The present findings show that CD72-deficient mice spontaneously produce autoantibodies and develop features of lupus-like autoimmune disease. Using the HEL anergic B cell model, we demonstrated that in the absence of CD72, anergic B cells inappropriately proliferate and survive in response to self-antigen stimulation. Biochemical analyses showed that CD72 dominantly down-regulates BCR signaling to limit the antigen-induced rise in [Ca2+]i and the activation of NFATc1, NF-κB, MAPK, and Akt in anergic B cells. Mechanistically, CD72 is associated with Cbl-b, suggesting that Cbl-b may mediate the negative effects of CD72 on BCR signaling in anergic B cells.
CD72 plays a role in discriminating tolerogenic signaling from immunogenic signaling. Normally, foreign antigens elicit an immunogenic response while self antigens elicit a tolerogenic response by mature B cells. The mature B cells from both IgHEL
-Tg control and IgHEL
mice transduce an immunogenic signal resulting in B cell activation after antigen stimulation (9
). In the present study we used the dTg anergic model to elucidate the function of CD72 in regulating B cell anergy and tolerogenic signaling. In anergic B cells, CD72 constitutively regulates BCR-induced signaling to limit proliferation and survival. As a result of a lack of cyclin D2 up-regulation and Rb phosphorylation, dTg anergic B cells have a near complete block in proliferation and fail to enter the S phase (). B cells from dTg CD72−/−
mice misinterpret self-antigen binding as an immunogenic signal, which results in a breakdown of B cell self tolerance and leads to B cell activation. The up-regulation of cyclin D2 and Bcl-xL
in antigen-stimulated dTg CD72−/−
B cells offers a molecular explanation for their altered proliferation and survival ().
Anergic B cells from the dTg model have been shown to be defective in the induction of the calcineurin/NFAT and NF-κ
B signaling pathways (30
). CD72 deficiency allows dTg anergic B cells to activate both of these immunogenic pathways. Our data directly assessing the activity of these pathways are supported by the induction of cyclin D2, the expression of which requires both calcineurin/NFAT and NF-κ
B signaling (41
). MAPK and Akt signaling are fundamental regulators of cell cycle and survival, and these pathways are also induced to a greater extent by self-antigen binding to dTg CD72−/−
CD72 may modulate the B cell tolerance via binding to its ligand, CD100. CD72 negatively regulates BCR-induced signaling, but binding of CD72 to CD100 can release this negative regulation (16
). CD100 is expressed on several immune cell types, including T cells, B cells, and antigen-presenting cells. It is likely that CD100 fine-tunes CD72-mediated negative regulation during T cell–dependent responses, which is consistent with the reduced T cell–dependent responses in CD100-deficient mice (17
). The observation that CD72−/−
mice develop autoimmunity may appear at odds with a recent report that CD100-deficient mice develop autoantibodies and autoimmune disease (43
). However, CD100 is expressed on several different cell types, has signaling properties of its own, and has ligands other than CD72, so the mechanisms that contribute to the development of autoimmune disease in aged CD100−/−
mice may be complex.
By utilizing the ability of an anti-CD72 mAb to mimic CD100 binding to surface CD72, we demonstrated that anergic B cells continuously require CD72 to maintain B cell tolerance. As shown in , coincubation of anti-CD72 mAb and HEL in B cell cultures revealed that CD72 ligation indeed releases the inhibitory effects of CD72 on cell cycle progression and survival, resulting in breakdown of self tolerance in anergic B cells. These findings suggest that proper regulation of CD72 is important to maintain B cell tolerance in the periphery.
CD72 down-regulates BCR signaling through Cbl-b to maintain B cell anergy. CD72 may anchor Cbl-b to the membrane in close proximity to the signalosome, and thus allow the appropriate regulation of Cbl-b function in anergic B cells (). Syk tyrosine kinase has been shown to be a target of Cbl-mediated ubiquitination upon antigen stimulation in mature B cells (44
). Preliminary findings (Li DH, et al: unpublished observations) indicate that Syk ubiquitination was significantly enhanced in WT B cells as compared with CD72-deficient B cells after antigen stimulation. This finding implies that Cbl-b may increase its E3 ubiquitin ligase activity after dephosphorylation by CD72 and, in turn, enhance the ubiquitination of Syk. The enhanced ubiquitination of Syk may augment turnover of the active phospho-Syk associated with Igα
, thereby decreasing Syk kinase activity (25
). Based on our observations, we propose a working model for CD72 function in immunogenic and tolerogenic signaling, as shown in .
Figure 6 Working model of the role of CD72 in maintaining B cell anergy. CD72 is essential for the maintenance of B cell tolerance through the interaction with Cbl-b and in the negative regulation of B cell receptor (BCR) signaling. During foreign antigen engagement (more ...)
Collectively, these results show that aged CD72-deficient mice develop lupus-like autoimmune disease, and that in the absence of CD72, anergic B cells inappropriately proliferate and survive in response to self-antigen stimulation in vitro. Biochemical analyses indicate that in anergic B cells, CD72 down-regulates BCR signaling. Thus, CD72, which normally functions to reduce mature B cell proliferation and differentiation, is an important modulator of peripheral B cell anergy. The enhanced understanding of cell surface receptors that critically maintain immune tolerance will aid in the development of targeted therapies to treat autoimmune disorders.