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Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

Mucosal Immunology (2018) | Download Citation



Notch ligand Delta-like ligand 4 (DLL4) has been shown to regulate CD4 T-cell differentiation, including regulatory T cells (Treg). Epigenetic alterations, which include histone modifications, are critical in cell differentiation decisions. Recent genome-wide studies demonstrated that Treg have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around the Treg master transcription factor, Foxp3 loci. Here we report that DLL4 dynamically increased H3K4 methylation around the Foxp3 locus that was dependent upon upregulated SET and MYDN domain containing protein 3 (SMYD3). DLL4 promoted Smyd3 through the canonical Notch pathway in iTreg differentiation. DLL4 inhibition during pulmonary respiratory syncytial virus (RSV) infection decreased Smyd3 expression and Foxp3 expression in Treg leading to increased Il17a. On the other hand, DLL4 supported Il10 expression in vitro and in vivo, which was also partially dependent upon SMYD3. Using genome-wide unbiased mRNA sequencing, novel sets of DLL4- and Smyd3-dependent differentially expressed genes were discovered, including lymphocyte-activation gene 3 (Lag3), a checkpoint inhibitor that has been identified for modulating Th cell activation. Together, our data demonstrate a novel mechanism of DLL4/Notch-induced Smyd3 epigenetic pathways that maintain regulatory CD4 T cells in viral infections.

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  1. 1.

    Curotto de Lafaille, M. A. et al. Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation. Immunity 29, 114–126 (2008).

  2. 2.

    Huang, H., Ma, Y., Dawicki, W., Zhang, X. & Gordon, J. R. Comparison of induced versus natural regulatory T cells of the same TCR specificity for induction of tolerance to an environmental antigen. J. Immunol. 191, 1136–1143 (2013).

  3. 3.

    Josefowicz, S. Z. et al. Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 482, 395–399 (2012).

  4. 4.

    Durant, L. R. et al. Regulatory T cells prevent Th2 immune responses and pulmonary eosinophilia during respiratory syncytial virus infection in mice. J. Virol. 87, 10946–10954 (2013).

  5. 5.

    Loebbermann, J. et al. Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection. Mucosal Immunol. 5, 161–172 (2012).

  6. 6.

    Fulton, R. B., Meyerholz, D. K. & Varga, S. M. Foxp3+ CD4 regulatory T cells limit pulmonary immunopathology by modulating the CD8 T cell response during respiratory syncytial virus infection. J. Immunol. 185, 2382–2392 (2010).

  7. 7.

    Chen, W. et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003).

  8. 8.

    Tone, Y. et al. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat. Immunol. 9, 194–202 (2008).

  9. 9.

    Rubtsov, Y. P. et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28, 546–558 (2008).

  10. 10.

    Maynard, C. L. et al. Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3- precursor cells in the absence of interleukin 10. Nat. Immunol. 8, 931–941 (2007).

  11. 11.

    Weiss, K. A., Christiaansen, A. F., Fulton, R. B., Meyerholz, D. K. & Varga, S. M. Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection. J. Immunol. 187, 3145–3154 (2011).

  12. 12.

    Loebbermann, J. et al. IL-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice. PLoS ONE 7, e32371 (2012).

  13. 13.

    Massoud, A. H. et al. An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells. Nat. Med. 22, 1013–1022 (2016).

  14. 14.

    Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).

  15. 15.

    Ohkura, N., Kitagawa, Y. & Sakaguchi, S. Development and maintenance of regulatory T cells. Immunity 38, 414–423 (2013).

  16. 16.

    Wilson, C. B., Rowell, E. & Sekimata, M. Epigenetic control of T-helper-cell differentiation. Nat. Rev. Immunol. 9, 91–105 (2009).

  17. 17.

    Wei, G. et al. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity 30, 155–167 (2009).

  18. 18.

    Miyao, T. et al. Plasticity of Foxp3(+) T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity 36, 262–275 (2012).

