MUTATIONAL ANALYSIS OF CTLA-4 FUNCTION IN VIVO

T细胞分子CTLA-4在下调T细胞免疫应答中起重要作用。缺乏CTLA-4的小鼠在几周龄时死亡，伴有大量多克隆T细胞扩增和暴发性自身免疫。CTLA-4调节T细胞反应的抑制信号仍不完全清楚。CTLA-4可以被酪氨酸磷酸化，具有两个细胞质Tyr残基（位置165和182），这两个残基在相关分子CD28中都是保守的。这两个保守的Tyr是CD28的磷酸化位点，它们都与CD28共刺激的传递有关。我们认为与Tyr 165和182的相互作用对CTLA-4的抑制功能至关重要。为了解决这些可能性，我们将使用基因靶向技术，在ctla-4基因的165或182号位置制造出具有Tyr突变的Phe的小鼠。我们将应用最近开发的策略在小鼠基因组中引入位点特异性突变。为此，我们将ctla-4的等基因基因组片段亚克隆到一个包含正选择和负选择的插入靶向载体中，并使用“hit and run”策略将Tyr替代突变所需的Phe引入ES细胞系的ctla-4外显子4。含有所需突变的胚胎干细胞亚克隆将用于制造携带所需突变的嵌合小鼠，然后用于建立杂合子和最终的纯合子。CTLA-4-/-小鼠的戏剧性表型将有助于分析这些突变的后果。

The T cell molecule CTLA-4 plays an important role in downregulating T cell immune responses. Mice deficient in CTLA-4 die at several weeks of age with massive polyclonal T cell expansion and fulminant autoimmunity. The inhibitory signals by which CTLA-4 regulates T cell responses remain incompletely understood. CTLA-4, which can be tyrosine-phosphorylated, has two cytoplasmic Tyr residues (positions 165 and 182), both of which are conserved in the related molecule, CD28. These two conserved Tyr are phosphorylation sites in CD28, and each has been implicated in the delivery of the CD28 costimulus. We propose that interactions with Tyr 165 and 182 are critical for the inhibitory function of CTLA-4. To address these possibilities we will use gene targeting to produce mice with Phe for Tyr mutations at either position 165 or 182 of the ctla-4 gene. We will apply recently developed strategies to introduce site specific mutations in the mouse genome. To this end we will subclone isogenic genomic fragments of ctla-4 into an insertional targeting vector containing positive and negative selection and use the "hit and run" strategy to introduce desired Phe for Tyr substitution mutations into exon 4 of ctla-4 in ES cell lines. ES subclones containing the desired mutations will be used to create chimeric mice carrying the desired mutation, which will then be used to establish heterozygotes and finally homozygotes. The dramatic phenotype of the CTLA-4-/- mice will facilitate analysis of the consequences of these mutations.