Fig. 4: Regulatory T cells in children with T1D.

A Reclustering of Treg clusters from the UMAP projection shown in Fig. 1F. Selected clusters were extracted and subjected to new normalization, scaling, integration, and dimensionality reduction. The resulting UMAP projection is shown on the right. n = 9890 cells from 43 donors. Cells are colored by cluster. B Same UMAP projection as in (A), colored by expression of a gene module upregulated in activated Tregs compared to resting Tregs from GSE15659⁵⁸. C Heatmap of selected marker genes characterizing the clusters shown in (A). Color represents row-scaled z-score of average gene expression per cluster. D Violin plots of the percentage of cells per cluster with non-zero expression of selected genes (87 samples). Bars show medians. E Density plots of Treg subtype abundance across healthy donors and children with T1D at T0 and T1. F Quantification of the percentage of naïve Tregs per sample among total Tregs, gated as shown in (E). P-values were calculated using a two-tailed Mann–Whitney test (healthy donors vs. children with T1D at T0) or a two-tailed paired Mann–Whitney test (children with T1D at T0 vs. T1). Bars indicate medians. n = 13 healthy donors, n = 30 T1D T0, n = 29 T1D T1. G Same UMAP as (A), colored by pseudotime values calculated using the slingshot package. The arrow indicates the identified lineage trajectory. H Tile plot of correlation between pseudotime and Treg activation score from (B), binned into 20 intervals. P-value by two-sided Pearson correlation. I Violin plots showing median pseudotime values in healthy donors and children with T1D at T0 and T1. P-values as in (F). Bars indicate medians. n = 13 healthy donors, n = 30 T1D T0, n = 29 T1D T1. J Same UMAP as (A), colored by scaled imbalance score estimated using the condiments package. Higher scores indicate greater differences in cell density between healthy donors and children with T1D at T0 or T1. K Density of cells from healthy, T1D T0, and T1 along pseudotime. L Violin plots showing the pseudotime value at which cells show maximum density in each condition. P-values as in (F). Bars indicate medians. n = 13 healthy donors, n = 30 T1D T0, n = 29 T1D T1. T1D – Type 1 Diabetes Mellitus, T0 – timepoint at diagnosis, T1 – timepoint one year after diagnosis.