Measurement of telomerase activity in Tcon and Treg. Telomerase activity was measured in Tcon and Treg by PCR ELISA and southern hybridization. The level of telomerase activity is shown in percentage as relative telomerase activity compared with that of control template measured in each kit. All samples were measured in duplicate. (A) Telomerase activity of Tcon and Treg in healthy subjects (HS) and patients after HSCT. P values indicate comparisons for each category. (B) Telomerase activity of Tcon in healthy subjects and patient subsets based on severity of cGVHD. N.S. indicates not significant. (C) Telomerase activity of Treg in healthy subjects and patient subsets based on severity of cGVHD. (B-C) P values indicate comparisons among patient subsets. (D) Representative southern hybridization experiment for telomerase activity. Internal standard indicates the control for PCR amplification of telomere extension product whose size is 216 bp. The value for relative telomerase activity (%) calculated by PCR-ELISA is 1150.78 (lane 1, Jurkat cells), 23.76 (lane 3, Treg in healthy subject), 47.91 (lane 4, Treg in patient with no cGVHD), 31.37 (lane 5, Treg in patient with mild cGVHD), 7.58 (lane 6, Treg in patient with moderate cGVHD), and 0.09 (lane 7, Treg in patient with severe cGVHD), respectively. (E-F) Relationship between telomerase activity and each cell number in Tcon (E) and Treg (F). Correlation analysis was calculated using Spearman rank test.