Abstract
Regulatory T (Treg) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving Treg cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral Treg cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral Treg cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor Treg cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of H3K9me2 demethylase, leading to robust interferon-γ production and tumor Treg cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral Treg cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor Treg cell fitness, and we present a specific JMJD1C inhibitor that can target tumor Treg cells without affecting systemic immune homeostasis.
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Data availability
scRNA-seq data have been deposited in the GEO database under accession code GSE223930. ATAC-seq and CUT&RUN-seq data have been deposited in GEO under accession code GSE224084. Publicly available datasets accessed for use in this manuscript include GSE139325, GSE108989, GSE98638 and GSE99254. Source data are provided with this paper.
Code availability
The codes for scRNA-seq, ATAC-seq and CUT&RUN data analyses reported in this study have been deposited at GitHub (https://github.com/YuliangWang316/JMJD1C_Treg). Any additional information required to re-analyze the data is available from the corresponding authors upon request.
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Acknowledgements
We thank Z. Qin (Institute of Biophysics, Chinese Academy of Sciences) for providing MCA205 cells. This study was supported by the National Natural Science Foundation of China (grant numbers 32330036 to X.W., T222502 to M.Z., 81825018 to J.Q., 82301970 to X. Long, 82130085 to J.Q., 82101827 to Jingjing Chen, 82273855 to M.Z., 82204278 to X. Li and 31970828 to X.W.), National Key Research and Development Program of China (2022YFC3400504 to M.Z.), Youth Innovation Promotion Association CAS (2023296 to S. Zhang), Lingang Laboratory (LG202102-01-02 to M.Z. and LG-QS-202204-01 to S. Zhang), China Postdoctoral Science Foundation (2022M721675 to X. Long, 2020M681665 to Jingjing Chen and 2022M720153 to X. Li), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX22_1784 to Z.Z. and JX10113833 to S.W.), Postdoc Research Program of Taizhou School of Clinical Medicine (TZBSHKY202203 to X. Long), Major Program of Wuxi Medical Center, Nanjing Medical University (WMCM202310 to X.W.), Jiangsu Provincial Key Research Development Program of China (BE2022770 to Y.C.) and Jiangsu Outstanding Young Investigator Program (BK20200030 to X.W.).
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Contributions
X.W., J.Q. and M.Z. conceived and directed the study. X. Long, Y.W., Jingjing Chen, Y.L., Y.C. and B.L. performed most of the animal and cellular biology experiments. S. Zhang, H.H., X. Li, C.S., R.Y., D.C., G.C. and D.W. designed and performed the experiments regarding the small compound. R.C. performed the pharmacokinetic study of the inhibitor. Y.W. performed the bioinformatic analysis. H.Z., S. Zhai, Z.Z., S.W., M.L., J.Z. and Junhong Chen helped with mouse care and some in vitro experiments. X.W., J.Q. and M.Z. wrote the manuscript with input from all authors.
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Nature Immunology thanks Axel Kallies and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team.
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Extended data
Extended Data Fig. 1 Slingshot analysis based pseudotime analysis.
Slingshot analysis based pseudotime analysis was derived for the three populations of Treg from Fig. 1g.
Extended Data Fig. 2 JMJD1C is dispensable for systemic Treg development under steady state.
a, Splenic Treg cells were sorted out from Jmjd1cTreg WT and Jmjd1cTreg KO, treated with TNF plus IL-6 for 3 days, and subject to western blot analysis with anti-JMJD1C. b, Comparison of Treg cell cellularity in spleen and lymph nodes between Jmjd1cTreg WT (n = 7) and Jmjd1cTreg KO (n = 6) mice. pLN, peripheral lymphoid nodes. c, Representative flow cytometric plots of splenic CD4+ T cell subsets (up) or CD8+ T cell subsets(down) : naïve T (CD44− CD62L+) and T effector memory (CD44+CD62L–) cells from Jmjd1cTreg WT (n = 7) and Jmjd1cTreg KO (n = 6) mice. d, Representative images of hematoxylin and eosin staining for the indicated tissues from Jmjd1cTreg WT or Jmjd1cTreg KO mice at the age of 6-month-old. Scale bars: spleen 500 µm, colon 500 µm, lung 200 µm, liver 100 µm, kidney 50 µm. e, Frequency of Treg cells in different tissues as indicated from 6-month-old mice. Data represent two (a) or three (d) independent experiments. Data were pooled from 2 independent experiments (b, c, e). Data in (b, c, e) are shown in means ± SD. Two-tailed unpaired Student’s t-test (b, c, e).
Extended Data Fig. 3 Characterization of tumor Treg expansion, apoptosis and suppressive function.
a, Gating strategy for tumor Treg. b,c, Representative flow cytometric plots of Ki67 (b) and active caspase3 (c) levels in splenic Treg cells or intratumoral Treg cells from MCA205-tumor-bearing Jmjd1cTreg WT (n = 6) and Jmjd1cTreg KO (n = 7) mice. d, e, Ex vivo suppression of CellTrace Violet (CTV)-labeled WT naïve CD8+ T cell proliferation by Jmjd1cTreg WT and Jmjd1cTreg KO Treg cells sorted from tumors (d) or spleens (e) with the annotated co-culture ratios. Teff, CD8+ T effector cells. n = 3 biological independent samples. Data represent three independent experiments (b-e). Data in (b-e) are shown in means ± SD. Two-tailed unpaired Student’s t-test (b-e).
