The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.     

Cutting edge: BLyS enables survival of transitional and mature B cells through distinct mediators

These studies characterize BLyS responsiveness and receptor expression among transitional and mature peripheral B cells. The results show a maturation-associated increase in BLyS binding capacity that reflects differential expression patterns of the three BLyS receptors. Accordingly, BLyS administration enlarges only late transitional and mature peripheral B (MB) cell compartments. Furthermore, bromodeoxyuridine labeling and cell cycle analyses show these effects are mediated through enhanced proportional survival of cells traversing the T2, T3, and MB cell stages, rather than by causing proliferation or slowing transit within these subsets. Despite similar effects on survival, BLyS up-regulates the antiapoptotic genes A1 and bcl-x(L) in MB cells but not immature B cells. Together, these findings show that, while BLyS influences B cell survival in several peripheral differentiation subsets, the downstream mediators differ, thus providing the first direct evidence for an established B lineage survival system whose intermediates change as B cells mature.     

Competition for BLyS-mediated signaling through Bcmd/BR3 regulates peripheral B lymphocyte numbers

Striking cell losses occur during late B lymphocyte maturation, reflecting BcR-mediated selection coupled with requisites for viability promoting signals. How selection and survival cues are integrated remains unclear, but a key role for B lymphocyte stimulator (BLyS(TM); trademark of Human Genome Sciences, Inc.) is suggested by its marked effects on B cell numbers and autoantibody formation as well as the B lineage-specific expression of BLyS receptors. Our analyses of the B cell-deficient A/WySnJ mouse have established Bcmd as a gene controlling follicular B cell life span, and recent reports show Bcmd encodes a novel BLyS receptor. Here we show that A/WySnJ B cells are unresponsive to BLyS, affording interrogation of how Bcmd influences B cell homeostasis. Mixed marrow chimeras indicate A/WySnJ peripheral B cells compete poorly for peripheral survival. Moreover, in vivo BrdU labeling shows that (A/WySnJ x BALB/c)F(1) B cells have an intermediate but uniform life span, indicating viability requires continuous signaling via this pathway. Together, these findings establish the BLyS/Bcmd pathway as a dominant mediator of B cell survival, suggesting competition for BLyS/Bcmd signals regulates follicular B cell numbers.     

TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r(-/-) mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r(-/-) mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r(-/-) B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r(-/-) mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.     

CD45-deficient mice accumulate Pro-B cells both in vivo and in vitro

Efficient generation of mature B lineage cells requires the participation of the BCR, the pre-BCR, accessory coreceptors, and growth factor receptors. Together these receptors integrate cell intrinsic signals with regulatory pathways initiated by surrounding cells and structures. CD45 is a receptor tyrosine phosphatase expressed at high levels on all hemopoietic cells, and has been shown to modulate many signaling cascades in both positive and negative manners. In the absence of B220, the B lineage isoform of CD45, differentiation to the mature B cell stage is incomplete. We demonstrate that CD45-deficient mice also accumulate pro-B cells in the bone marrow. In vitro differentiation is altered in that B lineage populations exhibit prolonged survival in the presence of high concentrations of IL-7. Cell lines derived from CD45-deficient animals experience prolonged JAK/STAT activation in response to IL-7 stimulation, and constitutively elevated levels of phosphorylated src kinases. Aberrant IL-7Ralpha expression is observed in vivo, and may be responsible for the skewed development present in CD45(-/-) animals. Demonstrating that CD45-deficient pro-B cells are affected by the absence of B220 highlights a previously unrecognized parallel in B and T lineage precursors, and emphasizes that the presence of normal numbers of peripheral B cells does not assure that the bone marrow compartment is intact.     

Regulation of A1/Bfl-1 expression in peripheral splenic B cells

We analyzed here the expression of the prosurvival Bcl-2 homologue A1 in peripheral B cell compartment. We observed that A1 mRNA are highly expressed in peripheral B cells as compared with other anti-apoptotic genes of the Bcl-2 family such as bcl-xl and bcl-2 itself. The expression of A1 is up-regulated in immature B cells at the transition between transitional type 1 (T1) and type 2 (T2) cells, and remained highly expressed in mature (M) B cells. We, therefore, analyzed the effect of B cell antigen receptor (BCR) and BAFF receptor (BAFF-R) engagement on the regulation of A1 in total B220(+) cells but also FACS-sorted immature T1, T2 and M B cells. We demonstrated that only BCR engagement up-regulated the expression of A1 mRNA and protein. These results suggest that A1 may play a key role in antigen-dependent signals that are required for survival and/or proliferation of peripheral B cells.     

