IL-17–producing CD4+ T (Th17) cells, along with IFN-γ–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 current study, we showed that Th17 cells from mice with EAE express high levels of IL-7Rα compared with Th1 cells. Using mice that constitutively express IL-7Rα 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-γ 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-γ–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.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the 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+ 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 that is essential for the development of EAE, because both IL-23p19 KO and IL-23 receptor-deficient (IL-23R KO) mice are resistant to the development of EAE (57). 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, although 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 (1214). 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 naive and memory T cell populations in the periphery (17). IL-7/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 IL-7Rα mRNA splicing and results in increased levels of soluble IL-7Ra, 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. 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.

In this study, 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 Ab (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, because large amounts of IL-7 are produced in the CNS of mice and humans with autoimmunity.

C57BL/6 (B6) mice were purchased from The Jackson Laboratory and bred in the Benaroya Research Institute animal facility. Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1 triple reporter mice have been described previously (12). These mice were generated by breeding IL-17A-GFP (Biocytogen), IFN-γ knock-in Thy1.1 (23), and Foxp3-RFP (24) reporter mice together. IL-23R knock-in and IL-23R homozygous knock-in (IL-23R−/−) GFP reporter mice were previously described (6). IL-7RαTg and ROR-γt-GFP reporter mice were previously described (25, 26). 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.

For T cell differentiation, naive CD4+CD62LhighCD44lowCD25 T cells were isolated by FACS sorting (FACSAria; BD Biosciences) and cultured with irradiated (4000 rad) CD4-depleted spleen cells from wild-type (WT) mice and anti-CD3 (2.5 μg/ml, clone 145-2C11) for 5 d in complete RPMI 1640. For Th17 differentiation, 2.5 ng/ml human rTGF-β (R&D Systems), 30 ng/ml murine rIL (rmIL)-6 (PeproTech), 10 μg/ml anti-IFN-γ, and 10 μg/ml anti–IL-4 (National Institutes of Health/National Cancer Institute Biological Resources Bank Preclinical Repository) were used. A total of 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).

For intracellular cytokine-staining analysis, cells were incubated 5 h in complete RPMI 1640 containing 50 ng/ml PMA, 1 μg/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 Ab (clone 2.4G2; Bio X Cell, West Lebanon, NH). Viability of the cells was assessed by staining with fixable dye eFluor780 (eBioscience). Cells were then stained with anti-CD4 Ab 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 Abs in permeabilization buffer. Cells were acquired on LSRII (BD Biosciences), and data were analyzed with FlowJo software. The following Abs were used in our experiments: Abs to CD4 (clone L3T4) conjugated to PerCP-Cy5.5 or Alexa700, IL-7Rα (CD127) (clone A7R34) conjugated to PerCP-Cy5.5, and IFN-γ (clone XMG1.2) conjugated to PE-Cy7 or PE were purchased from eBioscience, and Ab to IL-17 (clone TC11-18H10.1) conjugated to allophycocyanin was purchased from BioLegend.

EAE was induced by s.c. immunization of mice into the flanks with an emulsion of myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (100 μg) emulsified in CFA supplemented with 4 mg/ml Mycobacterium tuberculosis extract H37Ra (Difco). In addition, the animals received 200 ng 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; and 5, moribund.

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 2.5 mg/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.

Mouse rIL-7 was purchased from eBioscience (San Diego, CA). M25 anti–IL-7 Ab was purchased from Bio X Cell. IL-7/M25 complexes (IL-7c) were generated, as previously described (27). Briefly, each mouse received complexes generated from a 30-min incubation at 37°C of 1.5 μg IL-7 with 15 μg mAb M25. WT mice immunized with MOG35–55 in CFA received three injections of IL-7c every other day starting at day 1 after immunization. ROR-γt-GFP mice were sacrificed 6 d after the first injection.

Statistical analyses were conducted with GraphPad Prism software. The p values were calculated with Student paired t test. The p values < 0.05 were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars denote ± SEM, as indicated.

Th17 and Th1 cells can induce EAE with different pathological phenotypes and distinct clinical signs (3, 4). Because high levels of IL-7R have been proposed to render cells more responsive to IL-7–mediated signaling (17), we examined IL-7R (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 (Fig. 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, whereas Foxp3+ T cells showed diminished expression of the receptor (Fig. 1B, 1C, and data not shown). Next, we examined the patterns of IL-7Rα expression on effector Th1 and Th17 cells after immunization with MOG35–55 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, whereas the expression of IL-7Rα on Th1 cells was significantly lower (Fig. 1D–F). The enrichment of IL-7Rα on Th17 cells was also observed at the peak of the disease (Fig. 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. (AI) Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1 triple reporter mice were immunized for EAE with MOG35–55 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 mean fluorescent intensity (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 [days 9–12 (D–F)] and peak [days 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 with isotype control (shaded gray histogram). (F and I) Summary of IL-7Rα expression quantified by 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 d after immunization with MOG35–55/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). Data are representative of at least two independent experiments with at least three mice each experiment. Statistics were performed with Student t test (*p < 0.05, **p < 0.01).

FIGURE 1.

