IL-10 Receptor Signaling Is Essential for TR1 Cell Function In Vivo

Leonie Brockmann, Nicola Gagliani, Babett Steglich, Anastasios D. Giannou, Jan Kempski, Penelope Pelczar, Maria Geffken, Bechara Mfarrej, Francis Huber, Johannes Herkel, Yisong Y. Wan, Enric Esplugues, Manuela Battaglia, Christian F. Krebs, Richard A. Flavell and Samuel Huber
J Immunol February 1, 2017, 198 (3) 1130-1141; DOI: https://doi.org/10.4049/jimmunol.1601045

Abstract

IL-10 is essential to maintain intestinal homeostasis. CD4+ T regulatory type 1 (TR1) cells produce large amounts of this cytokine and are therefore currently being examined in clinical trials as T cell therapy in patients with inflammatory bowel disease. However, factors and molecular signals sustaining TR1 cell regulatory activity still need to be identified to optimize the efficiency and ensure the safety of these trials. We investigated the role of IL-10 signaling in mature TR1 cells in vivo. Double IL-10eGFP Foxp3mRFP reporter mice and transgenic mice with impairment in IL-10 receptor signaling were used to test the activity of TR1 cells in a murine inflammatory bowel disease model, a model that resembles the trials performed in humans. The molecular signaling was elucidated in vitro. Finally, we used human TR1 cells, currently employed for cell therapy, to confirm our results. We found that murine TR1 cells expressed functional IL-10Rα. TR1 cells with impaired IL-10 receptor signaling lost their regulatory activity in vivo. TR1 cells required IL-10 receptor signaling to activate p38 MAPK, thereby sustaining IL-10 production, which ultimately mediated their suppressive activity. Finally, we confirmed these data using human TR1 cells. In conclusion, TR1 cell regulatory activity is dependent on IL-10 receptor signaling. These data suggest that to optimize TR1 cell–based therapy, IL-10 receptor expression has to be taken into consideration.

This article is featured in In This Issue, p.977

Introduction

Interleukin-10 plays an essential role in controlling inflammation. IL-10–deficient mice spontaneously develop inflammatory disease, and patients with mutations in either IL10 or IL10R suffer from severe early onset colitis (1, 2). Thus, IL-10 plays a fundamental role in maintaining intestinal immune homeostasis (3, 4). Resetting the physiological concentration of IL-10 in the intestine is considered the Holy Grail for all therapies, which aim to cure inflammatory-mediated diseases, especially in the intestine.

CD4+ T regulatory type 1 (TR1) cells secrete high levels of IL-10 and are known to play a major role in maintaining immune tolerance through their strong immune regulatory activity (5–10). Owing to the strong immune regulatory potential of TR1 cells, which has been proven in several preclinical mouse models (6–10), they are a major target of new approaches in the field of T cell–based therapy (11–13).

TR1 cells are characterized by coexpression of CD49b and LAG-3, high secretion of IL-10, and the lack of Foxp3 expression (14, 15). The expression of granzyme B and TGF-β1 also contributes to the suppressive capacity of TR1 cells, and the expression of CTLA-4 allows a cell–cell contact-dependent suppression of T cells by TR1 cells (16, 17). Chronic stimulation of CD4+ Th cells with IL-10 is sufficient to induce in vitro functional mouse and human TR1 cells. However, IL-10 is dispensable to induce mouse TR1 cells in vivo (18). Indeed, IL-27 was found to promote the differentiation of TR1 cells in vitro and in vivo. Consistent with this observation, mice deficient in IL-27R (WSX1−/−) have a strong reduction of TR1 cells upon infection (19). IL-27 induces the differentiation of mouse TR1-like cells by activating STAT1 and STAT3, which promotes LAG-3 expression and IL-10 production through the transcriptional factors Egr-2 and Blimp1 (encoded by Prdm1) (20). Other factors, such as Ahr, c-Maf, Nfil3, and retinoic acid–related orphan receptor-α, play a key role in the differentiation of TR1 cells (20–22). Although several transcriptional factors have been shown to drive the differentiation of TR1 cells, whether this requires one master regulator factor or a finely tuned network comprising several factors still remains unknown.

On the basis of global transcriptomic analysis, it has been shown that TR1 cells are distinct, not unlike how the other Th cell subsets are from each other (23, 24). Recently, their peculiar metabolic activity and their sp. act. during the night/day cycle have further highlighted the distinction of TR1 cells from TH17 and Foxp3+ regulatory T cells (Tregs) (25). As a consequence of all of these differences, the protective and anti-inflammatory functions of TR1 cells are what ultimately distinguishes them from other subsets such as TH1, TH2, and TH17 cells (17, 26, 27).

Several preclinical studies and mouse models of immune-mediated disease have sustained the translation of TR1 cell–based therapy into the clinic (8, 9, 28, 29). Human TR1 cells are being tested in clinical trials to treat autoimmune diseases, such as inflammatory bowel disease (IBD), or to limit donor-versus-host reactivity (graft-versus-host disease) after hematopoietic stem cell transplantation (11, 13). Treatment of Crohn disease patients with Ag-specific TR1 cells showed good tolerability and high potential. Also, a proof-of-concept trial with hematopoietic stem cell transplantation patients showed a positive outcome for patients who were treated with IL-10–anergized T cells containing TR1 cells (11, 13, 30). A new clinical trial for kidney transplanted patients is planned (31).

Although TR1 cell–based therapies are ongoing, it is unknown which molecular signal maintains TR1 cell regulatory activity, a basic biological aspect that is fundamental to design successful therapies. IL-10, the signature cytokine produced by TR1 cells, has been shown to regulate Foxp3+ Tregs by maintaining their regulatory function (32). However, it is still unclear whether TR1 cells also express IL-10R and whether IL-10 signaling is responsible for maintaining the functional stability of these cells.

The best studied IL-10 signaling pathway is the activation of JAK1 and STAT3 (33). PI3Ks as well as p38 MAPK pathways can also act downstream of IL-10 by binding to the activated IL-10 receptor complex (34, 35). Whether mature TR1 cells respond to IL-10 and, if so, through which signaling pathway are currently unknown.

