Deregulated CD40 signaling has been described in several human malignancies (18
). Yet, the question of whether aberrant CD40 signaling is sufficient to promote tumorigenesis remains unresolved. To investigate biological and pathological roles of constitutive CD40 signaling in B cells, we used the transmembrane domain of LMP1 as a surrogate to induce ligand-independent aggregation of the CD40 intracellular signaling domain. We show that constitutive CD40 signaling in B cells leads to a significant increase in peripheral mature B cells and, ultimately, to the development of mature B cell lymphomas.
Previous in vitro experiments have shown that the LMP1/CD40 fusion protein acts similar to LMP1 in terms of self-aggregation and is able to mimic ligand-triggered CD40 signaling with respect to the activation of NF-κB and stress-activated protein kinases (23
). In this study, we show that B cell–specific LMP1/CD40 expression in transgenic mice can replace CD40–CD40L interaction to promote immunoglobulin class switching, thus indicating that LMP1/CD40 indeed acts as a constitutively active CD40 receptor. B cell–specific expression of LMP1/CD40 in mice caused splenomegaly with an accumulation of both follicular and marginal zone B cells. The expansion of transgenic B cells may depend on their prolonged survival and/or increased proliferation as demonstrated by our ex vivo studies and in vivo BrdU incorporation assays ( and unpublished data). Despite their activated status, transgenic B cells were strongly impaired in GC formation. This is in accordance with a previous study showing that LMP1/CD40 is able to down-regulate bcl6
, which is a key regulator of the GC reaction (30
). We observed not only an accumulation of B cells but also of activated CD4+
T cells in young LMP1/CD40 mice. It is unlikely that this T cell expansion is caused by a leakiness of Cre recombinase expression because the same T cell expansion was observed in crossings with two independent B cell–specific Cre lines (CD19-Cre and CD21-Cre). Additionally, deletion of the stop cassette in T cells of LMP1/CD40 mice could not be detected by PCR (unpublished data). Therefore, the observed T cell expansion might instead be caused by an antigen-independent bystander effect induced by LMP1/CD40-expressing B cells. It has been shown that production of cytokines, as well as cell surface molecules, expressed on antigen-presenting cells during an immune response can activate T cells nonspecifically (31
), and that CD40-activated antigen-presenting cells promote the expansion of memory T cells (33
), suggesting that LMP1/CD40-expressing B cells may be able to generate an activating environment for T cells.
Deregulated CD40 signaling induced the development of lymphomas in >60% of mice older than 1 yr. Consistent with the B cell–specific expression of LMP1/CD40 and the effects of constitutive CD40 signaling on B cell survival and proliferation, most of these tumors were of clonal B cell origin. Only 1 out of 11 lymphomas was T cell derived. In all other tumors, the expanded population carried clonal IgH rearrangements and expressed B cell markers. The nodular growth pattern of the lymphomas was reminiscent of follicular lymphomas (35
). However, in contrast to follicular lymphomas, the variable regions of the immunoglobulin genes cloned from LMP1/CD40-expressing B lymphoma cells were unmutated, indicating a pre-GC origin. The late onset of tumor development, the heterogeneity in surface marker expression, and the activity of distinct signaling pathways suggest that multiple independent secondary oncogenic hits cooperate with CD40 activation to promote lymphomagenesis.
