Research ArticleIMMUNE REGULATION

Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19

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Science Immunology  25 Oct 2019:
Vol. 4, Issue 40, eaaw2707
DOI: 10.1126/sciimmunol.aaw2707
  • Fig. 1 AS2863619 is a potent Foxp3 inducer in Tconv cells.

    (A) Chemical structure of AS2863619, hereafter designated AS. (B) In vitro induction of Foxp3 expression in AS-treated mouse effector/memory and naïve CD4+ T cells as CD44highCD62Llow and CD44lowCD62Lhigh cells, respectively, and also in CD8+ T cells. Cells were stimulated with anti-CD3/CD28 mAb-coated beads and IL-2 in the presence or absence of AS (1.0 μM) or TGF-β (2.5 ng/ml) for 72 hours. Representative Foxp3 staining and percentages of Foxp3+ cells among CD4+ or CD8+ T cells after respective stimulation are shown (n = 3). (C and D) Foxp3+ cells generation by AS under TH1-, TH2-, TH17-, or TH9-inducing conditions. Naïve CD4+ T cells were anti-CD3/CD28–stimulated in the presence of IL-12 (10 ng/ml; TH1 condition, n = 5), IL-4 (10 ng/ml; TH2 condition, n = 4), IL-6 (20 ng/ml) + TGF-β (2.5 ng/ml; TH17 condition, n = 4), or IL-4 (10 ng/ml) + TGF-β (2.5 ng/ml; TH9 condition, n = 3). (E and F) Foxp3+ T cell generation from antigen-stimulated T cells. DO11.10 naïve CD4+ T cells were cocultured with APCs, 5 μM OVA peptide, and 1.0 μM AS and assessed for Foxp3 and DO11.10 TCR expression by flow cytometry (n = 3). (G) Expression of Treg signature molecules in AS- or TGF-β–treated anti-CD3/CD28–stimulated T cells assessed by flow cytometry. Data are representative of three independent experiments. (H) In vitro suppression assay using in vitro activated nTreg cells and TGF-β– or AS-induced iTreg cells (n = 3). Treg versus responder T cell ratio was 1:10. Vertical bars indicate means ± SD. **P < 0.01 by SNK method. ns, not significant.

  • Fig. 2 CDK8/19 as a key target of AS.

    (A) Candidate target molecules of AS revealed by tandem mass spectrometry (also see fig. S6 for chemical proteomics workflow). m/z, mass/charge ratio. (B) Correlation of Treg induction with the degree of CDK8/19 kinase inhibition. Five different kinase inhibitors including AS were examined for their Treg-inducing potency and their inhibitory activity of various kinases. Kinase inhibition was assessed in vitro using recombinant CDK8/cyclin C complex, CDK19/cyclin C complex, GSK3α, or GSK3β. IC50 was defined as the concentration of an inhibitor that reduced phosphorylation by half compared with DMSO control. EC150 for Treg induction was defined as the concentration of the compound that induced Foxp3+ Treg cells to 150% of DMSO control when mouse naїve CD4+ T cells were treated with anti-CD3/CD28 for 44 hours in the presence of the compound. Values are geometric mean of three (Treg induction, CDK19, GSK3α, and GSK3β) or four (CDK8) independent experiments. Pearson r, Pearson’s correlation coefficient (versus Treg induction). (C) Expression of CDK8, CDK19, and cyclin C after stimulation. Mouse CD4+ T cells stimulated with anti-CD3/CD28 were lysed for immunoblotting of these molecules at various time points. Data are representative of three independent experiments. (D) Effects of CDK8 or CDK19 knockdown on Foxp3 mRNA expression. Mouse CD4+ T cells were transfected with siRNA for CDK8 or CDK19; stimulated with anti-CD3/CD28, TGF-β, and IL-2; and examined for CDK8 and CDK19 expression by immunoblotting (left) or Foxp3 mRNA expression by quantitative RT-PCR (n = 3) (right). (E) Effects of retroviral expression of WT or KD CDK8 or CDK19 on Foxp3+ cell generation. Mouse CD4+ T cells infected with retroviruses harboring GFP (mock control), WT or KD CDK8, and WT or KD CDK19 were stimulated with anti-CD3/CD28 and IL-2 and examined for CDK8 and CDK19 expression by immunoblotting (left) and for the percentages of Foxp3+ cells among CD4+ T cells by flow cytometry (n = 5) (right). (F) Luciferase assay using CDK8 and/or CDK19 KO EL4 cell lines and a Foxp3 promoter plasmid (triplicate). Data are representative of two independent experiments. RLU, relative light unit. (G and H) Foxp3 induction in CDK8-KD–transfected CD4+ T cells by antigenic stimulation. Naїve CD4+ T cells isolated from DO11.10Rag2−/−Foxp3-eGFP reporter mice were stimulated with anti-CD3/CD28 for 22 hours, infected with mock or CDK8-KD or CDK8-WT–carrying retroviruses, incubated for another 22 hours in the absence of IL-2, and rested for 10 days in the presence of IL-2 (100 U/ml). LNGFR was used as a marker of infection. BALB/c-nu/nu mice were transferred with infected T cells on day 0 and treated with IL-2/anti–IL-2 Ab complexes on day 7 and then immunized with OVA/CFA on day 14. Draining lymph nodes were collected and examined for expression of Foxp3 by flow cytometry on day 20. Representative staining (G) and percentages of Foxp3+ cells among live LNGFR+ or LNGFRCD4+ cells in lymph nodes are shown (n = 6) (H). Vertical bars indicate means ± SD. ***P < 0.001, **P < 0.01, and *P < 0.05 (Dunnett’s test).