The aim of our study was to analyze the role of IL-10 signaling for the anti-inflammatory activity of mature mouse and human TR1 cells. Our data show that IL-10 plays a key role in sustaining the function of murine and human TR1 cells. Mouse TR1 cells with impaired IL-10 signaling are unstable and lose their regulatory activity in vivo in a T cell transfer–mediated IBD model (13). Finally, in vitro–induced human TR1 cells, which can potentially be employed in a cell therapy approach, also require IL-10 receptor signaling to maintain IL-10 production.

Materials and Methods

Mice

C57BL/6, C57BL/6 Rag1−/−, and C57BL/6 Rag1−/− CD45.1+ mice were obtained from the The Jackson Laboratory. CD4-dominant negative (DN) IL-10R transgenic mice and Foxp3RFP, IL-17AeGFP, and IL-10eGFP reporter mice are described elsewhere (26, 36–39). Age- and sex-matched littermates between 8 and 16 wk of age were used.

Flow cytometry

Anti-CD4, anti-CD62L, anti-CD44, anti-CD45.1, anti-CD45.2, anti-CD45RB, anti–TCR-β, anti–IL-10Rα (clone 1B1.3a, PE) and isotype control (rat IgG1,κ, PE) were purchased from BioLegend. Anti-STAT3 (pY705) and anti–pp38 MAPK were purchased from BD Biosciences. To identify dead cells, 7-aminoactinomycin D (BioLegend) staining was performed.

Anti-human anti-CD4, anti-CD45RA, and anti-CD49b (clone P1E6-C5) were purchased from BioLegend. Anti–LAG-3 was purchased from eBioscience (clone 3DS223H). The staining for LAG-3 and CD49b was performed at 37°C for 30 min.

For intracellular pSTAT3 and pp38 MAPK staining, cells were fixed with Phosflow Lyse/Fix Buffer (BD Biosciences) for 10 min at 37°C and permeabilized with Perm Buffer III (BD Biosciences) for 30 min on ice. The cells were stained for pSTAT3 or pp38 MAPK and extracellular markers for 1 h at room temperature before they were acquired on an LSR II flow cytometer (BD Biosciences).

In vitro TR1 cell and TH17 cell differentiation

CD4+ T cells were enriched from splenocytes of IL-10eGFP Foxp3RFP double reporter mice with CD4 microbeads using MACS (Miltenyi Biotec). For naive T cell enrichment, CD44+ and CD25+ T cells were depleted using biotinylated Abs and streptavidin beads (Miltenyi Biotec). For TR1 cell differentiation, naive T cells were cultured for 5 d at a density of 106 cells/ml with plate-bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (2 μg/ml) in medium (Click’s medium supplemented with 10% FCS, l-glutamine, penicillin, streptomycin, and 2-ME) under TR1-inducing conditions (0.5 ng/ml TGF-β1, 30 ng/ml IL-27). IL-10 (eGFP) and Foxp3 (mRFP) expression were determined by flow cytometry. For TH17 cell differentiation, naive T cells were cultured for 5 d at a density of 106 cells/ml with soluble anti-CD3 (3 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence of irradiated APCs (ratio 1:4) in medium (Click’s medium supplemented with 10% FCS, l-glutamine, penicillin, streptomycin, and 2-ME) under TH17 polarizing conditions (0.5 ng/ml TGF-β1, 10 ng/ml IL-6, 20 ng/ml IL-23, 10 ng/ml IL-1β). IL-17A (eGFP) expression was determined by flow cytometry.

In vitro suppression assay

Responder T cells were isolated from C57BL/6 mice and labeled with 5 μM violet dye. The cells were activated in the presence of irradiated APCs and 1.5 μg/ml anti-CD3 Ab and cultured either alone or in the presence of IL-10RαWT or IL-10RαImpaired TR1 cells at a 1:2 (TR1/responder) ratio. After 72 h the proliferation of the responder T cells was measured via flow cytometry.

In vitro kinase inhibition

SB203580, PD98059, JNK inhibitor II, or STAT3 inhibitor VI in DMSO was added to the culture medium in the indicated concentrations every 24 h (Calbiochem, Darmstadt, Germany). DMSO was added to control cultures at equivalent concentrations.

In vitro IL-10 receptor blocking

In vitro–differentiated wild-type (WT) TR1 cells were restimulated (CD3/CD28 Abs) in the presence of 50 μg/ml IL-10R Ab (clone 1B1) or isotype control Ab (rat IgG1,κ).

In vivo T cell stimulation and intestinal lymphocyte isolation

Mice were injected with anti-CD3 (clone 2C11, 15 μg) i.p. twice every other day and sacrificed 4 or 48 h after the second injection. As controls, mice were injected with isotype-matched Ab or PBS. We collected intraepithelial lymphocytes after the dissection of the Peyer’s patches by incubating the small intestine in the presence of 5 mM EDTA at 37°C for 30 min. Lamina propria lymphocytes were collected by digesting gut tissue with collagenase IV (100 U; Sigma-Aldrich) at 37°C for 45 min. The cells were further separated with a Percoll gradient (GE Healthcare).

Adoptive transfer models

Splenocytes were collected from 8- to 12-wk-old IL-17AeGFP reporter mice (CD45.1/2), and CD4+ T cells were enriched by a MACS system (Miltenyi Biotec). The CD4+ T cells were further sorted to collect CD45RBhiFoxp3RFP− cells using a FACSAria II. CD45RBhi cells (1.5 × 105) were i.p. injected either alone or together with in vitro–differentiated 1.5 × 105 WT or IL-10RImpaired TR1 cells into Rag1−/− mice. For generating highly pathogenic TH17 cells, 4 × 105 CD45RBhi cells were injected i.p. into Rag1−/− mice (CD45.1). The mice were weighed once a week to monitor IBD development. After establishment of colitis as determined by endoscopy, mice were sacrificed and lymphocytes were isolated from the colon and mesenteric lymph nodes. The cells were further FACS sorted to purify IL-17AeGFP+ T cells [(e)TH17 cells)]. (e)TH17 cells (3 × 104) were transferred, and isolated WT or transgenic TR1 cells (3 × 104) from the small intestine of anti-CD3–treated IL-10eGFP reporter mice were transferred either alone or with (e)TH17 cells into CD45.1 Rag1−/− mice. Colitis development was monitored using endoscopy.

Endoscopic procedure

We performed colonoscopy in a blinded fashion for colitis scoring using the Coloview system (Karl Storz, Tuttlingen, Germany) (40). Colitis scoring was based on the granularity of the mucosal surface, stool consistency, vascular pattern, translucency of the colon, and number of fibrins visible (0–3 points for each).

