研究者詳細 - 曲　正樹

論文 - 曲　正樹

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ニワトリB細胞株DT40-SWを用いたin vitro抗体作製システムの高機能化抗体遺伝子への変異導入効率の増強

金広優一, 曲正樹, 藤堂景史, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成20年度 109 - 109 2008年7月

ニワトリB細胞株を用いたin vitro抗体作製システムの高機能化変異様式の転換の高性能抗体作製への応用

梶田真道, 藤堂景史, 曲正樹, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成20年度 109 - 109 2008年7月

Analysis of B cell selection in the germinal center reaction during a T-dependent antibody response at a single cell level 査読

Takahiro Okazawa, Masaki Magari, Takafumi Kimoto, Emi Kouyama, Hitoshi Ohmori, Naoki Kanayama

IMMUNOLOGY LETTERS 117 ( 1 ) 96 - 105 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The quasimonoclonal mouse is useful to examine B cell selection during T-dependent antibody (Ab) responses because of its limited B cell populations mainly expressing the knockin 17.2.25 V-H-encoded H chain (VHT) paired with the lambda 1 or lambda 2 L chain. It has been reported that both two VHT/lambda 1 and VHT/lambda 2 B cell populations responded to a T-dependent antigen conjugated with a hapten p-nitrophenylacetyl (pNP), but only VHT/lambda 2 B cells differentiated to secrete high affinity anti-pNP IgG Abs by acquiring a critical mutation (T313A) in the VHT. The VHT/lambda 2 B cells may be more potent in migrating to the germinal centers (GCs) due to about 50-fold higher affinity for pNP than VHT/lambda 1 B cells. Here, to uncover how VHT/lambda 2 B cells were preferentially recruited for affinity maturation during the anti-pNP Ab response, we examined the L chain usage and mutation frequency of VHT+ GC B cells at a single cell level. VHT/lambda 2 B cells bearing the unmutated VHT gene were found in the GCs more frequently than VHT/lambda 1 and mutated VHT/lambda 2 counterparts in an early phase of the Ab response. In the course of the GC reaction, the number of VHT/lambda 2 B cells that mutated their VHT genes preferentially expanded, and finally VHT/lambda 2 B cells bearing the T313A mutation occupied VHT+ GC B cell population. Thus, it is suggested that B cells with a higher affinity were selected not only for entry to the GCs but also in the affinity maturation process during a T-dependent Ab response. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2008.01.002

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ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発抗原特異的クローンのセルソーターによる効率的単離

岡澤貴裕, 藤堂景史, 梶田真道, 曲正樹, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成19年度 149 - 149 2007年8月

ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発 Pax5発現抑制による抗体産生の増強

曲正樹, 綾高宏, 藤堂景史, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成19年度 149 - 149 2007年8月

ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発変異導入の促進による迅速な抗体ライブラリーの構築

金広優一, 曲正樹, 藤堂景史, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成19年度 148 - 148 2007年8月

Analysis of antigen-stimulated B cell migration into germinal centers during the early stage of a T-dependent immune response 査読

Emi Kouyama, Yumiko Nishikawa, Takahiro Okazawa, Masaki Magari, Hitoshi Ohmori, Naoki Kanayama

IMMUNOLOGY LETTERS 109 ( 1 ) 28 - 35 2007年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The quasimonoclonal (QM) mouse provides a model to analyze B cell selection because major B cell antigen receptors (BCR) are composed of the knockin V(H)DJ(H) 17.2.25 (VHT) encoded H chain and the lambda 1 or lambda 2 L chain, thereby being specific for (4-hydoxy-3-nitrophenyl)acetyl (NP). We have reported that during a T-dependent antibody (Ab) response for a low-affinity NP analog p-nitrophenylacetyl (pNP), although VHT/lambda 1 and VHT/lambda 2 IgM were equally produced, VHT/lambda 2 IgG almost exclusively underwent affinity maturation toward pNP. The initial affinity of VHT/lambda 2 B cells for pNP was approximately 50-100-fold higher than that of VHT/lambda 1 B cells, suggesting a role of BCR affinity in recruiting B cells to affinity maturation processes. Here, we investigated whether the intensity of BCR signals could contribute to the selection of VHT/lambda 2 B cells for affinity maturation. VHT/lambda 2 B cells were more responsive to pNP than VHT/lambda 1 B cells in vitro. When CFSE-labeled QM B cells were transferred into the wild type mice where T cells had been primed with chicken gamma-globulin (CGG), QM B cells challenged by pNP-conjugated CGG could be observed to get activated and migrate to GCs in the early phase of the T-dependent response to pNP-CGG. Adoptive transfer of sorted populations revealed that the VHT/lambda 2 B cell population was more potent in migration into GCs than the VHT/lambda 1 counterpart. Thus, it is suggested that the higher BCR affinity of VHT/lambda 2 B cells may be an initial cue for their recruitment to GCs during a T-dependent Ab response. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2006.12.011

