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. 2024 Feb 22;9(7):e167457. doi: 10.1172/jci.insight.167457

Figure 2. Interaction with inflamed but not control BBB-ECs causes loss of function in human Tregs.

Figure 2

(A) Schematic representation of Boyden chamber migration assay. hCMEC/D3 cells were stimulated with or without (control) IFN-γ and TNF-α (inflamed) for 24 hours followed by washing. Next, human Tregs were sorted by FACS from HD-derived PBMCs and used immediately as ex vivo Tregs or loaded on the chamber for migration assay. After 24 hours, migrated and nonmigrated Tregs were collected for further analysis. (B–G) Suppressive capacity of ex vivo Tregs, nonmigrated Tregs, or migrated Tregs after interaction with inflamed (B) or control (E) BBB-ECs. Representative dilution plots of CellTraceViolet of ex vivo and nonmigrated (C and F; 1:1) or ex vivo and migrated (D and G; 1:1) Tregs. Percentage proliferation represents CellTrace dilution of Teff in suppression assays with different ratios of Teff and Tregs (given as Teff/Treg). Relative proliferation is normalized to 1:0 condition (100%). Dilutions plots are normalized to the maximum count of the represented conditions. Gating strategy in Supplemental Figure 4. n = 4–8; 2-way ANOVA with Bonferroni’s multiple-comparison test compared with ex vivo condition per ratio. **P < 0.01; ***P < 0.001.