (5, 23). In this report, we directly tested the hypothesis that IL-7/IL-7R signaling could promote Th17 expansion and pathogenic activity independently of IL-23. We found that constitutive expression of IL-7Rα on T cells does not compensate for the lack of IL-23R expression, that IL-7 does not impact Th differentiation in the absence of IL-23R, and that IL-23 does not modulate IL-7Rα expression. Moreover, we determined that IL-7 enhanced the differentiation and expansion of Th1 cells, and induced the plasticity of Th17 cells by converting them into IFN-γ-producing Th17 cells.

IL-7 receptor is rapidly downregulated on T cells after ligation and/or activation via the T cell receptor but is subsequently re-expressed, particularly after the cells have differentiated into an effector/memory phenotype (17). Because memory T cells require IL-7, upregulation of this receptor is necessary for their survival (32). In IL-23R-deficient mice, Th17 cells are generated early after immunization, but they fail to expand, rendering mice resistant to the development of EAE (5, 6, 30). Based on these observations and a study showing that IL-7 promotes Th17 cell expansion (23), it was therefore reasonable to speculate that IL-23R-deficient T cells failed to upregulate IL-7Rα receptor, making them less sensitive to IL-7 and less likely to survive (5). In this report, we determined that IL-7Rα expression is more highly expressed on Th17 cells and on IL-23R⁺ T cells compared to Th1 cells. These results may explain why low expression of IL-7Rα on T cells could be detected in IL-23R-deficient mice, which failed to generate stable Th17 cells (5). By providing IL-7Rα expression on IL-23R-deficient T cells, we investigated whether IL-7/IL-7R signaling could promote Th17 expansion and stability, thereby inducing EAE. Against our expectations, IL-7Rα expression on T cells from IL-23R^−/− mice did not restore EAE susceptibility and did not promote the expansion of Th17 cells. We further determined that IL-7Rα expression was not modulated by the IL-23/IL-23R signaling axis (data not shown). To conclusively rule out the possibility that IL-23R-mediated IL-7/IL-7R signaling is essential for Th17 differentiation, we provided exogenous IL-7 and found that Th17 cell differentiation was not impacted after administration of IL-7, but IFN-γ expression was enhanced. Hence, we investigated the reasons behind these observations and our data help reconcile several published reports exploring the role of IL-7 during EAE development.

Several lines of evidence point to a disease-stimulating effect of IL-7: complete deficiency of IL-7Rα or selective deficiency of IL-7Rα on peripheral hematopoietic cells prevent or reduce clinical signs of EAE (22, 33). Furthermore, treatment of WT mice with an anti-IL-7Rα antibody limits EAE severity (21, 33). While IL-7 has been implicated in affecting both Th1 and Th17 cells, it remains unclear whether these two pathogenic T cell populations are equally affected by IL-7 in vivo. Indeed, the effects of IL-7 on T cell differentiation have been controversial. Liu et al. had reported that IL-7 did not affect Th1 cells and instead served a crucial role in the survival and expansion of Th17 cells, since blockade of the IL-7/IL-7R signaling pathway with an anti-IL-7Rα antibody altered the Th17 cell response (23). These results were questioned in light of another study, which reported that IL-7 promoted Th1 differentiation in human and murine naïve CD4⁺ T cells, while it had no effect on IL-17 production in vitro (21). The in vitro data obtained on Th differentiation contrasted with in vivo data presented in the same report and showed that treatment of mice afflicted with EAE with an anti-IL-7Rα antibody limited both Th1 and Th17 responses (21). However, the anti-IL-7Rα blocking antibody has a broad depleting effect on the T cell compartment, making it difficult to investigate how IL-7 affects T helper cell differentiation (21). In our study, we took the approach of providing sustained IL-7/IL-7R signaling via the use of IL-7/M25 complexes. One major obstacle for addressing the effects of IL-7 in vivo is the presence of an IL-7 sink, whereby IL-7 is rapidly consumed after exogenous delivery, making IL-7 activity very brief. By complexing IL-7 to the monoclonal anti-IL-7 clone M25 antibody, the cytokine is more potent and stable (34). Our data show a preferential expansion and proliferation of Th1 cells over Th17 cells after IL-7/IL-7R complex injection in MOG_35–55-immunized mice. In addition, in vitro differentiation of Th1 cells, but not, Th17 cells is enhanced in the presence of IL-7 (Figure 3A). Because Th1 cells are more prone to apoptosis than Th17 cells (35, 36), and because IL-7 can sustain Bcl-2 expression and promote cell survival (37), it is also possible that IL-7 prevents apoptosis in Th1 cells and therefore promote their preferential expansion (Figure 4F).