Th17 cells express high levels of IL-7Rα. (AI) Foxp3-RFP/IL-17A-GFP/IFN-γ-Thy1.1 triple reporter mice were immunized for EAE with MOG35–55 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 mean fluorescent intensity (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 [days 9–12 (D–F)] and peak [days 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 with isotype control (shaded gray histogram). (F and I) Summary of IL-7Rα expression quantified by 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 d after immunization with MOG35–55/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). Data are representative of at least two independent experiments with at least three mice each experiment. Statistics were performed with Student t test (*p < 0.05, **p < 0.01).

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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 (Fig. 1J, 1K), 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 with 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.

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 MOG35–55 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) (25). Because 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 MOG35–55/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 (Fig. 2A, 2B). Furthermore, IL-7/IL-7R signaling was highly functional, because cells from IL-23R−/−/IL-7RαTg mice were also capable of phosphorylating STAT5 at higher levels in response to IL-7 compared with cells from IL-23R−/− mice (Fig. 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. (AC) IL-7RαTg, IL-23R−/−/IL-7RαTg, and IL-23R−/− mice were immunized with MOG35–55 in CFA. Eight to 10 d 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 d 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 two independent experiments with n ≥ 2 mice in each experiment. (EH) 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 d 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 three 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. Statistics were performed using Student t test (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).

FIGURE 2.

IL-7– and IL-23–mediated signaling events act independently on T cells. (AC) IL-7RαTg, IL-23R−/−/IL-7RαTg, and IL-23R−/− mice were immunized with MOG35–55 in CFA. Eight to 10 d 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 d 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 two independent experiments with n ≥ 2 mice in each experiment. (EH) 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 d 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 three 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. Statistics were performed using Student t test (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).

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To determine how constitutive expression of IL-7Rα affects the proliferation and expansion of CD4+ T cells, we sorted naive (CD62LhighCD44low) 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 with IL-23R−/−/IL-7RαTg cells (Fig. 2D), showing that increased expression and signaling through the IL-7R induce 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 MOG35–55/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 (Fig. 2E, 2F). 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 (Fig. 2E, 2F). 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 MOG35–55/CFA. Whereas IL-7RαTg mice developed classical signs of EAE to the same extent as WT mice (Fig. 2G), IL-23R−/− and IL-23R−/−/IL-7RαTg mice did not develop any disease (Fig. 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 (Fig. 2H). However, we found an increase in GM-CSF–producing CD4+ T cells in the CNS of IL-7RTg mice afflicted with EAE (Fig. 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.

Although 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 Th cell differentiation of Th1 and Th17 cells. We activated WT naive CD4+ T cells with anti-CD3 and irradiated 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 (Fig. 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 (Fig. 3). In contrast, IL-7 did not promote, but rather impaired, Th17 cell differentiation in vitro (Fig. 3). 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 and B) Sorted naive (CD44lowCD62LhighCD25) CD4+ T cells were stimulated with 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 four independent experiments. Statistics were performed with Student t test (*p < 0.05, **p < 0.01).

FIGURE 3.

IL-7 enhances Th1, but inhibits Th17 differentiation in vitro. (A and B) Sorted naive (CD44lowCD62LhighCD25) CD4+ T cells were stimulated with 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 four independent experiments. Statistics were performed with Student t test (*p < 0.05, **p < 0.01).

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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 with IL-7 alone, and boosts lymphocyte numbers, particularly T cells, 2- to 3-fold (27). 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 (26) 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 2–2.5 times in IL-7c–treated mice compared with PBS-treated mice (Fig. 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 with PBS-treated groups (Fig. 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 (28).

FIGURE 4.

Expansion of IFN-γ+ T cells by IL-7c in vivo after immunization with MOG35–55/CFA. (AD) ROR-γt-GFP reporter mice were treated with IL-7/M25 complexes (IL-7c) or PBS three times at 2-d 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 MOG35–55/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 two to five mice per group, from at least two independent experiments (mean ± SEM). (G) Clinical scores of WT and IL-23R−/− mice immunized with MOG35–55/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 two combined experiments are shown. Statistics were performed with Student t test (*p < 0.05, **p < 0.01, ns, not significant).

FIGURE 4.

Expansion of IFN-γ+ T cells by IL-7c in vivo after immunization with MOG35–55/CFA. (AD) ROR-γt-GFP reporter mice were treated with IL-7/M25 complexes (IL-7c) or PBS three times at 2-d 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 MOG35–55/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 two to five mice per group, from at least two independent experiments (mean ± SEM). (G) Clinical scores of WT and IL-23R−/− mice immunized with MOG35–55/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 two combined experiments are shown. Statistics were performed with Student t test (*p < 0.05, **p < 0.01, ns, not significant).

