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Sci Immunology | 上海市免疫學(xué)研究所葉菱秀團(tuán)隊(duì)揭示抗體多樣化過程中抗體基因DNA片段缺失和插入產(chǎn)生的分子機(jī)制
發(fā)布日期：2023-03-25

科研進(jìn)展

2023年3月24日,，上海市免疫學(xué)研究所葉菱秀團(tuán)隊(duì)在Science Immunology期刊在線發(fā)表了題為“DNA Repair Mechanisms That Promote Insertion-Deletion Events During Immunoglobulin Gene Diversification”的研究論文,。該論文鑒定了在抗體多樣化過程中低頻率DNA片段插入事件和±1bp缺失或插入事件，揭示了±1bp和>1bp缺失或插入產(chǎn)生機(jī)制的差異,。在此基礎(chǔ)上,，通過外切酶或聚合酶的遺傳學(xué)操作獲得了能夠增加抗體基因缺失或插入的小鼠模型，為廣譜中和抗體的篩選提供了新的思路和方法,。

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DNA片段缺失或插入是基因組中單個(gè)或多個(gè)核苷酸的缺失或插入,。在免疫球蛋白即抗體的基因可變區(qū)外顯子中，片段缺失或插入(<50 bp)通常會(huì)導(dǎo)致B細(xì)胞受體編碼錯(cuò)誤,。然而,，“塞翁失馬，焉知禍?！?，長(zhǎng)片段缺失或插入又是罕見抗病毒廣譜中和抗體(broadly neutralizing antibody, bnAbs)獲得廣譜中和能力的關(guān)鍵。例如,，發(fā)生在抗HIV廣譜中和抗體基因上的片段插入大大提高了抗體與HIV包膜三聚體表位的結(jié)合力,。但是，由于片段缺失或插入在抗體多樣化過程中發(fā)生頻率較低,，很難用傳統(tǒng)模型和方法檢測(cè)得到,。因此，片段缺失或插入在抗體多樣化中的分子機(jī)制仍知之甚少,。本研究利用一種抗體無功能等位基因系統(tǒng),，同時(shí)結(jié)合超深度測(cè)序克服了片段缺失或插入檢測(cè)的困難，成功捕捉到生理?xiàng)l件下真實(shí)的片段缺失或插入事件（圖1）,。通過對(duì)片段缺失或插入圖譜的分析發(fā)現(xiàn)±1bp缺失或插入發(fā)生最頻繁,，占總?cè)笔Щ虿迦胧录?5%左右（圖2）。對(duì)>1bp堿基插入的序列特征進(jìn)行分析發(fā)現(xiàn)大部分的堿基插入為鄰近序列的復(fù)制插入,。發(fā)生在CDR3上的復(fù)制插入能夠增加CDR3的長(zhǎng)度,，可能促進(jìn)廣譜中和抗體的產(chǎn)生,。

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圖1 小鼠模型與實(shí)驗(yàn)設(shè)計(jì)流程
Schematic of the study design.

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圖2 抗體無功能等位基因上1bp和>1bp缺失或插入的占比
1 bp indels events are prevalent.

在體細(xì)胞高頻突變（Somatic Hypermutation, SHM）過程中，胞苷脫氨酶AID能夠作用于抗體可變區(qū)基因,，引起DNA損傷,。該損傷可以通過堿基切除修復(fù)途徑（base excision repair, BER）或錯(cuò)配修復(fù)途徑（mismatch repair, MMR）進(jìn)行修復(fù)，產(chǎn)生堿基突變以及DNA片段的缺失或插入,。為了探究DNA片段缺失或插入產(chǎn)生的分子機(jī)制,，本研究在抗體無功能等位基因小鼠模型基礎(chǔ)上構(gòu)建了15種具有代表性的DNA損傷應(yīng)答修復(fù)因子缺陷小鼠模型（圖1），并對(duì)其片段缺失或插入圖譜進(jìn)行了系統(tǒng)的描繪（圖3）,。研究發(fā)現(xiàn)±1bp與>1bp缺失或插入產(chǎn)生的分子機(jī)制截然不同（圖3-4）,，1bp缺失通過BER途徑產(chǎn)生，1bp插入既可以通過BER途徑產(chǎn)生,，也可以通過MMR途徑產(chǎn)生,，產(chǎn)生過程需要核酸外切酶ExoI和易錯(cuò)DNA聚合酶Polη的參與。>1bp缺失或插入都是通過BER途徑產(chǎn)生的,，并且都需要DNA雙鏈斷裂損傷修復(fù)因子53BP1的參與,。此外，>1bp插入還需要非同源末端連接途徑和DNA聚合酶Polλ的參與,。對(duì)片段缺失或插入分子機(jī)制的全面解析有助于理解廣譜中和抗體產(chǎn)生的機(jī)制,，為廣譜中和抗體的生產(chǎn)提供了新的思路，為HIV等疫苗的設(shè)計(jì)提供了新的理論基礎(chǔ),。

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圖3：抗體基因SHM中DNA片段缺失或插入機(jī)制的系統(tǒng)解析
Summary of the AID-initiated mutagenic outcomes of the different genetic models used in this study.

