Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis

CD40, a member of the tumor necrosis factor (TNF) receptor family, plays an essential role in T cell–dependent immune responses. Because CD40 is widely expressed on the surface of tumor cells in various B cell malignancies, deregulated CD40 signaling has been suggested to contribute to lymphomagenesis. In this study, we show that B cell-specific expression of a constitutively active CD40 receptor, in the form of a latent membrane protein 1 (LMP1)/CD40 chimeric protein, promoted an increase in the number of follicular and marginal zone B cells in secondary lymphoid organs in transgenic mice. The B cells displayed an activated phenotype, prolonged survival and increased proliferation, but were significantly impaired in T cell-dependent immune responses. Constitutive CD40 signaling in B cells induced selective and constitutive activation of the noncanonical NF-κB pathway and the mitogen-activated protein kinases Jnk and extracellular signal–regulated kinase. LMP1/CD40-expressing mice older than 12 mo developed B cell lymphomas of mono- or oligoclonal origin at high incidence, thus showing that the interplay of the signaling pathways induced by constitutive CD40 signaling is sufficient to initiate a tumorigenic process, ultimately leading to the development of B cell lymphomas

Rapid conditional knock-down–knock-in system for mammalian cells

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down–knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein

LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1.

The Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell-specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells

B cell expansion and lymphomagenesis induced by chronic CD40 signaling is strictly dependent on CD19.

CD40, a member of the Tumor necrosis factor receptor family, is expressed on all mature B-cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40-signaling in B-cells induced by a fusion protein consisting of the transmembrane part of the Epstein-Barr viral Latent Membrane Protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Since LMP1/CD40-expressing B-cells showed an upregulation of CD19, we investigated CD19 function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced life span of mature B-cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B-cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B-cells is dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival- and proliferation-signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K-dependent Erk-phosphorylation in human Diffuse Large B Cell Lymphoma cell lines. Our data provides evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40-signaling

(A) Lymphocytes of spleen (SP) and inguinal LNs were analyzed for the expression of IgM and IgD by flow cytometry

Numbers indicate the mean percentages and SD of gated populations. SP: follicular (FO) B cells (IgMIgD), marginal zone (MZ), and transitional B cells (IgMIgD); LN: IgMIgD. Values were calculated from seven independent experiments. (B) Flow cytometric analysis of follicular B cells (FO; CD21CD23) and marginal zone B cells (MZ; CD21CD23) in the spleen. Numbers indicate mean percentages and standard deviations of B220 B cells displaying a MZ B or FO B cell phenotype. Values were calculated from seven independent experiments. (C) Absolute numbers of FO and MZ B cells in the spleens of LMP1/CD40 and control mice. (D) Splenocytes were stained with antibodies specific for the indicated activation and adhesion markers. Histograms show surface expression of the indicated markers on gated B220 B cells from LMP1/CD40 (continuous line) and control (dotted line) mice. Lymphocytes isolated from LNs showed a similar extent of activation (not depicted).<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p

(A) Targeting strategy for the insertion of a conditional allele into the murine -locus

Cre-mediated recombination leads to deletion of the stop cassette and expression of under transcriptional control of the endogenous -promoter. The scheme of the targeting construct shows the EcoRI recognition sites and the location of the probe. The expected fragments after EcoRI digestion and hybridization with the labeled probe are indicated by the thin lines. SAS, splice acceptor site; STOP, Stop cassette, XbaI, site of insertion. (B) Southern blot analysis of EcoRI-digested genomic DNA showing the deletion efficiency of the stop cassette in B cells of the spleen (SP; lane 3) and the BM (lane 6) of LMP1/CD40 mice. DNA from B cells of control mice (lanes 2 and 5) and mice heterozygous for (lane 1 and 4) was included. B cells were purified by using magnetic beads against CD19. After deletion of the stop cassette, the rosa26 probe detects a 5.2-kb fragment (LMP1/CD40). The 15- and 7.1-kb fragments represent the wild-type allele ( locus) and the targeted allele (), respectively. The deletion efficiency was ∼50% in the BM and almost complete in the spleen. (C) LMP1/CD40 protein expression. Western blots were prepared from spleen lysates of LMP1/CD40;CD19-Cre (lanes 1 and 2) and control mice (lane 3). As control, protein lysates of 293 cells transiently transfected with a LMP1/CD40 expression vector were included in the analysis (lane 4). The 56-kD LMP1/CD40 chimeric protein was detected by an anti–human CD40 antibody. ns, nonspecific band.<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p

(A) Whole-cell extracts of splenic B cells of LMP1/CD40 (L/C) and control (co) mice from three independent preparations (1–3) were loaded on one gel and analyzed for Jnk (Jnk2, 54 kD; Jnk1, 46 kD), Erk (Erk1

44kD; Erk2, 42 kD), p38/MAPK (38 kD) and the corresponding phosphorylated forms with specific antibodies. Equal protein loading was controlled by Ponceau S staining. The graph shows the mean values of the fold induction of the signals from the unphosphorylated and phosphorylated forms of Jnk, Erk, and p38/MAPK compared with the signals in control cells. Mean values and SDs were calculated from at least five independent experiments. SDs are shown by error bars. *, P < 0.05; **, P < 0.001, calculated by the two-tailed Student's test. (B) After CD40 triggering, LMP1/CD40-expressing cells are damped in activation compared with control cells. Splenic B cells of LMP1/CD40 and control mice were stimulated with α-CD40 antibody for the indicated time points, and Western blots were performed as described in A. Equal protein loading was controlled by tubulin staining. One representative experiment out of three is shown. (C) The improved survival of LMP1/CD40-expressing B cells is dependent on continuous Erk phosphorylation. Splenic B cells of LMP1/CD40 and control mice (CD19-Cre) were cultured for up to 5 d with the Mek1/2 inhibitor U0126 (dotted line) and the Jnk-inhibitor SP600125 (dashed line; triangle and square, respectively). As control, B cells from LMP1/CD40 and control mice were cultured in the presence of DMSO (continuous lines, rectangle, and circle, respectively). Percentages of living cells (Topro negative) were determined by flow cytometry at day 1, 3, and 5. The bars show mean percentages of living cells of three independent experiments. Error bars show the SD.<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p

(A) Southern blot analysis to examine IgH gene configuration with an IgH-specific probe

Genomic DNA was prepared and digested with EcoRI from splenic cells of LMP1/CD40 mice that developed neoplasias (34, 142, 176, 197, 230, 247, 365, and 221), one young LMP1/CD40 mouse (900; 3 mo of age), as well as a control mouse (905). The sequence analysis of the amplified IgH genes is shown in Fig. S6. (B) Representative histological analyses of diseased LMP1/CD40 mice and age matched control mice. (1) HE-stained normal spleen. (2) HE-stained spleen representative for diseased LMP1/CD40 mice, showing prominent nodular tumor infiltrates. (3) Lymphoma infiltrate within the spleen of a representative diseased LMP1/CD40 mouse. B cells were visualized by immunohistochemistry using an antibody specific for B220. (4) Higher magnification of section described in 3. (5) Dispersed reactive T cells within the lymphoma infiltrate were detected by an anti-CD3–specific antibody. A representative highly magnified section is shown. Bars: (1 and 2) 400 μm; (3) 100 μm; (4, 5, and inset) 25 μm. (C) Flow cytometric analysis of splenic cells for the expression of IgM and IgD. The gates were set according to the age-matched controls. For the diseased LMP1/CD40 mice, a shift toward one or two main populations could be observed, as shown for three representative samples. Fig. S6 is available at .<p><b>Copyright information:</b></p><p>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1317-1329.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413030.</p><p></p