CD25^High CD4⁺ T cells originate from natural CD25⁺ CD4⁺ T cells.

<p>(<b>A</b>) Histogram showing CD25 expression pattern of unstimulated CD4⁺ T cells (day 0; top). Isolated CD25⁻ and CD25⁺ CD4⁺ T cells were co-cultured in a cross-over setup with only one of the subsets carrying CFSE label. Three days after stimulation with 0.1 µg/ml anti-CD3 mAb and irradiated TdAC, CD25 expression and CFSE dye dilution were measured using flow cytometry and displayed as density plots (bottom). (<b>B</b>) CFSE-labeled total CD4⁺ T cells (top) or CD25⁺-depleted CD4⁺ T cells (bottom) were stimulated as in (<b>A</b>). Shown are flow cytometry density plots of CFSE dye dilution versus CD25 expression gated on viable CD4⁺ T cells. Percentages indicate the percentage of CD25^High or CD25^Int cells within all CD25⁺ positive CD4⁺ T cells. (<b>C</b>) Contour plots showing CD25 versus Foxp3 expression of unstimulated and day 4-stimulated CD4⁺ T cells. Numbers on the right reflect the percentages within the gates 1–5, gated on viable CD4⁺ T cells. (<b>D</b>) CFSE-labeled total CD4⁺ T cells (shaded) or CD25⁺-depleted CD4⁺ T cells (solid line) were stimulated with anti-CD3 (0.1 µg/ml) and irradiated TdACs for 3 days. Shown are histogram overlays gated on viable CD4⁺ T cells and bar graph of proliferation index mean±SEM. (<b>E</b>) Total CD4⁺ T cells or CD25-depleted CD4⁺ T cells were stimulated with anti-CD3 (0.1 µg/ml) and irradiated TdACs for indicated time and proliferation was measured via ³H-thymidine incorporation. (<b>B, D, E</b>) Bar graphs represent the mean±SEM. Statistical significance was calculated by student t-test, ns: not significant, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. Experiments performed at least 3 times with similar results.