Total and secreted Trx-1 is involved in the maintenance of surface thiols and can be regulated by proinflammatory stimuli, mainly TNF-α. (A) This representative (of 4 independent donors) histogram (i) of a flow cytometric analysis represents the assessment of ALM-633-stained surface thiols on purified Tregs treated with the thiol-depleting agent NEM (2.5 or 5μM). (ii) Purified Tregs (n = 4) were treated overnight with H₂O₂ (10 or 20μM) with or without NEM (2.5μM) followed by flow cytometric evaluation of the specific cell death. (Bi) A representative (of 6 independent donors) histogram of the flow cytometric analysis of surface thiols on purified Tregs treated overnight with auranofin (25nM). (ii) Surface thiols were quantified based on the ALM-633 MFI and compared between treated (25nM auranofin) and untreated Tregs (n = 6). (C) Purified Tregs (n = 10) were cultured overnight with or without 1 μg/mL of a neutralizing anti-Trx-1 antibody (ATRX-03, IMCO) for blocking of secreted Trx-1. Surface thiols were quantified by flow cytometry. (D) A representative (of 6 independent donors) flow cytometric cell death analysis (viable cells are 7-amino-actinomycin D and annexin-V negative) of untreated Tregs (i) as well as cells treated overnight with 5μM H₂O₂ (ii), 1 μg/mL anti-Trx-1 antibody (iii), or both H₂O₂ and Trx-1 neutralizing antibody (iv). Specific cell death of Tregs (n = 6) induced by H₂O₂ with or without 1 μg/mL anti-Trx-1 antibody was quantified (v). (E) Relative gene expression of Trx-1 in purified Tregs (n = 6) was assessed on overnight culture with anti-CD2, anti-CD3, and anti-CD28 beads in a 1:1 cell to bead ratio, 5μM H₂O₂, 50 ng/mL interferon-γ, or 50 ng/mL TNF-α. The graph represents the ratio of Trx-1 gene expression in stimulated/unstimulated Tregs. (F) Furthermore, secreted Trx-1 was measured in the supernatants of Tregs (n = 7) treated as described in panel E, and results are shown with Trx-1 secretion in untreated cells set as 100%. (G) Purified Tregs were cultured overnight in the presence of 50 ng/mL TNF-α and/or 1 μg/mL anti-Trx-1 antibody. Alterations in (i) surface thiols (n = 7) and (ii) specific cell death (n = 10) induced by H₂O₂ (5μM) treatment were evaluated by flow cytometry. (H) The role of NF-κB for the TNF-α-mediated effects was evaluated by overnight treatment of Tregs with 50 ng/mL TNF-α with or without 10μM of the NF-κB inhibitor PS1145. The percentage (in relation to untreated Tregs) changes in (i) surface thiols (n = 10) and (ii) Trx-1 secretion (n = 5) were assessed by flow cytometry and ELISA, respectively. (I) Purified Tregs (n = 6) were cultured overnight with 50 ng/mL TNF-α and/or 100 IU/mL IL-2 followed by an ELISA measurement of Trx-1 in the supernatants. Bars represent SD. *P ≤ .05. **P ≤ .01. ***P ≤ .001.