A Genome-Wide Analysis Identifies a Notch–RBP-Jκ–IL-7Rα Axis That Controls IL-17–Producing γδ T Cell Homeostasis in Mice

Masataka Nakamura, Kensuke Shibata, Shinya Hatano, Tetsuya Sato, Yasuyuki Ohkawa, Hisakata Yamada, Koichi Ikuta and Yasunobu Yoshikai
J Immunol January 1, 2015, 194 (1) 243-251; DOI: https://doi.org/10.4049/jimmunol.1401619

Abstract

Notch signaling is an important regulator for the development and function of both αβ and γδ T cells, whereas roles of Notch signaling in T cell maintenance remain unclear. We reported previously that the Notch–Hes1 pathway was involved in the intrathymic development of naturally occurring IL-17–producing (IL-17+) γδ T cells. To gain insight into additional roles for the Notch axis in the homeostasis of γδ T cells, we performed a genome-wide analysis of Notch target genes and identified the novel promoter site of IL-7Rα driven by the Notch–RBP-Jκ pathway. Constitutive Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro, whereas conditional deletion of RBP-Jκ abrogated IL-7Rα expression, but not Hes1 expression, by γδ T cells and selectively reduced the pool size of IL-7Rαhigh IL-17+ γδ T cells in the periphery. In the absence of IL-7Rα–mediated signaling, IL-17+ γδ T cells were barely maintained in adult mice. Addition of exogenous IL-7 in vitro selectively expanded IL-17+ γδ T cells. Thus, our results revealed a novel role for the Notch–RBP-Jκ–IL-7Rα axis that is independent of Hes1 for homeostasis of IL-17+ γδ T cells.

Introduction

Notch signaling plays essential roles in T cell development, such as lineage commitment and maturation, in the thymus (1, 2). In mammals, four Notch receptors (Notch1, Notch2, Notch3, and Notch4) are bound to five ligands: Δ-like (Dll)1, Dll3, Dll4, Jagged1, and Jagged2. The thymus transmits important signals to generate mature T cells through Dll4, which is expressed on thymic epithelial cells (1). It was reported that Dll and Jagged expression on APCs is associated with Th1 and Th2 cell differentiation, respectively (3). Dll4 on dendritic cells induced the differentiation of Th17 cells through the Th17-specific transcription factor RORγt (4). Furthermore, Notch3 signaling induced by Jagged2 was reported to promote the expansion of regulatory T cells (5, 6). Thus, Notch signaling plays critical roles in αβ T cell differentiation and function in the periphery. Unlike conventional αβ T cells, which are exported from the thymus as naive cells and acquire effector functions upon Ag encounter in the periphery, some murine γδ T cells, so-called naturally occurring γδ T cells, harbor innate functions to produce inflammatory cytokines, such as IFN-γ and IL-17, within thymus. They are disproportionately distributed and persist in mucosal epithelia, such as skin, intestine, uterus, and lung, as tissue-associated, long-lived self-renewing cells that are important players in mucosal immunity in infections, wound healing, autoimmune disorders, and tumors (7–11). Interaction between Notch1 and Dll4 is essential for γδ T cell development from T cell precursors, and Notch1 expression is maintained on γδ T cells (12–14). We reported previously that the Notch1–Hes1 pathway is involved in the development of IL-17+ γδ T cells in the fetal thymus (15). Thus, it would be interesting to determine the additional roles of the Notch axis in the development, functional differentiation, and maintenance of the “naturally occurring” γδ T cells in the thymus and periphery.

IL-7Rα is expressed on naive αβ T cells. IL-7Rα is downregulated after stimulation through αβTCR and is not expressed on terminally differentiated effector αβ T cells, whereas IL-7Rα is re-expressed on effector/memory and central memory αβ T cells for their homeostasis mediated by IL-7 signaling, leading to the establishment of immunological memory, which is the hallmark of adaptive immunity (16–18). IL-7Rα transmits two important signals in T cells: for survival of naive and memory αβ T cells, which is mediated primarily by upregulation of antiapoptotic molecules (e.g., BCL-2 and MCL-1) (19–22) and for their proliferation via the PI3K–AKT pathway (23). Recently, it was shown in human T cell precursors that RBP-Jκ, a downstream molecule of Notch signaling, directly bound in the upstream and enhancer regions of the IL-7Rα gene and induced IL-7Rα expression (24, 25). IL-7Rα expression was highly enriched in IL-17+ γδ T cells in neonates and adult mice (26, 27). However, it was unclear whether Notch signaling controlled the homeostasis of γδ T cells via IL-7Rα expression.

In the current study, we demonstrated using genome-wide chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis, together with a mutational reporter assay, that the Notch–RBP-Jκ pathway directly induced IL-7Rα expression on γδ T cells through a novel promoter located −5.4 kb upstream of the transcription start site (TSS) of IL-7Rα. Constitutive Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro, whereas the lack of RBP-Jκ abrogated IL-7Rα expression, but not Hes1 expression, and selectively reduced IL-17+ γδ T cells. Addition of exogenous IL-7 in vitro selectively expanded IL-17+ γδ T cells. Thus, our results revealed a novel role for the Notch–RBP-Jκ–IL-7Rα axis, independent of Hes1, for homeostasis of IL-17+ γδ T cells.

