8-OHdG^hiT cells display signs of increased activation. (A-C) Levels of the T-cell activation marker CD25, CD69, and CD137 based on the MFI are shown for 8-OHdG^hi (n = 22) and 8-OHdG^lo (n = 22) CD3⁺ T cells, as measured at different time points after allo-SCT, using FACS. (D) t-SNE analysis based on CD4, CD8, CD69, and CD137 was performed on concatenated 17 000 CD3⁺CD56^neg T cells gated as representatively shown (top) from HDs and patients who underwent 8-OHdG^hi/lo allo-SCT (at all time points after allo-SCT). HD (black) and individual time points of patients who underwent 8-OHdG^hi (red) and 8-OHdG^lo (blue) allo-SCTs were then plotted based on the calculated variables t-SNE 1 and t-SNE 2 (middle). Representative histograms of CD4, CD8, CD69, and CD137 are shown for characteristic populations of HDs and patients who underwent 8-OHdG^hi and 8-OHdG^lo allo-SCT (for day +120 after allo-SCT; bottom). (E) HD-derived CD3⁺ T cells were activated for 72 hours with anti-CD2, anti-CD3, and anti-CD28 beads and treated twice per day with 10 μM H₂O₂. Expression of CD25, CD69, and HLA-DR was measured as the MFI using FACS (n = 5). Data are expressed as the fold change of expression in T cells pretreated with H₂O₂ relative to untreated T cells (+H₂O₂/−H₂O₂). P value: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. “n” indicates the sample number; bars represent the standard error of the mean.