Figure 3. Human BBB-EC–transmigrated Tregs present a dysfunctional, proinflammatory phenotype.

HD- and uRRMS-derived Tregs were loaded on an inflamed Boyden chamber migration assay as represented in Figure 2A. Tregs were also cultured alone for 24 hours in the same EC medium (untouched Tregs). After 24 hours, untouched, migrated, and nonmigrated Tregs were collected and bulk RNA-Seq was performed. 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. Benjamini-Hochberg adjusted P value (FDR < 0.05) was used to determine DEGs. n = 5 (HD) and n = 4 (uRRMS).