  • Fig. 3 Interaction of CDK8/19 and STAT5 in inducing Foxp3 expression.

    (A) Mouse CD4+ T cells were mock-infected or infected with retrovirus harboring the WT CDK8 and STAT5b genes, stimulated with anti-CD3/CD28 and IL-2, and subjected to immunoprecipitation and immunoblotting for CDK8, STAT5b, and MED12. Data are representative of two independent experiments. (B) STAT5 serine phosphorylation by CDK8. Recombinant GST-STAT5b incubated with recombinant WT or KD CDK8 in the presence or absence of 1.0 μM AS with 100 μM ATP and 10 mM MgCl2 was subjected to immunoblotting for phosphoserine (pS) of STAT5b. Data are representative of two independent experiments. (C) Control of serine and tyrosine phosphorylation by AS in activated T cells. Mouse CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of TGF-β for 22 hours and in the absence (DMSO) or presence of 100 nM AS, lysed, and subjected to immunoblot analysis for STAT5b, pS-STAT5b, or pY-STAT5. Signal intensity was quantified and normalized by GAPDH (n = 3 or 4). **P < 0.01 (Student’s t test). AU, arbitrary units. (D and E) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/28 for 22 hours, and PLA was performed to assess interaction between CDK8 and STAT5. Images were obtained using an LSM710 confocal microscope. Data were presented as maximum intensity projection (n = 3). Each red spot represents a single interaction, and DNA was stained with DAPI. (F) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/CD28 for 22 hours and examined for expression of CDK8 and STAT5. DNA was stained with Hoechst33342. Images were obtained using an LSM710 confocal microscope. Data are representative of two independent experiments. (G) Mouse CD4+ T cells infected with retrovirus encoding WT or S730A mutant STAT5b were stimulated with anti-CD3/CD28 and IL-2, without TGF-β, and subjected to immunoblotting for STAT5b (left), or assessed for the percentage of Foxp3+ cells among live virus–infected (i.e., GFP+) CD4+ T cells by flow cytometry (n = 7) (right). ***P < 0.001 (Student’s t test).

  • Fig. 4 Genome-wide enhancement of STAT5-dependent gene expression by AS.

    (A) Mouse CD4+ T cells stimulated with anti-CD3/CD28 with or without 1.0 μM AS were analyzed at the Foxp3 gene locus for STAT5 binding, H3K27ac, and chromatin status by ChIP-seq and ATAC-seq. (B) Mouse CD4+ T cells stimulated with anti-CD3/CD28 and TGF-β with or without 1.0 μM AS were subjected to ChIP-qPCR assay for pY-STAT5 binding at Foxp3 CNS0, CNS2, and core promoter regions (n = 3). *P < 0.05. (C) Density of STAT5 binding and indicated histone modifications in AS-treated or untreated T cells. Normalized ChIP-seq signal density is plotted for AS–up-regulated STAT5-binding sites ±2 kb. (D) STAT5-binding peaks were separated into two groups, i.e., unchanged or up-regulated peaks after AS treatment, and the frequency of the peaks located in promoter, intron, exon, intergenic, 3′ untranslated region (3′UTR) or 5′UTR of mRNAs, noncoding RNAs (ncRNA), and others was calculated in each group by using annotatePeaks.pl. of homer v4.8 in default settings. (E) Cumulative histogram of fold change on average (AS–up-regulated versus AS unchanged STAT5 peak sites) of three independent RNA-seq results. STAT5-binding peaks were classified into proximal promoter or enhancer regions (within 1000 bases in the H3K4me1 or H3K27ac-positive region). Statistical significance was determined by the Kolmogorov-Smirnov test.

