No abstract

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.

Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-μ and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.

Acquired immunity depends on proliferation and differentiation of antigen (Ag)-specific B cells in germinal centers (GCs) of lymphoid follicles in response to T cell-dependent Ags. Here, we studied the function of GC-associated nuclear protein that is selectively upregulated in GC-B cells. B cell-specific ganp-deficient mice were compromised in affinity maturation of hapten-specific antibodies against T cell-dependent Ags with retarded development of GCs. B cell numbers and development, serum Ig levels, mitogen-induced B cell proliferation in vitro, and responses to T cell-independent Ag were nearly normal; however, the mutant B cells showed a decrease in anti-CD40-induced proliferation and an increased susceptibility to B cell apoptosis in vitro and in vivo. B cell-specific ganp-deficient mice showed a decreased frequency of variable-region somatic mutations, especially of the high-affinity type (W 33 3 L) in the VH186.2 region against nitrophenyl-chicken gamma globulin, whereas the class switching was normal. We conclude that GC-associated nuclear protein is necessary for generation or maintenance of B cells with high-affinity B cell Ag receptors during the maturation in GCs. SHM is induced by the stimulation of BCR complexes containing BCR, CD19, and CD21 and by costimulatory signals through CD40 and CD154 (7,8). These stimuli provided in GCs probably upregulate molecules required for SHM. An RNA-editing molecule, activation-induced cytidine deaminase, expressed specifically in GCs is necessary for generation of SHM and CSR (9-11). Uracil DNA-glycosylase is up-regulated in GCs and is also involved in generation of SHM in a B cell line in vitro (12). The initial modification of DNA by activation-induced cytidine deaminase is probably followed by uracil DNA-glycosylase action, which may account for the generation of SHM in V regions during the immune response (12, 13). Various DNA polymerases were shown to be involved in generation of SHM in Ig V regions by experiments using cell lines of various DNA polymerases (14-18).Because the mutations generated by these molecular events are probably random, it is not clear how the high-affinity B cells are predominantly generated and selected during immune responses. This process is mostly undertaken in Ag-driven B cells in GCs, and the B cells with high-affinity BCRs are presumably selected for the higher binding activity to the Ags presented by the immune complex through CR1 expressed on the surface of follicular dendritic cells. Such B cells might undergo clonal expansion and differentiation into Ab-producing cells with the help of costimulatory activities provided by T helper 2 cells through cognate interactions such as CD40͞CD154 (CD40L) (19) and CD80͞CD28 (20) or mediated by cytokines such as IL-4 (21), IL-6 (22), BAFF͞Blys (23), and lymphotoxin-␣ (24). B cells with low-affinity, irrelevant, and selfreactive BCRs are eliminated or not stimulated for subsequent proliferation (25, 26). However, B cells with high-affinity BCRs are rescued from death in GCs by secreted...