Daisuke Kitamura

A critical role of λ5 protein in B cell development.

The lambda 5 gene is a homolog of immunoglobulin J lambda-C lambda genes, expressed specifically in immature B-lineage cells. Lambda 5-encoded molecules form membrane complexes with mu or D mu proteins in association with an additional protein specifically expressed in immature B cells that is encoded by the Vpre-B gene. We have generated mice in which the lambda 5 gene is inactivated by targeted gene disruption in embryonic stem cells. In these mice, B cell development in the bone marrow is blocked at the pre-B cell stage. However, the blockade is leaky, allowing B cells to populate the peripheral immune system at a low rate. These cells are allelically excluded and able to respond to antigen.

Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice.

The Src family protein-tyrosine kinase Lyn associates physically with the BCR and has been suggested to play an important role in BCR-mediated signaling. Studies with lyn-/- mice showed that the number of B cells decreased by half in their peripheral tissues. In addition, these B cells do not respond normally to a number of stimuli, including BCR cross-linking and CD40 ligand. Induction of tyrosine phosphorylation on a variety of cellular proteins, such as Vav, Cbl, and HS1, upon BCR cross-linking was also abolished in these B cells. Despite the impaired BCR-mediated signaling, concentrations of IgM and IgA in sera were remarkably elevated, and production of autoantibodies was detected in lyn-/- mice. Histological study showed splenomegaly and enlargement of lymph nodes that became evident with age in the mutant mice. The spleen contained significant number of plasma cells as well as unusual lymphoblast-like cells carrying Mac1 antigen and cytoplasmic IgM. These cells spontaneously secreted a large amount of IgM in vitro. Finally, significant number of lyn-/- mice show glomerulonephritis, an indication of autoimmune disease. From these data, we conclude that Lyn plays a role in signal transduction for not only clonal expansion and terminal differentiation of peripheral B cells but also elimination of autoreactive B cells.

Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development

The compartment of mouse B cell progenitors can be resolved into five developmentally related fractions by multicolor flow cytometry. Using this system and employing mutant mice in which the membrane exon of the mu chain, the lambda 5 gene, or the JH locus was inactivated by gene targeting, we found that expression of the pre-B cell receptor complex is necessary for the transition from the large CD43+ to the small CD43- pre-B cell stage. We report the occurrence of immunoglobulin heavy and light chain gene rearrangement at the stage of large B cell precursors. We show that neither the pre-B cell receptor complex nor any gene rearrangement in the heavy chain locus is required for the induction of kappa light chain gene rearrangement in early B cell progenitors.

Tyrosine Kinases Btk and Tec Regulate Osteoclast Differentiation by Linking RANK and ITAM Signals

Certain autoimmune diseases result in abnormal bone homeostasis, but association of immunodeficiency with bone is poorly understood. Osteoclasts, which derive from bone marrow cells, are under the control of the immune system. Differentiation of osteoclasts is mainly regulated by signaling pathways activated by RANK and immune receptors linked to ITAM-harboring adaptors. However, it is unclear how the two signals merge to cooperate in osteoclast differentiation. Here we report that mice lacking the tyrosine kinases Btk and Tec show severe osteopetrosis caused by a defect in bone resorption. RANK and ITAM signaling results in formation of a Btk(Tec)/BLNK(SLP-76)-containing complex and PLCgamma-mediated activation of an essential calcium signal. Furthermore, Tec kinase inhibition reduces osteoclastic bone resorption in models of osteoporosis and inflammation-induced bone destruction. Thus, this study reveals the importance of the osteoclastogenic signaling complex composed of tyrosine kinases, which may provide the molecular basis for a new therapeutic strategy.

B cell development regulated by gene rearrangement : arrest of maturation by membrane-bound Dμ protein and selection of DH element reading frames

In productively rearranged murine VH-DH-JH genes (encoding immunoglobulin heavy chain variable regions), the DH elements are preferentially used in one particular reading frame (RF1), although the recombination breakpoints at the DH-JH border vary. Despite this variability, the bias of RF usage is not due to cellular selection by antigen but is quantitatively established at the stage of DH-JH rearrangement: RF3 is counterselected on the basis of stop codons. RF2 allows the expression of a truncated mu chain (D mu protein) from most DH-JH joints. Using B cells in which the membrane exon of the mu chain is disrupted by homologous recombination on one of the two homologous chromosomes, we obtain evidence that membrane-bound D mu signals arrest of differentiation, presumably by preventing VH-DHJH joining. In addition to RF3 and RF2 counterselection, promotion of DH-JH joining in areas of sequence homology further enforces RF1 usage.

Cell Cycle Arrest and Apoptosis Induced by Notch1 in B Cells

Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Recently, we found that Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1 caused growth suppression of the cells, accompanied by a cell cycle inhibition at the G1 phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induced the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G1 cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1.

SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast

Commitment of the germ cell lineage during embryogenesis depends on zygotic gene expression in mammals, but little is known about the signaling molecules required for germ cell formation. Here we show that the intracellular signaling molecule SMAD1, acting downstream of bone morphogenetic protein (BMP) receptors, is required for the commitment of germ cell lineage from epiblast in early mouse embryos. Smad1 homozygous mutant embryos (Smad1-/-) were generated by in-frame insertion of lacZ gene into an exon of the Smad1 gene. Most of the Smad1-/- embryos contained no primordial germ cells (PGCs) and had short allantois, while histological analysis and in situ hybridization for the mesoderm marker genes revealed that early mesoderm induction was normal in those embryos. Smad1 expression was observed in epiblast and in visceral endoderm during gastrulation, while only a few alkaline phosphatase-positive PGCs at 7.5 and 8.5 days post coitum (E7.5 and E8.5) expressed Smad1. Phosphorylated SMAD proteins were localized in the proximal region of epiblast at E6.0-6.5, where the progenitors of PGCs and of allantois reside. Single-cell reverse transcription-polymerase chain reaction analysis revealed that the expression of Smad1, -5 and -8 were sporadic and mutually independent in proximal epiblast cells. We also found that BMP4-induced differentiation of PGCs from epiblast in vitro was fully dependent on the existence of phosphorylated SMAD1. These results indicate that SMAD1 signaling possesses a critical and non-redundant function in the initial commitment of the germ cell lineage.

Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.

BLNK suppresses pre–B-cell leukemogenesis through inhibition of JAK3

Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK(-/-) mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27(kip1) expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27(kip1) induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7-dependent proliferation and survival of pre-B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre-B-cell leukemia.

In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo

In response to T cell-dependent antigens, B cells proliferate extensively to form germinal centres (GC), and then differentiate into memory B (B(mem)) cells or long-lived plasma cells (LLPCs) by largely unknown mechanisms. Here we show a new culture system in which mouse naïve B cells undergo massive expansion and isotype switching, and generate GC-phenotype B (iGB) cells. The iGB cells expressing IgG1 or IgM/D, but not IgE, differentiate into B(mem) cells in vivo after adoptive transfer and can elicit rapid immune responses with the help of cognate T cells. Secondary culture with IL-21 maintains the proliferation of the iGB cells, while shifting their in vivo developmental fate from B(mem) cells to LLPCs, an outcome that can be reversed by withdrawal of IL-21 in tertiary cultures. Thus, this system enables in vitro manipulation of B-cell fate, into either B(mem) cells or LLPCs, and will facilitate dissection of GC-B cell differentiation programs.