In this study, we describe a novel congenital disorder featuring the first known germline mutation in CARD11
in a family affected by polyclonal expansion of naive B cells, splenomegaly, and potential predisposition to B cell malignancy (Darte et al., 1971
). A separate patient with highly similar disease manifestations harbors a germline G116S CARD11
mutation, which was previously characterized in DLBCL as a gain-of-function somatic mutation (Lenz et al., 2008a
). Although both mutations cause constitutive NF-κB activity in unstimulated lymphocytes, our findings suggest that the germline mutation does not transform a B cell clone outright. Instead, in addition to the oncogenic potential of constitutive NF-κB hyperactivation, germline CARD11
mutants may promote B cell malignancy by dramatically increasing the number of naive B cells that may undergo secondary genomic alterations and become malignant (Lenz et al., 2008b
; Lenz and Staudt, 2010
), as exemplified in P1 (see Supplemental text). It is well established that genomic instability inherent in the GC reaction is a primary driver of mutagenesis and B cell transformation (Küppers, 2005
). Indeed, a monoallelic deletion of tumor suppressor genes located in chromosome 13q14.3, associated with ~50% of B-CLL cases, was detected in P1 B-CLL cells and may have contributed to oncogenesis (see Supplemental text; Pekarsky et al., 2010
). However, preliminary evidence suggests that the P1 tumor does not harbor recurrent mutations recently associated with B-CLL (Fabbri et al., 2011
; Puente et al., 2011
). Our studies are ongoing to determine the nature of secondary mutations in B-CLL tumor cells from P1 and the frequency of CARD11
mutations specifically in B-CLL.
Our analysis indicated patient B cells did not appear to proliferate excessively in the periphery, but instead may have accumulated from increased B cell output from the bone marrow, as indicated by elevated CD10+
transitional B cells in the blood and consistent with hyperplasia reported in P1 bone marrow (Darte et al., 1971
). Patients’ peripheral blood B cells died more rapidly ex vivo, consistent with heightened sensitivity of transitional B cells to cell death (Palanichamy et al., 2009
). However, survival of transitional and/or naive B cells that complete maturation in peripheral lymphoid tissues may have been enhanced, explaining the continuous increase in spleen size over time (). This phenotype resembles mice expressing constitutively active IKK-β in the B cell lineage, which results in accumulation of longer-lived, resting mature B cells that are no longer dependent on BAFF for survival (Sasaki et al., 2006
). BAFF normally acts as a sensitive limiting factor for controlling peripheral B cell numbers; indeed, excess BAFF in circulation is associated with higher B cell production, autoimmune disease, and B cell malignancy (Khan, 2009
). However, neither serum BAFF levels nor surface expression of BAFF receptor (BR3) was elevated in our patients, suggesting B cell accumulation occurs independently of increased BAFF signaling through the alternative NF-κB pathway. Increased levels of unprocessed p100 in patient cells further suggest the alternative NF-κB activity is less engaged. Instead, we speculate that constitutive, canonical NF-κB signaling via mutant CARD11 drives greater output of immature/transitional B cells from the bone marrow and may activate prosurvival programs (e.g., BCL2 up-regulation) that permit the accumulation of noncycling and perhaps self-reactive immature B cells in vivo that would otherwise be deleted or anergized in response to chronic BCR engagement (Healy and Goodnow, 1998
). It will be interesting to determine the extent of autoreactivity in the patients’ B cell repertoire given that the transitional B cell stage is thought to represent a key negative selection checkpoint for self-reactive B cell clones (Carsetti et al., 1995
The fact that prominent lymphocytosis is largely restricted to the B cell compartment is surprising in our patients, because CARD11 mediates AgR-triggered activation of NF-κB in both B and T cells (Thome et al., 2010
). Although a recent study reported that adoptive transfer of bone marrow cells expressing constitutively active CARD11 resulted in Th2-mediated inflammation in mice (Blonska et al., 2012
), we did not observe a similar phenotype in our patients. In fact, patient T cells were hyporesponsive to AgR stimulation and did not proliferate in vitro unless stronger co-stimulation was provided. A modestly elevated proportion of CD4−
DN T cells in patient samples, which are typically unresponsive and die rapidly in vitro, cannot explain this phenomenon alone. More importantly, our investigation marshals clinical evidence consonant with conclusions from a recent study by Krishna et al. (2012)
. They showed that chronic NF-κB signaling, triggered by transgenic expression of constitutively active IKK-β (ca–IKK-β) in mice, also renders T cells hyporesponsive to Ag stimulation, including defects in calcium flux, ERK phosphorylation, induction of activation markers, proliferation, and IL-2 secretion. Unlike ca–IKK-β mouse T cells, however, our patients’ T cells did not show evidence of increased apoptosis or exhaustion (e.g., increased PD-1 expression; unpublished data). Consistent with the classical two-signal paradigm of T cell activation, we speculate that T cell hyporesponsiveness in mutant CARD11 patients may reflect a mild form of anergy induced by a chronic AgR-like signal 1 driven by E127G CARD11 in the absence of a strong co-stimulatory signal 2 (Chappert and Schwartz, 2010
). Rescued proliferation in the presence of CD2 ligation or exogenous IL-2 lends credence to this model. In contrast, B cell proliferation can be triggered by BCR cross-linking alone, mimicked by mutant CARD11. Impaired T cell responses may be linked to increased infections in mutant CARD11 patients, particularly in P4.
Our findings logically suggest overactive CARD11/NF-κB signaling may skew lymphocyte development and function in favor of selective B cell expansion. However, we also observed defects in Ig secretion and plasma cell differentiation in vitro for P2 and P3 mature B cells. The presence of mutant CARD11 throughout B cell development may paradoxically enlarge the pool of transitional and naive B cells that are nonetheless hampered in their ability to differentiate and function beyond the naive stage, including maintenance of a memory B cell pool. We hypothesize mutant CARD11 signaling during thymocyte development may also select for hyporesponsive T cell clones. Defects in T cell help to B cells may partly explain the paucity of GCs and few autoimmune manifestations in P2 and P3 to date. In contrast, deficiencies in T cell–independent humoral responses to polysaccharide Ags reinforce the idea of intrinsic dysregulation of B cell responsiveness/function with mutant CARD11 present, even if more self-reactive clones escape negative selection in transitional stages. Further studies are required to distinguish the differential influence of E127G versus G116S CARD11 expression on AgR signaling in B and T cells, selective induction of NF-κB–dependent genes, and lymphocyte development. Indeed, dynamic changes in these processes may also reflect a variable equilibrium in the translation and self-induced, ubiquitin-mediated degradation of different CARD11 mutants (Moreno-García et al., 2010
), although we did not observe abnormal CARD11 protein expression in our patients’ cells.
In conclusion, our findings reveal the genetic basis of a hereditary disorder of B cell lymphocytosis first described in an unsolved case report four decades ago. Our study of these patients illuminates how AgR signaling must be regulated differently by CARD11 in B and T cells, even though the proximal signaling machinery is nearly identical. Because of the contrasting features in lymphocytes, we designate this condition “B cell expansion with NF-κB and T cell anergy” (BENTA) disease. Although B cells are generally more susceptible to malignant transformation because of AID-induced genomic instability, the novel disease we describe here may help to explain the preponderance of B cell rather than T cell malignancies specifically associated with CARD11 activating mutations.