Histopathology procedure

Colons were fixed in Bouin’s fixative solution or 4% paraformaldehyde in PBS and embedded in paraffin. H&E-stained sections were evaluated by a semiquantitative criterion-based method (score 0–5) as described before (41).

Cytokine assay

We stimulated 4 × 104 mouse T cells/ml for 60 h with plate-bound CD3 Abs (10 μg/ml) and soluble CD28 Abs (10 μg/ml) in medium (Click’s medium supplemented with 10% FCS, l-glutamine, penicillin, streptomycin, and 2-ME). Cytokines were quantified by cytometric bead array (mouse TH1/TH2/TH17 cytokine kit; BD Biosciences) following the manufacturer’s instructions.

We stimulated 3 × 104 human T cells/200 μl for 96 h with CD3/CD28 beads (Dynabeads human T-activator CD3/CD28 for T cell expansion and activation; Life Technologies) in a 1:1 ratio in medium (Click’s medium supplemented with 10% FCS, l-glutamine, penicillin, streptomycin, and 2-ME). As indicated, 50 μg/ml human IL-10Rα Ab (R&D Systems) was added to the cell culture. Cytokines were quantified by a LEGENDplex assay (human Th cytokine panel; BioLegend).

Relative gene expression analysis

RNA from cells was isolated with TRIzol LS reagent (Life Technologies) in accordance with the manufacturer’s protocol. RNA was subjected to reverse transcription with SuperScript II (Invitrogen) with oligo(dT) primer in accordance with the manufacturer’s protocol. cDNA was semiquantified using commercially available primer/probe sets (Applied Biosystems) and analyzed with the change in cycle threshold method. All results were normalized to hypoxanthine phosphoribosyltransferase (Hprt) quantified in parallel amplification reactions during each PCR quantification.

Western blot

To analyze STAT3 activation, total cell lysates of indicated cell populations were separated in a 10% SDS-PAGE assay, transferred to polyvinylidene difluoride membranes (Merck Millipore), and probed with anti–phospho-Stat3 (Tyr705) or STAT3 Ab (Cell Signaling Technology), followed by incubation with the appropriate HRP-conjugated secondary Abs (Dako) and were visualized with the ECL substrate (Merck Millipore).

Immunofluorescence

In brief, cells were fixed for 10 min in 4% paraformaldehyde at room temperature. Cells were washed with PBS and incubated in PBS–Triton X-100 (0.3%) for 5 min. After washing, cells were incubated for 60 min in blocking buffer. Samples were stained overnight with biotinylated IL-10Rα Ab (primary rat anti-mouse Ab, 1:100, clone 1B1.3a; BioLegend) at 4°C. After washing secondary Ab (Alexa Fluor 568 goat anti-rat IgG; Invitrogen), staining was performed (1 h, room temperature) followed by 5 min staining with Hoechst 33258 (1:5000). For isotype control, the primary Ab was omitted.

Isolation of circulating human TR1 cells

PBMCs were isolated from buffy coats of healthy donors using the Biocoll separating solution (Biochrom). CD4+ T cells were enriched with a human CD4+ T cell enrichment kit in accordance with the manufacture’s protocol (Stemcell Technologies). The CD4+ T cells were further sorted to collect CD4+CD45RAloLAG-3+CD49b+ TR1 cells using a FACSAria II.

Generation of human TR1 cells in vitro

TR1 cells were generated in vitro as previously described (42). Briefly, naive CD4+ cells were cultured for 10 d with tolerogenic DC-10 in an allogeneic setting, in the presence of exogenous recombinant human IL-10 to produce a TR1-enriched product (TR1 cells) or with myeloid dendritic cells (mDC) in the absence of exogenous recombinant human IL-10 as control (non–TR1 cells).

ELISPOT

Dual IFN-γ/IL-10 ELISPOT (Diaclone, Besançon, France) was performed according to the manufacturer’s instructions, with slight modifications. Briefly, the filter-bottom plate was wetted and then coated with both anti–IFN-γ and anti–IL-10 capture. TR1 and non–TR1 cells were plated after thawing and overnight resting in serum-free X-VIVO 15 (tissue culture grade; Lonza). Stimulation was performed with anti-CD3 (2 μg/ml)/12-O-tetradecanoylphorbol-13-acetate (20 ng/ml) or with allogeneic mDC in the presence of rat anti-human IL-10R blocking Ab (50 μg/ml) or its isotype control (BD Biosciences). After a 48-h incubation, detection Abs were added and then visualization was performed with 3-amino-9-ethylcarbazole and Vector Blue (Vector Labs). The plate was acquired on an A.EL.VIS four-plate ELISPOT reader. Analysis was performed using ImageJ (version 1.48) to quantify IFN-γ–producing cells (red spots) and IL-10–producing cells (blue spots).

Microarray analysis

Gene expression analysis.

Affymetrix mouse gene 1.0ST array data were analyzed using R and the Bioconductor package “affy.” Samples were normalized by robust multiarray average with standard parameters. To define genes functionally related to mouse TR1 signature genes, genes interacting with their human orthologs with an interaction probability of at least 80% were extracted from the ImmuNet database (43) (downloaded on May 11, 2016). Mouse orthologs were then found for these functionally related genes. Data are available at National Center for Biotechnology Information GEO (https://www.ncbi.nlm.nih.gov/geo/, accession no. GSE89080).

Gene ontology analysis.

Gene Ontology (GO) term analysis was performed using GOrilla (44). GO terms enriched for TR1 signature genes (p value threshold of 0.001) were exported. Any genes >2-fold differentially expressed also mapping to these terms were retrieved using BioMart (http:\\www.biomart.org). Enrichment of these genes in the GO term category was calculated as described in Eden et al. (44). Log2 values of enrichment were calculated to better visualize the spread of the data.

Statistics

The Mann–Whitney U test, paired t test, or one-way ANOVA (posttest Tukey) was used to calculate statistical significance. A p value <0.05 was considered significant. Statistical calculations were performed using Prism program 5.0 (GraphPad Software).

Study approval

All experiments involving animals were carried out in accordance with the Institutional Animal Care and Use Committee of Yale University or in accordance with the Institutional Review Board “Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz” (Hamburg, Germany).

