Open Access

Cutting Edge: Inhibition of Glycogen Synthase Kinase 3 Activity Induces the Generation and Enhanced Suppressive Function of Human IL-10+ FOXP3+–Induced Regulatory T Cells

Hao Cheng, Lingbiao Wang, Biaolong Yang, Dan Li, Xiaoxia Wang, Xinnan Liu, Na Tian, Qianru Huang, Ru Feng, Zhengting Wang, Rui Liang, Sheng-Ming Dai, Ling Lv, Ji Wu, Yuan-Sheng Zang and Bin Li
J Immunol September 15, 2020, 205 (6) 1497-1502; DOI: https://doi.org/10.4049/jimmunol.2000136

Key Points

  • GSK3 inhibition promotes human IL-10+ FOXP3+ iTreg generation.

  • Human IL-10+ FOXP3+ iTregs exhibited enhanced suppressive features and function.

  • GSK3 inhibition upregulated the transcription of IL-10 in human FOXP3+ iTregs.

Abstract

IL-10 is critical for Foxp3+ regulatory T cell (Tregs)–mediated immune suppression, but how to efficiently upregulate IL-10 production in Tregs remains unclear. In this article, we show that human IL-10+ FOXP3+–induced regulatory T cell (iTreg) generation can be dramatically promoted by inhibiting GSK3 activity. IL-10+ FOXP3+ iTregs induced by GSK3 inhibition exhibit classical features of immune-suppressive T cells. We further demonstrate that IL-10+ iTregs exhibit enhanced suppressive function in both IL-10–dependent and –independent manners. The enhanced suppressive function of IL-10+ Tregs is not due to a single factor such as IL-10, although IL-10 may mediate this enhanced suppressive function to some extent. Mechanistically, the increased transcriptional activity of IL-10 promoter and the enhanced expression of C-Maf and BLIMP1 coordinately facilitate IL-10 expression in human iTregs under GSK3 inhibition. Our study provides a new strategy to generate human immune-suppressive IL-10+ FOXP3+ Tregs for immunotherapies.

Introduction

Thymus-derived natural regulatory T cells (nTregs) and periphery induced regulatory T cells (iTregs) are major subsets of regulatory T cells (Tregs). nTregs and iTregs have different suppressive function for immunotherapy, and in some cases iTregs exhibit more stability and enhanced suppressive activity than nTregs (1).

IL-10 is a powerful immune mediator with various physiological functions. Although IL-10 can be produced by many different cells, IL-10 derived from different cell sources is likely to be nonredundant. For instance, IL-10 produced by CD4+ effector T cells is an vital mechanism for their self-regulation, and IL-10 produced by Tr1 is crucial for maintaining immune tolerance (2, 3). IL-10 plays a critical role in the major contact-independent modes of Foxp3+ Treg suppression (4, 5). IL-10 derived from Tregs and IL-10 signaling are vital for Foxp3+ Treg function (6).

GSK3 is a growth signaling-sensitive kinase. Two isoforms of GSK3, GSK3α and GSK3β, have been identified with similar but nonredundant functions. GSK3 plays an important role in regulating T cell activation, exhaustion, and IL-10 production in effector and memory CD4+T cells (2, 7). Recent studies show that GSK3β inhibition promotes the differentiation of both murine and human iTregs and enhance their suppressive function through increasing Foxp3 stability and iTreg survival (8–10). As an important immune regulator, the production of IL-10 by human iTregs remains uncertain. In this study, we investigated whether GSK3 inhibition can promote the generation of IL-10+ FOXP3+ Tregs in human iTregs and further examined how GSK3 inhibition enhanced the human iTreg suppressive function.

Materials and Methods

Mice

B-NDG(NOD-PrkdcscidIL-2rgtm1/Bcgen) mice were from Biocytogen and housed in a specific pathogen-free facility. Mice were treated according to the committee-approved guidelines of the Shanghai Jiao Tong University School of Medicine.

