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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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Infidelity of BAC-Foxp3Cre-GFP-dependent GFP expression.(A-C) Tracking Foxp3+ cells that lack GFP expression during thymic Treg cell lineage commitment. (A) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3Cre-GFP mice before and after magnetic bead enrichment of CD25+ cells, as indicated. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression (right), as revealed by Foxp3 ICS, among indicated postsort populations. (B) Percentages and (C) numbers of Foxp3+ cells (ICS) among GFP− and GFP+ cells that had been sorted from DP and CD4SP thymocyte compartments. (D-F) Tracking Foxp3+ Treg cells that lack GFP expression in peripheral lymphoid tissues. (D) Representative dot plots depict presort analysis of CD25 and GFP expression among gated CD4+ T cells from LNs before and after magnetic bead enrichment of CD25+ cells. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression, as revealed by Foxp3 ICS (right), among indicated postsort populations. (E) Percentages and (F) numbers of Foxp3-expressing (ICS) CD4+CD25+ T cells among GFP− and GFP+ cells that had been isolated by flow cytometry from pooled scLNs of BAC-Foxp3Cre-GFP mice. All mice were six weeks old. Lines with arrowheads in dot plots illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively.
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pone-0041971-g004: Infidelity of BAC-Foxp3Cre-GFP-dependent GFP expression.(A-C) Tracking Foxp3+ cells that lack GFP expression during thymic Treg cell lineage commitment. (A) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3Cre-GFP mice before and after magnetic bead enrichment of CD25+ cells, as indicated. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression (right), as revealed by Foxp3 ICS, among indicated postsort populations. (B) Percentages and (C) numbers of Foxp3+ cells (ICS) among GFP− and GFP+ cells that had been sorted from DP and CD4SP thymocyte compartments. (D-F) Tracking Foxp3+ Treg cells that lack GFP expression in peripheral lymphoid tissues. (D) Representative dot plots depict presort analysis of CD25 and GFP expression among gated CD4+ T cells from LNs before and after magnetic bead enrichment of CD25+ cells. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression, as revealed by Foxp3 ICS (right), among indicated postsort populations. (E) Percentages and (F) numbers of Foxp3-expressing (ICS) CD4+CD25+ T cells among GFP− and GFP+ cells that had been isolated by flow cytometry from pooled scLNs of BAC-Foxp3Cre-GFP mice. All mice were six weeks old. Lines with arrowheads in dot plots illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively.

Mentions: We isolated highly pure populations of GFP-expressing CD25+ DP and CD25+ CD4SP thymocytes from BAC-Foxp3Cre-GFP mice (Fig. 3A) to directly assess Foxp3 protein expression by ICS with mAbs to Foxp3 (Foxp3 ICS). This approach demonstrated that GFP expression in both DP and CD4SP thymocytes from BAC-Foxp3Cre-GFP mice faithfully reflected Foxp3 expression (Fig. 3B, Fig. 4A,B). Similar results were obtained with GFP+ DP thymocytes from Foxp3IRES-GFP and BAC-Foxp3DTR-GFP mice (data not shown). However, up to 50% of BAC-Foxp3Cre-GFP transgenic CD25+ CD4SP thymocytes lacking GFP expression exhibited expression of Foxp3 (Fig. 4A,B), corresponding to up to 38.1±7.1% of the overall pool of Foxp3+ thymocytes in BAC-Foxp3Cre-GFP mice, as calculated based on absolute numbers of GFP? and GFP+ cells among Foxp3+CD4+CD25+ T cells (Fig. 4C). Similarly, although GFP expression in CD4+CD25+ T cells from peripheral lymphoid organs of BAC-Foxp3Cre-GFP mice closely reflected the expression of Foxp3 protein (Fig. 4D,E), we consistently observed that up to 60% of peripheral CD4+CD25+GFP? T cells expressed Foxp3 protein (Fig. 4D,E). Based on the numbers of GFP? and GFP+ cells depicted in Fig. 4F, we estimated the relative contribution of Foxp3+GFP? cells to the overall pool of peripheral Foxp3+ Treg cells in adult BAC-Foxp3Cre-GFP mice to be 11.3±2.7%.


