Fig 2: IRF4 re-expression results in apoptosis and differentiation of leukemia cells.a–c T8 and T11 cells were transduced with IRF4-RV or control empty vector (EV)-RV. a GFP+ cell frequency normalized to 24 h after transduction was recorded. One-Way ANOVA, Sidak post hoc for RV effect per tumor. Mean ± SD of n = 3 independent experiments. b Representative histogram of ?5 surface expression of GFP+ cells at 48 h. c Pooled Quantification of ?5Hi cells for three (24, 72 h) to five (48 h) independent experiments for T8 and T11. d–h T8 cells were collected in duplicates at 24 h after EV-RV and IRF4-RV transduction and subjected to bulk RNAseq. d MDS plot of top 100 gene ontology (GO) gene-sets varying between EV and IRF4 transduced T8. Representative gene sets annotated. Size of circles = number of genes, color = z score. e Analysis strategy for GO gene-set clustering using Markov clustering. f Gene-sets from Markov cluster 3 and corresponding P values and z scores. g Volcano plot of B-cell genes from Markov cluster 3 (red) highlighted within all differentially regulated genes (black). h Heat map of B-cell receptor signaling GO gene-set. Immunoglobulin genes and the tumor suppressor Blnk are marked. Color = z score.

Fig 3: Irf4-/- B lymphopoiesis is preleukemically altered.a–c flow cytometric analysis of BM cells for Hardy markers as in Fig. 1j. a tSNE of BM cells gated on B220+ cells. Colors correspond to Hardy fractions identified by the markers detailed in the legend. b and c quantification of Hardy fraction frequencies for n = 3 mice per genotype. d–h BM cells from Irf4-/- and wt mice were cultured in the presence of 10 ng/mL rmIL-7 for 6 days and d counted every two days. e, f After 4 days, cells were stained as in a–c and Hardy fractions quantified. g frequency and h absolute cell counts of ?5+ cells on day 4. i spleen cells from Irf4-/- (“y” = young: 6–10 weeks and old: >6 months) and wt mice were analyzed for the presence of CD2–/dimsIgµ– cells within the B220+ gate. one-way ANOVA, Tukey post hoc. j–m 7 µm cryosections from Irf4-/-Il-7eGFP and wt Il-7eGFP mice were stained for B220, CD2, GFP, and DAPI. j exemplary regions of BM cryosections. Arrowheads indicate B220+CD2–/dim cells. Scale bars = 15 µm. k automated B220+ cell detection: gray spheres indicate B220+ cells, larger spheres B220+CD2–/dim cells, color-coded for their distance to GFP+ cells. Rectangles indicate magnified areas in j. Scale bars = 40 µm. l, m quantification of distances to IL-7+ cells for l all B220+ and m B220+CD2–/dim cells. (n = 4 mice per genotype, one cryosection from femur metaphysis per mouse analyzed). Box and whiskers indicate mean and 95-IQR, dots indicate cells outside 95-IQR. n BM cells from Irf4-/- and wt mice were gated on B220+sIgµ– fr.A-D cells and analyzed for CXCR4 expression (left panels as representative staining). Data is presented for n = 7 (wt) and n = 6 (Irf4-/-) mice as the ratio of geometric mean for CXCR4 to isotype staining. o MACS-purified fr.A-D cells from BM were placed in the top insert of a Boyden chamber and left to migrate towards differing concentrations of CXCL12 for 16 h. Dots represent n = 4 biologically independent experiments, presented as migrated percentage of input cells. Two-Way ANOVA, Sidak post hoc for (b, c, e, f, o), Two-tailed unpaired t test for (g, h, l–n).

Fig 4: Spontaneous emergence of preB-I cell BCP-ALL in adult Irf4-/- mice.a A cohort of 80 Irf4-/- and wt mice was observed over 500 days for tumor development. Kaplan–Meier plot of survival. Box and whisker plot indicates minimum, maximum and median age at tumor appearance of the 14 affected mice. b Macroscopic appearance of an exemplary tumor (asterisk) and LNs (arrowheads) in an Irf4-/- mouse. Right: Hematoxylin-Eosin (HE) staining from the tumor. Scale bars: top: 50 µm, bottom: 20 µm. c Longitudinal spleen (S) axis (mm) of Irf4-/- mice with (n = 8) or without (n = 5) tumor and control wt mice (n = 6). tum = tumor. d HE stainings of lung, liver, and BM of tumor mouse T8 and a healthy Irf4-/- mouse. Scale Bars: 50 µm (lung and liver), 20 µm (BM). e IHC-stainings of T8 mouse spleen for Igµ, B220, and Ki67. Scale bars: 100 µm. f Schematic representation of gross Hardy fractioning by surface B220 and Igµ expression. g Whole BM cells from wt, Irf4-/-, and tumor mice were stained for B220 and sIgµ expression and analyzed by flow cytometry. h Quantification of cell frequencies gated as in (g) for BM, S, LN, and pB (peripheral blood) of n = 3 mice per group. i Tabular representation of Hardy fr. A-D by size, CD43-, CD24-, and BP-1-surface expression. j Surface expression of markers as in (i) of in vitro cultured T8 tumor cells k quantification of cell frequencies gated as in (j) for three tumors (T8, T11, TD3) l surface ?5 expression on T8, T11, and TD3 by flow cytometry in comparison to whole BM cells from Irf4-/- mice (BM) as a negative control. Statistical significance testing was performed with (c) one-way Welch-ANOVA followed by Dunnet’s T3 multiple comparison test and (h) with two-way ANOVA followed by pair-wise Tukey corrected comparisons within each organ. Bars depict mean ± SD, dots indicate mice (h) or distinct Irf4-/- tumors (k).