Materials and Methods

Mice

Lox-stop-lox-RosaNICD-ires-GFP, Mx-1-Cre mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Rag-1-Cre mice were provided by T. Rabbitts (Leeds Institute of Molecular Medicine, Leeds, U.K.) courtesy of K. Akashi (Kyushu University). IL-7Rα−/− mice were kindly provided by K.I. RBP-Jκflox/flox mice (RBRC01071) were provided by RIKEN BRC through the National Bio-Resource Project of the Japanese Ministry of Education, Culture, Sports, Science and Technology. γTg mice were generated by injection of functionally rearranged Vγ6Jγ1Cγ1, originated from a hybridoma clone (15) with the IRES-EGFP cassette derived from the pIRES2-EGFP vector (Clontech). The transgene was driven by a chicken β-actin promoter from the pCAGGS vector (kindly provided by J. Miyazaki, Osaka University, Osaka, Japan). Fetal Mx-1-Cre Lox-stop-lox-RosaNICD-ires-GFP mice were obtained from timed matings; the day of finding a vaginal plug was designated as day 0 of embryonic development. For conditional ablation by inducing Cre recombinase driven by an IFN-inducible Mx-1 promoter, 250 μg polyinosinic-polycytidylic acid (poly-IC; P1530; Sigma) was injected i.p. twice into pregnant mice on embryonic days 18 and 19. Mice were maintained at our institute in specific pathogen–free conditions. This study was approved by the Committee on the Ethics of Animal Experiments of the Faculty of Medicine, Kyushu University. Experiments were performed under the Guidelines for Animal Experiments.

Flow cytometric analysis

Peritoneal exudate cells were collected by an extensive wash of the peritoneal cavity with HBSS. Single-cell suspensions were prepared from the adult thymus and spleen using pairs of slide glasses. Cells from fetal and neonatal thymi were dissociated using a 1-mm syringe. Lung and ear skin were minced into 1–2-mm pieces and incubated with 1 mg/ml collagenase (Life Technologies, Grand Island, NY) and 20 mg/ml DNase (DN25; Sigma) in RPMI 1640 containing 10% FCS for 90 min at 37°C with vigorous vortexing every 15 min. Mononuclear cells were further purified with 40 and 70% Percoll by centrifugation at 600 × g for 20 min. FITC-conjugated anti-TCRδ (eBioGL3), anti-CD45.2 (104), and anti-MHC class II (M5/114.15.2) mAb; PE-conjugated anti-TCRδ (eBioGL3), anti–IL-7Rα (A7R34), anti-CD4 (L3T4), and anti–MHC class II (M5/114.15.2) mAb; and allophycocyanin-conjugated anti-TCRδ (eBioGL3) mAb, PerCP–eFluor 710–conjugated anti-TCRδ (eBioGL3) mAb, and anti–MHC class II (M5/114.15.2) mAb were purchased from eBioscience (San Diego, CA). FITC-conjugated anti–MHC class II (M5/114.15.2) mAb, allophycocyanin-conjugated anti-CD3ε (145-2C11) mAb, Alexa Fluor 647–conjugated anti–BCL-2 (BCL/10C4) mAb, PE-Cy7–conjugated anti-TCRδ (GL3) mAb, and PerCP-Cy5.5–conjugated anti-MHC class II (M5/114.15.2) mAb were purchased from BioLegend (San Diego, CA). FITC-conjugated anti–MHC class II (2G9) mAb, PE-conjugated anti–IFN-γ (XMG1.2) mAb, allophycocyanin-conjugated anti-CD8α (53.6.7) mAb, Alexa Fluor 647–conjugated anti–IL-17A (TC11-18H10) mAb, and V500-conjugated anti–MHC class II (M5/114.15.2) mAb were purchased from BD Biosciences (San Jose, CA). Lineage+ cells were stained using Lineage cell detection mixture (biotinylated Abs of CD5 [53-7.3], B220 [RA3-6B2], CD11b [M1/70], Gr-1 [RB6-8C5], 7-4, Ter-119, CD3ε [145-2C11], and DX5; Miltenyi Biotec). rIL-7 was purchased from PeproTech (Rocky Hill, NJ). We added propidium iodide (1 μg/ml) to the cell suspension just before running on the flow cytometer to detect and exclude dead cells for analyzing surface staining. To detect cytokine production, cells were stimulated with 25 ng/ml PMA (P8139) and 1 μg/ml ionomycin (I0634; both from Sigma) for 4 h at 37°C; 10 μg/ml Brefeldin A (B7651; Sigma) was added for the last 3 h of incubation. After the cells were stained with various mAbs for 20 min at 4°C, intracellular staining was performed according to the manufacturer’s instructions (BD Biosciences). A total of 100 μl BD Cytofix/Cytoperm solution (BD Biosciences) was added to the cell suspension with mild mixing and kept at 4°C for 20 min. Fixed cells were washed twice with 250 μl BD Perm/Wash solution (BD Biosciences) and then stained intracellularly for 30 min at 4°C. For in vivo proliferation analysis by 5-ethynyl-2′-deoxyuridine (EdU), neonatal mice were injected i.p. with 20 μl EdU (10 mM) in PBS. After 9 h, neonatal thymocytes were stained with mAb specific for TCRδ and IL-7Rα. EdU staining was performed with a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies), according to the manufacturer’s protocol. Stained cells were run on a FACSCalibur or FACSVerse flow cytometer (BD Biosciences). The data were analyzed using CellQuest (BD Biosciences) or FlowJo (Tree Star, Ashland, OR) software.

