Satoru Fujimura

Generation of High-Affinity Antibody against T Cell-Dependent Antigen in the Ganp Gene-Transgenic Mouse

Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (GanpTg). GanpTg displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (KD ∼ 2.50 × 10−7 M) of IgG1/λ1 and contained the VH186.2 region with W33L mutation. GanpTg generated much higher affinity (KD > 1.57 × 10−9 M) by usage of VH186.2 as well as noncanonical VH7183 regions. GanpTg also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (KD > 9.90 × 10−11 M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser502 that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.

Increased Expression of Germinal Center–Associated Nuclear Protein RNA-Primase Is Associated with Lymphomagenesis

Lymphomas arise containing abnormalities of various differentiation stage-specific molecules. In the study reported here, we have shown abnormal up-regulation of germinal center B cell-associated GANP in various human lymphomas including mantle cell, diffuse large B cell, and Hodgkin lymphoma, by immunohistochemical analysis. To study the role of GANP in lymphomagenesis, we generated mutant mice (ganp-Tg) that express the transgenic ganp gene under immunoglobulin enhancer and promoter control. Ganp-Tg mice showed a high incidence of lymphomagenesis (29.5%) after aging with a non-B/non-T cell surface phenotype having slight CD45R/B220 expression and Ig transcripts of rearranged VH-DH-JH IgH loci. Lymphomas generated in ganp-Tg mice displayed similar pathologic characteristics to mouse reticulum cell neoplasm or Hodgkin lymphoma-like lesions. The VH sequences of individual mice showed that the tumors proliferated from a single clone or oligoclones, as is found in human diffuse large B-cell lymphomas and Hodgkin lymphoma. These results suggest that GANP overexpression is a causative factor in the generation of B lymphomas.

Cutting Edge: Double-Stranded DNA Breaks in the IgV Region Gene Were Detected at Lower Frequency in Affinity-Maturation Impeded GANP−/− Mice

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken γ-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP−/− B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.

Involvement of GANP in B Cell Activation in T Cell-dependent Antigen Response

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210 kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.

Spontaneous increase of plasma-like cells with high GANP expression in the extrafollicular region of lymphoid organs of autoimmune-prone mice

Autoimmune-prone mice bear a hyper-active B cell population generated spontaneously in peripheral lymphoid organs. Expression of beta RNA-primase GANP was shown to be an activation marker in lymphoid follicle germinal center (GC) B cells after immunization with T cell-dependent antigen (TD-Ag) in normal mice. In this study, we examined the expression of GANP in lymphoid tissues of autoimmune-prone mice. GANP expression was up-regulated in GC-B cells after stimulation with TD-Ags; however, highly GANP-positive (GANP(hi)) cells were also observed in lymph nodes of non-immunized MRL/lpr mice. GANP(hi)cells in lymph nodes as well as in spleens of the different autoimmune-prone strains, MRL/lpr, NZB, (NZBxNZW)F1 and BXSB, gradually increased with age. This population was detected only in small numbers in the red pulp region of the spleen after immunization with TD-Ag in normal C57BL/6 and BALB/c mice. GANP(hi)cells had a B220(-)IgM(+)Syndecan-1(+)phenotype, but were negative for PAS-staining and bromo-deoxyuridine incorporation. These results demonstrate that GANP(hi)plasma-like cells appear in lymph nodes of autoimmune mice during aging, suggesting that the new plasma cell population might be generated after hyper-activation of B cells during the course of autoimmune disease.

Three cases of severe hepatitis or fulminant hepatitis which are thought to be originated from cytomegalovirus infection.

健常成人でのサイトメガロウイルス (CMV) 肝炎の重症化例の報告はこれまで数例しかなく, 黄疸の出現も稀とされている. 今回CMV感染に起因すると思われた, 健常者にみられた劇症肝炎と重症肝炎, およびコンプロマイズドホストにみられた重症肝炎の各1例, 計3例を経験した. 非A非B非C型の, いわゆる原因不明の重症ないし劇症肝炎の中には, CMV感染に起因するものも潜在している可能性があり, IgM-CMV抗体の測定, リンパ球中CMV抗原や血清CMV-DNAの測定, さらには肝生検などを含めた積極的な検索を行う必要がある.