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的小鼠在几周大时死亡，伴随着大量的多克隆T细胞增殖和暴发性自身免疫。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，在细胞质中有两个Tyr残基(165和182位)，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中的磷酸化位点，每一个都与CD28共刺激序列的传递有关。我们认为与Tyr 165和182的相互作用是CTLA-4抑制功能的关键。为了解决这些可能性，我们将使用基因打靶技术来培育具有CTLA-4基因165或182位Tyr突变的Phe小鼠。我们将应用最近开发的策略在小鼠基因组中引入特定位置的突变。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、负选择的插入靶向载体中，并使用“打了就跑”的策略将所需的TYR替换突变的Phe引入到ES细胞系CTLA-4的外显子4中。含有所需突变的ES亚克隆将被用来创造携带所需突变的嵌合小鼠，然后将被用来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.