Results

Intestinal TR1 cells express IL-10Rα and can respond to IL-10

We used Foxp3RFP IL-10eGFP double reporter mice and a model of anti-CD3–specific Ab-mediated transient intestinal inflammation to test whether a pure population of intestinal TR1 cells can respond to IL-10 (36, 37). This murine model has already been largely validated by others and by us as being capable of inducing CD4+Foxp3RFP−IL-10eGFP+CD49b+LAG-3+ bona fide TR1 cells in the small intestine (26). First, we found that ex vivo–isolated TR1 cells express IL-10Rα at a comparable level to intestinal Foxp3+ peripheral Tregs and levels ∼6-fold higher than splenic naive T cells (Fig. 1A, 1B).

FIGURE 1.
FIGURE 1.

In vivo–differentiated TR1 cells can respond to IL-10. (A) IL-10Rα expression and mean fluorescence intensity (MFI) of indicated cell populations. Black area represents the isotype control. Four independent experiments were performed. (B) Immunofluorescence staining (two independent experiments) of IL-10Rα on WT TR1 cells. (C and D) Change in MFI (ΔMFI) compared with unstimulated cells) of pSTAT3 levels. (C) Naive T cells, Foxp3+ T cells, and IL-10RαWT or IL-10RαImpaired TR1 cells were stimulated with IL-10 (100 ng/ml) for the indicated time points. Black area represents the unstimulated control. (D) IL-10RαWT or IL-10RαImpaired TR1 cells were stimulated for 20 min with the indicated concentrations of IL-10 or IL-6. Data are representative of three independent experiments.

We then wanted to test whether IL-10Rα was functional on TR1 cells. We therefore checked the level of phosphorylated STAT3 (45), the main downstream molecule of the IL-10R signaling, in intestinal TR1 cells upon in vitro stimulation with IL-10 (45).

By testing STAT3 phosphorylation at different times and in response to different doses of IL-10, we found that intestinal TR1 cells respond to IL-10 (Fig. 1C, 1D). As controls, splenic naive T cells, in which IL-10R was very low, did not show an elevated level of pSTAT3 after stimulation with IL-10 (Fig. 1C), whereas Foxp3+ Tregs responded to IL-10 with a distinct increase of pSTAT3 level (Fig. 1C). To further strengthen our data, we used CD4–DN IL-10R transgenic (IL-10RImpaired) TR1 cells, which overexpress a DN IL-10Rα–chain and therefore have largely impaired IL-10 signaling (37). We detected that these IL-10RImpaired TR1 cells only slightly upregulated pSTAT3 upon IL-10 stimulation (Fig. 1C, 1D). STAT3 can also be phosphorylated by proinflammatory cytokine IL-6. As a control, to confirm that STAT3 activation in CD4–DN IL-10R transgenic TR1 cells is not impaired per se, we stimulated WT and IL-10RImpaired TR1 cells with IL-6. In contrast to what we observed with IL-10 stimulation, the activation of pSTAT3 by IL-6 was normal in IL-10RImpaired TR1 cells (Fig. 1D).

Finally, we wanted to test whether the capacity of TR1 cells to respond to IL-10 is restricted to the mouse model used or whether it is a more general feature of TR1 cells. We therefore used the standard protocol (i.e., IL-27 plus TGF-β1) (21, 46) to induce bona fide TR1 cells in vitro. Initially, we confirmed IL-10Rα expression on in vitro–differentiated WT and IL-10RImpaired TR1 cells using immunofluorescence (Supplemental Fig. 1A). Next, we FACS sorted a pure population of differentiated TR1 cells and stimulated them at different times and with different doses of IL-10 and measured STAT3 phosphorylation via FACS and Western blot analysis. As for the in vivo–induced intestinal TR1 cells, we observed that also mature TR1 cells induced in vitro respond to IL-10 by activating STAT3 (Supplemental Fig. 1B–E).

Collectively, these data show that in vivo– and in vitro–differentiated murine TR1 cells express functional IL-10Rα and are, in principle, able to respond to IL-10 stimulation.

No phenotypically and early functional defect among WT and DN IL-10R transgenic TR1 cells

The above results show that TR1 cells express functional IL-10R. However, what exactly the role of this receptor is in TR1 cell biology remained elusive. To investigate this, WT and CD4–DN IL-10R transgenic (IL-10RImpaired) mice were treated with anti-CD3 mAb. Shortly after the second injection, the induction of intestinal TR1 cells was evaluated without any in vitro manipulation due to the use of the Foxp3RFP IL-10eGFP double reporters. Surprisingly, we did not observe any changes in the frequency and number of intestinal TR1 cells between WT and CD4–DN IL-10R transgenic mice upon treatment (Fig. 2A).

FIGURE 2.
FIGURE 2.

IL-10 signaling in T cells is not essential for the differentiation of TR1 cells. IL-10RαWT or IL-10RαImpaired Foxp3RFP IL-10eGFP double reporter mice were treated with anti-CD3. (A) Frequency of TR1 cells in the small intestine and CD49b and LAG-3 expression by TR1 cells are shown (IL-10RαWT, n = 4; IL-10RαImpaired, n = 5) (B) Gene expression analysis, comparing IL-10RαWT TR1 cells and IL-10RαImpaired TR1 cells. Fold change expression of TR1 cell signature genes of IL-10RαImpaired TR1 cells compared with IL-10RαWT TR1 cells is shown. (C) Maf, Ahr, Prdm1, Tgfb1, Ctla4, and Gzmb mRNA expression normalized to Hprt (data pooled of three independent experiments). (D) TR1-mediated in vitro suppression. Data are representative of five independent experiments.

