Design of Treg transcriptome studies. (A) Primary Treg transcriptome study. Blood samples were taken at regular intervals at 30, 60, 90, 120, 150, and 180 days after SCT, and CD4⁺CD25^hiCD127^lo/− Tregs were isolated by FACS. Samples were assigned to the no GVHD or aGVHD/cGVHD group according to clinical and histologic diagnoses and analyzed by microarray technology. Comparative analysis of Treg transcriptomes was performed as indicated by the arrows. (B) Primary and confirmatory Treg transcriptome studies. Microarrays of the primary Treg transcriptome study are red framed, analyzing patients in the no GVHD group at day 90 (array A) and day 150 (array C), in the GVHD group at day 90 (array F), with aGVHD manifestation (array G), at day 150 (array J), and with cGVHD manifestation (array K). Red lines demonstrate comparisons of the primary Treg transcriptome study, which also included a comparative analysis with healthy, age- and sex-matched donors (Table 2). Confirmatory microarray experiments are blue-framed, analyzing patients in the no GVHD group before 100 days (array B) and after 100 days (arrays D and E), in the GVHD group before 100 days (arrays H and I) and after 100 days (arrays L and M). In each array, CD4⁺CD25^hiCD127^lo/− Tregs with a purity more than 95% of n = 3 independent patients with exception of array D (n = 2) were analyzed. FC1-FC8 indicates the comparison of 2 arrays and resulting fold change values are summarized in Table 3.