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Foxp3 expression in induced T regulatory cells derived from human umbilical cord blood vs. adult peripheral blood

Bone Marrow Transplantation (2018) | Download Citation

Abstract

Foxp3 is essential for T regulatory cell (Treg) function. Broad complex-Tramtrack-Bric-a-brac domain (BTB) and Cap‘n’collar (CNC) homology 1, transcription factor 2 (BACH2) stabilizes Treg immune homeostasis in murine studies. However, little is known regarding what role, if any, BACH2 may have in Foxp3 regulation in human-induced Treg (iTreg). We examined Foxp3 expression and regulation comparing iTreg differentiated from umbilical cord blood (UCB) vs. adult blood (AB) naive CD4+ T-cells. Foxp3 expression was higher in UCB vs. AB-derived iTreg, and was sustained during 21-day expansion in vitro. The number of Foxp3+ iTreg generated from UCB vs. AB naive CD4+ T-cells was higher in iTreg differentiation conditions. In addition, UCB iTreg were more potent in suppressing T-cell proliferation compared to AB iTreg. Naive UCB CD4+ T-cells highly expressed BACH2 protein compared to AB. Putative transcriptional BACH2 binding sites were identified at the Foxp3 promoter, using BACH2 consensus sequence. Cross-linking chromatin immunoprecipitation (ChIP) showed that BACH2 binds to the Foxp3 proximal promoter in UCB iTreg, but not AB iTreg. BACH2 was transcriptionally active, as shRNA-mediated BACH2 knockdown resulted in reduction of Foxp3 gene transcription in UCB CD4+ T-cells. In summary, BACH2 serves to stabilize robust Foxp3 expression in UCB CD4+ T-cell-derived iTreg.

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References

  1. 1.

    Brunstein CG, Miller JS, McKenna DH, Hippen KL, DeFor TE, Sumstad D, et al. Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect. Blood. 2016;127:1044–51.

  2. 2.

    Brunstein CG, Miller JS, Cao Q, McKenna DH, Hippen KL, Curtsinger J, et al. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. Blood. 2011;117:1061–70.

  3. 3.

    Di Ianni M, Falzetti F, Carotti A, Terenzi A, Castellino F, Bonifacio E, et al. Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood. 2011;117:3921–8.

  4. 4.

    Hippen KL, Merkel SC, Schirm DK, Nelson C, Tennis NC, Riley JL, et al. Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease. Am J Transplant. 2011;11:1148–57.

  5. 5.

    Schmitt EG, Haribhai D, Williams JB, Aggarwal P, Jia S, Charbonnier LM, et al. IL-10 produced by induced regulatory T cells (iTregs) controls colitis and pathogenic ex-iTregs during immunotherapy. J Immunol. 2012;189:5638–48.

  6. 6.

    Beres A, Komorowski R, Mihara M, Drobyski WR. Instability of Foxp3 expression limits the ability of induced regulatory T cells to mitigate graft versus host disease. Clin Cancer Res. 2011;17:3969–83.

  7. 7.

    Koenecke C, Czeloth N, Bubke A, Schmitz S, Kissenpfennig A, Malissen B, et al. Alloantigen-specific de novo-induced Foxp3+ Treg revert in vivo and do not protect from experimental GVHD. Eur J Immunol. 2009;39:3091–6.

  8. 8.

    Bennett CL, Ochs HD. IPEX is a unique X-linked syndrome characterized by immune dysfunction, polyendocrinopathy, enteropathy, and a variety of autoimmune phenomena. Curr Opin Pediatr. 2001;13:533–8.

  9. 9.

    Fontenot JD, Rudensky AY. Molecular aspects of regulatory T cell development. Semin Immunol. 2004;16:73–80.

  10. 10.

    Mantel PY, Ouaked N, Ruckert B, Karagiannidis C, Welz R, Blaser K, et al. Molecular mechanisms underlying FOXP3 induction in human T cells. J Immunol. 2006;176:3593–602.

  11. 11.

    Tone Y, Furuuchi K, Kojima Y, Tykocinski ML, Greene MI, Tone M. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat Immunol. 2008;9:194–202.

  12. 12.

    Jana S, Jailwala P, Haribhai D, Waukau J, Glisic S, Grossman W, et al. The role of NF-kappaB and Smad3 in TGF-beta-mediated Foxp3 expression. Eur J Immunol. 2009;39:2571–83.

  13. 13.

    Popmihajlov Z, Smith KA. Negative feedback regulation of T cells via interleukin-2 and FOXP3 reciprocity. PLoS ONE. 2008;3:e1581.

  14. 14.

    Roychoudhuri R, Hirahara K, Mousavi K, Clever D, Klebanoff CA, Bonelli M, et al. BACH2 represses effector programs to stabilize T(reg)-mediated immune homeostasis. Nature. 2013;498:506–10.