Extended Data Fig. 4 scRNA-seq analysis of tumor Treg cells from MCA205-tumor-bearing mice.
a, Unsupervised clustering of tumor-infiltrating Treg cells from MCA205-tumor-bearing Jmjd1cTreg WT (4 mice) and Jmjd1cTreg KO (7 mice) mice. b, Dot plot showing the expression of marker genes for each cluster. c, Tumor Treg cells from MCA205-tumor-bearing Jmjd1cTreg WT (n = 6 mice) and Jmjd1cTreg KO mice (n = 7 mice) were stained for 4-1BB expression by flow cytometry. d, Tumor-infiltrating Treg cells cells were sorted from MCA205-tumor-bearing Jmjd1cTreg WT and Jmjd1cTreg KO mice, the protein levels of H3K9me2 and H3K9me1 were assessed by western blot. e, Volcano plot showing the genes differentially expressed in Jmjd1c WT and KO tumor Treg cells from the scRNA-seq data. P values were computed with two-sided Wilcoxon rank-sum test in Seurat and adjusted with Bonferroni method. f, Gene Set Enrichment Analysis (GSEA) showing the significantly enriched signatures among all the 50 hallmark gene sets in Jmjd1c WT versus Jmjd1c KO tumor Treg cells. P values were computed with empirical phenotype-based permutation tests (GSEA), and adjusted for multiple comparisons using the Benjamini–Hochberg method (FDR). Data represent two independent experiments (c, d). Data are shown in means ± SD (c). Two-tailed unpaired Student’s t-test (c).
Extended Data Fig. 5 Cytokine production in tumor effector T cells from WT and Stat3CATreg OE mice.
a, Representative flow cytometric plots of IFNγ and TNF production in Foxp3−CD4+ and CD8+ tumor-infiltrating T cells (left). Percentage of cytokine-producing T cells in dLN and tumor were plotted on the right. n = 5 mice. b, Representative flow cytometric plots of IL-17A production in Foxp3-CD4+ tumor-infiltrating T cells (left). Percentage of IL-17A-producing T cells were plotted on the right. n = 5 mice. Data represent two independent experiments. Data in (a, b) are shown in means ± SD. Two-tailed unpaired Student’s t-test (a-b).
Extended Data Fig. 6 193D7 treatment suppresses tumor growth.
C57BL6/J mice were subcutaneously inoculated with B16-F10, LLC or Hepa1-6 tumor cells. BALB/C mice were subcutaneously inoculated with CT26 tumor cells. When tumor grew to measurable size (~100 mm3), the tumor bearing mice were intraperitoneally treated with DMSO control or 193D7 at 25 mg/kg once a day, tumor growth was monitored. n = 6 mice. Data represent two independent experiments. Data are shown in means ± SD. Two-way ANOVA was used.
Extended Data Fig. 7 193D7 treatment shows no evidence of toxicity in mice.
C57BL6/J mice were intraperitoneally treated with DMSO control or 193D7 at 25mg/kg once a day for 14 days. n = 6 mice per group. a, Body weight was monitored over time. b, Quantification of alanine aminotransferase (ALT), alkaline phosphatase (ALP) in the serum at day 14. c, Representative images of hematoxylin and eosin staining for the indicated tissues from DMSO control or 193D7-treated mice at day 14. Scale bars: colon 500 µm, lung 200µm, liver 100 µm, kidney 50 µm. Data represent two independent experiments (a-c). Data are shown in means ± SD (a, b). Two-way ANOVA (a); two-tailed unpaired Student’s t-test (b).
Extended Data Fig. 8 193D7 treatment increased tumor effector T cell number and cytokine production by in wildtype mice.
C57BL6/J mice bearing MCA205 tumor were orally treated with DMSO control or 193D7 at 25 mg/kg once a day for 12 days as in Fig. 6j. DMSO, n = 7 mice; 193D7, n = 8 mice. a, Cell number of CD4+ and CD8+ T cells per gram tumor in MCA205-tumor-bearing mice. b, Percentage of cytokine-producing T cells in dLNs and tumors. Data represent two independent experiments. Data are shown in means ± SD. Two-tailed unpaired Student’s t-test (a, b).
Extended Data Fig. 9 193D7 treatment did not alter tumor effector T cell number and cytokine production in Jmjd1cTreg KO mice.
Jmjd1cTreg KO mice bearing MCA205 tumor were orally treated with DMSO control or 193D7 at 25mg/kg once a day for 12 days as in Fig. 6p. n = 5 mice. a, Cell number of CD4+ and CD8+ T cells per gram tumor in MCA205-tumor-bearing mice. b, Percentage of cytokine-producing T cells in tumors. Data represent two independent experiments. Data are shown in means ± SD (a, b). Two-tailed unpaired Student’s t-test (a, b).
Extended Data Fig. 10 Analysis of the effect of 193D7 treatment on tumor Treg cells in mixed bone marrow chimeras.
a, The experimental design of mixed bone marrow chimeras. b, The Treg frequency in tumor was analyzed by flow cytometry in tumor-bearing mixed chimeras. n = 6 mice. c, Lymphocytes were isolated from tumor tissues, stimulated and stained for IFNγ in tumor Treg cells. n = 6 mice. Data represent two independent experiments. Plotting data in (b, c) are all shown in means ± SD. Two-tailed unpaired Student’s t-test (b, c).
Supplementary information
Supplementary Information
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Supplementary Tables
Supplementary Table 1: list of genes with alterations in H3K9me2 signal and mRNA expression. Supplementary Table 2: top 100 candidate JMJD1C inhibitors.
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Long, X., Zhang, S., Wang, Y. et al. Targeting JMJD1C to selectively disrupt tumor Treg cell fitness enhances antitumor immunity. Nat Immunol 25, 525–536 (2024). https://doi.org/10.1038/s41590-024-01746-8
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DOI: https://doi.org/10.1038/s41590-024-01746-8
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