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

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

Infidelity of BAC-Foxp3Cre-GFP-dependent GFP expression.(A-C) Tracking Foxp3+ cells that lack GFP expression during thymic Treg cell lineage commitment. (A) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3Cre-GFP mice before and after magnetic bead enrichment of CD25+ cells, as indicated. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression (right), as revealed by Foxp3 ICS, among indicated postsort populations. (B) Percentages and (C) numbers of Foxp3+ cells (ICS) among GFP− and GFP+ cells that had been sorted from DP and CD4SP thymocyte compartments. (D-F) Tracking Foxp3+ Treg cells that lack GFP expression in peripheral lymphoid tissues. (D) Representative dot plots depict presort analysis of CD25 and GFP expression among gated CD4+ T cells from LNs before and after magnetic bead enrichment of CD25+ cells. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression, as revealed by Foxp3 ICS (right), among indicated postsort populations. (E) Percentages and (F) numbers of Foxp3-expressing (ICS) CD4+CD25+ T cells among GFP− and GFP+ cells that had been isolated by flow cytometry from pooled scLNs of BAC-Foxp3Cre-GFP mice. All mice were six weeks old. Lines with arrowheads in dot plots illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412838&req=5

pone-0041971-g004: Infidelity of BAC-Foxp3Cre-GFP-dependent GFP expression.(A-C) Tracking Foxp3+ cells that lack GFP expression during thymic Treg cell lineage commitment. (A) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3Cre-GFP mice before and after magnetic bead enrichment of CD25+ cells, as indicated. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression (right), as revealed by Foxp3 ICS, among indicated postsort populations. (B) Percentages and (C) numbers of Foxp3+ cells (ICS) among GFP− and GFP+ cells that had been sorted from DP and CD4SP thymocyte compartments. (D-F) Tracking Foxp3+ Treg cells that lack GFP expression in peripheral lymphoid tissues. (D) Representative dot plots depict presort analysis of CD25 and GFP expression among gated CD4+ T cells from LNs before and after magnetic bead enrichment of CD25+ cells. Histograms show postsort analysis of GFP expression (left) and Foxp3 expression, as revealed by Foxp3 ICS (right), among indicated postsort populations. (E) Percentages and (F) numbers of Foxp3-expressing (ICS) CD4+CD25+ T cells among GFP− and GFP+ cells that had been isolated by flow cytometry from pooled scLNs of BAC-Foxp3Cre-GFP mice. All mice were six weeks old. Lines with arrowheads in dot plots illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively.
Mentions: We isolated highly pure populations of GFP-expressing CD25+ DP and CD25+ CD4SP thymocytes from BAC-Foxp3Cre-GFP mice (Fig. 3A) to directly assess Foxp3 protein expression by ICS with mAbs to Foxp3 (Foxp3 ICS). This approach demonstrated that GFP expression in both DP and CD4SP thymocytes from BAC-Foxp3Cre-GFP mice faithfully reflected Foxp3 expression (Fig. 3B, Fig. 4A,B). Similar results were obtained with GFP+ DP thymocytes from Foxp3IRES-GFP and BAC-Foxp3DTR-GFP mice (data not shown). However, up to 50% of BAC-Foxp3Cre-GFP transgenic CD25+ CD4SP thymocytes lacking GFP expression exhibited expression of Foxp3 (Fig. 4A,B), corresponding to up to 38.1±7.1% of the overall pool of Foxp3+ thymocytes in BAC-Foxp3Cre-GFP mice, as calculated based on absolute numbers of GFP? and GFP+ cells among Foxp3+CD4+CD25+ T cells (Fig. 4C). Similarly, although GFP expression in CD4+CD25+ T cells from peripheral lymphoid organs of BAC-Foxp3Cre-GFP mice closely reflected the expression of Foxp3 protein (Fig. 4D,E), we consistently observed that up to 60% of peripheral CD4+CD25+GFP? T cells expressed Foxp3 protein (Fig. 4D,E). Based on the numbers of GFP? and GFP+ cells depicted in Fig. 4F, we estimated the relative contribution of Foxp3+GFP? cells to the overall pool of peripheral Foxp3+ Treg cells in adult BAC-Foxp3Cre-GFP mice to be 11.3±2.7%.

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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