Reagents, flow cytometry, and cell isolation

Abs were purchased from BD Biosciences, eBioscience, or BioLegend. Data were collected on an LSR Fortessa (BD) and analyzed using FlowJo software. Human studies were conducted in accordance with the Ruijin Hospital Ethics Committee. Colonic lamina propria mononuclear cells were isolated as previously described (5, 11).

Human iTreg differentiation

Human naive CD4+T cells were activated with anti-CD3 and CD28 beads (3:1; Life Technologies), TGF-β (5 ng/ml; R&D Systems), and IL-2 (100 IU/ml; R&D Systems) for 7 d in complete X-Vivo 15 medium (Lonza).

In vitro suppression assay and wound-healing assay

In vitro suppression assays and wound-healing assays were done as previously described (11).

Quantitative PCR

Primers used for real-time PCR are shown in Supplemental Table I.

Chromatin immunoprecipitation analysis

Chromatin immunoprecipitation (ChIP) assays were performed by using the SimpleChIP Chromatin IP Kit (Cell Signaling Technology). Abs used for ChIP were from Merck.

IL-10 capture assay

IL-10 capture assay was performed as previously described (11). Human iTregs were pretreated by GSK3 inhibitor (GSK3i) for 36 h and then stimulated with PMA and ionomycin for 5 h. IL-10–producing Tregs were then labeled using the IL-10 Secretion Assay-Cell Enrichment and Detection Kit (Miltenyi Biotec) according to the manufacturer’s instructions and then sorted into IL-10–producing and –nonproducing cells by FACS. The purity of the cell population after sorting was >98%.

Severe xenograft-versus-host disease model

Severe xenograft-versus-host disease (xeno-GvHD) model was performed as described previously (11).

Statistical analyses

Statistical analysis was performed using GraphPad Prism 8. The p values <0.05 were considered statistically significant.

Results and Discussion

GSK3 inhibition promotes human IL-10+ FOXP3+ iTreg generation and enhances the suppressive function of human iTregs

Previous data showed that iTreg differentiation triggers GSK3β phosphorylation. We confirmed the dynamics of GSK3 during iTreg differentiation. Unlike the previous data, iTreg differentiation activated GSK3 and reduced the inactive form of GSK3 (Supplemental Fig. 1A, 1B). Then, we treated iTregs with GSK3i SB216763 and detected the expression of Treg signature genes and cytokines by FACS. Human iTregs showed a great increase in IL-10 production after GSK3 inhibition. GSK3 inhibition decreased the mean fluorescence intensity of FOXP3 and increased classic Treg-associated gene expression: CD39, CD73, CD137, and CTLA-4 (Fig. 1A, 1B, Supplemental Fig. 1C). Surprisingly, GSK3 inhibition did not affect the expression of TGF-β as previously reported (10) and decreased generation of the proinflammatory iTregs that expressed TNF-α and IFN-γ (Fig. 1C). To confirm the impact of GSK3 inhibition on iTreg function, an in vitro Treg suppression assay was performed. Results showed that GSK3i-pretreated iTregs displayed enhanced suppressive function (Fig. 1D). It was noteworthy that cultured T conventional cells and iTregs in the presence of GSK3i abrogated the inhibition of proliferation of T conventional cells by iTregs (Fig. 1D). GSK3 inhibition also increased the expression of CTLA-4 and Granzyme B (GZMB), which may also account for the enhanced suppressive function of human iTregs. In our experiment setting, we used CD3 and CD28 beads instead of APCs; thus, CTLA-4 may not affect the Treg suppressive function. We detected the viability of the effector T cells in the standard suppression assay, and results showed that Tregs showed no effect on the viability of the responder cells (Supplemental Fig. 2A). Collectively, these findings demonstrated that GSK3 inhibition enhanced the suppressive function of human iTregs by promoting human IL-10+ FOXP3+ iTreg generation and decreasing proinflammatory iTregs.

FIGURE 1.
FIGURE 1.