We further analyzed the surface markers, which typically characterize TR1 cells. The FACS analysis on WT and IL-10RImpaired TR1 cells showed similar levels of CD49b and LAG-3 expression (Fig. 2A). To further broaden our data, we performed a microarray analysis of IL-10RWT TR1 and IL-10RImpaired TR1 cells. The gene expression profiles between IL-10RWT TR1 and IL-10RImpaired TR1 cells were largely similar (Pearson correlation coefficient r = 0.989). For all transcriptional factors, extracellular receptor and cytokines known to be crucial for TR1 cell differentiation and function (TR1 signature genes), such as Prdm1, Maf, and Ahr, as well as Gzmb, Tgfb1, and Ctla4 mRNA expression, were unaffected in WT TR1 cells compared with IL-10RImpaired TR1 cells (Fig. 2B). The similar expression of some of these genes was also confirmed by conventional mRNA analysis (Fig. 2C). We further expanded our analysis to those genes that were found to be functionally related to the TR1 signature genes (related genes) based on the ImmuNet database (43): none of those genes was >2-fold differentially expressed among the two genotypes. Although most genes were similarly expressed, we found 300 genes >2-fold differentially expressed among IL-10RImpaired TR1 cells compared with WT TR1 cells (National Center for Biotechnology Information GEO; https://www.ncbi.nlm.nih.gov/geo/, accession no. GSE89080). We therefore asked whether any of these genes are functionally related to the genes known to be involved in TR1 cell differentiation. A GO analysis revealed 379 GO terms enriched for TR1 signature genes. Within these GO categories, enrichment of TR1 signature genes was consistently higher than enrichment of the genes >2-fold differentially expressed between IL-10RImpaired TR1 cells and WT TR1 cells (Supplemental Fig. 2). Based on the current knowledge about TR1 cells, we could not reveal any significant defect of IL-10RImpaired TR1 cells. However, we cannot exclude that among the differentially expressed genes are some genes that could affect TR1 cells. Thus, we tested the functional activity of WT and IL-10RImpaired TR1 cells isolated shortly after the in vivo induction using anti-CD3 Ab treatment to further exclude a defect of IL-10RImpaired TR1 cells. The in vitro suppressive assay showed that both WT and IL-10RImpaired TR1 cells have similar suppressive capacities (Fig. 2D). These data indicate that shortly after TR1 cell induction, IL-10RImpaired TR1 cells do not show any obvious defect compared with WT TR1 cells, suggesting that IL-10 signaling is not essential for TR1 cell differentiation.

IL-10 signaling in TR1 cells maintains their suppressive function in vivo in a murine IBD model

Although IL-10R signaling is dispensable for acquiring a TR1-like phenotype and in vitro suppressive function, we wondered whether IL-10R signaling influences the long-term functional stability of TR1 cells, as is the case with Foxp3+ Tregs (32, 47). We tested the functionality of IL-10RImpaired TR1 cells and WT TR1 in the CD45RBhi T cell transfer colitis model. Transfer of CD4+Foxp3−CD45RBhi cells caused severe disease that could be prevented by the cotransfer of in vitro–differentiated wild type TR1 cells. IL-10RImpaired TR1 cells showed a significantly reduced regulatory activity compared with WT TR1 cells. Interestingly, we observed that IL-10RImpaired TR1 cells still have the ability to reduce the colitis caused by the transfer of CD4+Foxp3−CD45RBhi T cells into Rag1−/− mice by trend (Supplemental Fig. 3). Nevertheless, in patients suffering from Crohn’s disease a possible TR1-based cell therapy is required to not only suppress the development of naive T cells into pathogenic effector T cells, but to control already differentiated cells. Thus, we wanted to test IL-10RImpaired TR1 cells and WT TR1 in the more challenging model of TH17 cell–induced colitis (26). CD4+IL-17AeGFP+ effector (e)TH17 cells were generated using the CD45RBhi transfer colitis model and isolated from the intestine and mesenteric lymph nodes of diseased mice. The (e)TH17 cells were then cotransferred into Rag1−/− recipients together with WT TR1 cells or IL-10RImpaired TR1 cells. Transfer of (e)TH17 cells caused severe disease, characterized by histological and endoscopic findings of colitis and weight loss. Transfer of WT TR1 cells prevented the development of colitis mediated by (e)TH17 cells. In contrast, IL-10RImpaired TR1 cells failed to block colitis mediated by (e)TH17 cells (Fig. 3A). These recipient mice showed similar weight loss and endoscopic and histological colitis scores to the experimental group of mice that received (e)TH17 cells alone (Fig. 3B, 3C). Adoptive transfer of IL-10RImpaired TR1 cells alone did not cause colitis, nor did the transfer of WT TR1 cells alone. The recipient mice showed no signs of weight loss (Fig. 3A) or colitis as evaluated by endoscopic and histological colitis scores (Fig. 3B, 3C).

FIGURE 3.
FIGURE 3.

IL-10 signaling in TR1 cells is essential for their in vivo function. In vivo induced IL-10RαWT or IL-10RαImpaired TR1 cells were injected alone or together with in vivo–differentiated effector (e)TH17 cells. (A) Mass loss, endoscopic, and histological colitis scores 5 wk upon transfer [(e)TH17, n = 7; (e)TH17 plus IL-10RαWT TR1, n = 7; (e)TH17 plus IL-10RαImpaired TR1, n = 10; IL-10RαWT TR1, n = 8; IL-10RαImpaired TR1, n = 8; lines indicate mean ± SEM). Representative endoscopic image (B) and histology (scale bars, 200 μm) (C) are shown. H&E staining was performed. Results are cumulative of two independent experiments. One-way ANOVA (posttest Tukey) was used to calculate significance. **p < 0.01, ***p < 0.001.

In summary, WT cells, but not TR1 cells with impaired IL-10 signaling, could suppress colitis development. However, 10RImpaired TR1 did not aggravate disease caused by (e)TH17. Furthermore, transfer of TR1 cells with impaired IL-10 signaling alone did not cause disease, backing up the argument for the safety of TR1 cells in human trials.

IL-10 receptor signaling in TR1 cells maintains IL-10 production

Although IL-10 receptor is dispensable for the development of TR1 cells, this receptor becomes fundamental to maintain the functional activity of TR1 cells in vivo.

However, the reason why TR1 cells require IL-10 receptor signaling to maintain their functional activity remained unresolved. Knowing that other CD4+ T cells, such as TH17, TH2, and Foxp3+ Tregs, are required to respond to their own cytokines to maintain their functional activity, we wondered whether TR1 cells are also required to respond to IL-10 to maintain the production of IL-10, which mediates their suppressive activity.

