Andrew Craxton

BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)–induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3–only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector–mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal–regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim−/− mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.

Syk and Bruton's Tyrosine Kinase Are Required for B Cell Antigen Receptor-mediated Activation of the Kinase Akt

Activation of Akt by multiple stimuli including B cell antigen receptor (BCR) engagement requires phosphatidylinositol 3-kinase and regulates processes including cell survival, proliferation, and metabolism. BCR cross-linking activates three families of non-receptor protein tyrosine kinases (PTKs) and these are transducers of signaling events including phospholipase C and mitogen-activated protein kinase activation; however, the relative roles of PTKs in BCR-mediated Akt activation are unknown. We examined Akt activation in Lyn-, Syk- and Btk-deficient DT40 cells and B cells from Lyn−/− mice. BCR-mediated Akt activation required Syk and was partially dependent upon Btk. Increased BCR-induced Akt phosphorylation was observed in Lyn-deficient DT40 cells and Lyn−/− mice compared with wild-type cells suggesting that Lyn may negatively regulate Akt function. BCR-induced tyrosine phosphorylation of the phosphatidylinositol 3-kinase catalytic subunit was abolished in Syk-deficient cells consistent with a receptor-proximal role for Syk in BCR-mediated phosphatidylinositol 3-kinase activation; in contrast, it was maintained in Btk-deficient cells, suggesting Btk functions downstream of phosphatidylinositol 3-kinase. Calcium depletion did not influence BCR-induced Akt phosphorylation/activation, showing that neither Syk nor Btk mediates its effects via changes in calcium levels. Thus, BCR-mediated Akt stimulation is regulated by multiple non-receptor PTK families which regulate Akt both proximal and distal to phosphatidylinositol 3-kinase activation.

Signal transduction pathways that regulate the fate of B lymphocytes

Publisher Summary This chapter discusses the signaling pathways that are involved in regulating B cell fate. The chapter begins with a brief overview of the rapidly advancing area of the programmed cell death. A major breakthrough occurred when studies using the Caenorhabditis elegans model revealed that key genes that control the onset of programmed death of 131 of the 1090 somatic cells are generated during the development of the worm. Two of these genes, crd-3 and cecl-4 are required for cell death to occur and a third gene— ced-9— is necessary to prevent cells (that normally survive) from undergoing apoptosis. B cells are capable of responding to signals initiated by the B cell receptor (BCR) in amazingly plastic ways. Few studies indicate that CD22 also has two functional cytoplasmic domains. Surface receptors with opposing signaling domains may be a common theme in regulatory molecules. In the case of FcγRIIB and perhaps CD22, the tendency to dampen signals initiated via the BCR may predominate over a signal-enhancing function. Many of the key players that determine the fate of B lymphocytes are now identified, although the rules of the “game” of cell life and cell death are poorly understood. The reason BCR signaling induces cell death in one context and promotes survival in another is perplexing.

Regulation of B-cell entry into the cell cycle.

Summary: B cells are induced to enter the cell cycle by stimuli including ligation of the B‐cell receptor (BCR) complex and Toll‐like receptor (TLR) agonists. This review discusses the contribution of several molecules, which act at distinct steps in B‐cell activation. The adapter molecule Bam32 (B‐lymphocyte adapter of 32 kDa) helps promote BCR‐induced cell cycle entry, while the secondary messenger superoxide has the opposite effect. Bam32 and superoxide may fine tune BCR‐induced activation by competing for the same limited resources, namely Rac1 and the plasma membrane phospholipid PI(3,4)P2. The co‐receptor CD22 can inhibit BCR‐induced proliferation by binding to novel CD22 ligands. Finally, regulators of B‐cell survival and death also play roles in B‐cell transit through the cell cycle. Caspase 6 negatively regulates CD40‐ and TLR‐dependent G1 entry, while acting later in the cell cycle to promote S‐phase entry. Caspase 6 deficiency predisposes B cells to differentiate rather than proliferate after stimulation. Bim, a pro‐apoptotic Bcl‐2 family member, exerts a positive regulatory effect on cell cycle entry, which is opposed by Bcl‐2. New insights into what regulates B‐cell transit through the cell cycle may lead to thoughtful design of highly selective drugs that target pathogenic B cells.

BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas‐mediated cell death

Microorganisms with pathogen‐associated molecular patterns (PAMP) activate B cells directly by binding to TLR and also indirectly by inducing APC to release cytokines such as BAFF that promote B cell survival. We found that murine B cells activated concomitantly with LPS (TLR‐4 ligand) and BAFF are protected from spontaneous apoptosis, but are more susceptible to Fas/CD95‐mediated cell death. This increased susceptibility to Fas‐induced apoptosis is associated with a dramatic coordinated up‐regulation of Fas/CD95 and IRF‐4 expression through a mechanism mediated, at least in part, by inhibition of the MEK/ERK pathway. Up‐regulation of Fas/CD95 by BAFF is restricted to B cells activated through TLR‐4, but not through TLR‐9, BCR or CD40. TLR ligands differ in the BAFF family receptors (R) they induce on B cells: BAFF‐R is increased by the TLR4 ligand, LPS, but not by the TLR9 ligand, CpG‐containing oligodeoxynucleotides, which, in contrast, strongly up‐regulates transmembrane activator and CAML interactor (TACI). This suggests the up‐regulation of Fas by BAFF is mediated by BAFF‐R and not by TACI. Consistently, APRIL, which binds to TACI and B cell maturation antigen but not BAFF‐R, did not enhance Fas expression on LPS‐activated B cells. Increased susceptibility to Fas‐mediated killing of B cells activated with LPS and BAFF may be a fail‐safe mechanism to avoid overexpansion of nonspecific or autoreactive B cells.

Bim regulates BCR‐induced entry of B cells into the cell cycle

BH3‐only Bcl‐2 homologs are key regulators of the intrinsic apoptotic pathway. In particular, Bim, is critical for mediating apoptosis of hematopoietic cells including B cells. While studies using Bcl‐2 Tg mice have defined an important role for Bcl‐2 in cell cycle control, the role of BH3‐only proteins is less clear. Using Bim KO mice, we show that Bim is required for B cells to enter the cell cycle normally. Bim KO B cells had reduced cell division compared to WT B cells in response to BCR, TLR3 or TLR4 signaling, whereas Bim deficiency did not affect TLR9‐induced B cell division. Cell cycle progression in BCR‐ and LPS‐stimulated Bim KO B cells was blocked at the G0–G1 stage. BCR‐induced p130 degradation and pRb hyperphosphorylation on Ser807/811, which are critical for G1 entry, were reduced in Bim KO compared to WT B cells. Likewise, BCR‐induced p27Kip1 degradation was decreased in Bim KO compared to WT B cells. These defects in BCR‐induced cell cycle entry correlated with a proximal defect in BCR‐mediated intracellular calcium release in Bim KO B cells. Our results suggest that the balance of pro‐ and anti‐apoptotic Bcl‐2 family proteins is critical for controlling both cell cycle progression and apoptosis in B cells.

A CD40 Bridge between Innate and Adaptive Immunity

In this issue of Immunity, Brodeur et al. show that C4b binding protein (C4BP), a regulator component of the classical complement (C) pathway, can bind to CD40 receptors on B cells and activate them. This suggests a novel way by which CD40 may function to bridge innate and adaptive immune responses.

Modulation and function of caspase pathways in B lymphocytes

Summary:  During their development, B‐lineage cells are selected to mature, to die, to divide, or to survive and wait, ready to respond to external signals. The homeostatic balance between growth, death, and survival is mediated by signaling pathways through the B‐cell antigen receptor (BCR) complex, cytokine and chemokine receptors or cell–cell coreceptor interactions. The BCR complex is a master regulator essential at key checkpoints during development. These checkpoints involve various processes, including negative selection (deletion), anergy, receptor editing, and positive selection. Without BCRs or downstream BCR‐signaling components, B‐lineage cells arrest during development. Removal of BCRs from mature B cells leads to their death. Here, we discuss signaling pathways in B cells that activate members of the caspase family of cysteine proteases. In some B‐cell subsets, BCR signaling activates caspases, which in turn induce a program leading to cell death. However, in other contexts, caspases are involved in the proliferation of B cells. The outcome depends in part on the presence or absence of modifiers that affect signaling thresholds and on which caspases are activated. These mechanisms allow the coordinated regulation of proliferation and apoptosis that is essential for lymphoid homeostasis.