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 na ï ve 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.

To investigate the function of NF-κB RelA (p65), we generated mice deficient in this NF-κB family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA−/− fetal liver transplants, similar to the relA+/+ and +/− cases. T cells were found to mature to Thy-1+/TCRαβ+/CD3+/CD4+ or CD8+, while B cells had the ability to differentiate to IgM+/B220+ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3+anti-CD28, LPS, anti-IgM, and PMA+calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.

We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions. AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region. Approximately 30 paralogues were identified in the Arabidopsis genome. Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif. Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding. AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin. Biochemically, AHL1 was also found in the nuclear matrix fraction. In the M phase, AHL1 was localized on the chromosomal surface. The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization. Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain. In addition, AHL1 is located on the surface of chromosomes during mitosis.

We found that two distinct antibody maturation pathways exist in the immune response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl and that the junctional amino acid introduced by a process far preceding somatic hypermutation determined the pathway of affinity maturation. Antibodies belonging to each pathway clearly separated into two separate branches of a phylogenic tree. We also constructed a three-dimensional fitness landscape for antibody evolution by introducing the association constants of the antibodies into the phylogenic tree as the third axis, allowing us to comprehend the significance of junctional diversity in the "evolvability" of antibodies. Thermodynamic analyses of the antigen-antibody interactions suggested that a high conformational versatility in the antigen-combining site allows for the enhanced evolvability of antibodies.