Roles of the Autoimmune Regulator (AIRE) Gene in Immune Response

1.AIRE基因的表达：本研究采用mRNA原位杂交和免疫组织化学方法研究了AIRE基因在人体不同组织中的表达。我们发现，AIRE在胸腺髓质中有不同的细胞表达，在淋巴结旁皮质和髓质中也有表达，在脾和胎肝中也有表达。RT-PCR不仅在胸腺、淋巴结、脾和睾丸中检测到AIRE基因，而且在所分析的大多数人和小鼠组织中也检测到了AIRE基因。我们还发现AIRE基因在外周血单核/树突状细胞系中有限制性表达。AIRE蛋白的结构和功能：我们发现，AIRE与异源DNA结合域融合后，可以激活报告启动子的转录。我们还发现，AIRE通过HSR结构域形成同源二聚体，并在体外与常见的转录共激活因子CREB结合蛋白(CBP)相互作用，并在酵母核中通过CBP.3的CH1和CH3保守域相互作用。APECED患者的AIRE基因突变：我们在两个APECED日本家系中发现了四个新的AIRE基因突变。AIRE表达细胞系的建立：为了建立稳定的AIRE表达细胞系，我们获得了一只由小鼠AIRE基因启动子驱动表达SV40T抗原的转基因小鼠。我们从转基因小鼠的胸腺中分离出细胞，并将它们保存在培养物中，从而建立了永生化细胞。这些永生化的AIRE细胞在研究AIRE基因功能的分子方面非常有用。

1. AIRE gene expression: We have investigated the expression of the AIRE gene by mRNA in situ hybridization and immunohistochemistry in various human tissues. We showed that AIRE is expressed in distinct cells in thymus medulla, and also in rare cells in lymph node paracortex and medulla, and in spleen and fetal liver. By RT-PCR AIRE mRNA was detected not only in thymus, lymph node, spleen and testis but also in most human and mouse tissues analyzed. We also found that the AIRE gene is restrictively expressed in the peripheral monocyte/dendritic cell lineage.2. Structure and function of AIRE protein: We showed that AIRE fused to a heterologous DNA binding domain activates transcription from a reporter promoter. We also showed that AIRE forms homodimers through the HSR domain and interacts with common transcriptional co-activator CREB-binding protein (CBP) in vitro and in yeast nuclei through the CH1 and CH3 conserved domains of CBP.3. AIRE gene mutations in APECED patients: We have identified four novel AIRE gene mutations in two Japanese families with APECED.4. Establishment of AIRE-expressing cell line: To establish permanent lines of "AIRE-expressing cells", we produced a transgenic mouse expressing SV40 T-antigens driven by mouse AIRE gene promoter. We isolated cells from thymus of the transgenic mouse and maintained them in culture from which we established immortalized cells. These immortalized AIRE cells are extremely useful in studies on the molecular aspects of AIRE gene functions.