The generation and characterization of mice lacking A20 specifically in B cells, Tnfaip3fl/fl CD19-Cre mice, has allowed us to unveil several novel functions for A20. We have discovered that B cell specific expression of A20 restricts CD40 and BCR responses, terminates CD40 triggered NF-κB signals, restricts B cell survival, and prevents autoimmunity. These studies provide unique molecular insights into B cell homeostasis, human SLE and B cell lymphomas.
mice are largely healthy, in marked contrast to mice lacking A20 expression in all cells or in all hematopoietic cells. This observation is consistent with our prior suggestion that the cachexia, myeloid dysregulation and perinatal lethality observed in globally A20-deficient mice is largely due to myeloid cell dysfunction. While Tnfaip3fl/fl CD19-Cre
mice contained mild expansion of T cell numbers, these perturbations are probably not due to aberrant A20 deletion as judged by our molecular analyses and by the published literature for this Cre strain (Rickert et al., 1997
; Schmidt-Supprian and Rajewsky, 2007
). Rather, these mice express increased quantities of splenic IL-4 mRNA (data not shown), and Tnfaip3fl/fl CD19-Cre
B cells produced more IL-6 and expressed higher amounts of co-stimulatory molecules upon stimulation. Thus, T cell expansion may be due to antigen-independent bystander effects induced by B cell derived cytokines and/or co-stimulatory molecules, as has been observed in other settings of B cell hyper-responsiveness (Homig-Holzel et al., 2008
; Hao et al., 2008
Our findings demonstrate that A20 performs important functions in adaptive immune cells in addition to previously described functions in innate immune cells (Lee et al, 2000
; Boone et al, 2004
; Hitotsumatsu et al, 2008
). A20’s roles in restricting CD40 and BCR triggered NF-κB signals add to the spectrum of signaling cascades regulated by this ubiquitin modifying enzyme. NF-κB signaling is important for regulating B cell homeostasis (Sen 2006
; Siebenlist et al, 2005
). The phenotypes of our mice lacking A20 expression in B cells reveal the importance of tightly regulating basal NF-κB signals in these cells.
B cell specific loss of A20 expression leads to increased numbers of autoantibody producing cells. B cells undergo several stages of negative selection to eliminate autoreactive cells both in the bone marrow and in peripheral lymphoid organs (Jacobi and Diamond, 2005
; Shlomchik, 2008
; Yurasov and Nussenzweig, 2007
; von Boehmer and Melchers, 2010
). In the bone marrow, immature B cells are selected as a consequence of BCR and BAFF signals, and intracellular cell survival factors. In the periphery, selection occurs during GC maturation. Germinal centers are sites where B cells undergo expansion, immunoglobulin class switching, somatic hypermutation, and affinity maturation (Klein and Dalla-Favera, 2008
). B cells with low affinity for antigen or reactivity for self antigens are negatively selected within GCs (Shlomchik, 2008
). Deletion of autoreactive B cells helps prevent autoimmunity, and defective GC selection of autoreactive B cells has been observed in human SLE patients (Cappione et al., 2005
). Fas (CD95) is highly expressed on GC B cells (Watanabe et al, 1995
). While the role of Fas mediated PCD in GC selection has been controversial (Smith et al., 1995
; Takahashi et al., 2001
; Mizuno et al., 2003
; Hoa et al., 2008
), Fas mediated PCD likely plays an important role in eliminating autoreactive B cells (Rathmell et al., 1995
; Fukuyama et alk., 2002
; William et al., 2002
). Thus, the accumulation of GC B cells in Tnfaip3fl/+ CD19-Cre
and Tnfaip3fl/fl CD19-Cre
mice may be due to the increased resistance of A20 deficient B cells to physiological PCD, leading to the escape of autoreactive B cells.