Close modal

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 d 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, whereas no change in the absolute number of ROR-γt+ T cells was observed between both groups (Fig. 4B). However, we found that IL-7c boosted the frequency and number of CXCR3+ Th1 cells compared with PBS-treated mice (Fig. 4B). Thus, IL-7 enhanced IFN-γ production and the number of Th1 cells, but did not expand Th17 or regulatory T 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 WT 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 with PBS-treated mice (Fig. 4C). In accordance with data from the ROR-γt-GFP reporter mice (Fig. 4A, 4B), IL-7c treatment significantly enhanced the absolute number of Th1 cells in WT and IL-23R−/− mice compared with PBS treatment (Fig. 4D, 4E). 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 (Fig. 4D, 4E). The generation of IFN-γ+IL-17+ T cells remained unchanged between PBS- and IL-7C–treated B6 and IL-23R−/− mice (Fig. 4D). These results, together with the results observed in IL-7RαTg mice (Fig. 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 (Figs. 3, 4D) and increasing the numbers of Th1 cells (Fig. 4B, 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 (Fig. 4F). The increased proliferation of Th17 cells, compared with 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 (Fig. 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 affect EAE development, we immunized WT and IL-23R–deficient mice with MOG35–55 to induce disease and treated them with IL-7c. We observed a slight increase, although not statistically significant, in the severity of the disease of IL-7c–treated compared with PBS-treated WT mice (Fig. 4G). This was associated with an increased number of IFN-γ+ CD4+ T cells generated in the spleen of WT mice treated with IL-7c (data not shown). In contrast, IL-23R–deficient mice, whether treated with IL-7c, were resistant to the development of EAE (Fig. 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.

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 with 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 (Fig. 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. (BD) 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 mean fluorescent intensity on Th1, Th1/17, and Th17 cells. (E) Naive 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 two independent experiments of n ≥ 2 mice/group. Statistics were performed using Student t test (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).

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. (BD) 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 mean fluorescent intensity on Th1, Th1/17, and Th17 cells. (E) Naive 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 two independent experiments of n ≥ 2 mice/group. Statistics were performed using Student t test (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).

Close modal

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 (Fig. 1), 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 (Fig. 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, whereas those that respond downregulate the IL-7Rα and express IFN-γ.

To address this hypothesis, we differentiated naive 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 (Fig. 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.

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, 29). In contrast, IL-7 is required for lymphopoiesis and in many aspects of naive and memory T cell survival and maintenance in the periphery (30). 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, 22). 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-7R is rapidly downregulated on T cells after ligation and/or activation via the TCR, 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 (31). 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, 29). Based on these observations and a study showing that IL-7 promotes Th17 cell expansion (22), it was therefore reasonable to speculate that IL-23R–deficient T cells failed to upregulate IL-7Rα, 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 with 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 prevents or reduces clinical signs of EAE (22, 32). Furthermore, treatment of WT mice with an anti–IL-7Rα Ab limits EAE severity (21, 32). Although 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. (22) had reported that IL-7 did not affect Th1 cells and instead served a crucial role in the survival and expansion of Th17 cells, because blockade of the IL-7/IL-7R signaling pathway with an anti–IL-7Rα Ab altered the Th17 cell response. These results were questioned in light of another study, which reported that IL-7 promoted Th1 differentiation in human and murine naive CD4+ T cells, whereas 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 Ab limited both Th1 and Th17 responses (21). However, the anti–IL-7Rα blocking Ab has a broad depleting effect on the T cell compartment, making it difficult to investigate how IL-7 affects Th 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 anti–IL-7 clone M25 mAb, the cytokine is more potent and stable (33). Our data show a preferential expansion and proliferation of Th1 cells over Th17 cells after IL-7/IL-7R complex injection in MOG35–55-immunized mice. In addition, in vitro differentiation of Th1 cells, but not Th17 cells, is enhanced in the presence of IL-7 (Fig. 3A). Because Th1 cells are more prone to apoptosis than Th17 cells (34, 35), and because IL-7 can sustain Bcl-2 expression and promote cell survival (36), it is also possible that IL-7 prevents apoptosis in Th1 cells and therefore promotes their preferential expansion (Fig. 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 (37). In addition, we determined that IL-7 induced IFN-γ production by Th17 cells (Fig. 5E). Our laboratory (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, whereas IL-7 does not promote IL-17 production or the expansion of Th17 cells, it most likely facilitates the plasticity of Th17 cells and their production of IFN-γ (Fig. 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, because IL-7 is highly expressed in the CNS during neuroinflammation (Fig. 5A). This could also be relevant in MS because IL-7 expression is increased in brain lesions (38, 39). 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, 40). Thus, our results further provide evidence that IL-7Rα may serve as a target in treatment strategies for Th1-mediated multiple sclerosis.

SNPs in the IL-7/IL-7R pathway are associated with an increased risk of 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 Th 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 (Fig. 1A–J), but also on Th17 cells present in the CNS during EAE (Fig. 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.

We thank Drs. Pamela Fink and Steve Ziegler for providing the IL-7RαTg and ROR-γt-GFP mice, respectively.

This work was supported by National Institutes of Health Grants R01 NS081687, R01 NS059996, and R21 NS077116 (to E.B.).

Abbreviations used in this article:

EAE

experimental autoimmune encephalomyelitis

LN

lymph node

MOG

myelin oligodendrocyte glycoprotein

MS

multiple sclerosis

rmIL

murine rIL

SNP

single-nucleotide polymorphism

WT

wild-type.

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The authors have no financial conflicts of interest.