In vitro activation of γδ T cells by a Notch ligand, Dll4

During the culture of 1,000 or 10,000 sorted CD117highCD25+ double-negative (DN)2 cells (embryonic day 17) on a layer of TSt-4 thymic stromal cells expressing murine Dll-4 (TSt-4/Dll4) or not expressing murine Dll-4 (TSt-4/no) in 24-well plates for 13 d, γδ T cells were differentiated and then activated. Culture was performed without additional cytokines, and half of the medium was changed once on day 7.

Quantitative RT-PCR

Total RNA from cells sorted using a FACSAria (BD Biosciences) was purified using an RNeasy Micro Kit (QIAGEN). First-strand cDNA synthesis was performed using Superscript II (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Quantitative RT-PCR was performed on an ABI PRISM thermal cycler (Applied Biosystems) using SYBR Premix Ex Taq (RP041A; Takara). The following gene-specific primers were used: Hes1, 5′-ACACCGGACAAACCAAAGAC-3′, 5′-ATGCCGGGAGCTATCTTTCT-3′ and β-actin, 5′-GGAATCCTGTGGCATCCATGAAAC-3′, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. The 2−ΔΔCt equation was used to calculate the relative expression of target genes against that of β-actin.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) was performed by modifying the Upstate Biotechnology protocol, as described previously (28). γδ T cells from thymocytes of C57BL/6 mice (0–3 d old) were purified with a FACSAria II (BD Biosciences) and fixed using 0.5% formaldehyde at room temperature for 5 min. After cross-linking was stopped by the addition of glycine to a final concentration of 0.125 M, cells were washed twice with cold PBS and pelleted by centrifugation. Cells were resuspended in ChIP buffer (10 mM Tris-HCl [pH 8], 200 mM KCl, 1 mM CaCl2, 0.5% Nonidet P-40) containing protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 μg/ml pepstatin A), followed by incubation on ice and brief sonication to obtain cell lysates. DNA was subsequently sheared by micrococcal nuclease to obtain 150-bp DNA fragments. A total of 20 μl Dynabeads (Invitrogen) coated with anti-rabbit IgG was prebound to the rabbit mAb against the intracellular Notch1 (ICN1) domain (D1E11; Cell Signaling) and then incubated with sheared DNA overnight on a rotator at 4°C. Normal rabbit IgG (cat. no. 2729; Cell Signaling) and anti-GFP polyclonal Ab (code no. 598; MEDICAL & BIOLOGICAL LABORATORIES) were used as negative controls. After magnetic separation, pellets were washed three times with ChIP buffer, wash buffer (ChIP buffer containing 500 mM KCl), and TE (pH 8). Immune complexes were eluted from the Dynabeads with elution buffer (50 mM Tris-HCl [pH 8], 10 mM EDTA, 1% SDS). After treatment with proteinase K, immunoprecipitated DNA was purified using a QIAquick PCR purification kit (QIAGEN) and analyzed by ChIP-seq and ChIP-quantitative PCR (qPCR). DNA sequencing of the ChIP library was performed using HiSeq1500 (Illumina, San Diego, CA). Sequence reads for ICN1 and input were aligned with the mouse reference genome (mm10) using Bowtie program (v1.0.0). Peak identification of binding sites of ICN1 was done using MACS program (v1.4.2) (29). Enriched motifs within the peak regions were determined using the MEME program (v4.9.0) (30). The data were deposited in the Gene Expression Omnibus under accession number GSE56756 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56756). ChIP-qPCR was performed using primer sets as follows: −1.8 kb upstream of TSS of IL7Rα, 5′-GATGGGCATCTCTGGGACTA-3′, 5′-TCCATATCCCCATTGCTAGG-3′; −5.4 kb upstream of TSS of IL7Rα, 5′-GCAGAGCAGGTGGTAAGTGA-3′, 5′-TCAGAGCCCTTGGACTGAG-3′; and −26 kb upstream of TSS of IL7Rα, 5′-TGTCCTGCATTTCACAGTGC-3′, 5′-ACATGGTAATCCTGCCCAAG-3′.

Luciferase reporter assay

Luciferase activity was measured with the Dual-Luciferase Reporter Assay Kit (Promega). A mouse T cell line, KKF (kindly provided by K.I.), was cotransfected with MigICN1 (150 ng/well) or MigR1 (150 ng/well) as a control vector together with an IL-7Rα–luc construct containing the wild-type (WT) sequence (CTGGGAA) or the mutated sequence (CGAAACC) of the putative RBP-Jκ binding site (200 ng/well) using Lipofectamine LTX & PLUS Reagent (Life Technologies). pGL4.74 vector (1 ng/well) was used as internal control. Firefly luciferase activity was normalized to Renilla luciferase activity, and fold change was calculated.

