Fig. 7

CLL T cells have altered membrane organization and disorganized lipid raft formation. A PBMCs from HD and CLL patients were stimulated with αCD3/αCD28 antibodies for 2 days. Immunofluorescence to assess the localization of lipid rafts on T cells was performed by staining samples with AF448-conjugated choleratoxin-B (CT-B), followed by incubation with AF594-conjugated antibodies against CD4 and CD8. DAPI was used as nuclear staining. Representative images of each experimental condition analyzed are shown (left). A schematic overview of lipid rafts localized around the T-cell receptor (TCR) in stimulated T cells and CT-B binding is provided (right). B CT-B intensity was quantified and plotted as total abundance (mean fluorescence, left) and clustering (maximal fluorescence, right) in HD and CLL T cells. Each dot represents the average value of CT-B fluorescence from all T cells in one field. C PBMCs from HD and CLL patients were either preincubated for 1 h with 2 mM methyl-β-cyclodextrin (MBCD), prior to a 2-day T-cell stimulation with αCD3/αCD28 antibodies in the continued presence of MBCD, or stimulated without MBCD present. Expression of CD25, CD69, and CD38 was measured on HD and CLL CD4+ T cells. D PBMCs from HD and CLL patients were stimulated with αCD3/αCD28 antibodies for 5 days in the same experimental conditions as in (C). Proliferation of CD4+ T cells is shown as percentage divided cells (left) and division index (right). Data are presented as mean ± SEM and differences were analyzed with two-way repeated measures ANOVA with Tukey’/Šidák’s s multiple comparison test (B) or with paired t-test (C, D). **** = p < 0.0001; *** = p < 0.001; ** = p < 0.01; * = p < 0.05