Furthermore, consistent with the idea that IL-7 modulates the differentiation of effector T cells, we found that in all Th-polarizing conditions tested IL-7 promotes GM-CSF production, a cytokine that has been implicated in the pathogenicity of T cells during EAE (9, 10). We also found an increase in GM-CSF production in the CNS of mice that constitutively expressed the IL-7R. These results were confirmed by an independent study published while this manuscript was in preparation (38). In addition, we determined that IL-7 induced IFN-γ production by Th17 cells (Figure 5E). Our lab (12) and others (14) have highlighted the pathogenicity of Th17 cells in the CNS of mice during EAE. We reported that IL-23 is a critical factor that leads to the generation of these cells (12). In this study, we show that while IL-7 does not promote IL-17 production or the expansion of Th17 cells, it likely facilitates the plasticity of Th17 cells and their production of IFN-γ (Figure 5E). Most Th1 cells in the CNS of mice afflicted with EAE have been recognized as ex-Th17 cells (Th17 cells that have extinguished IL-17 expression and express IFN-γ) (14). Our data suggest that although IL-23 plays an essential role in the emergence of IL-17⁺ IFN-γ⁺ (12), IL-7 could also participate in this process and sustain the plasticity of these cells, since IL-7 is highly expressed in the CNS during neuroinflammation (Figure 5A). This could also be relevant in MS since IL-7 expression is increased in brain lesions (39, 40). Together, our results suggest that IL-7, which is abundantly expressed in the CNS, could participate in EAE pathogenesis by converting Th17 cells into Th1 cells and expanding Th1 cells. These data build upon previous findings linking IL-7/IL-7R signaling to the promotion of Th1 cells and higher levels of serum IL-7 with a therapeutic benefit of IFN-β (21, 41). Thus, our results further provide evidence that IL-7Rα may serve as a target in treatment strategies for Th1-mediated multiple sclerosis.

Single-nucleotide polymorphisms (SNPs) in the IL-7/IL-7R pathway are associated with an increased risk of multiple sclerosis (MS). In particular, the skewed levels of IL-7 and IL-7Rα expression, resulting from specific SNPs, are believed to influence responsiveness of IL-7Rα⁺ cells. Because the level of IL-7Rα expression has traditionally been proposed to correlate with the level of responsiveness of the cells, it was therefore of particular interest to analyze the expression of IL-7Rα on different subsets of T helper cells in lymphoid organs and CNS during the course of EAE. We detected high expression levels of IL-7Rα on Th17 cells. This enhanced expression of IL-7Rα on IL-17⁺ ROR-γt⁺ Th17 cells was not only observed in the periphery (Figure 1A–J) but also on Th17 cells present in the CNS during EAE (Figure 5B–D), indicating that IL-7Rα, thus, serves as marker of Th17 cells. It was therefore surprising to observe a limited effect of IL-7 on Th17 cells, which are higher for IL-7Rα expression, and a promoting effect of IL-7 on Th1 cells, which express lower levels of the receptor. Our data support the novel notion that IL-7Rα is enriched on Th17 cells because they are refractory to IL-7 signaling and therefore IL-7-mediated downregulation of the IL-7Rα. Indeed, because IL-7 complexes do not expand Th17 cells in vivo and Th17 cells maintain a high level of expression of the IL-7Rα, we propose that most Th17 cells do not readily respond to IL-7, or if they do, they lose IL-17 expression and acquire IFN-γ expression, whereas Th1 cells respond to IL-7/IL-7R signaling and downregulate IL-7Rα. In summary, we provide a model in which IL-7R is highly enriched on Th17 cells during EAE and is not involved in the protection of IL-23R-deficient mice to EAE development. As Th17 cells are converted to IFN-γ-producing cells, they downregulate the receptor. Thus, this study provides a novel mechanism by which Th17 cells can be converted to Th1 cells in response to chronic IL-7 stimulation in the CNS during EAE.