To test this, we adoptively transferred in vivo–generated WT and IL-10RImpaired TR1 cells, which had been isolated from the small intestine of Foxp3RFP IL-10eGFP double reporter mice, into congenic Rag1−/− mice. Five weeks later, transferred cells were isolated from different organs and IL-10eGFP expression was analyzed. Approximately 45% of the previously transferred WT TR1 cells isolated from the colon or mesenteric lymph nodes still expressed IL-10. However, only 10% of the previously transferred CD4–DN IL-10R transgenic TR1 cells still demonstrated IL-10 production (Fig. 4A). To balance out the bias resulting from the transfer of TR1 cells into immune-deficient mice, we continued testing this aspect. However, this time we used an in vitro reductionist approach to investigate whether intestinal TR1 cells require IL-10 receptor signaling to sustain the expression of IL-10 upon in vitro reactivation. In line with the in vivo experiments, in vitro–restimulated IL-10RImpaired TR1 cells produce significantly lower amounts of IL-10 as compared with WT TR1 cells. As expected based on our previous results, which show that IL-10 suppresses IL-17A but not IFN-γ production by T cells in vivo (26), we observed in vitro that the production of IFN-γ was similar, whereas IL-10RImpaired T cells produced more IL-17A (Fig. 4B). Notably, the mRNA levels of Tgfb1, Ctla4, and Gzmb, which are involved in the suppressive function of TR1 cells, were not altered in IL-10RImpaired TR1 cells compared with WT TR1 cells (Fig. 4C). Collectively, these data suggest that IL-10 is fundamental to sustain IL-10 production in TR1 cells over time.

FIGURE 4.
FIGURE 4.

IL-10 signaling in TR1 cells sustains IL-10 expression. (A) In vivo–induced IL-10RαWT or IL-10RαImpaired TR1 cells were injected into Rag1−/− mice. Cells were isolated 5 wk after transfer. Representative dot plots of IL-10eGFP expression of four pooled mice per group are shown. Data are representative of three independent experiments. (B) Cytokine production of TR1 cells was quantified using a cytometric bead array. Means ± SEM from three independent experiments are shown. A Mann–Whitney U test was used to calculate significance. (C) Tgfb1, Ctla4, and Gzmb mRNA expression normalized to Hprt. Data are cumulative of three independent experiments.

When cells are not synchronized in vivo, it is difficult to clearly dissect early IL-10 receptor–related biological effects occurring during the cell differentiation phase from later effects happening when cells have already acquired their phenotype. We had to find a solution when faced with this technical limitation, so once more we took advantage of a better controllable in vitro experimental setting. We therefore differentiated TR1 cells starting with naive WT and IL-10RImpaired T cells in the presence of IL-27 and TGF-β1. In line with the in vivo experiment, we did not observe any difference after the first 5 d of TR1 differentiation, even when IL-10 was added to the culture (Fig. 5A). Then, we FACS sorted the TR1 cells and monitored IL-10 expression over time after in vitro polyclonal TCR stimulation. To exclude that the observed defect in IL-10 production by IL-10RImpaired TR1 cells was caused by side effects due to the overexpression of a DN IL-10Rα, we included an additional control. We restimulated WT TR1 cells either in the presence of a blocking IL-10R Ab or isotype control. Thus, we were able to assess the influence of IL-10 signaling on the IL-10 production of mature TR1 cells starting from the same pool of cells. The resulting data confirmed our in vivo findings that IL-10RImpaired TR1 cells showed a faster decrease of IL-10 expression over time compared with WT TR1 cells (Fig. 5B). Likewise, TR1 cells restimulated in the presence of blocking IL-10R Ab demonstrated a strong decrease of IL-10 expression over time compared with TR1 cells in the presence of an isotype Ab (Fig. 5B).

FIGURE 5.
FIGURE 5.

IL-10 signaling is dispensable for the in vitro differentiation of TR1 cells with IL-27 and IL-10 sustains IL-10 production in in vitro–differentiated TR1 cells. (A) In vitro differentiation of IL-10RαWT and IL-10RαImpaired TR1 cells. Five independent experiments were performed. (B) IL-10eGFP change in mean fluorescence intensity (ΔMFI) of restimulated IL-10RαImpaired TR1 cells and IL-10RαWT TR1 cells plus anti–IL-10R Ab compared with IL-10RαWT TR1 cells are shown. Results are cumulative of three independent experiments.

IL-10 maintains IL-10 production in TR1 cells via activation of p38 MAPK

It has previously been shown that IL-10 production by Foxp3+ Tregs is controlled in a STAT3-dependent manner following IL-10R signaling. Furthermore, induction of IL-10 during in vitro differentiation of TR1 cells with IL-27 also depends on STAT3 activation (48, 49). However, p38 MAPK signaling has also been linked to IL-10 production in cell types other than T cells, such as human monocytes and macrophages, and it has also been linked to the regulatory function of induced Tregs (50–53). To address the role of STAT3 and p38 MAPK in maintaining IL-10 production in TR1 cells, we compared the phosphorylation status of STAT3 and p38 MAPK in a pure population of in vitro–induced WT and IL-10RImpaired TR1 cells over time upon restimulation.

We could not observe a reduction of pSTAT3 in IL-10RImpaired TR1 cells compared with WT TR1 cells over time (Fig. 6A). In contrast, phosphorylation of p38 MAPK was reduced by trend in IL-10RImpaired TR1 cells compared with WT TR1 cells (Fig. 6A). These results suggested a possible correlation between IL-10 production and the activation level of p38 MAPK in TR1 cells. To further test the functional role of STAT3 and p38 MAPK signaling in TR1 cells, kinase inhibitors were used. As a control, we also tested inhibitors of other major MAPK pathways, ERK1/2 and JNK, because these kinases have also been linked to IL-10 expression in other immune cell types, such as TH1 and TH2 cells or human monocytes and macrophages (54–56). WT TR1 cells were differentiated in vitro from CD4+ T cells, isolated from Foxp3RFP IL-10eGFP double reporter mice, and reactivated for 48 h. To test the role of p38 MAPK signaling in the stability of TR1 cells, a specific p38 inhibitor (SB203580), which has previously been shown by others and us to selectively block p38 MAPK signaling in T cells (57, 58), was added during the reactivation (Fig. 6B). The frequency of IL-10eGFP+ cells was tested. Both STAT3 and JNK inhibitors slightly reduced the frequency of IL-10eGFP+. However, high concentrations of STAT3 or JNK inhibitor also decrease the viability of TR1 cells (data not shown). Inhibition of ERK1/2 activation did not have any effect on the IL-10 production of TR1 cells. Strikingly, inhibition of p38 MAPK resulted in a significant and strong reduction in the frequency of IL-10eGFP+ in a dose-dependent manner (Fig. 6B). p38 MAPK inhibitor neither compromised viability of the cells nor resulted in overgrowth of IL-10eGFP− cells in culture (Supplemental Fig. 4A). Moreover, in line with our previous in vivo data, in vitro–differentiated WT TR1 cells, reactivated in the presence of p38 MAPK inhibitor, did not show significant changes in the expression of Gzmb, Tgfb1, and Ctla4 genes (Fig. 6C).