In vitro generation of functionally immunosuppressive human IL-10+ FOXP3+ iTreg by inhibiting GSK3 activity. (A) Human iTregs cultured with the GSK3i SB216763 and the expression of FOXP3 and IL-10+ FOXP3+ iTregs were detected by FACS. (B) The mean fluorescence intensity (MFI) of FOXP3 and percentage of FOXP3+ iTregs (n = 3) and IL-10+ FOXP3+ iTregs (control n = 4, DMSO and GSK3i n = 7) were assessed as shown in (A). Data are means ± SD. *p < 0.05, ****p < 0.0001 by t test. (C) The expression of TGF-β, GZMB, TNF-α, and IFN-γ by FOXP3+ iTregs was detected by FACS. (D) In vitro suppression assay was performed in iTregs pretreated with or without GSK3i or cocultured with GSK3i. ns, not significant.

Features and suppressive activity of IL-10+ FOXP3+ iTregs

Because of the low percentages of IL-10+ FOXP3+ iTregs and the difficulty of detection, IL-10+ FOXP3+ Treg features were rarely reported. The gene expression pattern and function of human IL-10+ FOXP3+ iTregs remain unknown. We examined the features and function of the IL-10+ FOXP3+ Tregs. IL-10+ and IL-10− iTregs were sorted for RNA sequencing (RNA-seq) (Supplemental Fig. 2B). Compared with the IL-10− FOXP3+ Tregs, IL-10+ FOXP3+ Tregs expressed higher levels of effector cytokines such as GZMA and IL-10 (Fig. 2A). We further detected classical Treg signature genes by FACS. Human IL-10+ FOXP3+ iTregs expressed slightly higher CCR5, consistent with previously reported mouse IL-10+ Treg data (12). IL-10+ iTregs also expressed more CTLA-4, GZMA, HLA-DR, and CD49a and slightly higher levels of GZMB, CD45RO, CD44, and CXCR5 compared with IL-10− iTregs. There was no difference in the expression of FOXP3 and KLRG1 between these two groups (Fig. 2B, Supplemental Fig. 2C). Highly expressed CTLA-4, CD44, and CD45RO indicated that there was different activation status in IL-10+ and IL-10− iTregs. IL-10+ Tregs had a memory phenotype rather than a terminal effector phenotype (Fig. 2B). However, whether these two human Treg subsets were representative of two stages of Treg differentiation or two distinct subpopulations remained unclear. According to the previous murine genetic tracing data (12), we claimed that IL-10+ Tregs represent IL-10–producing subset, whereas IL-10− iTregs represent IL-10–nonproducing subset. We next tested the suppressive ability of IL-10+ FOXP3+ iTregs. In vitro wound-healing assay and suppression assay suggested that compared with IL-10− iTregs, IL-10+ FOXP3+ iTregs exhibited enhanced suppressive function (Fig. 2C, 2D). IL-10 neutralization slightly reduced the function of IL-10+ iTregs in wound-healing assay. But anti–IL-10R treatment failed to totally abolish the suppressive function of IL-10+ iTregs in suppression assay. We also checked the cytotoxic activity of IL-10+ iTregs and IL-10− iTregs, and these two subsets had similar cytotoxicity (Supplemental Fig. 2D, 2E). The low expression of Perforin may account for the limited cytotoxicity. These results indicated that enhanced suppressive function of IL-10+ iTregs is likely not due to a single factor. IL-10+ iTregs’ enhanced suppressive function was partially IL-10 dependent. We confirmed the phenotype in vivo by examining the colon tissue samples from IBD patients, and the percentage of IL-10+ FOXP3+ Tregs/FOXP3+ Tregs was higher at uninflamed sites than inflamed sites (Fig. 2E), and this was consistent with the previous data that IL-10–deficient Tregs are abundant in the colonic tissue but unable to restrain inflammation (5). To further verify the suppressive ability of IL-10+ FOXP3+ iTregs in vivo, we used a humanized mouse model of severe xeno-GvHD on B-NDG mice. Unlike previous data that showed that directly overexpressed exogenous human IL-10 in NOD-scid IL-2rγcnull mice exacerbates xeno-GvHD (13), mice that received IL-10+ iTregs lived significantly longer than mice injected with PBMCs alone or IL-10− iTregs (Fig. 2F). We had not directly blocked IL-10 by using anti–IL-10 Abs in vivo because of their severe side effects and the fact that not only IL-10+ Tregs but also other immune cells could produce IL-10. Further investigations were still required to confirm this point by using more-specific Ab-neutralization methods or IL-10 conditional knockout mice. Furthermore, IL-10+ iTregs were isolated after PMA/ionomycin stimulation in our study. Despite the maximum stimulatory ability for cytokine induction by PMA/ionomycin, it also had many other effects on gene expression. A better method is needed for isolating IL-10–producing human iTregs in the future. Collectively, our data suggested that IL-10+ FOXP3+ iTregs had more classical features of Tregs and exhibited enhanced suppressive activity in both IL-10–dependent and –independent manners.

