B cells diversify their immunoglobulin (Ig) genes that encode B-cell antigen receptors (BCR) and antibodies during their early development in the bone marrow. Among the newly generated B cells, those survived through the selection for BCR integrity and against self-reactivity, further maturate into either follicular B cells or marginal zone B cells in the peripheral lymphoid tissues such as lymph nodes and spleen. These mature B cells express BCR of IgM and IgD classes, both having the same specificity. BCR expression and its ligand-independent signaling are necessary for the B cell maturation and survival in the periphery.

Upon encountering antigens through BCR, mature B cells are activated and, with the aid of ‘T-cell help’, they proliferate and undergo class switching to IgG or IgE. Then some of them differentiate into short-lived plasma cells in the extrafollicular region and others form germinal centers (GC) in the B-cell follicles, in which somatic hypermutations (SHM) in Ig V region genes further diversifie the Ig repertoire. Among the diversified GC B cells, those expressing BCR (typically of IgG classes) that binds the immunized antigen with high affinity are selected, and they differentiate into memory B cells or long-lived plasma cells (LLPC), both contributing to the long lasting humoral immunological memory.

During the T-cell dependent (TD) immune responses, BCR plays important roles: 1) antigen-mediated BCR signals activate B cells to initiate cell-cycling and to upregulate the expression of MHC class II and T-cell stimulating molecules such as CD80 and CD86; 2) BCR internalizes bound antigens which are then digested and presented on MHC class II to helper T (Th) cells; 3) The affinity of BCR determines the survival and differentiation of GC B cells into memory B cells or LLPCs.

BCR signal transduction pathways have been extensively studied in the past two decades, and major players have been identified and their roles elucidated, such as protein tyrosine kinases, Syk, Lyn, Btk, an adaptor molecule BLNK (also termed SLP65 or BASH), and effector molecules such as phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinase (PI3K). BCR downstream pathways leading to activation of transcription factors such as AP-1, NF-AT and NF-κB have also been elucidated. We identified BLNK (we originally named BASH; Goitsuka et al. 1998, J. Immunol. 161:5804) among other groups and have studied its function using the mice deficient for this molecule. BLNK plays a key role in pre-BCR-mediated progression of early B-cell development including induction of L-chain gene rearrangement (Hayashi et al. 2000, PNAS 97:2755; 2004, Immunity 18:825; 2004, J. Immunol. 173:5980; Yamamoto et al. Blood 108:2703; Oda et al. 2008, Int. Immunol. 20:1417). We also found that BLNK regulates IL-7-receptor signaling, which partly accounts for the development of pre-B cell leukemia in BLNK-deficient mice (Nakayama et al. 2009, Blood 113:1483).

In B cells, BLNK associates with several signaling molecules such as PLCγ2, Vav, Grb2, Nck, Btk and Syk, and function as a scaffold protein to recruit them into a BCR signaling complex at the plasma membrane. In BLNK-deficient mice, the number of mature B cells in the peripheral lymphoid organs is severely reduced, although it gradually recovers with age. The BLNK-deficient B cells scarcely proliferate or up-regulate B7-2 in response to BCR-ligation in vitro. Accordingly, BLNK-deficient mice produce little antibodies in response to T-independent antigens and show a delayed response to TD antigens with sufficient affinity maturation and memory formation (Yamamoto et al. 2004, Int. Immunol. 16:1161).

It has been postulated that BLNK relocates to BCR upon its ligation and tethers signaling molecules under the plasma membrane, but the mechanism of how BLNK is recruited to BCR is not fully understood. The SH2 domain of BLNK has been shown to bind to a cytoplasmic domain of Igα, a signaling subunit of surface IgM complex. In addition, we found that the SH2 domain binds to HPK1, one of the MAP4-kinases, upon its phosphorylation by BCR crosslinking (Tsuji et al. 2001, J. Exp. Med. 194:529). Alternatively, a leucine zipper-like motif (LZ) in the N-terminal domain of BLNK has been shown to be responsible for its plasma-membrane recruitment. Using the N-terminal domain of BLNK as bait for the yeast two-hybrid system, we identified novel membrane-spanning proteins, BNAS1 and BNAS2. The two proteins are unlike each other but both contain the LZ and recruits BLNK where they express in cells (Imamura et al. 2004, J. Biol. Chem. 279:26425; Katahira et al. 2006, Int. Immunol. 18:545). The functions of these proteins are under investigation. We also identified H-Ras in the same screening, and found that the binding of BLNK and an active form of H-Ras promotes BCR-mediated capping and ERK activation (Imamura et al., 2009, J. Biol. Chem. 284:9804).

Recently, our interest has shifted to immune responses of B cells and the mechanisms of their diversification, selection and differentiation in GC. To study them at molecular levels, we have established a B-cell culture system in which B cells exhibiting a GC-B-cell phenotype proliferate extensively, undergo class-switching and differentiate into precursors of memory B cells or LLPCs depending on culture conditions (Nojima et al. 2011, Nat. Commun. 2:465). This system will be useful to elucidate several unsolved questions including mechanisms of AID-mediated mutagenesis, affinity selection, negative selection of self-reacting GC B cells, identity of transcription factors that determine the fate of GC B cells toward memory B cells, requirements for survival of memory B cells and LLPCs, and regulation of memory recall response.

By combining the classic mouse immunization protocols and the modern flow cytometric technologies, we found that memory B cells induced by the primary immunization with a TD antigen cannot elicit recall response to a T-independent type II antigen carrying the same epitopes as the primary antigen, but they are tolerized presumably through clonal deletion. This phenomenon may account for the maintenance of self-tolerance of memory B cells that may have acquired self-reactivity during diversification in GC (Haniuda et al. 2011, J. Immunol. 186:5620).