FIGURE 6.
FIGURE 6.

IL-10 signaling sustains IL-10 production in TR1 cells via activation of p38 MAPK. (A) pSTAT3 and pp38 MAPK changes in mean fluorescence intensity (ΔMFI) of restimulated IL-10RαImpaired TR1 cells compared with IL-10RαWT TR1 cells are shown. Results are cumulative of three independent experiments. A paired t test was used to test significance. (B) Frequency of IL-10eGFP of restimulated TR1 cells in the presence of indicated inhibitors are shown (mean ± SEM of three independent experiments). One-way ANOVA (posttest Tukey) was used to calculate significance. *p < 0.05. (C) Tgfb1, Ctla4, and Gzmb mRNA expression normalized to Hprt. Results are cumulative of three independent experiments.

STAT3 has been previously reported to be important during the differentiation of TR1 cells in the presence of IL-27 (59, 60). We therefore tested the effect of STAT3 on the differentiation of TR1 cells from naive T cells. As expected, the addition of STAT3 inhibitor blocked TR1 cell differentiation, demonstrating the activity of this compound (Supplemental Fig. 4B). We also tested p38 MAPK inhibitors and we observed that this compound also blocks the differentiation of TR1 cells almost completely, whereas inhibition of ERK1/2 or JNK did not affect the differentiation of TR1 cells in vitro (Supplemental Fig. 4B).

In summary, our findings suggest that IL-27 initiates the differentiation of naive CD4+ T cells into TR1 cells through STAT3 and p38 MAPK activation. Additionally, differentiated TR1 cells, which express functional IL-10R, are required to respond to IL-10 to maintain p38 MAPK activation and, in turn, sustain IL-10 production.

IL-10 maintains IL-10 production in human TR1 cells

Human TR1 cells are of great interest for Treg-based immunotherapy because of their strong potential to induce immune homeostasis (14, 61, 62). Thus, we aimed to investigate the role of IL-10 signaling in mature human TR1 cells. Using CD49b and LAG-3 as markers, we isolated circulating human TR1 cells from PBMCs of healthy donors (Fig. 7A). We restimulated the isolated TR1 cells in vitro together with IL-10Rα blocking Ab (or isotype) and measured cytokine release. In line with the data obtained with mouse TR1 cells, we did not observe differences in the production of IFN-γ, IL-4, or IL-6 between TR1 cells upon blocking IL-10R compared with control. IL-10 production of human TR1 cells was, however, decreased by >30% in the presence of IL-10Rα blocking Ab, highlighting the importance of IL-10 signaling to maintain the production of IL-10 (Fig. 7B and data not shown). In line with our murine data, we saw no significant difference in the mRNA expression of GZMB, TGFB1, and CTLA4 between restimulated TR1 cells in the presence or absence of IL-10Rα blocking Ab (Fig. 7C).

FIGURE 7.
FIGURE 7.

IL-10 signaling sustains IL-10 production in human TR1 cells. (A–C) Circulating human TR1 cells (CD4+CD45RAloCD49b+LAG-3+) were FACS sorted from PBMCs of healthy donors (n = 5) (A). TR1 cells were restimulated with anti-CD3 and anti-CD28 for 96 h with either 50 μg/ml human IL-10Rα or isotype control Ab and the indicted cytokines were quantified. A paired t tested was used to calculate significance (B). TGFB1, CTLA4, and GZMB mRNA expression normalized to HPRT (C). (D) In vitro–differentiated TR1 and non–TR1 cells were restimulated with anti-CD3/12-O-tetradecanoylphorbol-13-acetate (TPA) or allogeneic mDC for 48 h with 50 μg/ml human IL-10Rα blocking Ab or isotype control. A dual IFN-γ/IL-10 ELISPOT was performed. Data are cumulative of two independent experiments.

Next, we used IL-10–anergized T cells containing TR1 cells that were generated in vitro with tolerogenic dendritic cells (DC-10) (42). This is the most efficient protocol to induce human TR1 cells that are going to be used in the upcoming clinical trials (63). We observed that differentiated TR1 cells reactivated in the presence of IL-10Rα blocking Ab showed reduced numbers of IL-10 producer cells compared with isotype-treated cells. Of note, the effect we observed was independent of the activation used: both polyclonal and allogeneic mDC stimulation in the presence of IL-10Rα blocking Ab led to a reduction of IL-10–producing cells (Fig. 7D), confirming our results obtained with circulating human TR1 cells regarding the IL-10 production. Interestingly, blockade of IL-10 signaling led to increased IFN-γ production in the pool of IL-10–anergized T cells. This difference might be due to the presence of non–TR1 cells in the IL-10–anergized T cell pool, which might be affected differently by the blocking of IL-10 signaling. CD4+ T cells generated in vitro in the absence of IL-10 (non–TR1 cells) were also tested and they did not show an increased IFN-γ production and only a slight reduction in IL-10 production upon blockage of IL-10 signaling (Fig. 7D).

In summary, IL-10 signaling is also essential to maintain IL-10 production by freshly isolated and in vitro–induced human TR1 cells.

Discussion

Chronic TCR stimulation in the presence of IL-10 has been shown to be sufficient to induce highly regulatory mouse and human TR1 cells (29, 64). However, findings by Maynard et al. (18) demonstrated that in a totally IL-10–deficient mouse, TR1 cells were still present in the intestine. Our study allowed us to test the direct effect of IL-10 on intestinal TR1 cells and therefore exclude possible extrinsic effects and side effects related to the use of a totally IL-10–deficient mouse model.

