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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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Genetic lineage tracing of Foxp3+ Treg cells in BAC-Foxp3Cre-GFP × R26Y mice.Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP−YFP− and GFP−/GFP+YFP+ populations among (A) CD25+ CD4SP thymocytes and (B) CD4+CD25+ T cells from LNs of BAC-Foxp3Cre-GFP × R26Y mice. Representative presort analysis of GFP and YFP expression among gated CD4+CD25+ cells and postsort analysis are shown as dot plots. Histograms depict Foxp3 expression (ICS) in sorted GFP−YFP− (left) and GFP−/GFP+YFP+ (right) cells. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Data are representative of three independent experiments including at least three mice. (C) Percentages of GFP+ cells among ex vivo populations of YFP−CD25+ CD4SP thymocytes (left) and peripheral YFP−CD4+CD25+ T cells (right). (D) mRNA expression of GFP, YFP and Foxp3 was determined by real-time RT-PCR in sorted GFP−YFP− and GFP−/GFP+YFP+ cells presented in (A and B).
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pone-0041971-g005: Genetic lineage tracing of Foxp3+ Treg cells in BAC-Foxp3Cre-GFP × R26Y mice.Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP−YFP− and GFP−/GFP+YFP+ populations among (A) CD25+ CD4SP thymocytes and (B) CD4+CD25+ T cells from LNs of BAC-Foxp3Cre-GFP × R26Y mice. Representative presort analysis of GFP and YFP expression among gated CD4+CD25+ cells and postsort analysis are shown as dot plots. Histograms depict Foxp3 expression (ICS) in sorted GFP−YFP− (left) and GFP−/GFP+YFP+ (right) cells. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Data are representative of three independent experiments including at least three mice. (C) Percentages of GFP+ cells among ex vivo populations of YFP−CD25+ CD4SP thymocytes (left) and peripheral YFP−CD4+CD25+ T cells (right). (D) mRNA expression of GFP, YFP and Foxp3 was determined by real-time RT-PCR in sorted GFP−YFP− and GFP−/GFP+YFP+ cells presented in (A and B).

Mentions: Possible reasons underlying the presence of significant Foxp3+GFP− T cell populations in both thymus and peripheral lymphoid tissues of BAC-Foxp3Cre-GFP mice include delayed up-regulation of GFP expression that lags behind up-regulation of Foxp3 expression in developing Treg cells, the failure to induce Foxp3 BAC transcriptional activity in some Foxp3+ Treg cell lineage-committed cells, and the down-regulation of Foxp3 BAC expression in mature Foxp3+ Treg cells. In an initial attempt to address these issues, we sought to track Foxp3 and GFP expression in BAC-Foxp3Cre-GFP mice that additionally expressed YFP under the control of the Rosa26 promoter only after excision of a loxP-flanked STOP cassette (R26Y) [39]. To this end, YFP-expressing cells among CD25+ CD4SP thymocytes (Fig. 5A) and peripheral CD4+CD25+ Treg cells (Fig. 5B) were FACS purified from BAC-Foxp3Cre-GFP × R26Y mice, irrespective of their GFP expression status. Postsort analysis revealed that the vast majority of YFP+ cells from thymus (97.0±1.28%) and periphery (97.9±0.21%) exhibited co-expression of GFP, suggesting a minor contribution of transcriptional down-regulation of the BAC-Foxp3Cre-GFP transgene in initially Foxp3+GFP+ T cells to the populations of Foxp3+GFP? cells (Fig. 4). As expected, Foxp3 ICS revealed that YFP expression closely correlated with the expression of Foxp3 protein (Fig. 5A,B) in both CD25+ CD4SP thymocytes (98.0±0.14%) and peripheral CD4+CD25+ Treg cells (98.3±0.9%). In addition, BAC-Foxp3Cre-GFP × R26Y mice allowed us to track sizable YFP? populations among CD25+ CD4SP thymocytes and peripheral CD4+CD25+ T cells, which were found to be either GFP? or GFP+ (Fig. 5A and B, presort; Fig. 5C). It appears reasonable to speculate that thymic and peripheral GFP+YFP? T cells represent developing Foxp3+ Treg cells that, after having successfully up-regulated expression of both Foxp3 protein and the Cre recombinase-GFP fusion protein, are in the process of Cre-mediated excision of the loxP-flanked STOP cassette and subsequent up-regulation of YFP expression. In fact, initially GFP+YFP? cells appear to gradually increase YFP expression until reaching maximum levels (see presort analysis, Fig. 5A and B). Unexpectedly, Foxp3 ICS revealed that the majority of GFP?YFP? cells among CD25+ CD4SP thymocytes (68.4±2.2%; Fig. 5A, bottom) and peripheral CD4+CD25+ cells (61.8±8.1%; Fig. 5B, bottom) exhibited Foxp3 protein expression. In addition to the absence of GFP and YFP protein, gfp and yfp mRNA expression was found to be below the level of detection in peripheral GFP?YFP?CD4+CD25+ T cells of BAC-Foxp3Cre-GFP × R26Y mice, whereas expression of Foxp3 mRNA was readily detectable (Fig. 5D). These data are consistent with our interpretation that the Foxp3 BAC transgene is transcriptionally inactive in the GFP?YFP? subset among peripheral CD4+CD25+Foxp3+ T cells from BAC-Foxp3Cre-GFP × R26Y mice.


