CAML is required for efficient EGF receptor recycling

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitous protein that has been implicated in signaling from the cell surface receptor TACI in lymphocytes, although its role and mechanism of action are unknown. To study its function in the mouse, we disrupted the CAML gene and found it to be required for early embryonic development, but not for cellular viability. CAML-deficient cells have severely impaired proliferative responses to the epidermal growth factor (EGF). Although EGF-induced activation of signaling intermediates and internalization of the EGF receptor (EGFR) are normal in the absence of CAML, the recycling of internalized receptors to the plasma membrane is defective, leading to its reduced surface accumulation. We demonstrate that CAML normally associates directly with the kinase domain of the EGFR in a ligand-dependent manner. These data implicate CAML in EGFR signaling and suggest that it may play a role in receptor recycling during long-term proliferative responses to EGF.     

BCR-mediated apoptosis associated with negative selection of immature B cells is selectively dependent on Pten

The molecular basis of B cell receptor (BCR)-induced apoptosis during the negative selection of immature B cells is largely unknown. We use transitional immature B cells that are highly susceptible to BCR-induced apoptosis to show that Pten is selectively required for BCR-mediated initiation of the mitochondrial death pathway. Specifically, deleting Pten, but not other pro-apoptotic molecules, abrogates BCR-elicited apoptosis and improves viability in wild-type immature B cells. We further identify a physiologically and significantly higher intracellular Pten level in immature B cells, as compared to mature B cells, which is responsible for low AKT activity and the propensity towards death in immature B cells. Restoration of AKT activity using a constitutive form of AKT or reduction of Pten to a level comparable with that seen in mature B cells rescues immature B cells from BCR-induced apoptosis. Thus, we provide evidence that Pten is an essential mediator of BCR-induced cell death, and that differential regulation of intracellular Pten levels determines whether BCR ligation promotes cell death or survival. Our findings provide a valuable insight into the mechanisms underlying negative selection and clonal deletion of immature B cells.     

The Importance of LAT in the Activation,Homeostasis, and Regulatory Function of T Cells

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T_reg cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.     

Differentiation of chronic lymphocytic leukemia B cells into immunoglobulin secreting cells decreases LEF-1 expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.     

IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production

Persistent cross-linking of hen egg lysozyme (HEL)-specific B cell membrane Ig (mIg) in double transgenic mice that express soluble HEL as a self Ag (HEL-Ig mice) decreases B cell mIgM expression, responsiveness, and life span. Because in vitro treatment with IL-4 inhibits T cell apoptosis through a Stat6-independent mechanism, increases mIg expression, and suppresses activation-induced B cell death, we studied IL-4 effects on B cell mIg expression, survival, and Ab secretion in Stat6-sufficient and deficient HEL-Ig mice. IL-4 treatment nearly normalized B cell number and greatly increased the percentage of mature B cells in HEL-Ig mice, but failed to normalize mIgM expression or spontaneous LPS-induced IgM secretion. IL-4 effects on B cell survival and maturation were CD4(+) T cell independent, but Stat6 dependent, and did not involve receptor editing. IL-4 had to be present while B cells were generated to have a detectable effect on autoreactive B cell survival; however, the survival of B cells generated in the presence of IL-4 was substantially increased even after IL-4 was withdrawn. These observations suggest that: 1) activation-induced B cell death and anergy are independent processes; 2) B cells that survive to maturity develop increased resistance to Ag-induced deletion; and 3) IL-4 promotes B and T cell survival through different mechanisms.     

Selective Preservation of Bone Marrow Mature Recirculating but Not Marginal Zone B Cells in Murine Models of Chronic Inflammation

Inflammation promotes granulopoiesis over B lymphopoiesis in the bone marrow (BM). We studied B cell homeostasis in two murine models of T cell mediated chronic inflammation, namely calreticulin-deficient fetal liver chimeras (FLC), which develop severe blepharitis and alopecia due to T cell hyper responsiveness, and inflammatory bowel disease (IBD) caused by injection of CD4⁺ naïve T cells into lymphopenic mice. We show herein that despite the severe depletion of B cell progenitors during chronic, peripheral T cell-mediated inflammation, the population of BM mature recirculating B cells is unaffected. These B cells are poised to differentiate to plasma cells in response to blood borne pathogens, in an analogous fashion to non-recirculating marginal zone (MZ) B cells in the spleen. MZ B cells nevertheless differentiate more efficiently to plasma cells upon polyclonal stimulation by Toll-like receptor (TLR) ligands, and are depleted during chronic T cell mediated inflammation in vivo. The preservation of mature B cells in the BM is associated with increased concentration of macrophage migration inhibitory factor (MIF) in serum and BM plasma. MIF produced by perivascular dendritic cells (DC) in the BM provides a crucial survival signal for recirculating B cells, and mice treated with a MIF inhibitor during inflammation showed significantly reduced mature B cells in the BM. These data indicate that MIF secretion by perivascular DC may promote the survival of the recirculating B cell pool to ensure responsiveness to blood borne microbes despite loss of the MZ B cell pool that accompanies depressed lymphopoiesis during inflammation.     