We found that IL-10 signaling is not essential to induce TR1 cells in vivo. Of note, the overexpression of a DN IL-10Rα suppressed IL-10–mediated STAT3 activation. Our data suggest that the differentiation of TR1 cells is dependent on STAT3 activation, but independent of IL-10 signaling. However, the activation of STAT3 by other factors, such as IL-27 and IL-6, is not affected in transgenic mice with impaired IL-10 signaling, which explains this finding. Furthermore, it is widely accepted that IL-27 is sufficient to induce TR1 cells in vivo (46, 48, 59, 60). Our data further support this as they show that TR1 cells with a strongly impaired IL-10 signaling do not show any obvious defects shortly after induction. However, we showed that mature TR1 cells can respond to IL-10. More importantly, IL-10 signaling is crucial for their function. By responding to IL-10, TR1 cells maintain the production of IL-10. This allows them to preserve their regulatory activity. When mouse intestinal TR1 cells do not respond to IL-10, they lose IL-10 production and therefore their suppressive function. Accordingly, IL-10 signaling in TR1 cells was crucial to maintain their high level of potential to prevent CD4+Foxp3−CD45RBhi T cell– and TH17 cell–mediated colitis. Interestingly, we observed that IL-10RImpaired TR1 cells still have the ability to reduce the colitis caused by CD45RBhi T cells by trend. In contrast, IL-10RImpaired TR1 cells were not able to improve colitis induced by the transfer of TH17 cells. This difference could be due to the different disease severity in the two colitis models or the different Th cell composition, because the CD45RBhi colitis model is dominated by TH1 and TH17 cells. Thus, it would be possible that IL-10 signaling in TR1 cells is more important to control TH17 cells than TH1 cells, as has been proposed for Foxp3 Tregs (32). Taken together, these studies with our present findings suggests that Foxp3+ Tregs and TR1 cells need to respond to IL-10 to sustain IL-10 production, which is then essential to directly control TH17 cells. Nevertheless, we cannot exclude that other TR1-associated suppressive mechanisms, such as the secretion of granzyme B or TGF-β, are more important to suppress other cell types.

TR1 cells can exert their regulatory function through several mechanisms (16, 17, 65). Among these, we found that TR1 cells that do not respond to IL-10 lose their IL-10 production and their capacity to suppress TH17–mediated colitis, but they still express normal levels of granzyme B and CTLA-4. All of these molecules have been considered as being involved in alternative regulatory mechanisms of TR1 cells (29, 66, 67). Nevertheless, IL-10 seems to be essential to control TH17 cells (26, 32). The possibility remains that in the absence of IL-10 secretion, other regulatory mechanisms of TR1 cells would be directed to other proinflammatory cells. For example, granzyme B secreted by TR1 cells could kill APCs (16).

Several signaling molecules have been identified to induce and maintain IL-10 expression in a variety of cells, for example, STAT3, p38 MAPK, and ERK1 and ERK2 (ERK1/2) (50–53, 68). Furthermore, it has been previously shown that IL-10 signaling sustains the production of IL-10 via activation of STAT3 in Foxp3+ Tregs. TR1 cells also depend on STAT3 signaling for differentiation (32, 48), but surprisingly we found that it is dispensable for the maintenance of IL-10 secretion in mature TR1 cells, whereas p38 MAPK plays a crucial role in this process. However, because addition of JNK and STAT3 inhibitors affected cell viability at high concentrations, we cannot completely exclude that JNK or STAT3 signaling in addition to p38 MAPK signaling might affect IL-10 production. However, STAT3 requires Foxp3 and histone acetyl transterase-1 to epigenetically modify the Il10 promoter region to allow for gene regulation via STAT3 in Foxp3+ Tregs (69). The absence of Foxp3 in mature TR1 cells could mitigate the role of STAT3 in this process and explain why p38 MAPK, which does not require Foxp3-mediated stabilization, sustains IL-10 expression in TR1 cells.

Considering the literature, we propose IL-27/Erg-2/Blimp1 in conjunction with STAT3, Ahr, and c-Maf as the driving forces for TR1 cell differentiation. However, IL-10 signaling, through p38 MAPK, is required for the stabilization and function of intestinal TR1 cells. Whether and how IL-10 and IL-21 (another cytokine that has been proposed to sustain the TR1 cell phenotype) synergistically sustain IL-10 production remain to be further investigated (21, 46, 59).

Human TR1 cells display a high level of potential to maintain and re-establish immune homeostasis. They therefore receive major focus in current immunological and clinical research into the design of TR1 cell–based therapies to treat human diseases such as IBD (61, 62). In such trials the success of a TR1-based therapy is strongly linked to high IL-10 production of TR1 cells (67). The functional stability of TR1 cells could be critical for the efficiency and safety of these cells as therapeutics.

We extended our key findings to human biology. We show that IL-10 is also essential for human TR1 cells to maintain their IL-10 production. The capacity of TR1 cells to sustain themselves by IL-10 is therefore key to determine the long-term success of the therapy. Currently, it has been proposed to select in vitro–induced TR1 cells based on the expression of CD49b and LAG-3 (67). Further enriching TR1 cells by high expression of IL-10 receptor could optimize the efficiency and ensure the safety of TR1 cell–based trials. Furthermore, measuring the expression of IL-10 receptor could serve as a marker to determine the efficacy of a TR1 cell therapy.

Collectively, we show that TR1 cells require the presence of their immunoregulatory cytokine IL-10 to maintain the cell function. Accordingly, TR1 cell stability is dependent on IL-10 signaling in TR1 cells themselves. Overall, our data indicate the crucial role of IL-10 signaling in TR1 cells and suggest that TR1-based T cell therapy is safe and efficient for the treatment of IBD.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Cathleen Haueis and Sandra Wende for excellent technical assistance. Furthermore, we thank the FACS Sorting Core Unit of the Universitätsklinikum Hamburg–Eppendorf for the excellent support.

Footnotes

  • ↵2 R.A.F. and S.H. are cosenior authors.

  • S.H. is supported by funding from Hofschneider Stiftung für Experimentelle Biomedizin and Ernst Jung-Stiftung. This work was supported in part by the Howard Hughes Medical Institute (to R.A.F.), Deutsche Forschungsgemeinschaft Grants HU 1714/3-1 (to S.H.), SFB841 (to J.H. and S.H.), and SFB1192 (to S.H. and C.F.K.), and by the 7th Framework Programme of the European Union (Marie Curie Actions Initial Training Network FP7-PEOPLE-2011-ITN), under Marie Skłodowska-Curie Grant 289903 (to B.M.).

  • The microarray data presented in this article have been submitted to the National Center for Biotechnology Information's Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE89080.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    DN
    dominant negative
    (e)TH17 cell
    IL-17AeGFP+ T cell
    GO
    Gene Ontology
    HPRT
    hypoxanthine phosphoribosyltransferase
    IBD
    inflammatory bowel disease
    mDC
    myeloid dendritc cell
    TR1
    T regulatory type 1
    Treg
    regulatory T cell
    WT
    wild-type.

  • Received June 20, 2016.
  • Accepted November 20, 2016.

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