Abstract
This chapter shows protocols for the differentiation of peripheral Treg (pTreg) from polyclonal and monoclonal CD4+ T cells. Polyclonal naïve CD4+ T cells can differentiate into pTreg upon adoptive transfer into Foxp3-diphtheria toxin receptor transgenic recipient mice in which endogenous Tregs are transiently depleted by administration of diphtheria toxin before adoptive transfer. Differentiation of monoclonal pTreg is induced through oral delivery of ovalbumin into RAG-deficient DO11.10 mice, in which T cells are ovalbumin specific. We show the isolation of naïve CD4+ T cells by flow cytometry, the administration of ovalbumin in drinking water, and the analysis tools, including an optional protocol for the enrichment of analysis samples in CD4+ T cells using a magnetic purification.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133(5):775–787
Abbas AK, Benoist C, Bluestone JA, Campbell DJ, Ghosh S, Hori S et al (2013) Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol 14(4):307–308
Savage PA, Klawon DEJ, Miller CH (2020) Regulatory T cell development. Annu Rev Immunol 38:421–453
Apostolou I, Von Boehmer H (2004) In vivo instruction of suppressor commitment in naive T cells. J Exp Med 199(10):1401–1408
Cobbold SP, Castejon R, Adams E, Zelenika D, Graca L, Humm S et al (2004) Induction of foxP3 + regulatory T cells in the periphery of T cell receptor transgenic mice Tolerized to transplants. J Immunol 172(10):6003–6010
Mucida D, Kutchukhidze N, Erazo A, Russo M, Lafaille JJ, Curotto De Lafaille MA (2005) Oral tolerance in the absence of naturally occurring Tregs. J Clin Invest 115(7):1923–1933
Curotto de Lafaille MA, Kutchukhidze N, Shen S, Ding Y, Yee H, Lafaille JJ (2008) Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation. Immunity 29(1):114–126
Kretschmer K, Apostolou I, Hawiger D, Khazaie K, Nussenzweig MC, von Boehmer H (2005) Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol 12:1219–1227
Zhou G, Drake CG, Levitsky HI (2006) Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood 107(2):628–636
Wang L, Pino-Lagos K, De Vries VC, Guleria I, Sayegh MH, Noelle RJ (2008) Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells. Proc Natl Acad Sci U S A 105(27):9331–9336
Alonso R, Flament H, Lemoine S, Sedlik C, Bottasso E, Péguillet I et al (2018) Induction of anergic or regulatory tumor-specific CD4+ T cells in the tumor-draining lymph node. Nat Commun 9(1):2113
Samstein RM, Josefowicz SZ, Arvey A, Treuting PM, Rudensky AY (2012) Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell 150(1):29–38
Nutsch K, Chai JN, Ai TL, Russler-Germain E, Feehley T, Nagler CR et al (2016) Rapid and efficient generation of regulatory T cells to commensal antigens in the periphery. Cell Rep 17(1):206–220
Curotto de Lafaille MA, Lino AC, Kutchukhidze N, Lafaille JJ (2004) CD25− T cells generate CD25+ Foxp3+ regulatory T cells by peripheral expansion. J Immunol 173(12):7259–7268
Paiva RS, Lino AC, Bergman ML, Caramalho Í, Sousa AE, Zelenay S et al (2013) Recent thymic emigrants are the preferential precursors of regulatory T cells differentiated in the periphery. Proc Natl Acad Sci U S A 110(16):6494–6499
Almeida-Santos J, Bergman M-L, Cabral IA, Demengeot J (2021) Interruption of Thymic activity in adult mice improves responses to tumor immunotherapy. J Immunol 206(5):978–986
Pratama A, Schnell A, Mathis D, Benoist C (2020) Developmental and cellular age direct conversion of CD4+ T cells into RORγ+ or Helios+ colon Treg cells. J Exp Med 217(1):e20190428
Tanaka A, Sakaguchi S (2017) Regulatory T cells in cancer immunotherapy. Cell Res 27(1):109–118
Almeida-Santos J, Bergman ML, Amendoeira Cabral I, Correia V, Caramalho Í, Demengeot J (2020) The multifaceted Foxp3fgfp allele enhances spontaneous and therapeutic immune surveillance of cancer in mice. Eur J Immunol 50(3):439–444
Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G et al (2007) Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 204:57–63. https://doi.org/10.1084/jem.20061852
Acknowledgments
We thank Ana Regalado for monoclonal antibody production, the teams of Marta Monteiro and Manuel Rebelo for flow cytometry and mouse husbandry, respectively, and Inês Amendoeira Cabral for technical assistance.
This work was supported by the Instituto Gulbenkian de Ciência–Fundação Calouste Gulbenkian and by the Portuguese Scientific Council (Fundação para a Ciência e a Tecnologia). Mouse experiments were also in part supported by the national infrastructure European Regional Development Fund CONGENTO LISBOA-01-0145-FEDER-022170 (FCT, Lisboa2020, Por2020).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Almeida-Santos, J., Bergman, ML., Demengeot, J. (2023). Differentiation of Peripheral Treg. In: Ono, M. (eds) Regulatory T-Cells. Methods in Molecular Biology, vol 2559. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2647-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2647-4_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2646-7
Online ISBN: 978-1-0716-2647-4
eBook Packages: Springer Protocols