Specific inhibition of PIM2 affects the generation of PBs. (A) Left: On D6, total blood B cells were differentiated into 2 different cell populations: CD23⁺ aBCs and PBs. Right: PIM2 mRNA expression and protein expression in CD23⁺ aBCs and PBs on D6. Data are presented as the median (range), n = 10. (B) Diagram illustrating the mode of action of SSO-PIM2. Specific inhibition of PIM2 was performed by specifically blocking PIM2 gene expression using a novel antisense RNA strategy based on a morpholino SSO. In line with a previously described forced-splicing–dependent nonsense-mediated decay approach to knockdown,^56,57 the SSO targets the 5′ splice site of exon 2 of PIM2, induces out-of-frame exon skipping, and generates a premature stop codon in exon 4. This latter eventually triggers nonsense-mediated decay or generates a truncated, nonfunctional protein. Details are provided in the supplemental Methods. (C) Activated B cells were treated with various doses of SSO-PIM2 from D4 to D6. Top, PIM2 mRNA expression levels were compared with those in a control experiment, set arbitrarily to 100% (mean ± SD, n = 4) (top section). The agarose gel from the PIM2 PCR shows the exon skipping (bottom section). Dose escalation experiments allowed us to select an SSO-PIM2 concentration of 2 µM for the subsequent experiments in the in vitro model. Bottom, PIM2 protein expression was assessed by western blot on D6 after PIM2 inhibition with an increasing dose of SSO-PIM2. (D) PIM2 mRNA expression (mean ± SD, n = 6) (left) and protein expression (right), as determined by immunoblotting and immunofluorescence in CD23⁺ aBCs and PBs after PIM2 inhibition with SSO-PIM2 (2 µM). Sytox (blue) stains the nucleus. Scale bar: 10 µm. (E-F) On D4, cells were treated with SSO-PIM2 (E) or increasing doses of PIMi (F). On D6, analysis of cell differentiation was performed in the model described in panel A. Proportion of CD23⁺ aBCs (CD23⁺/CD38^low cells) and PBs (CD38^high/CD23^– cells) and the absolute number of each cell population were assessed by flow cytometry according to the gating strategy described in supplemental Figure 2D. Left: The absolute number of PBs and CD23⁺ aBCs obtained on D6 after PIM2 inhibition, compared with controls. Data are presented as the mean ± SD, n = 8. Right: One representative result of the proportion of PBs on D6 (evaluated by using flow cytometry) after PIM2 inhibition, compared with controls. Statistical significance was evaluated by using Mann-Whitney U (panel A), Wilcoxon (panel E), and Friedman (panel F) tests. *P < .05, **P < .01, ***P < .001, ****P < .0001. CpG, cytosine guanine dinucleotide; D0, day 0; D2, day 2; DMSO, dimethyl sulfoxide; NT, no treatment; ns, not significant. Further details are presented in supplemental Figure 2.