Fig. 3: Human CLL cells with loss-of-function NFKBIE mutations are positively selected in vitro by microenvironmental signals that activate the NF-κB pathway.

A Analysis of effects of microenvironmental signals involved in NF-κB pathway activation on the growth of NFKBIE-mutated primary human CLL cells. NFKBIE mutations were introduced in primary leukemic cells from 11 CLL patients with wild type NFKBIE by CRISPR/Cas9 editing. The cells were cultured for 3 days with 3T3-CD40L fibroblasts (1:10 ratio) +IL-4/IL-21 before being split and cultured for additional 48 h alone or in the presence of 3T3-CD40L fibroblasts, CpG ODN2006 (1 μM), or anti-IgM (20 μg/ml). NFKBIE mutant allele frequency was determined at day 5 of culture. The upper left panel provides a schematic representation of the experimental approach. The graph in the upper right panel represents the summary of the results with the 11 different CLL samples. Statistical analysis was done using the Wilcoxon signed-rank test. The results of two representative samples (G398 and G392) are shown in the bottom panels. B Analysis of effects of CD40L-stimulation on the growth of CXCR4- or CD19-edited primary human CLL cells. The same experimental approach was used as in part A, except for the use of guide RNAs targeting human CXCR4 or CD19 instead of NFKBIE. Capillary plots of the targeted region of the CXCR4 and CD19 genes from one representative sample out of 3 analyzed are shown. Wild-type alleles are indicated by a red arrow; mutant alleles are indicated by a black arrow. C Flow cytometry analysis of one CLL sample transfected with GFP-labeled Cas9 protein. Analysis was done after 1 h and after 72 h from the transfection.