Tregs in human secondary lymphoid are in a late activation stage and are polyclonally activated. (A) CpG methylation in the Treg cell-specific demethylation region of the FOXP3 gene in FACS-sorted, human SPL-derived CD4^posCD25h^pos Tregs and CD4^posCD25^neg Tconvs. Methylation status of the indicated CpG positions is indicated by circles: ○, hyperdemethylated (<95% methylation) or ●, methylated (>95% methylation). Complete demethylation was indicated by >95% conversion of C to T in the sequenced product; sites of complete methylation were indicated when >95% of the sequence peak heights indicated C (N = 3 male donors, a representative example is shown). (B) Mononuclear cells were obtained from healthy human PB and SPL samples and analyzed for expression of Helios and FoxP3 on CD4^pos cells by flow cytometry (N = 3 for each tissue, representative examples are shown). (C) Flow cytometry of Ki67 expression in human PB, BM, liLNs, inLNs, and SPL Tregs (left panel) and in CD4^posCD25^posCD69^pos liLN, inLN, and SPL Tregs (right panel; N = 5 for each tissue). Data were analyzed using a random-effect logistic regression model and no significant differences were found. (D) Flow cytometry of TCR-Vβ expression of CD69^neg and CD69^pos Tregs in liLN, inLN, and SPL samples. Representative examples are shown (N = 2-6 for each tissue). (E) TCR gene rearrangement patterns of CD25^posCD69^pos and CD25^posCD69^neg sorted T cells from PB and SPL, showing polyclonal TRB gene rearrangement patterns, both the complete VDJ-rearranged TRB genes (tubes A and B) and the incomplete DJ-rearranged TRB genes (tube C). A polyclonal sample is shown as control. A representative example is shown (N = 2); duplicates revealed similar patterns (not shown).