Absolute reconstitution of CD25⁺ and FOXP3⁺ populations in CD4⁺ cells. Scatterplots indicate absolute cell numbers in patients and their donors; horizontal bars represent the median of each group. Red circles refer to patients with clinically significant (≥ grade II) aGvHD development. The gray-shaded area represents the interquartile (25%-75%) range of cell counts in donors. ALC indicates absolute lymphocyte count (as determined by the department of laboratory medicine by flow analysis and lymphocyte gating, includes T and B lymphocytes but also NK cells and a fraction of monocytes). After immunophenotyping of frozen cell specimen, absolute numbers for CD3⁺, CD3⁺ CD4⁺, and CD3⁺ CD8⁺ cells were calculated from ALCs. (A) Pre indicates patients before transplantation (n = 10); Don, donor (n = 13); d30, d60, and d90, patients 30 (n = 13), 60 (n = 8), and 90 (n = 4) days after transplantation. (B-F) Pre (n = 11); Don (n = 13); d30 (n = 13), d60 (n = 8), and d90 (n = 5).