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

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

Genetic lineage tracing of Foxp3+ Treg cells in BAC-Foxp3Cre-GFP × R26Y mice.Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP−YFP− and GFP−/GFP+YFP+ populations among (A) CD25+ CD4SP thymocytes and (B) CD4+CD25+ T cells from LNs of BAC-Foxp3Cre-GFP × R26Y mice. Representative presort analysis of GFP and YFP expression among gated CD4+CD25+ cells and postsort analysis are shown as dot plots. Histograms depict Foxp3 expression (ICS) in sorted GFP−YFP− (left) and GFP−/GFP+YFP+ (right) cells. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Data are representative of three independent experiments including at least three mice. (C) Percentages of GFP+ cells among ex vivo populations of YFP−CD25+ CD4SP thymocytes (left) and peripheral YFP−CD4+CD25+ T cells (right). (D) mRNA expression of GFP, YFP and Foxp3 was determined by real-time RT-PCR in sorted GFP−YFP− and GFP−/GFP+YFP+ cells presented in (A and B).
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pone-0041971-g005: Genetic lineage tracing of Foxp3+ Treg cells in BAC-Foxp3Cre-GFP × R26Y mice.Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP−YFP− and GFP−/GFP+YFP+ populations among (A) CD25+ CD4SP thymocytes and (B) CD4+CD25+ T cells from LNs of BAC-Foxp3Cre-GFP × R26Y mice. Representative presort analysis of GFP and YFP expression among gated CD4+CD25+ cells and postsort analysis are shown as dot plots. Histograms depict Foxp3 expression (ICS) in sorted GFP−YFP− (left) and GFP−/GFP+YFP+ (right) cells. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate. Data are representative of three independent experiments including at least three mice. (C) Percentages of GFP+ cells among ex vivo populations of YFP−CD25+ CD4SP thymocytes (left) and peripheral YFP−CD4+CD25+ T cells (right). (D) mRNA expression of GFP, YFP and Foxp3 was determined by real-time RT-PCR in sorted GFP−YFP− and GFP−/GFP+YFP+ cells presented in (A and B).
Mentions: Possible reasons underlying the presence of significant Foxp3+GFP− T cell populations in both thymus and peripheral lymphoid tissues of BAC-Foxp3Cre-GFP mice include delayed up-regulation of GFP expression that lags behind up-regulation of Foxp3 expression in developing Treg cells, the failure to induce Foxp3 BAC transcriptional activity in some Foxp3+ Treg cell lineage-committed cells, and the down-regulation of Foxp3 BAC expression in mature Foxp3+ Treg cells. In an initial attempt to address these issues, we sought to track Foxp3 and GFP expression in BAC-Foxp3Cre-GFP mice that additionally expressed YFP under the control of the Rosa26 promoter only after excision of a loxP-flanked STOP cassette (R26Y) [39]. To this end, YFP-expressing cells among CD25+ CD4SP thymocytes (Fig. 5A) and peripheral CD4+CD25+ Treg cells (Fig. 5B) were FACS purified from BAC-Foxp3Cre-GFP × R26Y mice, irrespective of their GFP expression status. Postsort analysis revealed that the vast majority of YFP+ cells from thymus (97.0±1.28%) and periphery (97.9±0.21%) exhibited co-expression of GFP, suggesting a minor contribution of transcriptional down-regulation of the BAC-Foxp3Cre-GFP transgene in initially Foxp3+GFP+ T cells to the populations of Foxp3+GFP? cells (Fig. 4). As expected, Foxp3 ICS revealed that YFP expression closely correlated with the expression of Foxp3 protein (Fig. 5A,B) in both CD25+ CD4SP thymocytes (98.0±0.14%) and peripheral CD4+CD25+ Treg cells (98.3±0.9%). In addition, BAC-Foxp3Cre-GFP × R26Y mice allowed us to track sizable YFP? populations among CD25+ CD4SP thymocytes and peripheral CD4+CD25+ T cells, which were found to be either GFP? or GFP+ (Fig. 5A and B, presort; Fig. 5C). It appears reasonable to speculate that thymic and peripheral GFP+YFP? T cells represent developing Foxp3+ Treg cells that, after having successfully up-regulated expression of both Foxp3 protein and the Cre recombinase-GFP fusion protein, are in the process of Cre-mediated excision of the loxP-flanked STOP cassette and subsequent up-regulation of YFP expression. In fact, initially GFP+YFP? cells appear to gradually increase YFP expression until reaching maximum levels (see presort analysis, Fig. 5A and B). Unexpectedly, Foxp3 ICS revealed that the majority of GFP?YFP? cells among CD25+ CD4SP thymocytes (68.4±2.2%; Fig. 5A, bottom) and peripheral CD4+CD25+ cells (61.8±8.1%; Fig. 5B, bottom) exhibited Foxp3 protein expression. In addition to the absence of GFP and YFP protein, gfp and yfp mRNA expression was found to be below the level of detection in peripheral GFP?YFP?CD4+CD25+ T cells of BAC-Foxp3Cre-GFP × R26Y mice, whereas expression of Foxp3 mRNA was readily detectable (Fig. 5D). These data are consistent with our interpretation that the Foxp3 BAC transgene is transcriptionally inactive in the GFP?YFP? subset among peripheral CD4+CD25+Foxp3+ T cells from BAC-Foxp3Cre-GFP × R26Y mice.

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.

Show MeSH
Related in: MedlinePlus