  15. 15.

    Kim EH, Gasper DJ, Lee SH, Plisch EH, Svaren J, Suresh M. Bach2 regulates homeostasis of Foxp3+ regulatory T cells and protects against fatal lung disease in mice. J Immunol. 2014;192:985–95.

  16. 16.

    Chen C, Rowell EA, Thomas RM, Hancock WW, Wells AD. Transcriptional regulation by Foxp3 is associated with direct promoter occupancy and modulation of histone acetylation. J Biol Chem. 2006;281:36828–34.

  17. 17.

    Milano F, Gooley T, Wood B, Woolfrey A, Flowers ME, Doney K, et al. Cord-blood transplantation in patients with minimal residual disease. N Engl J Med. 2016;375:944–53.

  18. 18.

    Gutman JA, Ross K, Smith C, Myint H, Lee CK, Salit R, et al. Chronic graft versus host disease burden and late transplant complications are lower following adult double cord blood versus matched unrelated donor peripheral blood transplantation. Bone Marrow Transplant. 2016;51:1588–93.

  19. 19.

    Kekre N, Antin JH. Cord blood versus haploidentical stem cell transplantation for hematological malignancies. Semin Hematol. 2016;53:98–102.

  20. 20.

    Malard F, Milpied N, Blaise D, Chevallier P, Michallet M, Lioure B, et al. Effect of graft source on unrelated donor hemopoietic stem cell transplantation in adults with acute myeloid leukemia after reduced-intensity or nonmyeloablative conditioning: a study from the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire. Biol Blood Marrow Transplant. 2015;21:1059–67.

  21. 21.

    Lu L, Zhou X, Wang J, Zheng SG, Horwitz DA. Characterization of protective human CD4CD25 FOXP3 regulatory T cells generated with IL-2, TGF-beta and retinoic acid. PLoS ONE. 2010;5:e15150.

  22. 22.

    Fujimaki W, Takahashi N, Ohnuma K, Nagatsu M, Kurosawa H, Yoshida S, et al. Comparative study of regulatory T cell function of human CD25CD4 T cells from thymocytes, cord blood, and adult peripheral blood. Clin Dev Immunol. 2008;2008:305859.

  23. 23.

    Mitchell CA, Jefferson AB, Bejeck BE, Brugge JS, Deuel TF, Majerus PW. Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets. Proc Natl Acad Sci USA. 1990;87:9396–9400.

  24. 24.

    Kim YC, Bhairavabhotla R, Yoon J, Golding A, Thornton AM, Tran DQ, et al. Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion. Blood. 2012;119:2810–8.

  25. 25.

    Park SW, Pyo CW, Choi SY. High-efficiency lentiviral transduction of primary human CD34(+) hematopoietic cells with low-dose viral inocula. Biotechnol Lett. 2015;37:281–8.

  26. 26.

    Barrilleaux BL, Cotterman R, Knoepfler PS. Chromatin immunoprecipitation assays for Myc and N-Myc. Methods Mol Biol. 2013;1012:117–33.

  27. 27.

    Wu Y, Borde M, Heissmeyer V, Feuerer M, Lapan AD, Stroud JC, et al. FOXP3 controls regulatory T cell function through cooperation with NFAT. Cell. 2006;126:375–87.

  28. 28.

    Sawant DV, Vignali DA. Once a Treg, always a Treg? Immunol Rev. 2014;259:173–91.

  29. 29.

    Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 2008;322:271–5.

  30. 30.

    Klocke K, Holmdahl R, Wing K. CTLA-4 expressed by FOXP3+ regulatory T cells prevents inflammatory tissue attack and not T-cell priming in arthritis. Immunology. 2017;152:125–37.

  31. 31.

    Park HJ, Park JS, Jeong YH, Son J, Ban YH, Lee BH, et al. Correction: PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells. J Immunol. 2015;195:5841–2.

  32. 32.

    Zheng J, Chan PL, Liu Y, Qin G, Xiang Z, Lam KT, et al. ICOS regulates the generation and function of human CD4+ Treg in a CTLA-4 dependent manner. PLoS ONE. 2013;8:e82203.

  33. 33.

    Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, et al. Role of LAG-3 in regulatory T cells. Immunity. 2004;21:503–13.

  34. 34.

    Gautron AS, Dominguez-Villar M, de Marcken M, Hafler DA. Enhanced suppressor function of TIM-3+ FoxP3+ regulatory T cells. Eur J Immunol. 2014;44:2703–11.

  35. 35.

    Milpied P, Renand A, Bruneau J, Mendes-da-Cruz DA, Jacquelin S, Asnafi V, et al. Neuropilin-1 is not a marker of human Foxp3+ Treg. Eur J Immunol. 2009;39:1466–71.