  19. 19.

    Zhou, X. et al. Foxp3 instability leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10, 1000–1007 (2009).

  20. 20.

    Lal, G. et al. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J. Immunol. 182, 259–273 (2009).

  21. 21.

    Yue, X. et al. Control of Foxp3 stability through modulation of TET activity. J. Exp. Med. 213, 377–397 (2016).

  22. 22.

    DuPage, M. et al. The chromatin-modifying enzyme Ezh2 is critical for the maintenance of regulatory T cell identity after activation. Immunity 42, 227–238 (2015).

  23. 23.

    Arvey, A. et al. Inflammation-induced repression of chromatin bound by the transcription factor Foxp3 in regulatory T cells. Nat. Immunol. 15, 580–587 (2014).

  24. 24.

    Hamamoto, R. et al. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat. Cell Biol. 6, 731–740 (2004).

  25. 25.

    Nagata, D. E. et al. Epigenetic control of Foxp3 by SMYD3 H3K4 histone methyltransferase controls iTreg development and regulates pathogenic T-cell responses during pulmonary viral infection. Mucosal Immunol. 8, 1131–1143 (2015).

  26. 26.

    Sandy, A. R. & Maillard, I. Notch signaling in the hematopoietic system. Expert Opin. Biol. Ther. 9, 1383–1398 (2009).

  27. 27.

    Maillard, I., Adler, S. H. & Pear, W. S. Notch and the immune system. Immunity 19, 781–791 (2003).

  28. 28.

    Radtke, F., Fasnacht, N. & Macdonald, H. R. Notch signaling in the immune system. Immunity 32, 14–27 (2010).

  29. 29.

    Bailis, W. et al. Notch simultaneously orchestrates multiple helper T cell programs independently of cytokine signals. Immunity 39, 148–159 (2013).

  30. 30.

    Fang, T. C. et al. Notch directly regulates Gata3 expression during T helper 2 cell differentiation. Immunity 27, 100–110 (2007).

  31. 31.

    Mukherjee, S., Schaller, M. A., Neupane, R., Kunkel, S. L. & Lukacs, N. W. Regulation of T cell activation by Notch ligand, DLL4, promotes IL-17 production and Rorc activation. J. Immunol. 182, 7381–7388 (2009).

  32. 32.

    Elyaman, W. et al. Notch receptors and Smad3 signaling cooperate in the induction of interleukin-9-producing T cells. Immunity 36, 623–634 (2012).

  33. 33.

    Samon, J. B. et al. Notch1 and TGFbeta1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells. Blood 112, 1813–1821 (2008).

  34. 34.

    Ting, H.-A. et al. Notch ligand delta-like 4 promotes regulatory T cell identity in pulmonary viral infection. J. Immunol. Baltim. Md. 1950 198, 1492–1502 (2017).

  35. 35.

    Huang, M.-T. et al. Notch ligand DLL4 alleviates allergic airway inflammation via induction of a homeostatic regulatory pathway. Sci. Rep. 7, 43535 (2017).

  36. 36.

    Charbonnier, L.-M., Wang, S., Georgiev, P., Sefik, E. & Chatila, T. A. Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling. Nat. Immunol. 16, 1162–1173 (2015).

  37. 37.

    Tu, L. et al. Notch signaling is an important regulator of type 2 immunity. J. Exp. Med. 202, 1037–1042 (2005).

  38. 38.

    Maillard, I. et al. Mastermind critically regulates Notch-mediated lymphoid cell fate decisions. Blood 104, 1696–1702 (2004).

  39. 39.

    Sandy, A. R. et al. Notch signaling regulates T cell accumulation and function in the central nervous system during experimental autoimmune encephalomyelitis. J. Immunol. 191, 1606–1613 (2013).

  40. 40.

