Role of SF Gene Cluster in Autoimmunity

我们以前已经证明S/el基因簇是启动自身免疫性疾病的关键因素， 在NZM 2410小鼠中导致致命性狼疮的级联反应。我们对Slelb的定位克隆分析鉴定了一个 CD 2家族基因的七基因簇（SF基因簇）作为免疫耐受性破坏的原因， 核抗原SF簇含有CD 48、CD 84、2B 4、SLAM、Ly 9、Lyl 08和CSI。各种 研究表明，这些基因调节几种细胞因子的激活阈值和效应子功能。 免疫细胞谱系。虽然，我们的遗传分析清楚地表明SF基因簇与系统性 自身免疫;这些基因介导疾病的机制尚不清楚。最强的单基因 SF簇中的候选物是Lyl 08，其中Ly 108 -1同种型在B6. Slel B B中组成性上调 细胞然而，自身免疫可能不是由单个基因引起的，而是由以下因素的综合作用引起的： SF基因簇中存在多个多态等位基因。在这里，我们提出的特点功能 这些候选基因在体外和体内的性质，并使用遗传操作，直接测试 这些基因的等位基因破坏免疫耐受的能力。我们有四个具体目标：1。评估 SF簇基因在体内的表达。对该簇中相关基因的详细描述将 需要生产单克隆抗体和具有表达构建体的转基因小鼠。2.到 定义Lyl 08在淋巴细胞功能中的作用。我们将评估Lyl 08在T和B细胞中的作用， 增殖、活化、耐受性和效应子功能。这些研究将使用体外和 用B6和B6.Slelb小鼠的体内测定。3.为了评估Lyl 08违反耐受性的能力， 体内核抗原。我们已经产生了一系列表达Lyl 08的特异性同种型的构建体， 确定B细胞中Ly 108 -1的过表达是否会破坏B6细胞对核抗原的耐受性 小鼠，而Ly 108 -2的过表达将抑制B6.Slelb中的自身免疫。我们还将生产类似的 表达Lyl 08反义mRNA（RNAi）的构建体，以评估破坏Lyl 08表达的影响 对免疫系统和免疫反应的发展。4.评估功能 SF簇在体内的性质。开发一个系统，可以分析 在SF成员中，我们将在129个ES细胞中整合侧翼LoxP位点并删除SF簇。SF 群集无效小鼠，与SF家族个体成员的BAC拯救策略相结合，将允许 该基因簇在体内的功能评估

We have previously demonstrated that the S/el gene cluster is a key element in initiating the autoimmune cascade that leads to fatal lupus in the NZM2410 mouse. Our positional cloning analysis of Slelb identified a seven-gene cluster of CD2 family genes (SF gene cluster) as causative for a breach in immune tolerance to nuclear antigens. The SF cluster contains CD48, CD84, 2B4, SLAM, Ly9, Lyl08, and CSI. A variety of studies indicate that these genes modulate the activation thresholds and effector functions of several immune cell lineages. Although, our genetic analyses clearly implicate the SF gene cluster with systemic autoimmunity; the mechanism by which these genes mediate disease is unknown. The strongest single gene candidate in the SF cluster is Lyl08, in which the Ly108-1 isoform is constitutively upregulated in B6.Slelb B cells. However, autoimmunity may not be caused by a single gene, but rather by the combined effects of multiple polymorphic alleles in the SF gene cluster. Here we propose to characterize the functional properties of these candidate genes in vitro and in vivo and use genetic manipulation to directly test the ability of alleles of these genes to breach immune tolerance. We have four specific aims: 1. To assess the expression of SF cluster genes in vivo. A detailed characterization of relevant genes in this cluster will require the production of monoclonal antibodies and transgenic mice with expression constructs. 2. To define the role of LylO8 in lymphocyte function. We will assess the role of Lyl08 in T and B cell proliferation, activation, tolerance and effector functions. These studies will be performed using in vitro and in vivo assays with B6 and B6.Slelb mice. 3. To assess the ability of LylO8 to breach tolerance to nuclear antigens in vivo. we have produced a series of constructs expressing specific isoforms of Lyl08 to determine whether over expression of Ly108-1 in B cells will breach tolerance to nuclear antigens in B6 mice, while over-expression of Ly108-2 will suppress autoimmunity in B6.Slelb. We will also produce similar constructs expressing Lyl08 anti-sense mRNA (RNAi) to assess the impact of disrupting Lyl08 expression on the development of the immune system and immune responsiveness. 4. To assess the functional properties of the SF cluster in vivo. To develop a system that will allow an analysis of the interactions among SF members, we will integrate flanking LoxP sites and delete the SF cluster in 129 ES cells. SF cluster null mice, combined with a BAC rescue strategy with individual members of the SF family, will allow an assessment of function of this gene cluster in vivo