WM cells enhance Treg expansion. (A) Treg expansion assay. CD4⁺CD25⁺ Tregs cultured with either CD19⁺ B cells derived from healthy donors or WM cells (BCWM.1; WMCL.1), for 72 hours using a transwell. FOXP3⁺ cells within the CD4⁺ cells are shown. Significantly higher Treg expansion was observed upon exposure to WM cells compared with B cells derived from healthy donors. A representative experiment is shown. The experiment was repeated 3 times. ∗∗∗P < .001 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. ∗∗∗P < .001. (C) Treg-mediated suppression as measured by CFSE dilution. CD4⁺CD25⁻ Tcons were isolated from HDs and labeled with 5 μM CFSE. Cells were activated with anti-CD3– and anti-CD28–coated beads and cultured either alone or in the presence of either CD4⁺CD25⁺ Tregs obtained from patients with WM or HDs, at the indicated Tcon-to-Treg ratio. After 72 hours, proliferation was determined by CFSE dilution and flow cytometric analysis. ∗∗P < .001 determined using two-way ANOVA. Error bars indicate mean ± SD. (D) Heatmap obtained from RNA-seq comparing WM-Tregs (n = 14) and HD-Tregs (n = 8) (EBI3: P = .03; FGL2: P < .001; GZMB: P < .0001; PRF1: P < .001; TIM3: P = .02). (E) WM-Tregs present with a significant enrichment of CTLA4-related genes, as assessed by GSEA. NESs were generated by comparing WM-Tregs and HD-Tregs. NES, nominal P value, and false discovery rate q values are shown.