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Methods Mol Biol. 2016;1371:43-78. doi: 10.1007/978-1-4939-3139-2_4.

Standardization, Evaluation, and Area-Under-Curve Analysis of Human and Murine Treg Suppressive Function.

Author information

1
Division of Transplant Immunology, Department of Pathology and Laboratory Medicine and Biesecker Center for Pediatric Liver Disease, Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. ttankmail@gmail.com.
2
Department of Surgery, Penn Transplant Institute, Hospital of the University of Pennsylvania and University of Pennsylvania, Philadelphia, PA, 19104, USA.
3
Division of Nephrology, Department of Pediatrics, Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
4
Division of Transplant Immunology, Department of Pathology and Laboratory Medicine and Biesecker Center for Pediatric Liver Disease, Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.

Abstract

FOXP3+ T-regulatory (Treg) cells have important roles in immune homeostasis, and alterations in their number and function can predispose to diseases ranging from autoimmunity to allograft rejection and tumor growth. Reliable identification of human Tregs remains a persistent problem due to a lack of specific markers. The most definitive Treg characterization currently involves combined assessment of phenotypic, epigenetic and functional parameters, with the latter typically involving in vitro Treg suppression assays. Unfortunately, suppression assays are frequently performed using differing methods and readouts, limiting comparisons between studies. We provide a perspective on our experience with human and murine Treg suppression assay conditions, including Treg data obtained in clinical transplant studies, Tregs isolated from healthy donors and treated with epigenetically active compounds, and Tregs from standard murine strains (C57BL/6 and BALB/c). We provide detailed descriptions and illustrations of typical problems, shortcomings and troubleshooting; describe new modifications and approaches; and present a new method for calculation of suppressive assay data using a modified area-under-curve (AUC) method. This method allows us to directly compare Treg suppressive function between multiple patients (such as in clinical transplant studies), to reliably track changes in Treg function from the same person over time, or compare effects of Treg-modulating compounds tested with different healthy donors Tregs in separate or combined experimental settings.

KEYWORDS:

FOXP3+ regulatory T cells; Suppression assay; Tregs

PMID:
26530794
PMCID:
PMC5554116
DOI:
10.1007/978-1-4939-3139-2_4
[Indexed for MEDLINE]
Free PMC Article

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