Concurrent activation of NF-κB and Notch signaling induces B-cell lymphomatous transformation in adult mice. (A) Reduced tumor-dependent survival of double transgenic TriB mice (coactivation of NF-κB and Notch2ICN) compared with single transgenic NIK mice (NF-κB activation only), single transgenic Notch mice (Notch activation only), and normal B6 mice (no genetic pathway activation) used as controls (Con; n ≥ 12 for each phenotype). Lymphoma development was fully penetrant (100% tumor incidence in TriB mice). (B) Flow cytometric analysis of changes in the mature B-cell compartment leading to lymphoma development in TriB mice. CD21 and CD23 expression of GFP⁺-gated B cells was measured at 5 time points ranging from 4 to 15 months of age. Progressive loss of, first, CD23 expression and, later on, CD21 expression leads to a largely homogeneous double-negative population of CD21⁻CD23⁻ lymphoma cells. B cells of normal mice (Con) gated on CD19⁺ were included as control. Mean frequencies ± standard error of cell fractions are indicated (n = 3). (C) Loss of B220 expression in the course of neoplastic B-cell development, using tumor-bearing 12-month-old TriB mice (n = 6) as an example. The flow result shown was confirmed by B220 immunostaining of tissue sections presented in supplemental Figure 2C. (D) Representative flow diagrams of expression levels of MZB markers CD1d, CD36 and CD25 in MZPs (red) and lymphoma cells (green) of TriB mice relative to MZPs in normal mice (wild type [WT]) used as control (black; n = 3 in both groups). (E) Flow diagram indicating limited potential for expression of plasma cell marker CD138 and lack of expression of myeloid markers Mac1/Gr1 in TriB lymphoma cells (n = 3). (F) Hematoxylin and eosin–stained tissue sections of liver and spleen of TriB mice diffusely infiltrated with neoplastic B cells (original magnification, ×500; n = 3). Residual hepatocytes in the liver are discernible to the right of the yellow dotted line. (G) Agarose gel containing ethidium bromide–stained PCR fragments of genomic VDJ rearrangements that indicate monoclonal growth (single band) of lymphomas obtained from TriB mice (right; panel, n = 3) but polyclonal B cells (multiple bands) in WBM or spleen (Spl) samples from TriB mice. Germ line JH1-3 fragments are denoted by arrow heads on the left, next to the size marker. A PCR indicator fragment of T-cell gene Thy1 served as positive control. (H) PCR products from TriB lymphoma shown in panel G were subcloned with the help of a TA cloning kit, DNA sequenced, and aligned to corresponding regions of the immunoglobulin heavy-chain germ line locus using the IgBLAST software tool (n = 5; supplemental Figure 3A). In the example shown, 9 of 10 PCR fragments were identified as V3D3J4 rearrangements. The remaining fragment used diversity gene D2 instead. (I) Ingenuity pathway analysis results for canonical pathways using differentially expressed genes comparing TriB lymphoma with TriB MZPs. Differentially expressed genes were defined as q value <.05 and log2-fold change ≥1 or ≤ −1. z score refers to the direction and magnitude of the enrichment. (J) z scores of selected significant canonical pathways that are enriched in TriB lymphoma (yellow) or TriB MZPs (blue). For all pathways listed, P < .05. **P < .01.