  36. 36.

    Delgoffe GM, Woo SR, Turnis ME, Gravano DM, Guy C, Overacre AE, et al. Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis. Nature. 2013;501:252–6.

  37. 37.

    Joller N, Lozano E, Burkett PR, Patel B, Xiao S, Zhu C, et al. Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses. Immunity. 2014;40:569–81.

  38. 38.

    Mahmud SA, Manlove LS, Schmitz HM, Xing Y, Wang Y, Owen DL, et al. Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells. Nat Immunol. 2014;15:473–81.

  39. 39.

    Gu J, Ni X, Pan X, Lu H, Lu Y, Zhao J, et al. Human CD39hi regulatory T cells present stronger stability and function under inflammatory conditions. Cell Mol Immunol. 2017;14:521–8.

  40. 40.

    Shevach EM, Thornton AM. tTregs, pTregs, and iTregs: similarities and differences. Immunol Rev. 2014;259:88–102.

  41. 41.

    Rudra D, deRoos P, Chaudhry A, Niec RE, Arvey A, Samstein RM, et al. Transcription factor Foxp3 and its protein partners form a complex regulatory network. Nat Immunol. 2012;13:1010–9.

  42. 42.

    Muto A, Tashiro S, Nakajima O, Hoshino H, Takahashi S, Sakoda E, et al. The transcriptional programme of antibody class switching involves the repressor Bach2. Nature. 2004;429:566–71.

  43. 43.

    Lesniewski ML, Haviernik P, Weitzel RP, Kadereit S, Kozik MM, Fanning LR, et al. Regulation of IL-2 expression by transcription factor BACH2 in umbilical cord blood CD4+ T cells. Leukemia. 2008;22:2201–7.

  44. 44.

    Afzali B, Gronholm J, Vandrovcova J, O’Brien C, Sun HW, Vanderleyden I, et al. BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency. Nat Immunol. 2017;18:813–23.

  45. 45.

    Miyagawa Y, Kiyokawa N, Ochiai N, Imadome K, Horiuchi Y, Onda K, et al. Ex vivo expanded cord blood CD4 T lymphocytes exhibit a distinct expression profile of cytokine-related genes from those of peripheral blood origin. Immunology. 2009;128:405–19.

  46. 46.

    Li B, Samanta A, Song X, Iacono KT, Bembas K, Tao R, et al. FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression. Proc Natl Acad Sci USA. 2007;104:4571–6.

  47. 47.

    Schmitt EG, Williams CB. Generation and function of induced regulatory T cells. Front Immunol. 2013;4:152.

  48. 48.

    Bacchetta R, Passerini L, Gambineri E, Dai M, Allan SE, Perroni L, et al. Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J Clin Invest. 2006;116:1713–22.

  49. 49.

    Rascle A, Johnston JA, Amati B. Deacetylase activity is required for recruitment of the basal transcription machinery and transactivation by STAT5. Mol Cell Biol. 2003;23:4162–73.

  50. 50.

    Kadereit S, Mohammad SF, Miller RE, Woods KD, Listrom CD, McKinnon K, et al. Reduced NFAT1 protein expression in human umbilical cord blood T lymphocytes. Blood. 1999;94:3101–7.

  51. 51.

    Katoh H, Qin ZS, Liu R, Wang L, Li W, Li X, et al. FOXP3 orchestrates H4K16 acetylation and H3K4 trimethylation for activation of multiple genes by recruiting MOF and causing displacement of PLU-1. Mol Cell. 2011;44:770–84.

  52. 52.

    Li L, Godfrey WR, Porter SB, Ge Y, June CH, Blazar BR, et al. CD4+ CD25+ regulatory T-cell lines from human cord blood have functional and molecular properties of T-cell anergy. Blood. 2005;106:3068–73.

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Acknowledgements

We thank Daniel Zwick for editorial assistance and Dr. Bruce Torbett for valuable suggestions. This work was supported by the Abraham J. & Phyllis Katz Foundation, Atlanta, GA.

Author contributions

J-sD, FZ, and MJL conducted experiments, literature search, developed study design, analyzed data, and wrote the manuscript. AYH, WJvHF, and MF contributed to the study design, data review and interpretation, as well as writing and approval of the manuscript.

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Affiliations

  1. Cleveland Cord Blood Center, Cleveland, OH, USA

    • Jeong-su Do
    • , Fei Zhong
    • , Wouter J. Van’t Hof
    • , Marcie Finney
    •  & Mary J. Laughlin
  2. Case Western Reserve University, Cleveland, OH, USA

    • Jeong-su Do
    • , Alex Y. Huang
    •  & Mary J. Laughlin

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The authors declare that they have no conflict of interest.

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Correspondence to Jeong-su Do.

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DOI

https://doi.org/10.1038/s41409-018-0205-6