  • Fig. 5 In vivo induction of antigen-specific pTreg cells by AS.

    (A) Flow cytometry of Foxp3+ cells in DO11.10RAG2−/−Foxp3-eGFP reporter mice with or without subcutaneous OVA immunization. CD4+ T cells in the draining lymph nodes of mice treated orally with AS (30 mg/kg) every day for 7 days were stained for Foxp3 on day 8. Representative staining (left) and percentages of Foxp3+ cells among CD4+ T cells (n = 6) (right). (B) Gene expression pattern of Foxp3+ T cells from OVA- and AS-treated DO11.10RAG2−/−Foxp3-eGFP reporter mice (designated as DO/Rag AS-pTreg), as shown in (A), compared with Foxp3+ T cells (DO nTreg) and Tconv cells (DO effector T cells) in OVA-immunized DO11.10 mice or Tconv cells (DO naїve T cells) in OVA-nonimmunized mice. Heat map shows the expression of Treg signature genes analyzed by RNA-seq. Hierarchical cluster analysis was conducted on all expressed genes. (C) Flow cytometry of Treg signature molecules expressed by AS-iTreg cells (AS-pTreg) in (A) or nTreg cells from OVA-immunized DO.11.10 mice. Itgb8 expression was assessed by mRNA staining. Data are representative of at least three experiments. (D) Bisulfite sequencing showing Treg-specific demethylated regions at Foxp3 CNS2, Eos int1b, and Helios int3a. Data are representative of two to four experiments. (E) Suppression assay performed by coculturing of VPD-labeled responder CD4+ T cells, APCs, and nTreg or pTreg cells, shown in (A), in the presence of soluble anti-CD3 mAb. Representative result (left) and total results (n = 3) (right). Vertical bars indicate means ± SD. Statistical significance was assessed by the SNK method, Student’s t test, or Kaplan-Meier method. **P < 0.01 and *P < 0.05.

  • Fig. 6 Therapeutic effects of AS on autoimmunity and allergy.

    (A to E) AS treatment of DNFB-induced contact skin hypersensitivity. Mice expressing diphtheria toxin receptor under the Foxp3 promoter were sensitized epicutaneously with DNFB on the abdominal skin on days 0 and 7 and challenged on day 14 by applying DNFB on the ear, whereas some of them were orally administered with AS (30 mg/kg) daily for 2 weeks (n = 4). A group of mice (n = 6) were also treated with diphtheria toxin daily from days 0 to 14 to deplete Foxp3+ cells. Ear swelling was measured 24 hours after challenge (A), with preparation of histology (hematoxylin and eosin staining) (B). Percentages of Foxp3+ cells (C) and IFN-γ+ cells (D) among CD4+ T cells and KLRG1+ cells among Foxp3+CD4+ T cells (E) in the regional lymph nodes on day 14 (n = 3 or 4). (F) AS treatment of NOD mice. NOD mice (n = 10) were treated daily with AS (30 mg/kg, p.o.) from 8 to 10 weeks of age and assessed for urinary glucose once a week (left). Histological severity of insulitis was scored from 0 to 2 at 10 weeks of age (n = 9 to 11) (middle and right). (G) Percentages of Foxp3+ T cells among CD4+ T cells and KLRG1+Foxp3+CD4+ T cells among Foxp3+CD4+ T cells in the regional lymph nodes of NOD mice (n = 4) were assessed by flow cytometry. (H) Percentages of IFN-γ+ T cells in the regional lymph nodes of 10-week-old NOD mice (n = 4) were assessed by flow cytometry. (I and J) EAE was induced by MOG immunization, and AS (30 mg/kg) was administered daily from day 0 to day 14 (n = 7 or 8 per group). (I) EAE clinical scores and histology of spinal cords were assessed from day 8 to 28 after immunization. Spinal cords from EAE-induced mice were stained with Luxol fast blue. Percentages of Foxp3+, KLRG1+, and IL-17+ cells (J) among CD4+ T cells in the regional lymph nodes were analyzed by flow cytometry on day 14 (n = 3 or 4). Vertical bars indicate means ± SD. Statistical significance was assessed by the SNK method, Student’s t test, or Kaplan-Meier method. **P < 0.01 and *P < 0.05. AUC, area under the curve.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/40/eaaw2707/DC1

    Fig. S1. AS dose, TGF-β, and IL-2 in AS-dependent in vitro induction of Foxp3+ T cells.

