Figure 2. Treg cells during autoimmune responses experience an impaired mitochondrial function.

(A)Seahorse analysis of oxygen consumption rate (OCR) in CD4⁺CD25⁺GITR⁺ Treg cells isolated from dLNs (inguinal) of naïve or immunized mice 9 d following s.c. Mog35-55/CFA injection. For B-F CD4⁺Foxp3⁺ Treg cells were isolated from dLNs (inguinal for pre-diseased or cervical for diseased) of naïve, pre-diseased or EAE induced with disease activity score > 3.5, Foxp3gfp.KI mice.

(B)Intracellular ATP values are depicted for naïve (n = 6), pre-diseased (n = 6) and diseased (n = 5) Foxp3⁺ Treg cells (****P < 0.0001).

(C) Mitochondrial membrane potential measured by flow cytometry using TMRE. Mean fluorescence intensity (MFI) of TMRE is depicted for naïve (n = 9), pre-diseased (n = 10) and diseased Treg cells (n = 8) (*P = 0.0423, *P = 0.05).

(D) MFI of cytochrome c in naïve or pre-diseased Treg cells. One representative experiment of two is depicted (n = 4 mice per group, *P = 0.0492).

(E) Representative immunofluorescence confocal microscopy images for Mitotracker (red) and DAPI (blue) and Mitotracker puncta/cell, (n = 5 mice per group, *P = 0.0111).

(F) Representative images of Foxp3⁺ Treg cells using transmission electron microscopy are depicted.

(G) Treg cells were isolated as CD4⁺CD127^-CD25⁺ cells from peripheral blood of healthy individuals (n = 14) and subjects with MS (n = 11). GSEA plot showing the enrichment of “GO Mitochondrial depolarization” (NES 1.42, FDR 0.23) gene set. The top enriched genes are listed to the right of each plot.

Results are presented as mean ± SEM. Statistical significance was obtained by One-way ANOVA or unpaired Student’s t-test. See also Figure S2