Statistics

Statistical significance was calculated by Prism software (GraphPad, San Diego, CA) using the Student t test. Differences with p values < 0.05 were considered statistically significant.

Results

ICN1 binding adjacent to putative Notch targets including IL-7Rα and Hes1

To search for the targets of the Notch pathway, neonatal γδ T cells were subjected to ChIP-seq analysis using the Ab against ICN1. ChIP-seq analysis yielded 5913 ICN1 peaks, of which 65% were located within or near genes (Fig. 1A). The genome-wide analysis also implicated the involvement of Notch signaling in diverse signaling pathways in γδ T cells, because ICN1 binding was observed adjacent to previously unreported, putative Notch targets, including Nr4a1, which is the downstream target of γδTCR signals (26), as well as several known Notch targets, such as Hes1, TCF1, CD25, and BCL11b, in T lineage cells (1) (Fig. 1B).

FIGURE 1.
FIGURE 1.

Notch signaling induces IL-7Rα expression on γδ T cells through RBP-Jκ. (A) Genomic distribution of ICN1 peaks in γδ T cells. Upstream and downstream regions were defined as −10 kb upstream of the TSS and +10 kb downstream of the 3′ end of genes, respectively. (B) ICN1 binding peaks around various genes are underlined in red. Upper numbers (arrows) indicate the distance from the TSS of each gene. Values for the y-axis were calculated by F-sEquation (58). (C) ChIP-qPCR was performed at −5.4, −26, and −1.8 kb upstream of the TSS of the IL-7Rα gene after immunoprecipitation with anti-ICN1 mAb or isotype-matched anti-GFP Ab. Bar graphs show the mean ± SD from triplicate wells. (D) An ICN1-expressing plasmid, MigICN1 (designated as ICN1), together with IL-7Rα–luc constructs containing the WT sequence or the mutated sequence of the putative RBP-Jκ binding site, were transiently cotransfected with KKF cells. Bar graph shows mean (± SD) fold changes of relative luciferase reporter activity in six to eight wells. Data in (C) and (D) are representative of at least two independent experiments. (E) IL-7Rα expression on γδ T cells was analyzed after activation of γδ T cells without (Dll4−) or with (Dll4+) Notch signaling in vitro. (F) After induction of constitutively active Notch signaling (GFP+) in vivo, IL-7Rα expression on γδTCR+ thymocytes was analyzed. (E and F) A representative graph or a zebra plot of IL-7Rα expression is shown after gating on γδTCR+ cells. Bar graph (right panel) shows the mean fluorescence intensity (MFI) ± SD of IL-7Rα expression on γδTCR+ cells in four wells (E) or four mice (F). Data are representative of four (E) and three (F) independent experiments. *p < 0.05, ***p < 0.001, Student t test.

Notably, between −100 kb upstream and +100 kb downstream of the TSS of IL-7Ra, we identified three ICN1-binding peaks: −5.4, −87, and −94 kb upstream. Among these sites, a consensus RBP-Jκ–binding motif (CTGGGAA) was found within the peak at the −5.4 kb upstream site (circled in red) but not at the other sites (Fig. 1B). ChIP-qPCR analysis was performed to further validate the specific binding of ICN1. In addition to the newly identified, putative RBP-Jκ binding site, sites at −1.8 kb [previously reported as the putative binding site for RBP-Jκ (24)] and −26 kb (no binding on the same chromosome) upstream of the TSS of IL-7Ra were tested. Consistent with ChIP-seq data, high ChIP enrichment was only detected in the −5.4 kb upstream region (Fig. 1C). Supporting this result, recent genome-wide epigenetic analysis of CCR6+ IL-17+ γδ T cells showed enrichment of active H3K4me2 marks, but no repressive H3K27me3 marks, in the putative RBP-Jκ binding site (−5.4 kb), suggesting a permissive epigenetic status (31). These results suggest that, in murine γδ T cells, ICN1 is specifically bound at the novel promoter site, −5.4 kb upstream of the TSS of IL-7Rα, possibly through the putative RBP-Jκ binding site. In contrast, we could not find the consensus binding motif of RBP-Jκ (CTGGGAA) within the peaks in the promoter region of Hes1.

Next, to examine the promoter activity, we transiently cotransfected a murine T cell line, KKF, with a vector carrying ICN1 and a luciferase reporter vector containing the newly identified promoter site of IL-7Ra. In the presence of the WT promoter site, luciferase activity significantly increased after activation of Notch signaling. In contrast, introduction of a mutation of the RBP-Jκ binding site abrogated the promoter activity (Fig. 1D). These results suggested that IL-7Rα expression on γδ T cells was directly induced by Notch signaling through the promoter.