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Novel in vitro screening system for monoclonal antibodies using hypermutating chicken B cell library 査読

Kagefumi Todo, Kenji Miyake, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 102 ( 5 ) 478 - 481 2006年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.

DOI： 10.1263/jbb.102.478

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B細胞免疫反応と免疫記憶超変異ニワトリB細胞株を用いたin vitro抗原生産システム(In vitro antibody generation system using a hypermutating chicken B cell line)

Kanayama Naoki, Todo Kagefumi, Okazawa Takahiro, Ikeda Mika, Fujita Risako, Magari Masaki, Ohmori Hitoshi

日本免疫学会総会・学術集会記録 36 55 - 55 2006年11月

Establishment of lymphotoxin β receptor signaling-dependent cell lines with follicular dendritic cell phenotypes from mouse lymph nodes. 査読

Yumiko Nishikawa, Masaki Hikida, Masaki Magari, Naoki Kanayama, M. Mori, Tomohiro Kurosaki, Hitoshi Ohmori

The Journal of Immunology 177 ( 8 ) 5204 - 5214 2006年10月

Suppressive effects of mometasone furoate on an antigen-specific IgE antibody response and production of IL-4 in mice 査読

Masaki Magari, Mika Ikeda, Miki Asakura, Naoki Kanayama, Masami Ogawa, Hitoshi Ohmori

IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 28 ( 3 ) 491 - 500 2006年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：TAYLOR & FRANCIS INC

A nasal formulation of mometasone furoate (MF) is advantageous in avoiding systemic activity characteristic of glucocorticoids when it is applied topically. To confirm anti-allergic effects of this glucocorticoid formulation elaborately, we investigated whether the drug can suppress the production of IgE antibodies and related cytokines. It we showed that IgE production induced in mice immunized via intranasal route was significantly reduced when the mice were administered MF intranasally. Further, MF was effective in inhibiting production of type-2 helper T cell cytokines in vivo and in vitro. These results provide a immunopharmacological basis for clinical efficacy of this drug.

DOI： 10.1080/08923970600928155

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Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line 査読

N Kanayama, K Todo, S Takahashi, M Magari, H Ohmori

NUCLEIC ACIDS RESEARCH 34 ( 2 ) e10 2006年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

DOI： 10.1093/nar/gnj013

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抗体遺伝子変換機構を利用した非抗体タンパクのDNAシャッフリングによる機能改変

藤堂景史, 高橋佐都子, 片岡大輔, 曲正樹, 金山直樹, 大森斉

日本免疫学会総会・学術集会記録 35 80 - 80 2005年11月

培養B細胞株の変異機能を利用する新規なin vitro抗体作製システム

三宅健司, 藤堂景史, 曲正樹, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成17年度 118 - 118 2005年9月

Immunogenicity of autologous IgG bearing the inflammation-associated marker 3-nitrotyrosine 査読

H Ohmori, M Oka, Y Nishikawa, H Shigemitsu, M Takeuchi, M Magari, N Kanayama

IMMUNOLOGY LETTERS 96 ( 1 ) 47 - 54 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