在機(jī)制研究基礎(chǔ)上,，本研究成功構(gòu)建了能夠增加抗體基因缺失或插入頻率的小鼠模型（圖3），核酸外切酶Trex2過表達(dá)的小鼠模型能夠促進(jìn)片段缺失的產(chǎn)生,，DNA聚合酶Polβ條件性敲除的小鼠模型能夠促進(jìn)片段插入的產(chǎn)生,。這兩種小鼠模型可以與人源化抗體小鼠模型聯(lián)合使用，用于疫苗的研發(fā)和廣譜中和抗體的生產(chǎn),。

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圖4：抗體基因SHM中DNA片段缺失或插入機(jī)制的系統(tǒng)解析

Distinct factors/pathway that generate the 1-bp or longer indel events.

上海交通大學(xué)醫(yī)學(xué)院上海市免疫學(xué)研究所葉菱秀研究員,，瑞典卡羅琳斯卡醫(yī)學(xué)院Qiang Pan-Hammarström教授和意大利都靈大學(xué)病理學(xué)系Roberto Chiarle教授為本論文的共同通訊作者，郝茜助理研究員和占傳棕博士研究生為本論文的共同第一作者,。本研究得到了國家重點(diǎn)研發(fā)計(jì)劃,、國家自然科學(xué)基金、上海交通大學(xué)醫(yī)學(xué)院國際科研合作項(xiàng)目以及中國博士后科學(xué)基金等資助,。我們衷心感謝所有參與本研究的合作團(tuán)隊(duì)成員,，同時(shí)感謝上海交通大學(xué)醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院和上海市免疫學(xué)研究所的公共技術(shù)平臺(tái)以及免疫所與瑞金醫(yī)院共建的免疫與疾病研究中心對(duì)本研究的大力支持。

葉菱秀,，研究員,，博士生導(dǎo)師。2010年博士畢業(yè)于劍橋大學(xué)， 2010～2015年于哈佛大學(xué)醫(yī)學(xué)院進(jìn)行博士后研究,，2016年加入上海交通大醫(yī)學(xué)院上海市免疫學(xué)研究所并擔(dān)任抗體多樣化課題組長(zhǎng),。獲得國家自然科學(xué)基金優(yōu)秀青年項(xiàng)目、國際合作項(xiàng)目等基金資助,。葉菱秀課題組長(zhǎng)期致力于B淋巴細(xì)胞抗體多樣化分子機(jī)制研究,，特別是廣譜中和抗體與自身抗體產(chǎn)生機(jī)制。課題組在體液免疫反應(yīng)和中和抗體發(fā)現(xiàn)上積累了多種技術(shù)和完善的研究體系,。實(shí)驗(yàn)室已培養(yǎng)“博新計(jì)劃”博士后,、博士研究生多名。歡迎對(duì)本課題組研究感興趣的學(xué)生及博士后的加入,，共同探討推進(jìn)B細(xì)胞抗體多樣化研究的進(jìn)展,。課題組主頁為http://dripwizz.com/sii/info/1053/1455.htm，歡迎感興趣的申請(qǐng)者來函咨詢[email protected],。

Research Progress

Study in Science Immunology reveals the mechanisms that generate insertion and deletion events during antibody diversification

Researchers from the laboratory of Leng-Siew Yeap at the Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Qiang Pan-Hammarström at the Karolinska Institute and Roberto Chiarle at Harvard University and the University of Torino have reported the results of their work to elucidate the mechanisms that generate rare mutagenic outcomes of antibody diversification process, with implications for the generation of antiviral broadly neutralizing antibodies and the development of autoantibodies.

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The study, published online on 24 March 2023 in Science Immunology, represents a major advance in understanding the mechanisms underlying the generation of insertions and deletions (indels), rare outcomes of a physiological process that generates mutations in DNA to produce good antibodies. Indels are generally undesirable in the genome, but they are common and have been shown to be functionally important in rare antibodies that can neutralize a wide range of viral strains. To study the mechanisms that generate insertions and deletions, the researchers take advantage of a powerful passenger-antibody gene knock-in mouse model, which allows the detection of unselected mutational events, and ultra-deep sequencing of a large numbers of germinal center B cells to gain insight into even relatively rare events. This led to the revelation that ±1 bp indel events, which are deleterious to the B cell receptor function, are the most common indel events, while longer indel events, which can generate productive antibodies, are rare. By analyzing a large number of DNA repair gene-deficient mice carrying the passenger allele, the researchers identified the different DNA repair pathways that generate indels of different sizes, e.g. the mechanisms that generate 1 bp events are not the same as those that generate longer indels. The study also generated mutant mouse models with either increased deletions or insertions, which could potentially be used to identify rare antibodies.

The co-first authors of this paper are Dr. Qian Hao, a research associate, and Chuanzhong Zhan, a PhD student in the Yeap Lab. This work was supported by funding from the National Key R&D Program of China, the National Natural Science Foundation of China, the NSFC-MAECI Italy-China Collaborative grant, the Joint Research Initiative of the School of Medicine, Shanghai Jiao Tong University, the Swedish Research Council, the Swedish Cancer Society, CIMED, the Alice Wallenberg Foundation, and the China Postdoctoral Science Foundation. We would like to thank all of our wonderful collaborators on this study who are not mentioned here individually. The authors also acknowledge the support of the Center for Immune-Related Diseases at Shanghai Institute of Immunology and Ruijin Hospital and Core Facilities at the Shanghai Institute of Immunology and the School of Basic Medical Science.

The Yeap lab welcomes enthusiastic students and postdoctoral fellows to join the group. Interested individuals can contact Dr. Yeap at [email protected].

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