CD4⁺CD25^hi T cells from rebound phase were suppressive in vitro. To fractionate CD4⁺CD25^hi and CD4⁺CD25^- T cells, purified CD4 T cells isolated from rebound were stained with anti-CD25 PE and subsequently with anti-PE MicroBeads and isolated over magnetic columns as described in “Patients and methods.” We developed and optimized this procedure to consistently enrich for CD4⁺CD25^hi T cells without FACS sorting. (A) The dot plots were gated on CD3⁺CD4⁺ T cells and represent the initial starting CD4 T-cell population (Unfractionated) and the subsequent purified CD4CD25 subsets. The numbers represent the percentage of CD4⁺CD25^hi T cells for each fraction. (B, C) Freshly isolated CD4⁺CD25^- T cells (50 000 cells/well) were cocultured alone (CD25^-) or with CD4⁺CD25^hi T cells at different ratios and stimulated with anti-CD3 and irradiated T-depleted PBMCs (250 000 cells/well). Proliferation was assessed by [³H]thymidine incorporation pulsed on day 4 and harvested 18 hours later. The results represent the average [³H]thymidine incorporation (CPM) from 5 replicate wells per culture with calculated standard error of the mean. The P values were calculated using 2-sample t test. (D) CD4 T cells were initially purified from cryopreserved PBMCs collected during rebound from another patient, and stained with PE-conjugated anti-CD25, allophycocyanin-conjugated anti-CD4, and FITC-conjugated anti-CD3 antibodies, and subsequently separated into CD4⁺CD25^hi and CD4⁺CD25^- subsets using FACSVantage DiVa sorter. The result is the mean of [³H]thymidine incorporation in triplicate cultures with standard error of the mean, pulsed on day 6 and harvested 18 hours later.