Figure 2.

CLL cells treated with CD40L+/gp350+ EVs upregulate costimulatory surface molecules and become immunogenic for gp350-specific CD4+ T cells.

(a) Western Blot of ERK1/2 (upper panel) and JNK (lower panel) phosphorylation in untreated CLL cells or cells treated with gp350+/CD40L+ EVs. Cell lysates were prepared after 20, 40 and 80 min of incubation. (b) PBMCs were isolated from seven patients diagnosed with CLL and stimulated with EVs from untransfected HEK293 cells and cells expressing CD40L and gp350 for 48 h. The upregulation of the immune accessory molecules CD80, CD86, CD54 and CD95 was studied by measuring the mean fluorescence intensity (MFI) by flow cytometry. Asterisks represent statistically significant different responses to EV treatment (Mann–Whitney–Wilcoxon test, **p < 0.01, ***p < 0.001). (c) CLL cells were labelled with CFMDA cell tracker dye and incubated with CD40L+/gp350+ EVs (upper right panel) or left untreated (upper left panel) overnight. The cells were mixed with untreated CFMDA-negative cells and CD54 expression was analysed by flow cytometry after 24 h (lower panel). (d) HLA-DR13+ mini-LCLs and primary CLL cells, as well as mismatched control cells, were used as antigen-presenting cells and incubated with 500 ng of different EVs, as indicated. After coincubation for 24 h with HLA-DR13-restricted gp350-specific CD4+ T cells, IFN-γ secretion was measured by ELISA. The results are shown as mean and SD of triplicates. P values were calculated with an unpaired t-test.