Figure 5. Inflammatory genes are upregulated in CNS-isolated Tregs.

EAE was induced in female FOXP3^Cre-GFP Rosa^RFP fate-mapping mice. Tissues were collected at peak (18 dpi; Supplemental Figure 1), and RFP⁺GFP⁺ FOXP3⁺ Tregs and RFP⁺GFP^– exFOXP3 T cells were sorted by FACS from spleen and pooled brain and spinal cord (CNS). Gating strategy in Supplemental Figure 1. Single-cell RNA-Seq was performed on CNS- and spleen-derived RFP⁺GFP^– exFOXP3 T cells and RFP⁺GFP⁺FOXP3⁺ Tregs. (A and B) Two-dimensional UMAP plot showing the clustering of 3,029 cells based on gene expression divided into 5 clusters. Point coordinates are based on UMAP dimensionality reduction of the top 15 principal components. Individual points correspond to single cells colored according to clusters (A) and samples (B). Sample representation in UMAP and top expressing genes per cluster in Supplemental Figure 6. (C) Hierarchical clustering showing changes in gene expression of the experimental conditions. Relative gene expression is indicated by color: upregulation in red and downregulation in blue. Genes and samples with similar expression were automatically grouped (left and top trees). Inflammation-, migration-, and regulation-related genes are highlighted. Expression values are shown as Z scores. Differential expression was analyzed using the Wilcoxon ranked-sum test.