8-OHdG^hiT cells are exhausted and functionally impaired. (A) Expression of exhaustion markers PD-1, KLRG1, CD152, and CD223 was semiquantified on 8-OHdG^hi (n = 6-10) and 8-OHdG^lo (n = 6-10) T cells using FACS based on the MFI. (B) 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. The MFI of PD-1, KLRG1, CD152, and CD223 was measured using FACS (n = 5). Data are expressed as the fold change of MFI in T cells pretreated with H₂O₂ relative to untreated T cells (+H₂O₂/−H₂O₂). (C) The percentages of IFN-γ (left) and GrzB (right) positive CD3⁺ T cells stimulated with anti-CD2, anti-CD3, and anti-CD28 beads for 72 hours were measured using FACS in 8-OHdG^hi and 8-OHdG^lo samples (n = 6). (D) 8-OHdG^hi/lo PBMCs were coincubated with the host’s bone marrow–derived AML blasts from time point of initial diagnosis (n = 3). Coculture was performed in different E:T ratios. During coculture for 48 hours, T cells were stimulated with anti-CD2, anti-CD3, and anti-CD28 beads. Specific cell death of AML blasts was assessed using FACS. (E) Elimination of AML blasts, from time point of diagnosis, by reconstituting T cells (n = 3) was assessed by real-time imaging using an IncuCyte Zoom device. Cocultures were performed for 24 hours in a E:T ratio of 1:1 and in the presence of anti-CD2, anti-CD3, and anti-CD28 antibodies. (F) T cells with/without H₂O₂ pretreatment (n = 5) were cocultured for 24 hours with OCI-AML3 and MOLM-13 AML cell lines in different E:T ratios in presence of anti-CD2, anti-CD3, and anti-CD28 antibodies. Specific cell death was assessed using FACS. P value: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. “; bars represent the standard error of the mean. E:T, effector to target; n, sample number; PBMC, peripheral blood mononuclear cell.