Fig 2: Dose-dependent synergy between IKK2ca and TCL1 expression in CLL-like lymphomagenesis.A Ex vivo analysis of young mice (9–15 weeks old) showing total splenocytes, absolute B1a and B2 cell numbers in spleen. B Scatter plot showing the percentage of B1a cells in peripheral blood (PB) over time, measured by flow cytometry analysis (% of living cells). The bold lines connect the median for each time point; the faint lines connect consecutive blood analyses from one mouse. C Kaplan-Meier survival curves, number of mice and median survival for each genotype are indicated. D Ex vivo analysis of spleens of burdened mice showing absolute numbers of B1a and B2 cells. E Ex vivo analysis of lymph nodes (LN), peritoneal cavity (PerC) and bone marrow (BM) from burdened mice showing absolute numbers of B1a cells measured by flow cytometry. D, E Age of burdened mice is indicated for each genotype. *p < 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal Wallis test followed by Dunn’s multiple comparison test. For full statistical analysis, see Table S2. B (CD19+), B1a (CD19hi B220low CD5+), B2 (B220hi CD19+).

Fig 3: Dose-dependent induction of SLL/CLL-like lymphomagenesis by constitutive IKK2 signaling.A, B Ex vivo analysis of spleens from aged CD19Cre IKK2ca and CD19Cre IKK2ca/ca mice compared to CD19Cre control mice (6–12 months old). A Photograph of representative spleens from different genotypes and spleen weight. Percentages of mice with spleen weight above 300 mg are indicated below the graph for each genotype. B Absolute numbers of B cells, marginal zone B cells (MZB), germinal center B cells (GCB) and B1a cells in spleens of mice of the indicated genotypes. Percentages of mice with splenic B1a cell numbers above 10 million are indicated below the graph for each genotype. C Kaplan-Meier survival curve of CD19Cre IKK2ca compound mice, number of mice, their genotypes and median survival are indicated. D Spleen weight and absolute splenic B1a cell numbers of aged mice (>12 months). Percentage of mice with spleen weight above 300 mg or B1a cell numbers above 10 million are indicated below each graph for each genotype. E Percentage of B1a cells in peripheral blood (PB, % of living cells) of mice at different time points measured by flow cytometry. F Histological analysis of CD19cre IKK2ca lymphomas. Representative HE and immunohistochemistry (IHC) stainings of spleen sections for samples with small lymphocytic lymphoma-like (SLL) morphology, as well as transformed diffuse large B cell lymphoma-like (t.DLBCL) and plasmacytoma-like (PC) are shown for the indicated genotypes. The incidences of lymphoma and Ki-67 scores calculated by IHC are shown for the indicated genotypes. G BCR clonality analysis. Representative BCR clonality plots are shown for the indicated genotypes. IgM BCR clonality was assessed by RNA sequencing of B1a cells from mice of the indicated genotypes. Each dot represents a unique CDR3 sequence supported by ≥5 reads; dot size reflects read count. Expanded clones (>5% of functional reads) are shown as larger colored dots; non-clonal sequences appear as small gray dots. BCR clone counts and dominant clone frequency (size of circle) are depicted for each animal per genotype and age cohort. H Clonality assessment by Southern blot analysis of StuI-digested DNA isolated from primary lymphomas from CD19cre IKK2ca and CD19cre IKK2ca/ca and lymphomas expanded in adoptive transfer experiments. The employed JH probe detects IgH VDJ rearrangements. Arrows indicate the transfer of primary lymphomas (L) to transferred lymphomas (TR) expanding in individual recipient mice. GL germline, Spl spleen, PC peritoneal cavity. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal Wallis test followed by Dunn’s multiple comparison test. For full statistical analysis, see Table S2. B (CD19+), B2 (B220hi CD19+), B1 (B220low CD19hi), B1a (B220low CD19hi CD5+), B1b (B220low CD19hi CD5-), GCB (CD19+ CD95hi CD38low) and MZB (CD19+ CD1dhi CD21+CD23low).