Fig 1: TCF1+and TCF1-Tregs Have Distinct Transcriptomes(A) Principle-component analysis (PCA) of the transcriptomes of the R1, R2, and R3 Treg subsets as defined in Figure 1.(B) Volcano plots showing the comparisons among the three Treg subsets. The T cell activation gene signature (red open circles) was superimposed onto each plot. The numbers of T cell activation signature genes that were over- or under-represented in each comparison and the p values (by ?2 test) were shown.(C and D) Heatmaps (row normalized) depicting the expression of the selected transcription factor (C) and key functional molecules (D) in the R1, R2, and R3 Treg subsets. Genes were ranked and clustered according to their correlation to Tcf7 (C) or Icos (D). r2, correlation coefficient; min, lowest expression in each row; max, highest expression in each row.(E and F) Flow cytometric analysis of surface molecule: ICOS, CD103, and KLRG1 (E) and transcription factors: IRF4 and Blimp1 (F) in the R1, R2, and R3 Treg subsets.(G) Graphic summary illustrating the revised model of the Treg pool in peripheral lymphoid organs.The data are representative of one experiment with n = 2 replicates for each Treg subsets (A–D) or n = 3 independent experiments (E and F). Shown are mean ± SEM (E and F). p values in (B) were calculated using the chi-square (?2) test. p values in (E) and (F) were calculated using the two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., non-significant.See also Figure S2 and Tables S1, S2, and S3.