    Lukacs, N. W. et al. Differential immune responses and pulmonary pathophysiology are induced by two different strains of respiratory syncytial virus. Am. J. Pathol. 169, 977–986 (2006).

  41. 41.

    Schaller, M. A. et al. Notch ligand Delta-like 4 regulates disease pathogenesis during respiratory viral infections by modulating Th2 cytokines. J. Exp. Med. 204, 2925–2934 (2007).

  42. 42.

    Miller, A. L., Bowlin, T. L. & Lukacs, N. W. Respiratory syncytial virus-induced chemokine production: linking viral replication to chemokine production in vitro and in vivo. J. Infect. Dis. 189, 1419–1430 (2004).

  43. 43.

    Amsen, D. et al. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117, 515–526 (2004).

  44. 44.

    Wen, H., Dou, Y., Hogaboam, C. M. & Kunkel, S. L. Epigenetic regulation of dendritic cell-derived interleukin-12 facilitates immunosuppression after a severe innate immune response. Blood 111, 1797–1804 (2008).

  45. 45.

    Collison, L. W. & Vignali, D. A. A. In vitro Treg suppression assays. Methods Mol. Biol. 707, 21–37 (2011).

  46. 46.

    Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

  47. 47.

    Anders, S., Pyl, P. T. & Huber, W. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinforma. Oxf. Engl. 31, 166–169 (2015).

  48. 48.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  49. 49.

    Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 6, e21800 (2011).

  50. 50.

    Ohkura, N. et al. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity 37, 785–799 (2012).

  51. 51.

    Zhou, L., Chong, M. M. W. & Littman, D. R. Plasticity of CD4+ T cell lineage differentiation. Immunity 30, 646–655 (2009).

  52. 52.

    Dillon, S. C., Zhang, X., Trievel, R. C. & Cheng, X. The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol. 6, 227 (2005).

  53. 53.

    Castel, D. et al. Dynamic binding of RBPJ is determined by Notch signaling status. Genes Dev. 27, 1059–1071 (2013).

  54. 54.

    Borchers, A. T., Chang, C., Gershwin, M. E. & Gershwin, L. J. Respiratory syncytial virus--a comprehensive review. Clin. Rev. Allergy Immunol. 45, 331–379 (2013).

  55. 55.

    Hashimoto, K. et al. Respiratory syncytial virus infection in the absence of STAT 1 results in airway dysfunction, airway mucus, and augmented IL-17 levels. J. Allergy Clin. Immunol. 116, 550–557 (2005).

  56. 56.

    Mukherjee, S. et al. IL-17-induced pulmonary pathogenesis during respiratory viral infection and exacerbation of allergic disease. Am. J. Pathol. 179, 248–258 (2011).

  57. 57.

    Stier, M. T. et al. Respiratory syncytial virus infection activates IL-13-producing group 2 innate lymphoid cells through thymic stromal lymphopoietin. J. Allergy Clin. Immunol. 138, 814–824.e11 (2016).

  58. 58.

    Hill, J. A. et al. Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity 27, 786–800 (2007).

  59. 59.

    Gavin, M. A. et al. Foxp3-dependent programme of regulatory T-cell differentiation. Nature 445, 771–775 (2007).

  60. 60.

    van der Veeken, J. et al. Memory of inflammation in regulatory T cells. Cell 166, 977–990 (2016).

  61. 61.

    Placek, K. et al. MLL4 prepares the enhancer landscape for Foxp3 induction via chromatin looping. Nat. Immunol. 18, 1035–1045 (2017).

  62. 62.

    Mazur, P. K. et al. SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer. Nature 510, 283–287 (2014).

  63. 63.

    Liu, H. et al. Elevated levels of SET and MYND domain-containing protein 3 are correlated with overexpression of transforming growth factor-β1 in gastric cancer. J. Am. Coll. Surg 221, 579–590 (2015).

  64. 64.

