CD40 ligation of CLL cells alleviates CLL-mediated T-cell suppression in a contact-dependent manner. PBMCs derived from patients with CLL were thawed and cultured for 2 days on a layer of CD40L expressing fibroblasts. Two days after CD40 stimulation, CLL cells were harvested and fresh PBMCs derived from patients with CLL and HDs were thawed, CTV labeled and cocultured with or without the CD40-stimulated CLL cells in a 1:1 ratio. (A) T cells in presence or absence of CD40-stimulated CLL cells were stimulated for 2 days with soluble stimulatory CD3/28 antibodies, after which expression of CD25 was measured (HD, n = 4; CLL, n = 3). (B) In a different experiment, PBMCs from HDs and patients with CLL were thawed and stimulated with CD3/28 antibodies in the presence of CD40L expressing fibroblasts. Fibroblasts were removed after 24 hours, and T-cell activation (CD25) was analyzed for 6 days (HD, n = 4; CLL, n = 6). (C) To analyze cell-cell contact dependency, PBMCs derived from patients with CLL were first cultured on a layer of CD40L-expressing fibroblasts and harvested after 2 days and were plated either in the transwell insert or not with autologous PBMCs (schematic overview: supplemental Figure 2). After plating the cells in the transwell or together, T cells were stimulated for 2 days with CD3/28 antibodies after which CD25 expression was analyzed (HD, n = 10; CLL, n = 10). Red bars indicate T cells present from the start of the experiment and blue bars indicate T cells that were added during the transwell set up. P values were calculated using a 1-way ANOVA for panels A,C or a t test for panel B. The data are presented as mean ± SEM; ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. Stim, stimulated.