To explore the link between inflammation and autoimmunity, we analyzed the immunogenicity of 3-nitrotyrosine(NT)-bearing self-proteins, an inflammation-associated marker that is formed by nitration of protein tyrosine residues with peroxynitrite generated during inflammation. An interesting feature of NT is its structural similarity to a synthetic hapten, 4-hydroxy-3-nitrophenylacetyl (NP), with which some anti-DNA antibodies (Abs) have been reported to show cross-reactivity. We confirmed that some of anti-DNA monoclonal Abs (mAbs) obtained from MRL/lpr mice also bound NT as well as NP. Based on these findings, we examined whether NIT-bearing autologous IgG (NT-1gG) as a model of NT-self proteins is immunogenic to induce a DNA-cross-reactive anti-NT Ab response in autologous normal mice. Anti-NT IgM and IgG Ab responses were elicited after the third immunization with NT-IgG. concomitant with an increase in anti-single stranded (ss)DNA titer. Interestingly, a part of anti-NT mAbs thus induced showed cross-reactivity with ssDNA. some of which used VH sequences that were highly homologous to those reported in anti-DNA Abs from NZB/WF1 mice. Splenic T cells primed with NT-IgG. but not with unmodified IgG. showed a proliferative response to the inducing antigen. Collectively, NT-IgG is immunogenic in autologous hosts. and can induce anti-NT Abs that are cross-reactive with ssDNA. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2004.07.004

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B細胞株の遺伝子改変能を利用したタンパク分子改変システムの構築

藤堂景史, 高橋佐都子, 曲正樹, 金山直樹, 大森斉

日本免疫学会総会・学術集会記録 34 263 - 263 2004年11月

Generation of IgM and IgG 1 monoclonal antibodies with the identical variable regions: comparison of avidity

Naoki Kanayama, Kimi Yamakoshi, Masaaki Kiyomi, Masaki Magari, Hitoshi Ohmori

Memoris of the Faculty of Engineering, Okayama University 38 91 - 96 2004年

B cell selection and affinity maturation during an antibody response in the mouse with limited B cell diversity 査読

N Kanayama, T Kimoto, K Todo, Y Nishikawa, M Hikida, M Magari, M Cascalho, H Ohmori

JOURNAL OF IMMUNOLOGY 169 ( 12 ) 6865 - 6874 2002年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC IMMUNOLOGISTS

The quasi-monoclonal mouse has limited B cell diversity, whose major (similar to80%) B cell Ag receptors are comprised of the knockin V-H 17.2.25 (VHT)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/lambda1 and VHT/lambda2 IgM were equally produced, VHT/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 Of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that VHT/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an,affinity maturation process.

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Role for complement receptors (CD21/CD35) in the regulation of recombination activating gene expression in murine peripheral B cells 査読

H Ohmori, M Magari, Y Nakayama, N Kanayama, M Hikida

IMMUNOLOGY LETTERS 83 ( 2 ) 95 - 99 2002年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect on immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-2478(02)00083-4

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Contribution of light chain rearrangement in peripheral B cells to the generation of high-affinity antibodies 査読

M Magari, T Sawatari, Y Kawano, M Cascalho, M Wabl, N Kanayama, M Hikida, H Ohmori

EUROPEAN JOURNAL OF IMMUNOLOGY 32 ( 4 ) 957 - 966 2002年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-V C H VERLAG GMBH

Recently, peripheral B cells have been shown to undergo secondary V(D)J rearrangement of immunoglobulin genes, but the physiological role of this event has not been fully elucidated. To investigate whether rearrangement of L chain genes in the periphery is involved in the generation of high-affinity antibodies (Ab), we used the 17.2.25 rearranged VHDJH gene (VHT)-knockin mouse whose B cell diversity is limited due to the expression of the site-directed transgene. Immunization of the mouse with p-nitrophenylacetyl (pNP)-conjugated chicken gamma-globulin preferentially led to the production of anti-pNP IgG Ab comprised of non-VHT-encoded H chains and lambda chains. lambda(+) IgG constituted a majority of high-affinity Ab to this hapten. RAG-2 mRNA and the recombination signal sequence break of the lambda1 gene increased in the draining lymph node of immunized mice, but not of nonimmunized animals. There was a close correlation between the levels of these parameters implicating X, gene rearrangement and the production of lambda(+) high-affinity anti-pNP IgG. These observations were reproduced in RAG-1-deficient mice that were reconstituted with the spleen cells of the knockin mouse. Thus, our findings suggest that L chain rearrangement that occurs in the periphery can contribute to affinity maturation of Ab.

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