    Kim, H. et al. Requirement of histone methyltransferase SMYD3 for estrogen receptor-mediated transcription. J. Biol. Chem. 284, 19867–19877 (2009).

  65. 65.

    Liu, C. et al. SMYD3 as an oncogenic driver in prostate cancer by stimulation of androgen receptor transcription. J. Natl Cancer Inst 105, 1719–1728 (2013).

  66. 66.

    Koch, M. A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat. Immunol. 10, 595–602 (2009).

  67. 67.

    Zheng, Y. et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses. Nature 458, 351–356 (2009).

  68. 68.

    Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 326, 986–991 (2009).

  69. 69.

    Jankovic, D. et al. In the absence of IL-12, CD4(+) T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10(-/-) setting. Immunity 16, 429–439 (2002).

  70. 70.

    Skokos, D. & Nussenzweig, M. C. CD8- DCs induce IL-12-independent Th1 differentiation through Delta 4 Notch-like ligand in response to bacterial LPS. J. Exp. Med. 204, 1525–1531 (2007).

  71. 71.

    Kojima, S., Nara, K. & Rifkin, D. B. Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells. J. Cell Biol. 121, 439–448 (1993).

  72. 72.

    Wang, Z. & Griffin, M. TG2, a novel extracellular protein with multiple functions. Amino Acids 42, 939–949 (2012).

  73. 73.

    Telci, D., Collighan, R. J., Basaga, H. & Griffin, M. Increased TG2 expression can result in induction of transforming growth factor beta1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide. J. Biol. Chem. 284, 29547–29558 (2009).

  74. 74.

    Cao, L. et al. Tissue transglutaminase links TGF-β, epithelial to mesenchymal transition and a stem cell phenotype in ovarian cancer. Oncogene 31, 2521–2534 (2012).

  75. 75.

    Shao, M. et al. Epithelial-to-mesenchymal transition and ovarian tumor progression induced by tissue transglutaminase. Cancer Res. 69, 9192–9201 (2009).

  76. 76.

    Huang, C.-T. et al. Role of LAG-3 in regulatory T cells. Immunity 21, 503–513 (2004).

  77. 77.

    Do, J.-S. et al. An IL-27/Lag3 axis enhances Foxp3+ regulatory T cell-suppressive function and therapeutic efficacy. Mucosal Immunol 9, 137–145 (2016).

  78. 78.

    Sega, E. I. et al. Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation. PLoS ONE 9, e86551 (2014).

  79. 79.

    Haribhai, D. et al. A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity. Immunity 35, 109–122 (2011).

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HT, DDN, MAS, and NWL designed the experiments. HT, AJR, DDN, and CM performed experiments. HT and NWL did data analysis and wrote the manuscript. We thank Dr. Matthew A Schaller and Consulting for Statistics, Computation, and Analytical Research (CSCAR) for consultations; Ivan Maillard for helpful discussions; Susan Morris, Lisa Riggs Johnson, for technical assistance; and Dr. Judith Connett for editing the manuscript. The manuscript was supported in part by NIH grant AI036302 (NWL).

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Author notes

    • Denise de Almeida Nagata

    Present address: Department of Cancer Immunology, Genentech, South San Francisco, CA, 94080, USA

    • Ivan P Maillard

    Present address: Department of Medicine, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA


  1. Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA

    • Hung-An Ting
    • , Denise de Almeida Nagata
    • , Andrew J Rasky
    • , Carrie-Anne Malinczak
    • , Matthew A Schaller
    •  & Nicholas W Lukacs
  2. Molecular and Cellular Pathology Program, University of Michigan, Ann Arbor, MI, 48109, USA

    • Hung-An Ting
    • , Carrie-Anne Malinczak
    •  & Nicholas W Lukacs
  3. Department of Internal Medicine, Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109, USA

    • Ivan P Maillard
  4. Department of Cell and Developmental Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109, USA

    • Ivan P Maillard


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Correspondence to Nicholas W Lukacs.

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