To investigate signals that control B cell selection, we examined expression of G5PR, a regulatory subunit of the serine/threonine protein phosphatase 2A, which suppresses JNK phosphorylation. G5PR is upregulated in activated B cells, in Ki67-negative centrocytes at germinal centers (GCs), and in purified B220+Fas+GL7+ mature GC B cells following Ag immunization. G5PR rescues transformed B cells from BCR-mediated activation-induced cell death by suppression of late-phase JNK activation. In G5PR-transgenic (G5PRTg) mice, G5PR overexpression leads to an augmented generation of GC B cells via an increase in non-Ag–specific B cells and a consequent reduction in the proportion of Ag-specific B cells and high-affinity Ab production after immunization with nitrophenyl-conjugated chicken γ-globulin. G5PR overexpression impaired the affinity–maturation of Ag-specific B cells, presumably by diluting the numbers of high-affinity B cells. However, aged nonimmunized female G5PRTg mice showed an increase in the numbers of peritoneal B-1a cells and the generation of autoantibodies. G5PR overexpression did not affect the proliferation of B-1a and B-2 cells but rescued B-1a cells from activation-induced cell death in vitro. G5PR might play a pivotal role in B cell selection not only for B-2 cells but also for B-1 cells in peripheral lymphoid organs.
MaterialDNA damage occurring during proliferation is detected by a sensor mechanism, and the subsequent cell cycling is regulated by checkpoint proteins. Mitotic checkpoint proteins, such as MAD2, MAD1, and BUBR1, strictly regulate the cell-cycle progression of DNA-damaged cells by arresting kinetochore tension during mitosis (10). Thus, the expression of mitotic checkpoint proteins might be altered according to the various B cell differentiation stages.The GC-associated nuclear protein GANP is homologous to Saccharomyces SAC3, which is a component of the ribonucleoprotein Thp1/SAC3 complex required for mRNA export in yeast cells (11). The lack of mammalian GANP does not affect the expression levels of cell-cycle-associated proteins, such as MAD2, BUBR1, and cohesin, but selectively impairs Shugoshin-1 expression, which is required to protect cohesion during mitosis (12), suggesting that the expression of mammalian mRNAs, particularly mRNAs essential for genomic stability, is selectively regulated during accelerated cell proliferation. Mammalian Pcid2 is a homolog of yeast Thp1 based on structural similarity with PAM (proteasome, COP9, initiation factor/PINT-associated module) and PINT (proteasome, Int-6, Nip-1, and TRIP-15)-like regions (13). Saccharomyces Thp1 is associated with RNAs as a ribonucleoprotein complex and is recruited to the nuclear pore docking proteins with SAC3 (14). We hypothesized that Pcid2 interacts with ribonucleoprotein complex in a manner similar to GANP and plays a role in B cell proliferation or survival.The findings of this study demonstrated that mammalian Pcid2 was selectively involved in the export of MAD2 mRNA and regulated the mitotic checkpoint function during cell proliferation. Pcid2 was expressed at high levels in pre-B, immature B, T1, and Materials and Methods Cell surface staining and sortingLymphoid cells from the bone marrow and spleen were stained by FITCconjugated mAb, PE-conjugated mAb, and biotin-labeled mAb with PerCPconjugated streptavidin (GE Healthcare, Buckinghamshire, U.K.), and analyzed by FACSCalibur (BD Biosciences, Mountain View, CA) using FlowJo software (Tree Star, Ashland, OR). mAbs were obtained from eBioscience (San Diego, CA) for B220, CD21, CD23, CD24, IgM, IgD, and CD93 (AA4.1). Anti-CD43 mAb was purchased from BD Biosciences. B220 + cells enriched by MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) were purified using a JSAN cell sorter (BayBioscience, Kobe, Japan) (15). For each fraction, 1.5 3 10 5 cells were collected with the purity of .95% by postsorting analysis. Real-time RT-PCRReal-time RT-PCR was performed using a LightCycler (Roche Molecular Biochemicals, Basel, Switzerland) with cDNAs after RT reaction using total RNA, oligo-dT primer (Invitrogen, Carlsbad, CA), and SuperScript III (Invitrogen). The data are presented as the relative ratio compared with control GAPDH. Specific oligonucleotide primers and probes of hPcid2, hMAD2, mPcid2, mMAD2, and GAPDH were purchased from Nihon Gene Research Laboratories (Sendai, Japan) (Supple...
Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn−/−) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn−/− mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenyl-chicken γ-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp33-to-Leu mutation in the IgVH-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs.
Receptor editing is believed to play a critical role in immunological tolerance by altering the specificity of autoreactive B cells that emerge in the bone marrow. To study whether receptor editing is altered in autoimmune disease, recombination activation gene 1 (rag1) transcription in B cells from New Zealand Black (NZB) mice was examined by introducing a GFP gene at the rag1 locus. Female NZB RAG1-GFP mice generated autoantibodies and glomerular B-cell population. In conclusion, the early B-cell subpopulation, which exhibits low RAG1 expression and presumably concomitantly low receptor editing, was found to be increased in NZB mice.
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