WM cells enhance Treg induction. (A) CD4⁺CD25⁻ non-Tregs were cultured either alone or in the presence of WM cells (BCWM.1; MWCL.1) or CD19⁺ B cells derived from healthy donors, for 72 hours, using a transwell. Significant Tregs induction was observed upon exposure to both BCWM.1 and WMCL.1. The percentage indicates the CD25⁺FOXP3⁺ cells gated on CD4⁺ cells. A representative experiment is shown. The experiment was repeated 3 times, using different normal CD19⁺ B cells (n = 4). ∗∗P < .01 determined using one-way ANOVA. Error bars indicate mean ± SD. (B) The proliferative rate of CD4⁺CD25⁺FOXP3⁺ Tregs was assessed by evaluating the percentage of Ki67⁺ cells. A significant increase in Ki-67 expression in Tregs was observed in the BCWM.1 and WMCL1 coculture groups compared with in the absence of cells or with CD19⁺ B-cell groups. A representative experiment is shown. The experiment was repeated 3 times. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, determined using one-way ANOVA. Error bars indicate mean ± SD. (C) CD4⁺CD25⁻ non-Tregs were cultured either alone or in the presence of CD19⁺ cells derived from the BM of patients with WM (WM-1; WM-2), or CD19⁺ B cells derived from healthy donors, for 72 hours, using a transwell. Significantly higher Treg induction was observed upon exposure to WM tumor cells. The percentage indicates the CD25⁺FOXP3⁺ cells gated on CD4⁺ cells. (D) The proliferative rate of CD4⁺CD25⁺FOXP3⁺ Tregs was assessed by evaluating the percentage of Ki67⁺ cells. WM and HD, as in panel C. P indicates P value; one-way ANOVA; Error bars indicate mean ± SD. (E) Tregs derived from patients with WM present with a significant enrichment of MAPK, PI3K/AKT-related genes, as assessed by GSEA. NESs were generated by comparing WM-Treg and HD-Tregs. NES, nominal P value, and false discovery rate q values are shown for each plot.