Altered homeostasis of CD4⁺FOXP3⁺ T cells in patients developing acute graft-versus-host disease after alloHSCT. Analysis of Treg subpopulations was performed in 20 consecutive alloHSCT recipients. Patients were analyzed at the time of engraftment (2-3 weeks after alloHSCT, see supplemental Table 2 for patient characteristics). In the control group (No aGVHD), a blood sample was collected at time of hematopoietic recovery (group 2, n = 7). In patients with aGVHD (n = 8), a blood sample was collected at diagnosis of aGVHD, before steroid treatment initiation. Patients in the group “aGVHD <1 mo” (n = 5) did not display symptoms of aGVHD at the time of analysis at the time of engraftment but developed aGVHD within 1 month after sampling (ie, 2 months after alloHSCT). (A) Expression of HLADR and CD45RA in the peripheral blood CD4⁺FOXP3⁺ T-cell compartment was analyzed by flow cytometry. (B-C) Plots represent the frequencies of the three Treg subpopulations within CD4⁺FOXP3⁺ T cells in 7 patients without aGVHD, 8 patients developing aGVHD, and 5 patients developing aGVHD within 1 month after sampling (B) and percentages of total CD4⁺FOXP3⁺ T cells within CD4⁺ T cells (C). (D) Expression of HLA-DR and CD45RA in the CD4⁺FOXP3⁻ T-cell compartment. P values were obtained using the 2-tailed nonparametric Mann-Whitney test. (E) Tregs and Tconvs were analyzed by flow cytometry for the expression of Helios and Ki-67. (F) Percentage of Ki-67⁺ (top panel) and Helios⁺ cells (bottom panel) in CD4⁺FOXP3⁺ Tregs and conventional CD4⁺FOXP3⁻ T cells in patients with aGVHD (n = 5) and patients not developing aGVHD (n = 6, No aGVHD). Five patients with aGVHD and 6 patients without aGVHD were analyzed. (G) Suppression assays were performed with Tregs and Tconvs from a patient with aGVHD. Ratios of 1:1 and 1:2 (Tresp:Treg) were used.