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Vagaries of fluorochrome reporter gene expression in Foxp3+ regulatory T cells.

Schallenberg S, Petzold C, Tsai PY, Sparwasser T, Kretschmer K - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Immunotolerance in Regeneration, CRTD/DFG-Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany.

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.

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Tracking Foxp3-dependent GFP expression during thymic Treg cell development.Representative flow cytometry of GFP and CD25 expression among DP and CD4SP thymocytes of Foxp3GFP, Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice, as indicated, (A) before and (B) after magnetic bead enrichment of CD25+ cells. All mice were six weeks old. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate.
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pone-0041971-g001: Tracking Foxp3-dependent GFP expression during thymic Treg cell development.Representative flow cytometry of GFP and CD25 expression among DP and CD4SP thymocytes of Foxp3GFP, Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice, as indicated, (A) before and (B) after magnetic bead enrichment of CD25+ cells. All mice were six weeks old. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate.

Mentions: Flow cytometric analysis of total thymocyte populations suggested that the population size of DP cells with Foxp3-dependent GFP expression in Foxp3GFP mice is near the detection limit of the method (Fig. 1A, top). Concurrent analysis of age-matched Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice indicated the existence of a small, albeit clearly discernable population of GFP+ DP cells, with the vast majority expressing high levels of CD25 (Fig. 1A). CD25 bead enrichment of total thymocytes prior to GFP expression analysis allowed us to visualize a minute Foxp3GFP+CD25+ DP cell population (Fig. 1B, top), although their proportional underrepresentation relative to Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice was maintained (Fig. 1B).


Vagaries of fluorochrome reporter gene expression in Foxp3+ regulatory T cells.

Schallenberg S, Petzold C, Tsai PY, Sparwasser T, Kretschmer K - PLoS ONE (2012)

Tracking Foxp3-dependent GFP expression during thymic Treg cell development.Representative flow cytometry of GFP and CD25 expression among DP and CD4SP thymocytes of Foxp3GFP, Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice, as indicated, (A) before and (B) after magnetic bead enrichment of CD25+ cells. All mice were six weeks old. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412838&req=5

pone-0041971-g001: Tracking Foxp3-dependent GFP expression during thymic Treg cell development.Representative flow cytometry of GFP and CD25 expression among DP and CD4SP thymocytes of Foxp3GFP, Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice, as indicated, (A) before and (B) after magnetic bead enrichment of CD25+ cells. All mice were six weeks old. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate.
Mentions: Flow cytometric analysis of total thymocyte populations suggested that the population size of DP cells with Foxp3-dependent GFP expression in Foxp3GFP mice is near the detection limit of the method (Fig. 1A, top). Concurrent analysis of age-matched Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice indicated the existence of a small, albeit clearly discernable population of GFP+ DP cells, with the vast majority expressing high levels of CD25 (Fig. 1A). CD25 bead enrichment of total thymocytes prior to GFP expression analysis allowed us to visualize a minute Foxp3GFP+CD25+ DP cell population (Fig. 1B, top), although their proportional underrepresentation relative to Foxp3IRES-GFP, BAC-Foxp3Cre-GFP and BAC-Foxp3DTR-GFP mice was maintained (Fig. 1B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Immunotolerance in Regeneration, CRTD/DFG-Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany.

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.

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