Vagaries of Fluorochrome Reporter Gene Expression in Foxp3+ Regulatory T Cells

Sonja Schallenberg,Pei-Yun Tsai,Cathleen Petzold,Karsten Kretschmer,Tim Sparwasser,Antonio A Freitas

doi:10.1371/journal.pone.0041971

Sonja Schallenberg, Pei-Yun Tsai + Show 4 more

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https://doi.org/10.1371/journal.pone.0041971

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Abstract

CD4+CD25+ regulatory T (Treg) cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4+CD8+ double-positive (DP) and CD4+CD8? single-positive stages of thymic development, as well as in postthymic CD4+ T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3+ Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC)-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3+ DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4+ T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3+ Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3+ Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene.

Highlights

Flow cytometric analysis of total thymocyte populations suggested that the population size of DP cells with Foxp3dependent green fluorescent protein (GFP) expression in Foxp3GFP mice is near the detection limit of the method (Fig. 1A, top)
In addition to exceedingly low cell numbers, and in agreement with previous reports [12,27], our attempts to FACS purify Foxp3+CD25+ DP thymocytes from Foxp3GFP mice was hampered by a propensity of GFP? DP cells to form doublets with GFP+ CD4+CD8? single-positive (CD4SP) cells, as revealed by post sort analysis of CD4, CD8 and GFP expression (Fig. 2A, top)
In contrast to Foxp3GFP mice, significant populations of GFP+CD25+ DP cells were detectable in Foxp3IRES-GFP mice, percentages and numbers failed to reach levels observed in bacterial artificial chromosome (BAC)-Foxp3Cre-GFP mice (Fig. 2C, top)

Summary

Introduction

A common hallmark of intra- and extrathymic CD4+CD25+ regulatory T (Treg) cell lineage commitment is the induction of Foxp expression as a consequence of appropriate T cell receptor (TCR) engagement with MHC class II:peptide ligands [1], resulting in stabilization and amplification of Treg cell-specific gene transcription [2,3] through Foxp occupancy of key target gene promoters [4,5].In the thymus, analysis at the single-cell level provided evidence for the induction of Foxp expression at the CD4+CD8+ doublepositive (DP) stage [6,7,8,9] and a precursor-progeny relationship between DP and CD4+CD8? single-positive (CD4SP) Foxp3+ thymocytes [8]. Analysis at the single-cell level provided evidence for the induction of Foxp expression at the CD4+CD8+ doublepositive (DP) stage [6,7,8,9] and a precursor-progeny relationship between DP and CD4+CD8? CD4SP thymocytes in non-TCR transgenic mice [11] These findings support a model of thymic Treg cell development, in which lineage commitment and subsequent induction of Foxp expression at both the DP and CD4SP stage can occur in parallel. The existence of CD4+Foxp3– precursors in lymph nodes (LNs) of non-TCR transgenic, non-manipulated mice that are precommitted to differentiate into Foxp3+ Treg cells has provided evidence on the relevance of peripheral Treg cell induction in the steady state [27].

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