A role for CD40 signaling in the pathogenesis of human B cell lymphomas and carcinomas has previously been suggested. We provide strong evidence that constitutive CD40 signaling exerts transforming activity in vivo. The oncogenic property of constitutive CD40 signaling appears in contrast with the previously reported therapeutic effects of CD40 triggering in hematopoietic malignancies (18
). Indeed, triggering the CD40 receptor can promote growth arrest and cell death of malignant CD40-expressing cells and can induce an autologous antitumor T cell response by up-regulating the costimulatory molecules B7.1 and B7.2 (37
). Our data suggest that this therapeutic approach might not be risk free. The level and duration of the CD40 signal may critically influence the balance between the apparently contrasting biological effects triggered by CD40 signaling. Indeed, low-level constitutive engagement of CD40 was shown to induce proliferation of lymphoma cells, whereas treatment of malignant cells with high doses of agonistic anti-CD40 antibodies or CD40L resulted in cell cycle arrest and apoptosis (20
Our gel retardation and nuclear fractionation experiments revealed that, compared to control B cells, RelB-containing complexes are increased in the nuclei of LMP1/CD40-expressing B cells, indicating constitutive activation of the noncanonical NF-κB pathway. We suggest that in LMP1/CD40-expressing B cells, RelB dimerizes with either p50 or p52, which were both found to be increased in the nuclear fraction. This is in accordance with previous reports showing that the noncanonical NF-κB pathway based on p100 processing leads to the nuclear translocation of not only RelB/p52 but also of RelB/p50 dimers (17
). In contrast to the noncanonical pathway, the canonical NF-κB pathway was not found to be activated in LMP1/CD40-expressing B cells, as indicated by similar p65 and c-Rel levels in the nuclear fractions of unstimulated LMP1/CD40-expressing B cells and control B cells. The reduced basal phosphorylation of IκB-α in LMP1/CD40-expressing B cells compared with control B cells suggests that the canonical NF-κB pathway is even less active in the mutant cells than in unstimulated control B cells. Additionally, in comparison to control B cells, p105 levels were increased, whereas c-Rel levels were decreased, in the cytoplasm of LMP1/CD40-expressing B cells. Whether this alteration is caused by the constitutive activation of CD40 or a direct consequence of the constitutively activated noncanonical NF-κB pathway remains to be determined. The question arises why LMP1/CD40 expression in B cells leads selectively to the activation of the noncanonical NF-κB pathway. In wild-type B cells, CD40 ligation results in a fast and robust activation of the canonical NF-κB activity, which is rapidly counteracted by multiple negative-feedback mechanisms, whereas the noncanonical NF-κB pathway responds more slowly and appears to lack strong negative-feedback mechanisms (39
). This might explain the preferential chronic activation of the noncanonical NF-κB pathway in LMP1/CD40-expressing B cells. Recently, two studies have provided compelling evidence that the noncanonical NF-κB pathway plays a critical role in multiple myeloma (MM) pathogenesis (40
). Thus, 20% of patients with MM were found to carry mutations in essential NF-κB components, leading mainly to the activation of the noncanonical NF-κB pathway (41
). Of note, in some cases of MM, a strong up-regulation of CD40 was observed. As shown for CD30 (42
), CD40 overexpression might lead to self-aggregation of CD40, resulting in ligand-independent constitutive CD40 signaling, and thus creating a primary oncogenic event similar to that seen in our experimental system.
In addition to the noncanonical NF-κB pathway, the MAPKs Erk and Jnk were constitutively phosphorylated in unstimulated LMP1/CD40-expressing B cells. The importance of Erk phosphorylation for the improved survival of ex vivo–isolated LMP1/CD40-expressing B cells was supported by the fact that the survival advantage by LMP1/CD40 expression was abrogated in the presence of the Mek1/2 inhibitor UO126. The Ras–Raf–Mek–Erk pathway has been implicated in malignant transformation of various cell types and has been associated with proliferation and survival of hematopoietic cells (43
). A basal constitutive activity of Erk, which has been attributed to CD40, CD30, and RANK signaling, has also been described in the Hodgkin-Reed Sternberg tumor cells of Hodgkin disease (44
). Treatment of Hodgkin disease cell lines with the MEK inhibitor UO126 resulted in the inhibition of proliferation, underlining the importance of constitutive Erk phosphorylation for the proliferation of Hodgkin lymphoma cell lines. Unlike premalignant LMP1/CD40 B cells, constitutive Erk phosphorylation was not consistently detected in all independent primary tumors derived from LMP1/CD40-expressing B cells. This result suggests the existence of distinct oncogenic partners involved in CD40-driven lymphomagenesis, some of which may bypass the need for Erk activation.
Our results provide direct evidence that constitutive CD40 signaling leads to the selective activation of the noncanonical NF-κB pathway and the MAPKs Jnk and Erk. The concerted action of these signaling pathways ultimately leads to B cell lymphomagenesis. The mouse model described in this paper provides a tool to dissect the contribution of these signaling pathways to lymphomagenesis in vivo.