人類免疫相關疾病研究中擬人化 B2M 小鼠模式之表徵與可行性

Abstract

自體免疫疾病普遍為多基因性（polygenic），涉及眾多易感性基因（susceptibility gene），當中較為著名的例子是 MHC 基因。近年來，透過基因組關聯分析（genome-wide association studies, GWAS）與次世代定序（next generation sequencing, NGS）的研究，我們已經深入了解特定 MHC 等位基因與自體免疫疾病的發生有顯著相關性。然而，MHC 等位基因並非誘發疾病的主因。為了釐清 MHC 等位基因與人類自體免疫相關疾病的致病機制，本實驗室發展出擬人化主要組織相容性複合體（major histocompatibility complex, MHC）之小鼠模式，目前已透過基於 CRISPR-Cas9 技術之同源臂重組方法建立出擬人化 β2-微球蛋白（β2-microglobulin, B2M）剔入小鼠模式，利用此小鼠模式作為實驗手段，將研究聚焦在我們感興趣之人類免疫相關疾病。 本文旨在探討擬人化 B2M 剔入（gene knock-in）小鼠模式之表徵與可行性。透過DNA、mRNA和蛋白質水平上的研究，該小鼠模型的建立已獲得證實。同時，透過定期體重測量與生理觀察，證明該小鼠模式具備良好生長發育狀況。然而，研究結果顯示雄性擬人化 B2M 剔入小鼠的肝臟重量指數顯著降低，其丙氨酸氨基轉移酶(alanine aminotransferase, ALT)與天門冬氨酸氨基轉移酶(aspartate aminotransferase, AST)的含量於血清中異常升高，說明雄性擬人化 B2M 剔入小鼠的肝臟受到一定的影響。值得注意的是，臟器外觀與蘇木精-伊紅染色結果判讀並未發現異常變化。 B2M 蛋白為 MHC-I 分子呈獻至細胞表面的重要基礎。透過分析 MHC-I 分子的呈獻狀態，擬人化 B2M 剔入小鼠展示出呈獻 MHC-I 分子之能力，並對其重鏈分子的呈獻有所偏好。鑒於 MHC 分子與 T 細胞具有密不可分的關聯性，本文評估該小鼠模式胸腺與次級淋巴器官中的淋巴球組成，結果顯示，擬人化 B2M 剔入小鼠之 T 細胞於胸腺中得以順利發育，並正常分布於周邊淋巴器官。然而，DN1（CD25- CD44+）階段之胸腺細胞比例降低與脾臟淋巴球數目異常，仍有待釐清。此外，擬人化 B2M 剔入小鼠之 T 細胞具備正常的活化能力與產生 Interferon-gamma （IFN-γ） 的功能，同時，在 T 細胞受體特徵中展示出與一般野生型（wild-type）小鼠相似的 V(D)J 基因使用模式，並具有相對平衡與多樣的 T 細胞受體CDR3 克隆型（clonotype）。 綜觀本研究結果，擬人化 B2M 剔入小鼠的整體健康狀態並未受到顯著影響，儘管雄性擬人化 B2M 剔入小鼠的肝臟出現一些問題，限制了其作為肝臟相關疾病研究的應用，總括而言，擬人化 B2M 剔入小鼠在 MHC-I 重鏈分子呈獻上的差異，不影響其 T 細胞發育、組成與活化，並具有多樣的 T 細胞受體庫（TCR repertoire），因此，擬人化 B2M 剔入小鼠具備作為研究模式的潛力。

Autoimmune diseases are generally considered to be polygenic, involving numerous susceptibility genes. Among these, the most well-known examples are the MHC genes. In recent years, through genome-wide association studies (GWAS) and next-generation sequencing (NGS) research, we have gained deeper insights into the significant correlation between specific MHC alleles and the occurrence of autoimmune diseases. However, MHC alleles are not the primary cause of disease induction. To elucidate the pathogenic mechanisms underlying the association between MHC alleles and human autoimmune-related diseases, our laboratory has developed a humanized mouse model of the major histocompatibility complex (MHC). Currently, using the homologous arm recombination method based on CRISPR-Cas9 technology, we have successfully established a humanized β2-microglobulin (B2M) knock-in mouse model. By utilizing this mouse model as an experimental tool, our research focuses on investigating the human immune-related diseases of interest. This study aims to explore the characterization and feasibility of the humanized β2-microglobulin (B2M) knock-in mouse model. The establishment of this mouse model has been confirmed through investigations at the DNA, mRNA, and protein levels. Additionally, regular measurements of body weight and physiological observations have demonstrated that the mouse model exhibits healthy growth and development. However, the research results indicate that the liver coefficient of male humanized B2M knock-in mice significantly decreased, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were abnormally elevated in the serum, suggesting the liver of male humanized B2M knock-in mice is affected to some extent. It is noteworthy that no abnormal changes were observed in organ appearance and histological analysis using hematoxylin-eosin staining. The B2M protein plays a crucial role in presenting MHC-I molecules on the cell surface. Through the analysis of MHC-I molecule presentation, the humanized B2M knock-in mouse model demonstrates the ability to present MHC-I molecules and exhibits a preference for the presentation of certain heavy chain molecules. Given the close association between MHC molecules and T cells, this study evaluates the lymphocyte composition in the thymus and secondary lymphoid organs of the mouse model. The results show that T cells in the humanized B2M knock-in mice undergo successful development in the thymus and exhibit normal distribution in the peripheral lymphoid organs. However, the decreased proportion of thymocytes at the DN1 (CD25- CD44+) stage and abnormal numbers of splenic lymphocytes still require further investigation. Additionally, the T cells of the humanized B2M knock-in mice possess normal activation capability and the ability to produce Interferon-gamma (IFN-γ). Furthermore, they exhibited similar V(D)J gene usage patterns in T cell receptor characteristics as the wild-type mice, along with a relative balance and diverse T cell receptor CDR3 clonotypes. Taking a comprehensive view of the research findings, the overall health of the humanized B2M knock-in mice was not significantly affected, although some issues were observed in the liver of male humanized B2M knock-in mice, limiting its application as a model for liver-related diseases. In summary, the differences in MHC-I heavy chain molecule presentation do not affect the development, composition, and activation of T cells in the humanized B2M knock-in mice, and they possess a diverse T cell receptor repertoire (TCR repertoire). Therefore, the humanized B2M knock-in mouse holds potential as a research model.