Memory B cells and CD27

Following antigen activation in germinal centers, B cells develop into memory B cells or plasma cells. Triggering via B-cell immunoglobulin receptors by antigens, cytokines and direct cell-to-cell contact by B and T cells plays an important role in the B cell differentiation into memory or plasma cells. Adult human peripheral blood B cells are separated into three subtypes by the expression of IgD and CD27, which belong to the tumor necrosis factor receptor (TNFR) family: IgD+ CD27- naive B cells, IgD+ CD27+ and IgD- CD27+ B cells. CD27+ B cells are larger cells with abundant cytoplasm carrying somatic hypermutation, and have an ability to produce immunoglobulin, indicating that CD27 is a memory marker of B cells. The ligation of CD27 yields crucial signals that positively control the entry of B cells into the pathway to plasma cells. We review observations on subpopulations and differentiation of mature B-cells by T/B cell interaction via CD27/CD70 as compared with CD40/CD154 interaction, and discuss about memory B cells.     

The extracellular membrane-proximal domain of human membrane IgE controls apoptotic signaling of the B cell receptor in the mature B cell line A20

Ag engagement of BCR in mature B cells can deliver specific signals, which decide cell survival or cell death. Circulating membrane IgE+ (mIgE+) cells are found in extremely low numbers. We hypothesized that engagement of an epsilonBCR in a mature isotype-switched B cell could induce apoptosis. We studied the role of the extracellular membrane-proximal domain (EMPD) of human mIgE upon BCR engagement with anti-Id Abs. Using mutants lacking the EMPD, we show that this domain is involved in controlling Ca2+ mobilization in immunoreceptors of both gamma and epsilon isotypes, as well as apoptosis in signaling originated only from the epsilonBCR. We mapped to the epsilonCH4 ectodomain the region responsible for apoptosis in EMPD-deleted receptors. Ca2+ mobilization was not related to apoptotic signaling. This apoptotic pathway was caspase independent, involved ERK1/2 phosphorylation and was partially rescued by CD40 costimulation. We therefore conclude that the EMPD of human mIgE is a key control element of apoptotic signaling delivered through engagement of epsilonBCR within the context of a mature B cell.     

TCR-mediated Notch signaling regulates proliferation and IFN-gamma production in peripheral T cells

Notch genes encode membrane receptors that regulate cell fate decisions in metazoa. Notch receptors and ligands are expressed in developing lymphoid tissue and mature lymphocytes and the role of Notch signaling in early T and B cell development has been studied extensively. However, its contribution to mature T cell function is unknown. TCR-mediated T cell activation is a fundamental process of the adaptive immune system that has been studied for decades; however, the details of this process are incompletely understood. In this study, we present evidence that Notch is required for TCR-mediated activation of peripheral T cells. Inhibition of Notch activation dramatically decreases T cell proliferation in both CD4 and CD8 cells and blocks both NF-kappaB activity and IFN-gamma production in peripheral T cells. Our data reveal a new, nondevelopmental function of Notch as a previously unknown key link in peripheral T cell activation and cytokine secretion.     

Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4(+) T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.     

RasGRP1 sensitizes an immature B cell line to antigen receptor-induced apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-kappaB and Bcl-x(L), which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-kappaB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-kappaB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-kappaB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.     

Essential role for CAML in follicular B cell survival and homeostasis

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.     

Transitional B cell fate is associated with developmental stage-specific regulation of diacylglycerol and calcium signaling upon B cell receptor engagement

Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton's tyrosine kinase and phospholipase C-gamma2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.     

NF-kappa B1 p50 is required for BLyS attenuation of apoptosis but dispensable for processing of NF-kappa B2 p100 to p52 in quiescent mature B cells

B lymphocyte stimulator (BLyS), a TNF family protein essential for peripheral B cell development, functions primarily through attenuation of B cell apoptosis. In this study, we show that BLyS activates NF-kappaB through both classical and alternative pathways with distinct kinetics in quiescent mature B cells. It rapidly and transiently enhances the p50/p65 DNA binding activity and induces phosphorylation of IkappaBalpha characteristic of the classical NF-kappaB pathway, albeit maintaining IkappaBalpha at a constant level through ongoing protein synthesis and proteasome-mediated destruction. With delayed kinetics, BLyS promotes the processing of p100 to p52 and sustained formation of p52/RelB complexes via the alternative NF-kappaB pathway. p50 is dispensable for p100 processing. However, it is required to mediate the initial BLyS survival signals and concomitant activation of Bcl-x(L) in quiescent mature B cells ex vivo. Although also a target of BLyS activation, at least one of the A1 genes, A1-a, is dispensable for the BLyS survival function. These results suggest that BLyS mediates its survival signals in metabolically restricted quiescent B cells, at least in part, through coordinated activation of both NF-kappaB pathways and selective downstream antiapoptotic genes.     

