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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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Comparative quantification of GFP+ DP cells.Flow cytometric isolation of GFP+ DP thymocytes from (A) Foxp3GFP, BAC-Foxp3Cre-GFP and (B) Foxp3IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25+GFP+ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (C) Quantification of GFP+ thymocytes. Percentages (left) and numbers (right) of GFP+ DP cells (top) and GFP+ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (A,B). All mice were six weeks old. (D) Numbers of GFP+ DP thymocytes from eleven-week-old Foxp3GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).
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pone-0041971-g002: Comparative quantification of GFP+ DP cells.Flow cytometric isolation of GFP+ DP thymocytes from (A) Foxp3GFP, BAC-Foxp3Cre-GFP and (B) Foxp3IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25+GFP+ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (C) Quantification of GFP+ thymocytes. Percentages (left) and numbers (right) of GFP+ DP cells (top) and GFP+ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (A,B). All mice were six weeks old. (D) Numbers of GFP+ DP thymocytes from eleven-week-old Foxp3GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).

Mentions: 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+ CD4SP cells, as revealed by post sort analysis of CD4, CD8 and GFP expression (Fig. 2A, top). In contrast, flow cytometric isolation yielded pure populations of GFP+CD25+ DP cells from thymi of BAC-Foxp3Cre-GFP (Fig. 2A, bottom), Foxp3IRES-GFP (Fig. 2B) and BAC-Foxp3DTR-GFP (data not shown) mice. To minimize the inclusion of doublets, we based our quantification of GFP+ thymocytes on the postsort analysis, after CD25 bead enrichment and flow cytometric isolation using stringent gating criteria. These experiments consistently revealed an approximately ten-fold increase in the population size of GFP+ DP thymocytes (Fig. 2C, top) in BAC-Foxp3Cre-GFP mice (0.019±0.006%; 2.05±0.24×104 cells), as compared to Foxp3GFP mice (0.0012±0.0002%; 0.19±0.03×104 cells). Similar results were obtained with GFP+ DP cells from age-matched Foxp3GFP mice on the C57BL/6 and BALB/c genetic backgrounds (Fig. 2D). In contrast to Foxp3GFP mice, significant populations of GFP+CD25+ DP cells were detectable in Foxp3IRES-GFP mice, although percentages and numbers failed to reach levels observed in BAC-Foxp3Cre-GFP mice (Fig. 2C, top). Thus, it appears that, in comparison with Foxp3 BAC transgenesis, the transgenic expression of GFP both as a Foxp3 fusion protein and from an IRES downstream of the Foxp3 gene results in considerably reduced Foxp3+CD25+ DP compartment sizes, albeit to different degrees. Importantly, the size of the GFP+CD25+ CD4SP cell population was largely comparable between Foxp3GFP, Foxp3IRES-GFP and BAC-Foxp3Cre-GFP mice (Fig. 2C, bottom).


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

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

Comparative quantification of GFP+ DP cells.Flow cytometric isolation of GFP+ DP thymocytes from (A) Foxp3GFP, BAC-Foxp3Cre-GFP and (B) Foxp3IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25+GFP+ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (C) Quantification of GFP+ thymocytes. Percentages (left) and numbers (right) of GFP+ DP cells (top) and GFP+ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (A,B). All mice were six weeks old. (D) Numbers of GFP+ DP thymocytes from eleven-week-old Foxp3GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412838&req=5

pone-0041971-g002: Comparative quantification of GFP+ DP cells.Flow cytometric isolation of GFP+ DP thymocytes from (A) Foxp3GFP, BAC-Foxp3Cre-GFP and (B) Foxp3IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25+GFP+ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (C) Quantification of GFP+ thymocytes. Percentages (left) and numbers (right) of GFP+ DP cells (top) and GFP+ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (A,B). All mice were six weeks old. (D) Numbers of GFP+ DP thymocytes from eleven-week-old Foxp3GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).
Mentions: 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+ CD4SP cells, as revealed by post sort analysis of CD4, CD8 and GFP expression (Fig. 2A, top). In contrast, flow cytometric isolation yielded pure populations of GFP+CD25+ DP cells from thymi of BAC-Foxp3Cre-GFP (Fig. 2A, bottom), Foxp3IRES-GFP (Fig. 2B) and BAC-Foxp3DTR-GFP (data not shown) mice. To minimize the inclusion of doublets, we based our quantification of GFP+ thymocytes on the postsort analysis, after CD25 bead enrichment and flow cytometric isolation using stringent gating criteria. These experiments consistently revealed an approximately ten-fold increase in the population size of GFP+ DP thymocytes (Fig. 2C, top) in BAC-Foxp3Cre-GFP mice (0.019±0.006%; 2.05±0.24×104 cells), as compared to Foxp3GFP mice (0.0012±0.0002%; 0.19±0.03×104 cells). Similar results were obtained with GFP+ DP cells from age-matched Foxp3GFP mice on the C57BL/6 and BALB/c genetic backgrounds (Fig. 2D). In contrast to Foxp3GFP mice, significant populations of GFP+CD25+ DP cells were detectable in Foxp3IRES-GFP mice, although percentages and numbers failed to reach levels observed in BAC-Foxp3Cre-GFP mice (Fig. 2C, top). Thus, it appears that, in comparison with Foxp3 BAC transgenesis, the transgenic expression of GFP both as a Foxp3 fusion protein and from an IRES downstream of the Foxp3 gene results in considerably reduced Foxp3+CD25+ DP compartment sizes, albeit to different degrees. Importantly, the size of the GFP+CD25+ CD4SP cell population was largely comparable between Foxp3GFP, Foxp3IRES-GFP and BAC-Foxp3Cre-GFP mice (Fig. 2C, bottom).

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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