Notch signaling directly induces IL-7Rα expression on γδ T cells

To test whether Notch signaling has the potential to induce IL-7Rα expression in mice, γδ T cells were cultured with Notch signaling on fetal thymic stromal cells expressing one of the Notch ligands, Dll4. After culture in the presence of Notch signaling, Notch1 expression on γδ T cells was downregulated (Supplemental Fig. 1A). Transcripts of Hes1, a target gene of Notch signaling (32), were significantly increased by continuous Notch signaling (Supplemental Fig. 1B). As shown in Fig. 1E, continuous Notch signaling via Dll4 significantly augmented IL-7Rα expression on γδ T cells in vitro. IL-17+ γδ T cells were mostly included in IL-7Rαhigh γδ T cells (Supplemental Fig. 1C). To examine this effect in vivo, we generated mice in which constitutively active Notch signaling was induced by poly-IC injection. After injecting poly-IC into pregnant mice, neonatal thymocytes of Mx-1-Cre Lox-stop-lox-RosaNICD-ires-GFP mice were analyzed. IL-7Rα expression on γδ T cells labeled with GFP, which indicated constitutively active expression of Notch signaling, was augmented significantly more than that on GFP− γδ T cells in the thymus (Fig. 1F). Thus, Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro.

RBP-Jκ is required for the maintenance of IL-17+ γδ T cells

Recent studies demonstrated that DNA binding of RBP-Jκ complexed with ICN1 showed canonical Notch signaling status, whereas noncanonical Notch signaling status was that ICN1 was bound to DNA in the absence of RBP-Jκ (2, 33). To ascertain whether RBP-Jκ induces IL-7Rα expression on γδ T cells, we disrupted the RBP-Jκ gene after γδ T cell development by crossing RBP-Jκ flox/flox mice with mice expressing Cre recombinase under control of the Rag-1 promoter. Consistent with a previous study (34), the transition of DN to double-positive (DP) cells was severely impaired in mice harboring a disrupted RBP-Jκ allele (Fig. 2A). However, DN2 and DN3 cells were clearly observed and, thereby, γδ T cell development was detected at a considerable level (Fig. 2A). Hes1 expression on γδ T cells was observed in the absence of RBP-Jκ (Fig. 2B). Strikingly, IL-7Rα expression on neonatal γδ T cells was abrogated by inactivation of RBP-Jκ (Fig. 2C).

FIGURE 2.
FIGURE 2.

Deletion of downstream target of Notch signaling, RBP-Jκ, abrogates IL-7Rα expression on γδ T cells. Neonatal (3-d-old, n = 5) thymocytes from RBP-Jκflox/flox (WT) or Rag1-CreRBP-Jk flox/flox (Deleted) mice were analyzed by flow cytometry. (A) Representative dot plots of DP (upper panels) and γδ T cells (lower panels) after gating on viable cells. Bar graphs show the mean ± SD of the absolute numbers of DP (top) and γδ T cells (bottom). (B) The bar graphs (right panels) show the mean ± SD of relative Hes1 expression normalized to β-actin expression (n = 3). (C) Representative graph of IL-7Rα expression after gating on γδTCR+ cells. Data are mean ± SD of five mice. All data are representative of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test.

We next examined whether RBP-Jκ was required for the maintenance of IL-17+ γδ T cells in adult mice. In RBP-Jκ–deficient γδ T cells, IL-17 producers were decreased ∼50-fold in the peritoneal cavities and thymus and 10-fold in the spleen, lung, and skin, whereas IFN-γ producers were relatively less affected in all tissues (Fig. 3A, 3B). Vγ4+ γδ T cells are a major subset of IL-17+ γδ T cells (9) [Heilig and Tonegawa’s (35) nomenclature for TCRγ genes was used]. Among Vγ4+ γδ T cells, IL-17+ cells were selectively decreased in the absence of RBP-Jκ (Fig. 3C). These results suggested that RBP-Jκ was required for the maintenance of IL-17+ γδ T cells but to a lesser extent in IFN-γ+ γδ T cells.

FIGURE 3.
FIGURE 3.

IL-17+ γδ T cells are selectively decreased in the absence of RBP-Jκ. IFN-γ+ and IL-17+ total (A) or Vγ4+ γδ T cells (C) in the thymus, spleen, peritoneal cavities, lung, and skin from RBP-Jkflox/flox (WT) or Rag-1-CreRBP-Jkflox/flox (Deleted) mice (4 wk-old) were analyzed after stimulation with PMA and ionomycin. Representative zebra plots are shown after gating on γδTCR+ cells. Numbers in the quadrants indicate the percentage of cells positive for IFN-γ or IL-17. Bar graphs show mean ± SD of the ratio of IL-17+/IFN-γ+ γδTCR+ cells. (B) Bar graphs show the mean ± SD of the absolute number of IFN-γ+ or IL-17+ γδ T cells (n = 3). The data are representative of three independent experiments. **p < 0.01, ***p < 0.001, unpaired Student t test.

