Conceptual models proposed a specific role for BH3-only proteins Puma and Bim in regulating lymphocyte apoptosis (1
). While Puma is a prime effector of apoptosis induced by p53-mediated cellular response to DNA damage, Bim is a critical mediator of apoptosis caused by growth factor withdrawal (1
). Yet, recent evidence has shown a certain degree of functional overlap between Bim and Puma, as single and combined loss of these BH3-only proteins delayed apoptosis induced by cytokine deprivation (8
). Herein, using complementary approaches, we tested a putative functional overlap between these BH3-only proteins in regulating apoptosis during B lymphopoiesis that is caused by the absence of IL-7. Our data provide evidence that loss of Bim, but not loss of Puma, can partially rescue the development and differentiation of B cells in an IL-7-deficient environment.
Three growth factors have been identified that promote B cell development in the mouse: IL-7, Fms-related tyrosine kinase 3 ligand (FLT3L) and thymic stromal lymphopoietin (TSLP) (2
). Whereas FLT3L and IL-7 are mandatory in governing B lymphopoiesis in fetal and adult life, TSLP is predominantly important during fetal life (29
). Accordingly, we found that adult B lymphopoiesis was significantly compromised in IL-7-deficient recipient mice upon reconstitution with WT hematopoietic progenitors. Strikingly, however, loss of Bim significantly enhanced B cell production under these circumstances (). Augmented B cell numbers resulted from increases in both B cell progenitors (pro-B/pre-B cells: sIgM−
B-lineage cells) and immature/mature (CD19+
) B cells, suggesting the existence of ongoing B lymphopoiesis in the absence of IL-7 (). Cells of non-hematopoietic origin (stromal cells) are the predominant source of IL-7 (31
). Although one cannot exclude the possibility that certain cells of hematopoietic origin may produce some minute amounts of IL-7, it is unlikely that those sustain B cell lymphopoiesis in our experimental system. We and others have shown that IL-7-proficient WT stem cell progenitors are not able to produce B cells in irradiated IL-7-deficient adult mice (29
). Hence, IL-7 produced by cells of hematopoietic origin, if any, is not sufficient to sustain adult B cell lymphopoiesis. In IL-7-sufficient recipients, lack of Puma or Bim did not significantly affect the number of B cell precursors, suggesting that B lymphopoiesis in the presence of IL-7 is not markedly affected by lack of Bim (). Our data provide evidence for the first time that lack of Bim can partially bypass the requirement for IL-7 during B cell development. Alternatively, the absence of Bim may promote an IL-7-independent pathway for B cell development that is normally concealed by the overt effects of IL-7. In this situation, TSLP, which receptor shares the IL-7Rα chain, emerges as a possible candidate promoting B cell development. Ectopic expression of TSLP appears to sustain adult B lymphopoiesis in the absence of IL-7 (32
), although the effects of exogenous TSLP seem to be restricted to the neonatal period in IL-7-sufficient mice (33
). Under normal circumstances, TSLP appears to be expressed at low levels in WT- and IL-7-deficient adult mice and these low levels fail to bypass the requirement of IL-7 for adult B lymphopoiesis (29
). Yet, the existing levels of TSLP, which may be elevated in lymphopenic conditions (e.g. upon whole-body gamma irradiation) as demonstrated for IL-7 (34
), may be sufficient to sustain B cell development in the absence of IL-7, providing that B cell progenitor survival is ensured by lack of Bim. Worth mentioning, the overexpression of Bcl-2 is insufficient to rescue B cell development in IL-7Rα KO mice, which lack both IL-7- and TSLP-mediated signaling (35
). These findings suggest that downstream signals of IL-7Rα may inhibit a Bcl-2-independent apoptosis pathway. Alternatively, the co-operative contribution of Bcl-2 with other critical survival Bcl-2-related protein, such as Mcl-1 (binding partner of Bim) (5
), may be necessary to inhibit this death cascade. From this viewpoint, it would be interesting to determine whether deficiency of Bim still permits B lymphopoiesis in the combined absence of TSLP and IL-7.
In a recent report, Marrack and colleagues. analyzed B cell development in Bim/IL-7 double-deficient mice. They found that absence of Bim failed to promote the differentiation of B cells (B220+
) in the absence of IL-7 (14
). In contrast, we find that Bim-deficient B cells can partially develop without IL-7. These differences may be explained in part by the distinct experimental models. In the of Oliver et al.
