Breg-like CLL cells promote conversion of naive Th cells into Tregs that are mediated by IL-10 and TGF-β. (A) Baseline proportion of CD4⁺CD25⁺CD127^lo and FoxP3⁺ Treg cells was compared within PBMCs isolated from healthy donors (n = 6) or patients with CLL (n = 12). (B) CD19⁺CD5⁺ cells and autologous naive Th cells (CD4⁺CD25⁻) were sorted from patients with CLL (n = 13). CD4⁺ T cells were also isolated from healthy donors (n = 6). Next, CLL cells were cocultured with either autologous or allogeneic (healthy donor-derived) naive Th cells in a 1:1 ratio that were prestimulated with anti-CD3 and anti-CD28 antibodies. After 3 days of incubation, cells were labeled for Treg markers and analyzed by flow cytometry. (C) To identify the mediators of conversion of naive Th cells to iTregs, a transwell coculture assay was conducted. The upper chamber contained either flow-sorted CD19⁺CD5⁺CD24⁺CD38⁺ (autologous Breg-like CLL cells) or CD19⁺CD5⁺CD24⁺CD38⁻ (autologous non-Breg) CLL cells from 8 patients with CLL, whereas the lower chamber contained autologous flow-sorted naive Th cells prestimulated with anti-CD3 and anti-CD28 antibodies. In addition, IL-10 or TGF-β neutralizing Ab (alone or in combination with one another) were added to the upper chamber and incubated for another 72 hours. The proportion (%) of Tregs was subsequently analyzed by flow cytometry. Contour or density 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; **P < .001.