How might A20 deficiency in B cells render them resistant to PCD? One possibility stems from the observation that NF-κB dependent proteins protect B cells against PCD. Indeed, both canonical and non-canonical NF-κB signaling downstream of BCR, CD40, BAFF, and TLR receptors are thought to promote B cell survival as well as proliferation and activation (Siebenlist et al., 2005
; Sen, 2006
; Homig-Holzel et al., 2008
). NF-κB has been suggested to be necessary for mediating BCR induced resistance to Fas mediated PCD (Mizuno and Rothstein, 2003
; Schram and Rothstein, 2003
). Our studies indicate that A20 directly restricts canonical NF-κB signals and suggest that these signals may lead to elevated non-canonical NF-κB signals. These increased NF-κB signals lead to increased expression of anti-apoptotic proteins such as Bcl-2 and Bcl-x. Deregulated expression of these proteins has been shown to cause altered GC B cell selection (Grillot et al., 1996
; Takahashi et al., 1999
). Hence, increased expression of Bcl-x and/or other NF-κB dependent proteins may provide a molecular underpinning for increased numbers of GC B cells in Tnfaip3fl/fl CD19-Cre
Heterozygous Tnfaip3fl/+ CD19-Cre
mice contain similarly increased numbers of GC B cells and autoantibodies as homozygous Tnfaip3fl/fl CD19-Cre
mice at young ages (i.e., 5–7 weeks old), suggesting that a high threshold of A20 expression must be maintained for properly selecting (or deleting) these cells. Reduced A20 expression in other cell types leads to increased production of NF-κB dependent gene products, so endogenous amounts of A20 protein appear to be limiting (O. Hitotsumatsu, S. Oshima, G. Hammer, unpublished data). Reduced (rather than absent) quantities of A20 expression or hypomorphic A20 proteins may also link A20 (TNFAIP3
) susceptibility SNPs with SLE in human patients (Musone et al, 2008
). Thus, mice expressing reduced amounts of A20 may prove to be highly relevant models of human autoimmune diseases.
While reduced A20 expression in B cells leads to accumulation of GC B cells and IgG autoantibodies, absent A20 expression also causes accumulation of immature B cells and IgM in Tnfaip3fl/fl CD19-Cre
mice and progressive activation of B cells with age. Hence, a lower amount of A20 is necessary to preserve selection of immature B cells and to restrict spontaneous B cell activation than the amount required for proper GC selection. As IgM autoantibodies may be protective against IgG mediated autoimmune disease, higher IgM amounts in homozygous Tnfaip3fl/fl CD19-Cre
mice may reduce the degree of autoimmune disease observed in these mice (Witte, 2008
). A20 amounts are dynamically regulated, largely in response to NF-κB signals (Krikos et al, 1992
). Thus, A20 expression amounts appear to be finely tuned to regulate NF-κB signaling and survival of distinct subsets of B cells.
Tnfaip3fl/fl CD19-Cre mice exhibit largely normal antigen specific B cell responses in vivo, despite the fact that mature Tnfaip3fl/fl CD19-Cre B cells exhibit increased responses to BCR, CD40, and TLR ligands in vitro. These findings suggest that B cell independent factors such as T cells and myeloid cells can properly restrict antigen specific B cell responses, even if they allow progressive accumulation of autoreactive B cells in Tnfaip3fl/fl CD19-Cre mice.
Our experiments indicate that A20 expression in B cells regulates GC B cell selection as well as B cell activation, thereby regulating key aspects of B cell tolerance. It is remarkable that B cell specific deletion of A20 alone is sufficient for autoimmunity in C57BL/6 mice. The appearance of IgG deposits suggests that abnormal B cells are sufficient for at least the initial stages of the autoimmunity and are part of the continuum to full blown disease. Hence, lupus prone Tnfaip3fl/fl CD19-Cre and Tnfaip3fl/+ CD19-Cre mice as well as genetic derivatives of these mice should be useful models for understanding human SLE. Heterozygous Tnfaip3fl/+ CD19-Cre mice may be particularly relevant, as reduced, rather than absent A20 expression may characterize this human condition.
Recent studies have shown that somatic loss of A20 in B cells causes several types of Hodgkin, non-Hodgkin, and marginal zone B cell lymphomas in humans (Kato et al., 2009
; Compagno et al., 2009
; Schmitz et al., 2009
; Novak et al., 2009
). Our findings that A20 deficient B cells express high amounts of Bcl-x and are resistant to Fas mediated PCD provide molecular insights into how A20 functions as a tumor suppressor in B cells. Remarkably, A20 is a pro-apoptotic protein in B cells even though it restricts TNF induced apoptosis in fibroblasts and hepatocytes (Lee et al., 2000
; Arvelo et al., 2002
). Hence, it is critical to analyze A20’s physiological functions in cell type-specific contexts. Future studies testing the potential of A20 deficiency to collaborate with other B cell oncogenes may reveal the spectrum of A20’s tumor suppressor functions in B cells.
In conclusion, we have demonstrated functions for A20 in regulating B cell responses, including the restriction of CD40 induced NF-κB signals. These cell autonomous functions are critical for B cell homeostasis and the prevention of autoreactive B cells and autoimmunity. In addition to unveiling new molecular mechanisms of B cell homeostasis, these studies provide critical insights into the pathogenesis of human SLE and B cell lymphomas.