CD38-targeting agents deplete Tregs (A) CD38 expression was measured as percentage expression and MFI on CD4⁺CD25⁺CD127^lo gated Tregs in PBMCs isolated from healthy donors (n = 6) or patients with CLL (n = 12). Gating strategy is shown, and compiled data of percentage CD38⁺ cells are presented as mean ± SEM. (B) Using a similar gating strategy, percentage of Tregs was measured ± treatment with daratumumab (1 µg/mL; 72 hours) in PBMCs from 13 patients with CLL. (C) The effect of daratumumab (Dara) or kuromanin (Kuro) on CLL cell-mediated conversion of CD4⁺CD25⁻ T cells to iTregs was tested. CD19⁺CD5⁺-sorted CLL cells were cocultured with CD4⁺ T cells from healthy donors with or without daratumumab (1 µg/mL) or kuromanin (30 µM) for 72 hours, followed by measurement of iTregs. (D) In a similar manner, the effect of daratumumab (D) or kuromanin (K) was assessed on cytokine induced iTregs (without any CLL cells). (E) The direct cytotoxic effects of daratumumab (D, 1 µg/mL, 24 hours) or kuromanin (K, 30 µM, 24 hours) were measured on Tregs from patients with CLL (n = 6 patients), using a fixable live dead assay. (F) Separately, CD4⁺CD25⁺CD127^lo sorted cells from 6 patients with CLL were treated with anti-CD38 agents (same concentrations and time as in panel H) and dual-stained with annexin V/PI followed by flow cytometry analysis. Contour plots are representative, and compiled data are presented as mean ± SEM with individual data points overlaid. Each experiment was performed at least twice in duplicate. *P < .05.