Role of SF Gene Cluster in Autoimmunity

描述（由申请人提供）：我们之前已经证明Sle1基因簇是启动自身免疫级联的关键因素，导致NZM2410小鼠的致命狼疮。我们对Sle1b的定位克隆分析发现，CD2家族基因的7个基因簇（SF基因簇）是导致对核抗原免疫耐受破坏的原因。SF集群包含CD48、CD84、2B4、SLAM、Ly9、Lyl08和CSI。各种研究表明，这些基因调节了几种免疫细胞系的激活阈值和效应功能。尽管我们的遗传分析清楚地表明SF基因簇与系统性自身免疫有关；这些基因介导疾病的机制尚不清楚。SF集群中最强的候选单基因是Ly108，其中Ly108-1亚型在B6中组成性上调。Sle1b B细胞。然而，自身免疫可能不是由单个基因引起的，而是由SF基因簇中多个多态性等位基因的共同作用引起的。在此，我们建议在体外和体内表征这些候选基因的功能特性，并利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。我们有四个具体目的：1)评估SF簇基因在体内的表达。该基因簇中相关基因的详细表征将需要生产单克隆抗体和具有表达构建的转基因小鼠。2)明确Ly108在淋巴细胞功能中的作用。我们将评估Ly108在T细胞和B细胞增殖、活化、耐受性和效应功能中的作用。这些研究将使用B6和B6的体外和体内试验进行。Sle1b老鼠。3)在体内评价Ly108对核抗原的破坏耐受能力。我们制作了一系列表达Ly108特异性异构体的构建体，以确定B细胞中过表达Ly108-1是否会破坏B6小鼠对核抗原的耐受性，而过表达Ly108-2是否会抑制B6. sle1b的自身免疫。我们还将产生表达Ly108反义mRNA （RNAi）的类似构建体，以评估破坏Ly108表达对免疫系统发育和免疫反应的影响。4)评估SF簇在体内的功能特性。为了开发一个能够分析SF成员之间相互作用的系统，我们将整合129个ES细胞中的侧翼LoxP位点并删除SF集群。SF基因簇缺失小鼠，结合SF家族个体成员的BAC拯救策略，将允许在体内评估该基因簇的功能。

DESCRIPTION (provided by applicant): We have previously demonstrated that the Sle1 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 Sle1b 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 Ly108, in which the Ly108-1 isoform is constitutively upregulated in B6.Sle1b 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 Ly108 in lymphocyte function. We will assess the role of Ly108 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.Sle1b mice. 3) To assess the ability of Ly108 to breach tolerance to nuclear antigens in vivo. We have produced a series of constructs expressing specific isoforms of Ly108 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.Sle1b. We will also produce similar constructs expressing Ly108 anti-sense mRNA (RNAi) to assess the impact of disrupting Ly108 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.