(A) Overview of the human FOXP3 gene locus with the exon/intron structure in blue and the TSDR region in red. The lower panel shows the methylation statues of TSDR in HD and AA expanded Tregs compared with non-Tregs. (B) Expanded Tregs were polyclonal in both AA and HD patients. The normalized Shannon entropy of expanded Treg repertoire was used to calculate the degree of clonality, which, on average, was 0.12. (C) Because we did not succeed in isolating enough Treg A and Treg B cells from AA patients for individual in vitro expansion because of the low number of Tregs in these patients, the composition of in vitro expanded Tregs were assessed by calculating the Euclidian distance between the mean expression for each parameter in Treg A and B, which was calculated in 24 Treg A and B (5 HD and 19 AA samples) and 5 (2 AA and 3 HDs) in vitro expanded Tregs. B cells were used as an irrelevant control. The following parameters, which showed the highest differences between subpopulations, were used for the Euclidian distance calculation: FOXP3, CD25, CD127, CD45RA, HLA-DR, CCR6, CCR4, CD69, CD27, CXCR3, CD45RO, CD4, CD20, CD95, CD161, CD28, CD152, CD7, CD279, and CD19. t-SNE1 and t-SNE2 were used for distance calculation (see also supplemental Figure 10). viSNE plot of expression centroids for all Treg cell subpopulations, B cells, and expanded Tregs across all samples. Treg A and Treg B cells were automatically gated from 24 individual samples (19 samples from AA patients, 5 HDs) using the automated clustering algorithm FLOCK on a subset of 700 000 cells proportionally selected from all samples. B cells were gated from the same 24 samples in Cytobank. Expanded Tregs were from 3 HDs and 2 AA patients. Expression centroids were computed for each cell population and used as input for the dimensionality algorithm t-SNE as implemented in the tool cyt (see “Methods” for more details). Each dot in the plot represents 1 particular cell population in a particular sample. Expression values were transformed using the asinh function in a cofactor of 5. Using a 1-tailed Welch’s 2-sample t test, we can reject the null hypothesis that the distance between Treg A and expanded Tregs is lower than in Treg B (P < 2.2 × 10⁻¹⁶), which suggests that expanded Tregs are more similar to subpopulation B than A.