Skip to main content

Identification of Foxp3+ T Follicular Regulatory (Tfr) Cells by Flow Cytometry

  • Protocol
T follicular Helper Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1291))

Abstract

Flow cytometry is a technology that allows multiparametric analysis of individual cells. As a consequence, it is among the most commonly used tools for the study of immune cells. It is useful both for the study of ex vivo cell populations isolated from experimental animals or human tissue and for characterizing the phenotype of cultured cells. The phenotypic analysis is based on antibodies associated to different fluorophores that specifically bind to key molecules. Genetically modified mouse strains that express a reporter gene under the control of a promoter of interest offer an important alternative for the staining of intracellular molecules without the need to permeabilize the cell membrane. In this chapter, we describe how Foxp3+ follicular regulatory T (Tfr) cells, a population of regulatory T (Treg) cells related to T follicular helper (Tfh) cells and involved in the regulation of germinal centers (GC), can be identified by flow cytometry.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wollenberg I, Agua-Doce A, Hernandez A, Almeida C, Oliveira VG, Faro J, Graca L (2011) Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells. J Immunol 187(9):4553–4560. doi:10.4049/jimmunol.1101328

    Article  CAS  PubMed  Google Scholar 

  2. Linterman MA, Pierson W, Lee SK, Kallies A, Kawamoto S, Rayner TF, Srivastava M, Divekar DP, Beaton L, Hogan JJ, Fagarasan S, Liston A, Smith KG, Vinuesa CG (2011) Foxp3+ follicular regulatory T cells control the germinal center response. Nat Med 17(8):975–982. doi:10.1038/nm.2425

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Chung Y, Tanaka S, Chu F, Nurieva RI, Martinez GJ, Rawal S, Wang YH, Lim H, Reynolds JM, Zhou XH, Fan HM, Liu ZM, Neelapu SS, Dong C (2011) Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat Med 17(8):983–988. doi:10.1038/nm.2426

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Sage PT, Francisco LM, Carman CV, Sharpe AH (2013) The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood. Nat Immunol 14(2):152–161. doi:10.1038/ni.2496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Vaeth M, Muller G, Stauss D, Dietz L, Klein-Hessling S, Serfling E, Lipp M, Berberich I, Berberich-Siebelt F (2014) Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression. J Exp Med 211(3):545–561. doi:10.1084/jem.20130604

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY (2005) Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22(3):329–341. doi:10.1016/j.immuni.2005.01.016

    Article  CAS  PubMed  Google Scholar 

  7. Haribhai D, Lin W, Relland LM, Truong N, Williams CB, Chatila TA (2007) Regulatory T cells dynamically control the primary immune response to foreign antigen. J Immunol 178(5):2961–2972. doi:10.4049/jimmunol.178.5.2961

    Article  CAS  PubMed  Google Scholar 

  8. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441(7090):235–238. doi:10.1038/nature04753

    Article  CAS  PubMed  Google Scholar 

  9. Wan YY, Flavell RA (2005) Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc Natl Acad Sci U S A 102(14):5126–5131. doi:10.1073/pnas.0501701102

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Komatsu N, Mariotti-Ferrandiz ME, Wang Y, Malissen B, Waldmann H, Hori S (2009) Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc Natl Acad Sci U S A 106(6):1903–1908. doi:10.1073/pnas.0811556106

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Bettini ML, Pan F, Bettini M, Finkelstein D, Rehg JE, Floess S, Bell BD, Ziegler SF, Huehn J, Pardoll DM, Vignali DA (2012) Loss of epigenetic modification driven by the Foxp3 transcription factor leads to regulatory T cell insufficiency. Immunity 36(5):717–730. doi:10.1016/j.immuni.2012.03.020

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Darce J, Rudra D, Li L, Nishio J, Cipolletta D, Rudensky AY, Mathis D, Benoist C (2012) An N-terminal mutation of the Foxp3 transcription factor alleviates arthritis but exacerbates diabetes. Immunity 36(5):731–741. doi:10.1016/j.immuni.2012.04.007

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by FCT grant: PTDC/SAU-IMU/120225/2010

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Luis Graca .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Maceiras, A.R., Graca, L. (2015). Identification of Foxp3+ T Follicular Regulatory (Tfr) Cells by Flow Cytometry. In: Espéli, M., Linterman, M. (eds) T follicular Helper Cells. Methods in Molecular Biology, vol 1291. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2498-1_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2498-1_12

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2497-4

  • Online ISBN: 978-1-4939-2498-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics