Figure 3. Mitochondrial oxidative stress is a hallmark of Treg cells in autoimmune environments.

For A-C 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.

(A) Mean Fluorescence Intenstity (MFI) of mtROS production measured by flow cytometry using mitosox Red in naïve (n = 13), pre-diseased (n = 9) and diseased Treg cells (n = 5) (*P = 0.0191, **P = 0.0075).

(B) Immunofluorescence confocal microscopy for 8-OHDG (red), TOM20 (green) and DAPI (blue) in isolated Treg cells. Representative fields (10 μm scale bar) from three independent experiments are depicted. Pearson correlation analysis for co-localization efficiency is depicted (***P < 0.0001).

(C) SOD2 (**P = 0.0069) and mCAT (*P = 0.0336) enzyme activity measured in isolated mitochondria from naïve (n = 4) or pre-diseased Treg cells (n = 3). Each sample was pooled from n = 4 - 5 mice.

(D) 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 Response to Oxidative Stress” (NES 1.43, FDR 0.23) gene set is depicted. The top enriched genes are listed to the right of each plot.

Results are expressed as mean ± SEM. Statistical significance was obtained by unpaired Student’s t- test (C) or One-way ANOVA (A, B).