PIM2 expression and regulation during normal B-cell differentiation. (A) Using a 2-step cell culture process, VPD-labeled naive B cells (NBCs) were differentiated into the cell populations P1, P2, and P3. P2 cells were subdivided into P2/CD23⁺ and P2/CD23^– subsets. The PB P1 population arose from the P2/CD23^– subset. (B) Left: PIM2 RNA-seq results on D4 or day 5 (D5) in the various cell populations. Data are presented as the mean ± SD, n = 3. Right: PIM2 mRNA expression in the various cell populations on D6. mRNA data are presented as the median (range); n = 6. Bottom, PIM2 protein expression in all cell populations, from day 0 (D0) to D6. (C) A single-cell quantitative RT-PCR heat map of the expression of 31 genes in P2/CD23^– vs P2/CD23⁺ cell subsets; the red arrow indicates PIM2 mRNA expression. (D) PIM2 mRNA expression (top) and protein expression (bottom) in different tonsil-derived B-cell populations. mRNA data are presented as the median (range), n = 7. (E) The Integrative Genomic Viewer genome browser window, showing several features around the PIM2 locus. Colored tracks at the top are our RNA-seq and ATAC-seq data.⁵ ATAC-seq showed that the open regions included the PIM2 promoter and an upstream enhancer that physically interact together. ATAC-seq regions close to PIM2 comprise transcription factor–binding sites for relevant factors such as STAT3, according to ENCODE data, and also BACH2, including one we have identified by chromatin immunoprecipitation.¹⁷ 5hmC marks are identified in ATAC-seq regions and the body of the PIM2 sequence in PBs.⁴ Details are provided in supplemental Figure 2A. (F) Levels of BACH2 and PIM2 mRNA expression (left) and protein expression (right) in D4^lo cells after transfection with siBACH2 on day 2 (D2). mRNA data are presented as the mean ± SD, n = 4. (G) On D5, PBs were sorted, starved, and then stimulated with the indicated cytokines. Left: PIM2 mRNA expression after 4 hours (pink) and 16 hours (red). For each time point, the results were normalized against the nonstimulated condition (set arbitrarily to 1 [in gray]). mRNA data are presented as the mean ± SD, n = 5. Right: PIM2, pSTAT3 and pSTAT5 protein expression after 16 hours. (H) On D5, PBs were sorted, starved, and then treated with increasing doses of a STAT3 inhibitor (Stat3i) 1 hour before the addition of IL-10. PIM2 mRNA expression (left) and protein expression (right) were assessed 4 hours after the addition of IL-10. mRNA data are presented as the mean ± SD, n = 5. (I) Regulation of PIM2 expression during B-cell differentiation. At the start of B-cell differentiation and despite the open chromatin on the promoter and on other regulatory regions, PIM2 expression is constrained by BACH2. BACH2 expression then declines throughout the differentiation process, whereas T follicular helper–driven molecules stimulate STAT3 signaling. Both events promote the burst in PIM2 expression in cells committed to differentiation into PBs. Statistical significance was evaluated by using the Mann-Whitney U test (panels C, F, G, and H) or the Kruskal-Wallis test (panel H). *P < .05, **P < .01, ***P < .001. CpG, cytosine guanine dinucleotide; GC, germinal center; IgM, immunoglobulin M; MBC, memory B cell; ns, not significant; siCTL, small interference (si) RNA control (CTL); TFs, transcription factors; VPD, violet cell proliferation dilution. Further details are presented in supplemental Figure 1.