Role of SF Gene Cluster in Autoimmunity

描述(由申请人提供)：我们先前已经证明，Sle1基因簇是启动NZM2410小鼠致死性狼疮的自身免疫级联反应的关键因素。我们对Sle1b的定位克隆分析发现，CD2家族基因的七个基因簇(SF基因簇)是导致对核抗原的免疫耐受性被打破的原因。SF群集包含CD48、CD84、2B4、SLAM、Ly9、Lyl08和CSI。各种研究表明，这些基因调节着几种免疫细胞系的激活阈值和效应器功能。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有关，但这些基因介导疾病的机制尚不清楚。SF簇中最强的候选单基因是Ly108，其中Ly108-1亚型在B6.Sle1b B细胞中结构性上调。然而，自身免疫可能不是由单个基因引起的，而是SF基因簇中多个多态等位基因的共同作用。在这里，我们建议在体外和体内表征这些候选基因的功能特性，并使用基因操作来直接测试这些基因的等位基因打破免疫耐受的能力。我们有四个具体的目标：1)评估SF簇基因在体内的表达。对这一簇中相关基因的详细描述将需要生产具有表达构建体的单抗和转基因小鼠。2)明确Ly108在淋巴细胞功能中的作用。我们将评估Ly108在T和B细胞增殖、激活、耐受和效应功能中的作用。这些研究将使用B6和B6.Sle1b小鼠的体外和体内试验进行。3)评价Ly108在体内打破对核抗原耐受性的能力。我们已经构建了一系列表达Ly108特定亚型的载体，以确定在B细胞中过度表达Ly108-1是否会打破B6小鼠对核抗原的耐受性，而在B6.Sle1b中过度表达Ly108-2将抑制自身免疫。我们还将生产类似的表达Ly108反义mRNA(RNAi)的构建物，以评估干扰Ly108表达对免疫系统发育和免疫应答的影响。4)体内评价SF簇的功能特性。为了开发一个能够分析SF成员之间相互作用的系统，我们将整合侧翼的loxP位点，并删除129个ES细胞中的SF簇。SF簇缺失小鼠，结合对SF家族个别成员的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.