Differential responsiveness of immature- and mature-stage murine B cells to anti-IgM reflects both FcR-dependent and -independent mechanisms.

Mature and immature B cells differ in their responses to antigen receptor crosslinking. Whereas mature B cells enter cell cycle in response to such stimulation, immature B cells exhibit proliferative unresponsiveness and undergo induced tolerance following surface immunoglobulin (sIg) engagement. Previous studies evaluating antigen receptor-mediated negative signaling have utilized intact goat anti-immunoglobulin (anti-Ig) antibodies as polyclonal ligands based upon observations that the Fc portion of these reagents does not interact with and mediate negative signaling through the FcR on mature B cells. Thus, the negative effects of goat anti-Ig on immature B cells have been attributed solely to signals mediated via their antigen receptors. In the studies reported here we show that the activation unresponsiveness inherent to immature B cells is FcR independent. However, we also show that immature B cells are sensitive to FcR-mediated inhibition and that these effects can be mediated by intact goat antibodies at concentrations that promote positive activation signals in mature B cells. Our results demonstrate that inhibition of immature B cell LPS responses by anti-Ig antibodies, used in previous studies as an in vitro model for B cell tolerance induction, is an FcR-mediated phenomenon. We show that developmentally associated anti-Ig-mediated inhibition of LPS requires the use of intact antibodies, and that this inhibition can be blocked by the anti-FcR monoclonal antibody 2.4G2. Flow cytometric analysis of FcR-positive B cells indicates that both mature and immature B cells express equivalent levels of FcR gamma. Therefore, the sensitivity of immature, but not mature, cells to intact goat anti-mu antibodies suggests that either FcRs or their associated inhibitory pathways change during B cell development.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

Ligation of CD28 in vivo induces CD40 ligand expression and promotes B cell survival.

Functional activation of T cells requires ligation of Ag receptors with specific peptides presented by MHC molecules on APCs concurrent with appropriate contacts of cell surface accessory molecules. Among these accessory molecules, interactions between CD28/CTLA-4 with B7 family members (CD80 and CD86) and CD40 with CD40 ligand (CD40L) play a decisive role in regulating the progression of balanced immune responses. However, most information regarding the role of accessory molecules in immune responses has been derived in the context of signals from the TCRs. Little understanding has been achieved regarding the consequence of ligation of costimulation molecules in absence of signals from the TCR. By employing an in vivo murine system, we show, herein, that ligation of CD28 alone with anti-CD28 Abs leads to a dramatic enlargement of the peripheral lymphoid organs characterized primarily by the expansion of B cells. B cells from anti-CD28-treated mice are resistant to spontaneous and anti-IgM-induced apoptosis. These cells are also unsusceptible to FasL-mediated apoptosis. Interestingly, this in vivo effect of CD28 on B cells is largely mediated by inducing the expression of CD40L, since coadministration of a blocking Ab against CD40L inhibited CD28-mediated B cell survival and expansion. Therefore, CD28-mediated expression of CD40L may play an important role in the regulation of lymphocyte homeostasis.     

TACI, an enigmatic BAFF/APRIL receptor, with new unappreciated biochemical and biological properties

BAFF is a B cell survival factor that binds to three receptors BAFF-R, TACI and BCMA. BAFF-R is the receptor triggering na?ve B cell survival and maturation while BCMA supports the survival of plasma cells in the bone marrow. Excessive BAFF production leads to autoimmunity, presumably as the consequence of inappropriate survival of self-reactive B cells. The function of TACI has been more elusive with TACI(-/-) mice revealing two sides of this receptor, a positive one driving T cell-independent immune responses and a negative one down-regulating B cell activation and expansion. Recent work has revealed that the regulation of TACI expression is intimately linked to the activation of innate receptors on B cells and that TACI signalling in response to multimeric BAFF and APRIL provides positive signals to plasmablasts. How TACI negatively regulates B cells remains elusive but may involve an indirect control of BAFF levels. The discovery of TACI mutations associated with common variable immunodeficiency (CVID) in humans not only reinforces its important role for humoral responses but also suggests a more complex role than first anticipated from knockout animals. TACI is emerging as an unusual TNF receptor-like molecule with a sophisticated mode of action.