    Fig. S2. In vitro induction of FOXP3 in human Tconv cells by AS.

    Fig. S3. Inability of AS to induce Treg-specific DNA hypomethylation in vitro in Tconv cells.

    Fig. S4. In vitro effects of AS on the proliferative activity and the phenotype of nTreg cells.

    Fig. S5. Effects of AS on gene expression by T cells, B cells, and DCs.

    Fig. S6. Identification of AS targets by chemical proteomics.

    Fig. S7. Dominant–negative type impaired kinase activity of KD CDK8 or CDK19 and Foxp3 induction in CD4+ T cells by their retroviral expression.

    Fig. S8. Generation of CDK8/19 KO EL4 cell lines.

    Fig. S9. Retroviral expression of KD or WT CDK8 in CD4+ T cells.

    Fig. S10. Effects of AS on serine phosphorylation of STAT5.

    Fig. S11. Effects of AS on genome-wide STAT5 binding.

    Fig. S12. Plasma concentration and body weight of mice treated with various doses of AS.

    Fig. S13. Induction of pTreg cells by AS treatment and antigen immunization.

    Fig. S14. RNA-seq analysis of AS-induced pTreg cells.

    Fig. S15. IL-2 augments in vivo AS-mediated pTreg induction.

    Fig. S16. Activation of DCs by AS.

    Fig. S17. Suppression of OVA-induced DTH by AS.

    Fig. S18. In vitro Foxp3-inducing effects of AS in the presence of various inhibitors.

    Fig. S19. Inhibition of STAT5 by CDK8/19 in activated Tconv cells and their Foxp3 expression induced by CDK8/19 inhibition via activating STAT5.

    Table S1. Kinase selectivity profiles of AS2863619 and AS3334366.

    Table S2. List of antibodies.

    Table S3. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. AS dose, TGF-β, and IL-2 in AS-dependent in vitro induction of Foxp3+ T cells.
    • Fig. S2. In vitro induction of FOXP3 in human Tconv cells by AS.
    • Fig. S3. Inability of AS to induce Treg-specific DNA hypomethylation in vitro in Tconv cells.
    • Fig. S4. In vitro effects of AS on the proliferative activity and the phenotype of nTreg cells.
    • Fig. S5. Effects of AS on gene expression by T cells, B cells, and DCs.
    • Fig. S6. Identification of AS targets by chemical proteomics.
    • Fig. S7. Dominant–negative type impaired kinase activity of KD CDK8 or CDK19 and Foxp3 induction in CD4+ T cells by their retroviral expression.
    • Fig. S8. Generation of CDK8/19 KO EL4 cell lines.
    • Fig. S9. Retroviral expression of KD or WT CDK8 in CD4+ T cells.
    • Fig. S10. Effects of AS on serine phosphorylation of STAT5.
    • Fig. S11. Effects of AS on genome-wide STAT5 binding.
    • Fig. S12. Plasma concentration and body weight of mice treated with various doses of AS.
    • Fig. S13. Induction of pTreg cells by AS treatment and antigen immunization.
    • Fig. S14. RNA-seq analysis of AS-induced pTreg cells.
    • Fig. S15. IL-2 augments in vivo AS-mediated pTreg induction.
    • Fig. S16. Activation of DCs by AS.
    • Fig. S17. Suppression of OVA-induced DTH by AS.
    • Fig. S18. In vitro Foxp3-inducing effects of AS in the presence of various inhibitors.
    • Fig. S19. Inhibition of STAT5 by CDK8/19 in activated Tconv cells and their Foxp3 expression induced by CDK8/19 inhibition via activating STAT5.
    • Table S1. Kinase selectivity profiles of AS2863619 and AS3334366.
    • Table S2. List of antibodies.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data.

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