), absence of Bim may generate defects in non-hematopoietic cells that are important to sustain B cell development (17
). Bone marrow stromal cells provide additional signals, besides IL-7, that are required for the maturation of B cell progenitors and one of these signals may be compromised in the absence of Bim. Moreover, in Bim.IL-7 DKO mice, the IL-7 deficiency is present throughout fetal and adult B cell development, such that hematopoietic precursors may compensate for the IL-7 deficiency. In contrast, we assessed Bim-deficient precursors that develop in an IL-7-sufficient environment upon transfer to an IL-7-deficient recipient, which might limit compensatory mechanisms. Transfer of IL-7.Bim DKO stem cells into Rag2.IL-7 DKO mice may help to validate this possibility. Finally, the time course was distinct in both studies. While bone marrow of IL-7.Bim DKO mice was analyzed at 6 weeks of age, our analysis was conducted at a later time point (14–17 weeks of age; 9–11 weeks after reconstitution of irradiated recipients). Worth mentioning here, and in line with our data, that Olivier et al.
) also noted an accumulation of B cells in the spleen of IL-7.Bim DKO mice at 12 weeks of age, which may be due to an increase in B lymphopoiesis and/or peripheral B cell survival (, and 6). Bone marrow analysis of older DKO animals might help to address this possibility.
Growth factor withdrawal engages a pro-apoptotic pathway in activated lymphocytes that entails de novo
transcription of Bim and Puma (5
). In the absence of growth factors (e.g. IL-3), PI3K–AKT/PKB signaling is attenuated leading to activation of forkhead transcription factor FoxO3A, which then translocate to the nucleus to mediate transcription of target genes, including the BH3-only proteins Puma and Bim (8
). In this scenario, one may expect that loss of Bim or Puma could provide a survival advantage to B cells that undergo development in the absence of IL-7. Yet, our data pointed to a dominant role of Bim in governing B cell homeostasis in the absence of IL-7, excluding Puma as an essential player in this process. This, however, does not exclude a role for Puma in B cell homeostasis once the B cell developmental program is achieved. Consistently, Bim.Puma DKO mice show a B cell hyperplasia that exceeds the one observed in Bim KO mice. On the other hand, loss of Bim did not restore mature B cells in IL-7−
recipients to levels found in IL-7+
mice reconstituted with WT LSK cells ( and ). This clearly indicates that at least one additional factor, presumably another BH3-only protein, may be targeted by IL-7 during B cell maturation. A good candidate for such molecule is the BH3-only protein Bmf. Mice lacking Bmf show B cell-restricted lymphadenopathy, beginning from the pre-B cell stage onwards. Moreover, Bim and Bmf are also found to be co-expressed throughout B cell development and show similar modes of post-translation regulation (37
The notion of competitive survival has been implicated in the control of peripheral B cell homeostasis, which relies on multiple environmental cues, including membrane-bound self-antigen and the tumor necrosis factor (TNF) cytokine family members, B cell activation factor from the tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) (38
). We observed an accumulation of immature as well as mature Bim-deficient B cells in the spleen of IL-7-sufficient and IL-deficient recipients suggesting that an IL-7-independent and Bim-dependent pathway governs peripheral B cell homeostasis. Accordingly, B cells accumulate in the spleen of mice lacking IL-7 (40
). The overt accumulation of Bim KO B cell subsets, including follicular and marginal zone B cells ( and ), indicates that Bim is a general regulator of B cell homeostasis in vivo
. The effector function of Bim-deficient B cells generated in the absence of IL-7, including the capacity to divide in response to LPS and IL-4 in culture or to produce serum soluble antibodies in vivo
, appeared intact (). Bim KO mice display abnormal elevated B cell numbers and serum Ig levels as consequence of disturbed B cell selection and extended B cell survival (11
). One may envision that mature B cells accumulating in the spleen of both IL-7+
as well as IL-7−
recipients may contain some deleterious autoreactive B cells that have escaped negative selection. BAFF is a critical regulator of peripheral B cell homeostasis, providing survival and maturation signals essential to the conversion of peripheral immature into mature recirculating B cells (38
). Interestingly, BAFF-mediated signals are able to thwart BCR-mediated apoptosis through suppression of Bim suggesting that concerted signaling through BCR and BAFF receptors modulates mature B cell homeostasis (41
). In this context, loss of Bim may also correct the defects in mature B cell homeostasis caused by deficiency in BAFF–BAFF-R signaling.
In conclusion, we provide evidence that adult B cell development could be partially rescued in the absence of IL-7 by the loss of Bim, but not by the loss of Puma, in hematopoietic progenitors. These data suggest that BH3-only proteins in B cell homeostasis operate in a hierarchical order, highlighting the chief role of Bim in governing this process.