FIGURE 2.
FIGURE 2.

Transcriptional and functional characteristics of human IL-10+ FOXP3+ iTregs generated by GSK3 inhibition. (A) Scatter plot of genes significantly upregulated (red), downregulated (blue), or stable (gray) in IL-10+ iTregs. (B) Expression of the Treg-associated functional genes in IL-10+ iTregs and IL-10− iTregs was detected by FACS. (C) In vitro wound-healing assay was performed by using IL-10+/IL-10− iTreg cellular supernatants with or without αIL-10, and open wound area was assessed by ImageJ (n = 3). Original magnification ×40. (D) In vitro suppression assay was performed by using IL-10+ iTregs, IL-10− iTregs, or IL-10+ iTregs with anti–IL-10R Ab. (E) Percentages of IL-10+ FOXP3+ Tregs from uninflamed (normal control [NC]) colon tissue or inflamed (IN) colon tissue isolated from human ulcerative colitis patients were assessed by FACS (n = 9). (F) xeno-GvHD with and without 3:1 PBMC/IL-10+/IL-10− Treg injection, showing survival plots (*p < 0.05, compared with PBMCs alone). Shown is one experiment from two independent experiments carried out with n = 4 mice in per group. Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by t test (D and E); means ± SEM, log-rank test (F).

GSK3 inhibition upregulated IL-10 transcription in human iTregs

All the above data suggested that GSK3 inhibition could regulate IL-10 expression and enhance Treg suppressive function. We then performed RNA-seq and examined the gene expression changes during GSK3 inhibition. RNA-seq results demonstrated that IL-10 is the top 3% gene among differentially expressed genes after GSK3 inhibition (Fig. 3A). The quantitative PCR (qPCR) results confirmed that GSK3 inhibition dramatically increases the IL-10 mRNA levels ∼300 times relative to control (Fig. 3B). The results also showed that decreased expression of TBX21 was responsible for the downregulation of IFN-γ (Fig. 3B). Previous data showed that GSK3 inhibition can promote Treg survival (8). To test this possibility, we examined whether GSK3 inhibition affects cell apoptosis. GSK3 inhibition in iTregs slightly increased apoptosis tendency (Supplemental Fig. 3A, 3B). Unlike the previous data (8, 9), GSK3 inhibition did not enhance FOXP3 stability (Fig. 1B). Both transcriptional and posttranscriptional regulation may mediate the upregulated IL-10 levels. Human IL-10 mRNA had a short half-life because of uridine-rich element–mediated decay. TTP was a uridine-rich element–binding protein that can promote IL-10 mRNA decay in RAW264.7 cells (14). GSK3 could phosphorylate TTP at Ser218 and regulate the TTP expression (15). We examined the IL-10 mRNA stability and found that IL-10 mRNA stability was not affected by GSK3 inhibition (Supplemental Fig. 3C). GSK3 inhibition slightly upregulated the expression of ZFP36 and downregulated HUR as potential negative feedbacks for the increased IL-10 mRNA expression (Supplemental Fig. 3D). Next, we further examined the mechanisms that regulate IL-10 transcription. IL-10 gene transcription in Th1, Th2, and Th17 cells was critically regulated by histone modifications (2). We investigated histone modifications at IL-10 promoter in human iTregs after GSK3 inhibition. Nine pairs of primer spanning the human IL-10 promoter (S1–S9) were used for the ChIP experiments. (Supplemental Fig. 3E). Results demonstrated that GSK3 inhibition dramatically increased histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 9 acetylation (H3K9ac), and histone H4 acetylation (H4ac) (Fig. 3C) in IL-10 promoter