Enumeration of CD4⁺25^high T cells in patients with and without cGVHD. (A) Unfractionated peripheral blood mononuclear cells (PBMCs) were stained with FITC-conjugated anti-CD4 and PC5-conjugated anti-CD25. Cells were gated on lymphocytes via their forward- and side-scatter properties. The gating strategy is shown for the analysis of CD4⁺CD25^high and CD4⁺CD25^- T cells and for flow sorting the respective fractions. The mean ± SEM purity of both CD4⁺CD25^high and CD4⁺CD25^- sorted fractions are shown (n = 3). Samples were processed in 2 batches to generate enumeration data, with a number of samples run twice to confirm reproducibility. (B) Composite box plots showing percentage of CD4⁺ lymphocytes that were CD25^high (left) and absolute numbers of CD4⁺CD25^high T cells (right). The top, bottom, and line through the middle of the box correspond to the 75th, 25th, and 50th percentiles, respectively. The bars on the bottom and top extend from the bottom 10th percentile and top 90th percentile, respectively. (C) Percentage of CD4⁺ T cells that were CD25^high according to time of sampling in months (mo) after transplantation in patients with (□) and without (▪) cGVHD. (D) Same data set is presented as absolute numbers of CD4⁺CD25^high T cells. Normal ranges, based on a healthy volunteer group (n = 10), are shown in panels C-D by the dotted lines. The normal frequency for CD4⁺ T cells that were CD25^high was 1.2% to 2.5% and the normal absolute number of CD4⁺CD25^high T cells was 7.4 to 20.4 × 10⁶/L.