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Vol 59, No 2 (2021)
Original paper
Submitted: 2021-04-26
Accepted: 2021-05-23
Published online: 2021-06-07
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Identification of circulating regulatory T lymphocytes with membrane markers — a new multiparameter flow cytometry protocol

Agnieszka Piekarska1, Michaël Pérès23, Magdalena Toton4, Malgorzata Kulczycka4, Krzysztof Lewandowski5, François Vergez23
DOI: 10.5603/FHC.a2021.0014
·
Pubmed: 34097299
·
Folia Histochem Cytobiol 2021;59(2):75-85.
Affiliations
  1. Department of Hematology and Transplantology, Medical University of Gdansk, 80-214 Gdansk, Poland
  2. Cancer Research Center of Toulouse, Inserm UMR 1037, CNRS ERL 5294, University of Toulouse, Oncopole, Toulouse, France
  3. Laboratoire de Génétique des Hémopathies, Institut Universitaire du Cancer Toulouse – Oncopole, Toulouse, France
  4. Laboratory of Hematology, University Clinical Centre, 80-214 Gdansk, Poland
  5. Department of Laboratory Medicine, Medical University of Gdansk, 80-214 Gdansk, Poland

open access

Vol 59, No 2 (2021)
ORIGINAL PAPERS
Submitted: 2021-04-26
Accepted: 2021-05-23
Published online: 2021-06-07

Abstract

Introduction. Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs.

Material and methods. Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed.

Results. The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs.

Conclusions. The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Abstract

Introduction. Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs.

Material and methods. Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed.

Results. The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs.

Conclusions. The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

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Keywords

T regulatory cells; enumeration; flow cytometry; 9-color panel; CD31

About this article
Title

Identification of circulating regulatory T lymphocytes with membrane markers — a new multiparameter flow cytometry protocol

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 59, No 2 (2021)

Article type

Original paper

Pages

75-85

Published online

2021-06-07

Page views

1551

Article views/downloads

1344

DOI

10.5603/FHC.a2021.0014

Pubmed

34097299

Bibliographic record

Folia Histochem Cytobiol 2021;59(2):75-85.

Keywords

T regulatory cells
enumeration
flow cytometry
9-color panel
CD31

Authors

Agnieszka Piekarska
Michaël Pérès
Magdalena Toton
Malgorzata Kulczycka
Krzysztof Lewandowski
François Vergez

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