IL-7Rα–mediated signaling is required for homeostasis of IL-17+ γδ T cells

Recent studies revealed that IL-17+ γδ T cells express high IL-7Rα (26, 27). Consistently, we found high IL-7Rα expression on IL-17+ γδ T cells after birth (Supplemental Fig. 2). We next questioned why IL-17+ γδ T cells maintained a relatively higher expression level of IL-7Rα than did IFN-γ+ γδ T cells. To this end, functions of γδ T cells generated in the absence of IL-7Rα–mediated signaling should be analyzed. However, because IL-7Rα–mediated signaling is indispensable for expression of the functional γ-chain, γδ T cells are completely absent in IL-7Rα−/− mice (Fig. 4A) (36, 37). To circumvent this effect, we used the previously established system that γδ T cell development in IL-7Rα−/− mice was rescued by introduction of a rearranged γ-chain (38). IL-7Rα−/− mice with the γ transgene (γTgIL-7Rα−/− mice) allowed γδ T cells to develop in the thymus (Fig. 4A). However, the number of γδ T cells containing IFN-γ and IL-17 producers was decreased ∼100-fold in the absence of IL-7Rα (Fig. 4B), suggesting insufficient γ gene expression, as described previously (38). Alternatively, because IL-7Rα–mediated signaling was important for the initial expansion of T cell progenitors (22), the incomplete restoration of γδ T cells could be explained, in part, by the fact that the γ transgene failed to proliferate IL-7Rα–deficient γδ T cell progenitors in DN2 and DN3 cells, whose populations were ∼100–500-fold smaller in size compared with IL-7Rα–sufficient γδ T cell progenitors (Supplemental Fig. 3A, 3B). In the periphery, γδ T cells were found only in the lung but were scarcely detected in the spleen and peritoneal cavity (Fig. 4A, Supplemental Fig. 3C). Similar to the case for the thymus, the number of pulmonary γδ T cells was significantly decreased in the absence of IL-7Rα (Fig. 4B). Thus, consistent with previous reports (38, 39), our data also suggested that IL-7Rα–mediated signaling was responsible for both the initial expansion and maintenance of γδ T cells.

FIGURE 4.
FIGURE 4.

IL-17+ γδ T cells are virtually absent in γTgIL-7Rα−/− mice. Cells of neonatal thymi (3 d old) and adult thymi and adult lung (4 wk old) from γTgIL-7Rα+/−, IL-7Rα−/−, and γTgIL-7Rα−/− mice were analyzed. (A) Representative dot plots are shown after gating on viable CD3ε+ I-A/I-E− cells. Expression of γ transgene was identified by EGFP expression. The number in each quadrant indicates the percentage of TCRδ+ cells within the gate. (B) Bar graphs show the mean ± SD of the absolute numbers of γδ T cells, IFN-γ+ γδ T cells, and IL-17+ γδ T cells. (C) IL-17+ and IFN-γ+ γδ T cells were analyzed after stimulation with PMA and ionomycin. Representative dot plots are shown after gating on EGFP+ TCRδ+ cells. Numbers in the quadrants indicate the percentage of IL-17+ or IFN-γ+ cells within γδTCR+ cells. Bar graphs show the mean ± SD of ratio of IL-17+/IFN-γ+ γδTCR+ cells. (D) Percentage of IFN-γ+ or IL-17+ γδ T cells in each γTgIL-7Rα−/− mouse relative to the mean of those in γTgIL-7Rα+/− mice. The data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test.

Next, we analyzed the functional properties of the partly restored γδ T cells in γTgIL-7Rα−/− mice. The pool size was restricted more severely in IL-17+ γδ T cells than in IFN-γ+ γδ T cells; thereby, effector γδ T cell populations were skewed to IFN-γ producers in both the thymus and the lung of γTgIL-7Rα−/− mice compared with those in γTgIL-7Rα+/− mice (Fig. 4C, 4D). These results suggested that the importance of IL-7Rα–mediated signaling for the maintenance of γδ T cells was more evident in IL-17+ γδ T cells than in IFN-γ+ γδ T cells.

IL-7 transmits essential signals for the proliferation and/or survival of T lymphoid cells (19, 20). We first examined the in vivo proliferation of neonatal γδ T cells after injection with EdU, which was incorporated in proliferating cells. EdU incorporation was significantly higher in IL-7Rαhigh γδ T cells than in IL-7Rαlow γδ T cells (Fig. 5A). To ascertain whether IL-7Rα−mediated signaling was truly functional and inducible for the proliferation of IL-17+ γδ T cells, neonatal γδ T cells were stimulated with 10 ng/ml of IL-7 in vitro. From days 1–3, both γδ T cell subsets increased slightly in number (Fig. 5C–E). However, after day 4, IL-17+ γδ T cells expanded to a greater degree than did IFN-γ+ γδ T cells. The PI3K–AKT pathway is a known downstream network of IL-7Rα–mediated signaling and is involved in promoting cellular metabolism of T cells (23). Upon IL-7 stimulation, persistent phosphorylation of AKT was specifically observed in CD27low γδ T cells that contained IL-7Rαhigh cells (Supplemental Fig. 4A). Thus, consistent with the previous study (27), IL-7 is an important factor in the regulation of the pool size of IL-17+ γδ T cells.

FIGURE 5.
FIGURE 5.

IL-7 induces expansion of IL-17+ γδ T cells more vigorously than that of IFN-γ+ γδ T cells in vitro. (A) After neonates (3 d old, n = 3) were injected i.p. with EdU for 9 h, the percentage of EdU+ cells in IL-7Rαhigh or IL-7Rαlow γδ T cells in the thymus was analyzed. Data are mean percentage ± SD of EdU+ IL-7Rαhigh or IL-7Rαlow γδ T cells. (B) BCL-2 expression in neonatal (3 d old) γδ T cells was analyzed. Representative graph after gating on IL-7Rαhigh (black line) or IL-7Rαlow (filled gray) γδ T cells. The dotted line indicates isotype-matched control mAb. (C–E) After 2 × 106 neonatal thymocytes (1 d old) were incubated with 10 ng/ml of IL-7 in vitro, IFN-γ+ and IL-17+ γδ T cells were analyzed at each time point. (C) Representative dot plots are shown after gating on γδTCR+ cells. Numbers in the quadrants indicate the percentage of IFN-γ+ or IL-17+ cells within the gate. Absolute numbers (D) or fold changes (E) of IFN-γ+ or IL-17+ γδ T cells. Data are mean ± SD of five individual wells at each time point. Data are representative of three (A and B) and four (C–E) independent experiments. *p < 0.05, ***p < 0.001, Student t test.

In clear contrast to IL-7–induced expansion, ex vivo analysis showed that the expression of BCL-2, an antiapoptotic molecule responsible for IL-7–mediated cell survival (19, 20), was even higher in IL-7Rαlow γδ T cells than in IL-7Rαhigh γδ T cells (Fig. 5B). The expression level of active caspase 3, a well-known indicator of early apoptosis, was comparable between IFN-γ+ and IL-17+ γδ T cells (Supplemental Fig. 4B). Therefore, the predominance of IL-17+ γδ T cells over IFN-γ+ γδ T cells in the neonatal thymus was not due to increased apoptosis of IFN-γ+ γδ T cells. After culture for 2 d without IL-7 or with a low dose of IL-7 (0.1 ng/ml), the number of γδ T cells decreased dramatically (Supplemental Fig. 4C, 4D). Thus, IL-7 had the potential to provide survival signals to both IFN-γ+ and IL-17+ γδ T cell subsets.

Discussion

Notch signaling directly regulated IL-7Rα expression on the IL-17+ γδ T cell subset, and IL-7Rα–mediated signaling was essential for the maintenance of this subset at least partly via induction of homeostatic proliferation. Thus, our findings highlight the importance of Notch signaling in IL-17+ γδ T cell homeostasis.

In this study, we demonstrated that the Notch–RBP-Jκ axis directly regulates IL-7Rα expression on IL-17+ γδ T cells. We previously found that the Notch–Hes1 axis was required for the development of IL-17+ γδ T cells in the fetal thymus (15). Similar to the case for RBP-Jκ–deficient DN cells (40), conditional deletion of RBP-Jκ mediated by Rag-1 promoter-driven Cre recombinase did not eliminate Hes1 expression on γδ T cells, suggesting that Notch signaling in the thymus regulated Hes1 expression independently of RBP-Jκ. The different signaling pathways explain why Notch signaling can be involved in both the intrathymic development and maintenance of IL-17+ γδ T cells. In contrast to Rag-1-Cre RBP-Jk f/f mice, the number of γδ T cells in the thymus of Lck-Cre RBP-Jk f/f mice was not decreased (40). Lck-GFP mice showed that the Lck protein was expressed in late DN2 (also known as DN2b) cells but not early DN2 (also known as DN2a) cells (41, 42). We recently found that γδ T cells were generated directly from both DN2a and DN2b stages (14). These results suggested that Cre recombinase driven by proximal Lck promoter might not delete RBP-Jκ in DN2a-derived γδ T cells, whereas Cre recombinase driven by the Rag-1 promoter almost completely inactivated RBP-Jκ in γδ T cells. The leaky DN2a-derived γδ T cells in Lck-Cre RBP-Jκf/f mice might contribute to maintain the γδ T cell pool to the same level as in WT mice. It is also interesting to know whether the Notch–RBP-Jκ axis is involved in the maintenance of IL-17+ αβ T cells via IL-7Rα. Th17 cells were shown to be long-lived cells resembling stem cell–like memory cells expressing IL-7Rα (43). In memory Th17 cells, IL-23R–mediated signaling was documented as being important for maintaining and/or re-expressing IL-7Rα (44), although the involvement of Notch–RBP-Jκ signaling has not been reported. Recently, innate lymphoid cells (ILCs) sharing innate functions with γδ T cells were identified both in human and mice. RBP-Jκ deletion selectively decreased IL-22–producing NKp46+IL-7Rα+ ILCs in the lamina propria but not in the Peyer’s patches (45). Epigenetic analysis revealed that IL-7Rα expression in ILC2 cells was directly regulated by TCF1, which was shown to be a direct target of the Notch–RBP-Jκ pathway in T cells (46, 47). In fact, we also found ICN1 binding on the promoter region (−31 kb upstream from the TSS) of TCF1 in γδ T cells (Fig. 1B). However, the development of IL-17+ γδ T cells increased in TCF1-deficient mice (48), suggesting the different expression mechanisms of IL-7Rα between ILCs and γδ T cells.

