B cell development and activation

B and T lymphocytes are characterized by the expression of diverse antigen receptors on their surface. B-cell antigen receptor (BCR) and antibody are produced from the same immunoglobulin (Ig) gene through alternative splicing. The Ig gene is diversified during B cell development through V(D)J recombination process in which D-J and V-DJ recombination of heavy (H) chain gene locus successively take place in pro-B cells, then V-J recombination of light (L) chain genes ( or ) in pre-B cells. After successful recombination of IgH gene, H chain product is assembled with surrogate light chains, VpreB and 5, forms a pre-B cell receptor complex (pre-BCR), and express on the surface of the cells now called large pre-B cells. The pre-BCR signals proliferation of the cells and then differentiation into the next stage called small pre-B cells where L gene rearrangement takes place.

After successful recombination of L chain gene, L chain products and H chain are combined into IgM that is the first BCR class to emerge on B cells. Depending on the affinity of the BCR to self antigens, B cells or BCR itself that bind to the self antigen are eliminated from the primary repertoire of B cells during the immature B cell stage, through apoptosis or receptor editing, respectively. This selection against self-reactivity of B cells is important to establish self-tolerance in B-cell system and to avoid auto-immunity. After the negative selection, the remaining B cells emigrate into the peripheral lymphoid organs and further maturate into so called mature B cells. Upon encountering   antigen through BCR, the mature B cells proliferate and, with the aid of T-cell help, undergo class switch recombination of H chain gene loci, then some of them differentiate into plasma cells and others form germinal centers in which somatic hypermutation in V region of H and L chains take place. From the germinal centers develop memory B cells and long-lived plasma cells equipped with IgG or other classes of Ig containing the affinity-selected mutations in V regions. Thus, immunoglobulin proteins not only serve as a receptor and antibody against numerous kinds of pathogens, but also drive B-cell development and selection of B cells.

Stimulation of B cells through the BCR provokes, depending on their developmental stage or situation, such diverse responses as proliferation, differentiation, apoptosis, or more specifically, induction or cessation of immunoglubulin gene rearrangements. BCR signaling is initiated by activation of cytoplasmic protein tyrosine kinases (PTKs) including Src-family kinases, Syk and Btk. Activated PTKs then phosphorylate and regulate the enzymatic activities of various signaling intermediates including phospholipase C2 (PLC, phosphoinositide 3-kinase (PI3K) and Vav, which in turn transmit the signals into distinct pathways leading to the activation of nuclear transcription factors such as AP-1, NF-AT and NF-B. Distinct signaling pathways appear to be activated selectively, depending on the developmental stage, activation state or tolerance status of B cells and the nature of the pertinent antigen. We are trying to elucidate the mechanism of the diversification and the selection of the receptor signal pathways by identifying and characterizing signaling molecules involving such processes and by analyzing how the molecules involved.

We previously identified the B-cell specific adaptor, BASH (also known as BLNK or SLP-65), belonging to an SLP-76 protein family.  BASH consists of an N-terminal basic domain, acidic domain containing tyrosine-based SH2-domain binding motifs, a central domain containing proline-rich motifs and a C-terminal SH2 domain. Shortly after BCR stimulation, BASH becomes phosphorylated primarily by Syk, translocates to the membrane fraction of cells and associates with PLC2, Vav, Nck and Btk. In addition, BASH constitutively associates with Grb2 and Syk. Experiments using normal or BASH-deficient B cell lines indicated that BASH mediates phosphorylation and activation of PLC2 by Syk and Btk, elevation of intracellular calcium concentration, and activation of NF-AT, NF-B, MAP kinases, ERK, JNK and p38, after BCR engagement. Thus, BASH has been proposed to function as a scaffold protein for various signaling effectors and to recruit them into a BCR signaling complex containing PTKs at the plasma membrane.

We have revealed a crucial role for BASH in B cell development and function as well as in BCR signal transduction through the analysis of BASH-knockout mice. In the BASH-/- mice, early B cell development was partially impaired and a normally rare population of large pre-B cells expressing pre-B cell receptor (pre-BCR) dominated in the bone marrow in place of small pre-B cells, although they were mostly non-cycling. The developmental arrest was complete in the mice additionally lacking CD19, a B-cell lineage-specific co-receptor of BCR, suggesting a partially compensatory role for CD19 in pre-BCR signal transduction. Despite the abundant expression of RAG2 in these mutant pre-B cells, these cells underwent less extent of L chain gene rearrangement. However, IgH gene rearrangement was normal and underwent allelic exclusion. These results indicate a pivotal role for BASH, with a partial compensation by CD19, in the pre-BCR signal transduction required for cell cycling, differentiation, activation of L chain gene loci for their rearrangement, and pre-BCR-downregulation.

Despite a proliferative defect in large pre-B cells in BASH knockout mice, such mice occasionally succumb to acute lymphoblastic leukemia (ALL) of pre-B cell phenotype at relatively early in life. Dominant preBCR-positive leukemic cells are detectable in blood as early as 4-weeks of age. The incidence of the ALL was ~5% of BASH-single- and ~15% of BASH/CD19- double knockout mice. Much higher incidence (~70%) was reported in BASH/Btk-double knockout mice that show complete arrest of B-cell development. We are trying to elucidate the mechanism of the leukemogenesis. We have derived several pre-B cell lines from the bone marrow of the affected mice. We have found that these cell lines are particularly useful to study the signaling requirement for the differentiation represented by the events such as L-chain gene rearrangement and pre-BCR downregulation, as described below.

In BASH-single knockout mice, the number of mature B cell populations in the peripheral lymphoid organs as well as peritoneal cavity was severely reduced, and serum immunoglobulin (Ig) was also reduced. The residual BASH-deficient B cells were phonotypically immature, scarcely proliferated or up-regulated B7-2 in response to BCR-ligation and weakly proliferated upon CD40-ligation or LPS-stimulation. This phenotype indicates that BASH is critical for BCR signaling inducing maturation and activation/ proliferation of peripheral B cells. The maturation block is also reported in mice deficient with BAFF, a TNF-related cytokine, and BAFF-receptor. We showed that the maturation block in BASH-knockout mice is not due to a defect in BAFF-receptor signaling, as described below.

We have been trying to identify novel signaling pathways downstream of BASH. We have shown that the SH2 domain of BASH mediates survival signal and identified a protein that interacts with the SH2 domain: a Ste20-related serine/threonine kinase of GCK family, HPK1. BCR-mediated activation of HPK1 was dependent on the interaction with BASH, and HPK1 was involved in the activation of IKK and NF-B. Physiological role of the interaction between BASH and HPK1 in vivo is now under investigation. In addition, we identified novel tetra-span membrane proteins, BNAS1 and BNAS2, which were bound with the N-terminal basic domain of BASH, through the yeast two-hybrid screening. Thus, a novel BCR signal pathway mediated through BASH is now being unveiled.