開發研究人類疾病的新免疫基因組學平台

Abstract

許多例子表明，HLA-B 基因的種系變異可能會影響個體的免疫反應和疾病發 生。據此，許多相關的基因轉殖鼠模型已被建立以研究相應的疾病。然而，大多數 小鼠模型都屬於“基因轉殖鼠”，它們將所需基因“隨機插入”到宿主基因組中， 並可能引起一些非預期的副作用。此外，目前為止還沒有 Graves’disease 的相關 HLA-B 小鼠模型。因此，本研究旨在建立具有重組酶介導的盒式交換（RMCE）系 統的 hHLA-B 外顯子 4 KI （knock-in）H-2D1 小鼠，它能方便地根據所需產生不同 的 HLA-B 等位基因。建立此小鼠後，我們將在小鼠中插入 Graves’disease 的風險 等位基因 HLA-B*46:01，並將其發展為 Graves’disease 小鼠模型。另外，我們還在 本研究中建立了人源化的β2 微球蛋白（hB2M）和 CD8 敲入小鼠，它們在抗原呈 現過程中均扮演重要角色。我們採用了 CRISPR-Cas9 或 ES 細胞靶向策略，將目標 基因插入小鼠體內，藉此將特定的突變精確地引入內源基因中，並建立小鼠品系。 我們的最終目標是配種這三種小鼠，以獲得具有三重敲入基因型小鼠。 目前我們只握有 hB2M KI 小鼠，其純系個體中 DNA、mRNA 和蛋白質的表現 已得到驗證。此外，我們發現 hB2M KI 小鼠無法表達 H-2K b 分子，但可以正常表達 H-2Db ，這足以滿足 CD4- CD8 + T 細胞的發育，但是這些細胞的功能需要進一步的實 驗來評估。總體而言，該小鼠品系似乎沒有任何異常或過早死亡的跡象，將有助於 研究免疫相關問題。 B 細胞受體（BCR）和 T 細胞受體（TCR）的集合形構了適應性免疫受體庫 （AIRR）。 BCR 和 TCR 的抗原結合位點由可變（V），多樣性（D）和連接（J） 片段組成。當發生 V（D）J 重組時，V（D）J 各自會有一個等位基因參與連接過 程，造就適應性免疫的極端多樣性。儘管已經提出了許多方法來識別免疫庫，在這 當中很少方法適合用於個人免疫受體庫的評估。在本篇研究中，我們建立了一種基 於探針捕獲的基因組 DNA（gDNA）次世代定序（NGS）方法，並結合了基於 BLASTn 的管線以辨別適應性免疫受體庫。我們根據 ImMunoGeneTics（IMGT）資料庫為 BCR 和 TCR 基因的每個已知的 V 和 J 等位基因設計了探針。 經由 Roche / NimbleGen 的客製化服務，針對每個 V 等位基因生成了三個探針、每個 J 等位基 因生成了一個探針，其中每個探針都是由連續的 60 bps 所組成。由於沒有標準可 以評估這項新提出的流程，因此我們將 IMGT 的 BCR / TCR 等位基因與 NA12878 和 NA24385 的全基因定序的 de-novo 組裝數據進行比對 [1]，並將那些完全匹配 的等位基因視為標準。 我們通過測試這兩種來自 Genome In A Bottle（GIAB, https://jimb.stanford.edu/giab）的參考材料（Reference Materials, RM）來驗證我們的 方法。結果顯示，對於 TCR 基因，NA12878 的所有 160 個 V 等位基因和 83 個 J 等位基因以及 NA24385 的所有 157 個 V 等位基因和 83 個 J 等位基因可以通過這 種方法精確分析，但是當使用源自 Epstein-Barr virus 轉化細胞株的基因組 DNA 時， 該分析流程無法鑑別 BCR 的所有 V 等位基因。 綜上所述，我們開發了一種新穎可靠的方法來分析 TCR 的 V 和 J 等位基因同 時標記了 NA12878 和 NA24385 的 TCR 等位基因。基因組 DNA 具有製備方便以 及易於保存的特點，並可隨時獲得大量樣品。我們的方法將來可以應用於人群研究 或個人精準醫療。

Numerous examples imply that germline variations of HLA-B genes may impact individuals’ immune response and diseases. Thus, many transgenic mouse models have been established to study corresponding diseases. However, most of the mice models belong to “transgenic mice” which “randomly integrates” the desired gene into the host genome and may cause some unexpecting side-effects. Moreover, no Graves’ diseaserelated HLA-B mouse model is available until this point. Accordingly, this study aims to establish an hHLA-B exon 4 KI (knock-in) H-2D1 mouse that carries a recombinasemediated cassette exchange (RMCE) system, which will enable convenient generation of different HLA-B alleles when needed. After this mouse is established, we will insert the risk allele of Graves’ disease, that is, HLA-B*46:01, into the mouse and develop it into Graves’ disease mouse model. Besides, we also want to establish humanized beta 2 microglobulin (hB2M) and CD8 KI mice, which do play important roles in the antigenpresenting process, in this study. We adopt CRISPR-Cas9 or ES-cell targeting strategy to specifically knock our desired gene into mice, which can precisely introduce specific mutations into endogenous genes and transmit them through the mouse germline. Our final goal is to breed these three strains of mice to get a mouse with triple knock-in genotype. We only have hB2M KI mice in hand at this moment, whose expressions of DNA, mRNA, and protein in the homozygous strain have been verified. In addition, we find that hB2M KI mice fail to express H-2K b molecule but can normally express H-2Db , which is enough for CD4- CD8 + T cells development, but the functionality of these cells requires further experiments to evaluate. Overall, this mouse strain does not appear to have any abnormalities or signs of premature death and will be helpful in studying immune-related issues. The collection of B cell receptor (BCR) and T cell receptor (TCR) form the adaptive immune receptor repertoire (AIRR). Antigen-binding sites of BCR and TCR are composed of variable (V), diversity (D), and joining (J) segments. When V(D)J recombination occurs, each gene in genomic DNA (gDNA) will have one allele participating in the joining process, which causes the extreme diversity of the adaptive immunity. Although many approaches have been proposed to identify immune repertoires, few of them are trustworthy for personal germline evaluation. In this study, we established a probe captured-based next-generation sequencing (NGS) method for genomic DNA (gDNA) combined with the BLASTn-based pipeline to determine the AIRR profiles. We designed probes for every identified V and J allele of BCR and TCR genes based on the ImMunoGeneTics (IMGT) database. Each probe is a continuous 60 bps sequence customized from Roche/NimbleGen, and each V allele is covered by three probes, while each J allele is covered by one probe. Since there is no benchmark to evaluate the newly proposed AIRR sequencing pipeline, we aligned the BCR/TCR alleles from IMGT to the whole-genome sequencing de-novo assembly data of reference materials (RMs) NA12878 and NA24385 [1] and annotate those perfectly matched alleles as standards. We verified our approach by testing these two RMs from the Genome In A Bottle (GIAB, https://jimb.stanford.edu/giab). For TCR genes, all V alleles and J alleles of NA12878 and NA24385 can be precisely assigned through this approach. However, this pipeline cannot call all V alleles of the BCR when the gDNA is originated from Epstein-Barr virus-transformed cell lines. To sum up, we develop a novel and reliable method to profile the V genes and J genes of TCR and annotate the TCR alleles of NA12878 and NA24385, which has not been achieved before. Genomic DNA has characteristics of handy preparation and convenient storage, with numerous samples readily available. Our approach can be applied in population studies or personal precision medicine in the future.