Recent studies using IL-7 reporter mice revealed that, similar to the predominance of IL-17+ γδ T cells in the thymus of young mice (8), the number of IL-7–expressing thymic epithelial cells was higher in neonatal and 1–2-wk-old mice than in adult mice (49). In addition to the thymus, IL-7–expressing cells were found in peripheral organs, such as abdomen, lung, skin, and intestine, where IL-17+ γδ T cells were localized (50). Such peripheral and ontogenic heterogeneity of IL-7–expressing cells might explain the distribution of IL-17+ γδ T cells, which receive important signals for their maintenance through IL-7Rα.

BCL-2 is a key factor involved in IL-7Rα–mediated survival in αβ T cells (19, 20). Interestingly, we found that IL-7Rαlow γδ T cells that contained IFN-γ producers expressed BCL-2 at even slightly higher levels than did IL-7Rαhigh γδ T cells. We previously reported that IFN-γ+ γδ T cells were virtually absent in mice deficient for IL-15 (8), which also induced BCL-2–mediated cell survival in γδTCR+ intraepithelial cells (51). Therefore, BCL-2 induced by IL-15 may contribute to survival of IFN-γ+ γδ T cells.

We also noticed that, in the absence of RBP-Jκ in γδ T cells, their functions were shifted to IFN-γ producers. This can be explained by the previous study using STAT5a-deficient mice, in which Th1 cell differentiation was increased (52). STAT5a, a major downstream target of IL-7Rα–mediated signaling, induced SOCS3, which inhibited Th1 cell polarization (53). Similarly, the loss of RBP-Jκ might impair the IL-7Rα–STAT5–SOCS3 signal and, thereby, increase the proportion of IFN-γ+ γδ T cells. However, in the absence of IL-7Rα–mediated signaling, IFN-γ–producing γδ T cells decreased in number, suggesting that unknown signals, rather than the inhibitory IL-7Rα–STAT5–SOCS3 signal, also might be involved.

We found the generation of IFN-γ+ IL-17+ γδ T cells after culture with IL-7 in vitro. A previous in vitro study showed that IFN-γ+ IL-17+ γδ T cells were generated from CD27− γδ T cells in which IFN-γ− IL-17+ γδ T cells were highly enriched (7). In an oral infection mouse model with Listeria monocytogenes, IFN-γ+ IL-17+ γδ T cells were detected postinfection (54). A recent genome-wide epigenetic analysis revealed that IL-17+ γδ T cells had the ability to become IFN-γ producers (31). These results suggested the possibility that IFN-γ+ IL-17+ γδ T cells were derived from IL-17+ γδ T cells via an unknown mechanism induced by IL-7. A fate-mapping study may be useful to answer this question (55).

We and another group reported that Vγ6+ γδ T cells were one of the major subsets of IL-17+ γδ T cells in the periphery, including the lung (9, 56). In adult mice, the introduction of the TCR Vγ6 gene to IL-7Rα−/− mice rescued γδ T cell development in the lung, but those cells were absent in the peritoneal cavity and spleen of γTgIL-7Rα−/− mice. Given that involvement of the δ-chain in skin Vγ5Vδ1+ γδ T cells was reported to be a factor that regulates tissue-specific migration (57), different γδTCRs might be expressed by pairing different δ-chains with a transgenic Vγ6 chain between IL-7Rα−sufficient and IL-7Rα–deficient γδ T cells in γTg mice. In fact, the introduction of a rearranged Vγ4 gene into IL-7Rα−/− mice restored γδ T cell development in the spleen, as well as in the thymus (38). The above results suggest the possibility that γδTCR-mediated signaling restricts peripheral localization.

In conclusion, the present work identified a novel role for Notch signaling in the maintenance of long-lived, self-renewing IL-17+ γδ T cells (9, 11), which play important roles in the first line of host defense against various pathogens. Furthermore, because a recent report showed that IL-7 also facilitates expansion of pathogenic IL-17+ γδ T cells in psoriasis (27), it also will be interesting to test whether the Notch1–RBP-Jκ–IL-7Rα axis can be a therapeutic target for treatment of the disease.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Mihoko Okubo, Miki Kijima, and Akiko Yano for secretarial assistance and Takako Ichinose and Shizue Taniichi for technical advice.

Footnotes

  • This work was supported by the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, which is a project launched and commissioned by the Japanese Ministry of Education, Culture, Sports, Science and Technology; a Grant-in-Aid from the Japan Society for Promotion of Science; and by grants from the Japanese Ministry of Education, Science and Culture (to Y.Y. and K.S.). K.S. received support from the Kaibara Morikazu Medical Science Promotion Foundation and the Takeda Science Foundation.

  • The sequences presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56756) under accession number GSE56756.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ChIP
    chromatin immunoprecipitation
    ChIP-seq
    chromatin immunoprecipitation followed by high-throughput DNA sequencing
    Dll
    Δ-like
    DN
    double negative
    DP
    double positive
    EdU
    5-ethynyl-2′-deoxyuridine
    ICN1
    intracellular Notch1
    ILC
    innate lymphoid cell
    poly-IC
    polyinosinic-polycytidylic acid
    qPCR
    quantitative PCR
    TSS
    transcription start site
    WT
    wild-type.

  • Received June 26, 2014.
  • Accepted November 2, 2014.

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