Spectratype and flow cytometry data demonstrating polyclonality of Tregs and expansion of the naive subset. (A) Flow cytometry data demonstrating the expanded subpopulation of Tregs. CD4⁺CD25^highFoxp3⁺ cells were labeled with CD27 and CD45RO, and naive and memory subsets of Tregs were identified. CD4⁺CD25^highFoxp3⁺CD27⁺CD45RO⁻ Tregs were considered to be naive Tregs, and CD4⁺CD25^highFoxp3⁺CD27⁺CD45RO⁺ were considered to be memory Tregs. (B) The percentage of naive Tregs was significantly higher in high-risk patients compared with low-risk patients and healthy age-matched controls (P = .032), whereas there was no significant difference in the percentage of memory Tregs (P = .28). (C) The complexity level of Tregs determined by spectratyping was not significantly different between the low-risk and high-risk cohorts of patients (P = .54). (D) A sample of the polyclonal spectratype of CD4⁺CD25^highFoxp3⁺ Tregs. Spectratyping has been done on CD4⁺CD25^high T cells to investigate the clonality of Tregs. The overall complexity of a Vβ subfamily was determined by counting the number of discrete peaks per Vβ subfamily. A score of 5 was given to a spectratype with 5 or more peaks. For the spectratypes with 1 to 4 peaks, a score of 1 to 4 was given, respectively. No spectratype signal was given a score of 0. The maximum complexity score for each patient would be 120 (5 × 24 = 120). (E) Standard sandwich ELISA performed on day 5 of culture. Reduced IFN-γ production by CD4⁺CD25^low cells from a patient with MDS stimulated with anti-CD3/CD28 beads (Dynal, Oslo, Norway) with or without a 1:1 ratio of Tregs to responder cells in 250 μL complete RPMI 1640 culture medium (Invitrogen, Paisley, United Kingdom) containing 2% FCS and supplemented with penicillin, streptomycin, and l-glutamine (PAA Laboratories GmbH, Haidmanweg, Austria). FACS plot generated by FlowTo (Tree Star Inc, Ashland, OR) for publication only.