regions. By contrast, GSK3 inhibition resulted in significantly deceased histone H3 lysine 27 trimethylation (H3K27me3) level (Fig. 3D). Intriguingly, H3K9ac, H4ac, and H3K4me3 were significantly enriched at the C-Maf and Sp1 binding sites, whereas GSK3 inhibition induced decreased enrichment of H3K27me3 at the C-Maf and Sp1 binding sites. Furthermore, expression of C-Maf was increased dramatically upon GSK3 inhibition, and GSK3 inhibition also slightly increased the expression of BLIMP1 (Fig. 3E). GSK3β interacted with C-Maf and regulated the function of C-Maf (16). C-Maf and Sp1 were key regulators of IL-10 expression in macrophages and Th17 cells (17, 18). BLIMP1 was critical to murine IL-10+ Treg generation (12). Histone modifications changed the chromatin structure and increased the accessibility of the chromatin. Loose chromatin structure in the IL-10 promoter cooperated with upregulated transcriptional factors to promote the generation of IL-10+ FOXP3+ iTregs. Although the application of IL-10 targeting therapy succeeded in many inflammatory diseases and inflammation-associated cancers, some failed trials had also been reported (13). GSK3 inhibition–induced IL-10+ FOXP3+ iTregs may afford better therapeutic interventions for autoimmune disease, graft-versus-host disease, and inflammation-associated cancers.

FIGURE 3.
FIGURE 3.

Chromatin remodeling and upregulation of C-Maf and BLIMP1 by GSK3 inhibition are critical for IL-10 production in human FOXP3+ iTregs. (A) Scatter plot of genes significantly upregulated (red), downregulated (blue), or stable (gray) in human FOXP3+ iTregs under GSK3 inhibition. (B) Treg lineage specific gene expression during GSK3 inhibition was detected by qPCR. (C and D) ChIP experiments were performed by using Abs that respectively bind H3K4me3, H3K9ac, pan-H4ac, and H3K27me3. qPCR was used to measure the relative enrichment of IL-10 promoter in each ChIP experiment (n = 3). (E) Expression of the C-Maf and BLIMP1 in FOXP3+ iTregs was examined by FACS during GSK3 inhibition (n = 3). Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t test.

Disclosures

B.L. is a cofounder of Biotheus Inc. and chairman of its scientific advisory board. The other authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Bo Chen for his instruction of ChIP assays.

Footnotes

  • This work was supported by grants from the National Natural Science Foundation of China (81830051, 31525008, 31961133011, 31670911, 81602558, and 31670885), the National Science Foundation for Young Scientist of China (31800744), the National Key Research and Development Project (2019YFA0906102), and the Shanghai Jiao Tong University–The Chinese University of Hong Kong Joint Research Collaboration Fund, and the Fundamental Research Funds for Central Universities.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ChIP
    chromatin immunoprecipitation
    GSK3i
    GSK3 inhibitor
    GZMB
    Granzyme B
    H4ac
    histone H4 acetylation
    H3K9ac
    histone H3 lysine 9 acetylation
    H3K4me3
    histone H3 lysine 4 trimethylation
    H3K27me3
    histone H3 lysine 27 trimethylation
    iTreg
    induced regulatory T cell
    nTreg
    natural regulatory T cell
    qPCR
    quantitative PCR
    RNA-seq
    RNA sequencing
    Treg
    regulatory T cell
    xeno-GvHD
    xenograft-versus-host disease.

  • Received February 10, 2020.
  • Accepted July 24, 2020.

This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles.

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