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.     

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.     

Protein Kinase Cδ Promotes Transitional B Cell-Negative Selection and Limits Proximal B Cell Receptor Signaling To Enforce Tolerance

Protein kinase Cδ (PKCδ) deficiency causes autoimmune pathology in humans and mice and is crucial for the maintenance of B cell homeostasis. However, the mechanisms underlying autoimmune disease in PKCδ deficiency remain poorly defined. Here, we address the antigen-dependent and -independent roles of PKCδ in B cell development, repertoire selection, and antigen responsiveness. We demonstrate that PKCδ is rapidly phosphorylated downstream of both the B cell receptor (BCR) and the B cell-activating factor (BAFF) receptor. We found that PKCδ is essential for antigen-dependent negative selection of splenic transitional B cells and is required for activation of the proapoptotic Ca²⁺-Erk pathway that is selectively activated during B cell-negative selection. Unexpectedly, we also identified a previously unrecognized role for PKCδ as a proximal negative regulator of BCR signaling that substantially impacts survival and proliferation of mature follicular B cells. As a consequence of these distinct roles, PKCδ deficiency leads to the survival and development of a B cell repertoire that is not only aberrantly autoreactive but also hyperresponsive to antigen stimulation.     

Cutting edge: Transitional T3 B cells do not give rise to mature B cells, have undergone selection, and are reduced in murine lupus

As the immediate precursors to mature follicular B cells in splenic development, immature transitional cells are an essential component for understanding late B cell differentiation. It has been shown that T2 cells can give rise to mature B cells; however, whether T3 B cells represent a normal stage of B cell development, which has been widely assumed, has not been fully resolved. In this study, we demonstrate both in vitro and in vivo that T3 B cells do not give rise to mature B cells and are instead selected away from the T1-->T2-->mature B cell developmental pathway and are hyporesponsive to stimulation through the BCR. Significantly reduced numbers of T3 B cells in young lupus-prone mice further suggest that the specificity of this subset holds clues to understanding autoimmunity.     

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.     

Survival of resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer

We previously showed that type I interferon-induced, Cre-mediated ablation of surface BCR expression in mature B cells through Ig-heavy chain deletion results in apoptosis of these cells. This led to the hypothesis that survival signals from the BCR are vital for mature B cells. Here, we test two critical assumptions of this model. First, we demonstrate loss of mature B cells upon induced mutation of a signaling module of the BCR, not precluding BCR surface expression. Second, we show that the cells are also lost upon BCR inactivation in the absence of an exogenous inducer like interferon, excluding that cell death depends on previous cellular activation by the latter. Kinetic data demonstrate that BCR-less mature B cells have a severely reduced lifespan, with a half-life of 3-6 days. Together these results establish that BCR signaling is required to keep resting mature B cells alive in vivo.     

Rapid B cell receptor-induced unfolded protein response in nonsecretory B cells correlates with pro- versus antiapoptotic cell fate

The adaptive unfolded protein response (UPR) is essential for the development of antibody-secreting plasma cells. B cells induced by lipopolysaccharide (LPS) to differentiate into plasma cells exhibit a nonclassical UPR reported to anticipate endoplasmic reticulum stress prior to immunoglobulin production. Here we demonstrate that activation of a physiologic UPR is not limited to cells undergoing secretory cell differentiation. We identify B cell receptor (BCR) signaling as an unexpected physiologic UPR trigger and demonstrate that in mature B cells, BCR stimulation induces a short lived UPR similar to the LPS-triggered nonclassical UPR. However, unlike LPS, BCR stimulation does not induce plasma cell differentiation. Furthermore, the BCR-induced UPR is not limited to cells in which BCR induces activation, since a UPR is also induced in transitional immature B cells that respond to BCR stimulation with a rapid apoptotic fate. This response involves sustained up-regulation of Chop mRNA indicative of a terminal UPR. Whereas sustained Chop expression correlates with the ultimate fate of the BCR-triggered B cell and not its developmental stage, Chop-/- B cells undergo apoptosis, indicating that CHOP is not required for this process. These studies establish a system whereby a terminal or adaptive UPR can be alternatively triggered by physiologic stimuli.     

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.