Genetic lineage tracing of Foxp3⁺ Treg cells in BAC-Foxp3^Cre-GFP × R26Y mice.

<p>Analysis of Foxp3 expression by Foxp3 ICS, in FACS purified GFP⁻YFP⁻ and GFP⁻/GFP⁺YFP⁺ populations among (<b>A</b>) CD25⁺ CD4SP thymocytes and (<b>B</b>) CD4⁺CD25⁺ T cells from LNs of BAC-Foxp3^Cre-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. (<b>C</b>) Percentages of GFP⁺ cells among <i>ex vivo</i> populations of YFP⁻CD25⁺ CD4SP thymocytes (left) and peripheral YFP⁻CD4⁺CD25⁺ T cells (right). (<b>D</b>) 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 (<b>A and B</b>).</p

Comparative quantification of GFP⁺ DP cells.

<p>Flow cytometric isolation of GFP⁺ DP thymocytes from (<b>A</b>) Foxp3^GFP, BAC-Foxp3^Cre-GFP and (<b>B</b>) Foxp3^IRES-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. (<b>C</b>) 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 (<b>A,B</b>). All mice were six weeks old. (<b>D</b>) Numbers of GFP⁺ DP thymocytes from eleven-week-old Foxp3^GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3^Cre-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).</p

Infidelity of BAC-Foxp3^Cre-GFP-dependent GFP expression.

<p>(<b>A-C</b>) Tracking Foxp3⁺ cells that lack GFP expression during thymic Treg cell lineage commitment. (<b>A</b>) Representative dot plots depict presort analysis of CD25 and GFP expression among gated DP and CD4SP thymocyte subsets from six-week-old BAC-Foxp3^Cre-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>B</b>) Percentages and (<b>C</b>) numbers of Foxp3⁺ cells (ICS) among GFP⁻ and GFP⁺ cells that had been sorted from DP and CD4SP thymocyte compartments. (<b>D-F</b>) Tracking Foxp3⁺ Treg cells that lack GFP expression in peripheral lymphoid tissues. (<b>D</b>) 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. (<b>E</b>) Percentages and (<b>F</b>) 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-Foxp3^Cre-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.</p

BAC-Foxp3^Cre-GFP-driven GFP expression faithfully reflects Foxp3 protein expression during thymic Treg cell lineage commitment.

<p>Direct assessment of Foxp3 protein expression using mAbs to Foxp3 after flow cytometric isolation of GFP⁺ cells from BAC-Foxp3^Cre-GFP mice. (A) Presort analysis of CD25 and GFP expression among gated DP and CD4SP cells from BAC-Foxp3^Cre-GFP mice after magnetic bead enrichment of CD25⁺ cells, as well as postsort analysis are depicted, as indicated. CD25^?GFP^? DP cells were included for comparison (left). (B) Foxp3 expression of sorted cells, as revealed by intracellular staining (ICS) with mAbs to Foxp3. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots and histograms indicate percentages of cells in the respective quadrant or gate.</p