CD40/CD40L blockade is functionally relevant in halting WM-cell–dependent Treg induction, even in the context of CXCR4^C1013G-mut WM cells. (A) CD4⁺CD25⁻ non-Tregs were cultured either alone or in the presence of WM cells (MWCL.1) for 72 hours, in presence or absence of CD40/CD40L-inhibitor (DRI-C21045; 8 μM), using a transwell. Inhibition of Treg induction was observed upon exposure to DRI-C21045. The percentage indicates the CD25⁺FOXP3⁺ cells gated on CD4⁺ cells. A representative experiment is shown. The experiment was repeated 3 times. P indicates P value, 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. Significant inhibition of Ki-67 in Tregs was observed. A representative experiment is shown. The experiment was repeated 4 times. P indicates P value, determined using one-way ANOVA. Error bars indicate mean ± SD. (C) Same experimental approach as in panel A, using MWCL.1-CXCR4^C1013G mutated cells. (D) Same experimental approach as in panel B, using MWCL.1-CXCR4^C1013G mutated cells. (E) Both induced and noninduced Tregs, prepared as in panel A, were harvested, and cell lysates were subjected to western blot analysis using antiphoshpo(p)-AKT, anti–total (tot)-AKT, anti–p-ERK, anti–tot-ERK, and anti–β actin.