Naturally occurring human Tregs express and secrete higher levels of Trx-1 compared with T4conv. (A) Intracellular (ICS) and extracellular (ECS) Trx-1 (iii-iv) was assessed by flow cytometry in freshly purified CD4⁺ T cells stained with Trx-1 before or after cell permeabilization on gating on CD25⁺CD127^low/negativeFOXP3⁺ Tregs and CD127^positiveFOXP3^negative T4conv (i-ii). (B) Trx-1 levels in Tregs and T4conv (n = 9) were quantified by flow cytometry based on the mean fluorescence index (MFI) of Trx-1 PE. (C) Trx-1 relative gene expression was determined in freshly purified Tregs and T4conv (n = 7). Each bar represents the Tregs/T4conv gene expression ratio from one donor. (D) Up-regulation of the Trx-1 relative gene expression was assessed in isolated Tregs and T4conv (n = 4) stimulated for 16 hours with activating anti-CD2, anti-CD3, and anti-CD28 beads. (E) Negatively selected CD4⁺ T-cells (i) were further subjected to a CD45RA⁺/CD45RO⁺ (ii-iii) and subsequent Treg (iv) isolation. (v-vi) MFI of Trx-1 PE and the Trx-1 relative gene expression was assessed in naive CD45RA⁺ (nTregs) and memory CD45RO⁺ (mTregs) Treg subsets (n = 6). (F) On 16-hour culture of Tregs and T4conv (n = 6) in AIM-V medium, full-length Trx-1 was quantified in the supernatants by ELISA. Parallel samples were treated overnight with 5mM of methylamine (MA), an inhibitor of the nonclassic, leaderless secretory pathway. (G) Freshly isolated Tregs and T4conv (n = 6) were cultured for 16 hours in the presence of H₂O₂ (0.5 or 5μM) with or without the TrxR inhibitor auranofin (25 or 50nM). (i) Specific cell death for all conditions was evaluated by flow cytometric analyses. (ii) The relative increase of specific cell death of Tregs/T4conv (n = 6) treated with H₂O₂ (5μM) on blocking of TrxR. The statistical analysis was performed by analysis of variance testing. Bars represent SD. *P < .05. **P < .01. ***P < .001. N.D. indicates not detected.