A search for genes that regulate allogeneic stem cell competition

描述(申请人提供)：免疫功能脊椎动物的移植物排斥反应可以利用T和NK细胞对造血细胞移植[HCT]和组织移植的反应。移植物抗宿主病(GVHD)是一种T细胞介导的综合征，即使供者和受者之间的MHC[人类，H2鼠]完全相同，也会在HCT后发展到免疫抑制的宿主。尽管移植物抗宿主病归因于T细胞对非人类白细胞抗原次要抗原的反应，但先天免疫系统可能的作用尚未得到很好的研究。我们将研究先天组织相容性系统在原脊索动物、小鼠和人类同种异体红细胞移植中的作用。在白僵菌(Botryllus Schlosseri)(一种与脊椎动物关系密切的原索动物)中，控制移植物接受或排斥的是一个单基因位点，即FuHC。共享一个等位基因的Botryllus群体形成嵌合体，而不共享一个等位基因的群体则排斥。Botryllus嵌合体的形成利用了一种类似于NK细胞的系统。然而，像哺乳动物的MHC一样，FuHC基因是高度多态的，种群携带数千个等位基因。我们推测，研究Botryllus中干细胞排斥或诱导耐受的机制将为哺乳动物诱导耐受的途径提供新的见解。我们已经鉴定、绘制和克隆了FuHC。FuHC与哺乳动物干细胞上高表达的基因(Tie1和Tek)在结构域上有同源性，与参与NK细胞信号和细胞黏附的基因(CD155和Igsf4)在序列和结构上也有同源性，这意味着NK样细胞是这一原始识别系统的介体。在Botryllus嵌合体中，其中一个伴侣的循环细胞可以竞争并取代另一个伴侣的生殖系和/或胞体。宿主组织的替换遵循“胜利者”与“失败者”品系的体细胞和生殖系干细胞竞争潜力(SCP)的遗传等级。我们的研究将这些捕食性细胞谱系定义为预期可分离的干细胞，并表明FuHC基因座和生殖系竞争遗传在移植的、纯化的干细胞水平上起作用。利用Botryllus的SCP特性，我们建议研究在宿主中调节同种异基因干细胞竞争的进化分子机制。我们计划：(I)确定可能调节Botryllus中干细胞竞争的基因，(Ii)研究选定的基因在干细胞移植植入和组织竞争性再繁殖中的作用，以及(Iii)研究Botryllus中与改变干细胞竞争潜力的基因同源的基因在哺乳动物移植免疫中的潜在作用。利用高通量测序平台，我们计划通过比较“赢家”和“输家”菌株之间的基因差异表达来研究SCP的分子调控。将对这些数据进行布尔分析，以确定候选的SCP基因。所选基因的表达模式将通过qRT-PCR和原位杂交进行验证，并通过基因敲除和体内植入研究来评估其功能。将分析小鼠和人类基因的等位基因多态性，以及与Botryllus中改变干细胞竞争潜力的基因的同源性；如果发现这些基因，我们将确定这些基因是否与移植结果有关。

DESCRIPTION (provided by applicant): Graft rejection in immune competent vertebrates can utilize both T and NK cell responses to hematopoietic cell transplants [HCT] and tissue grafts. Graft-versus-host disease (GVHD), a T-cell mediated syndrome, develops following HCT to immunosuppressed hosts, even when MHC [HLA humans, H2 mice] between donor and recipient are identical. Although GVHD is attributed to T-cell responses to non-HLA minor antigens, the possible contribution by the innate immune system has not been well studied. We will study the role of the innate histocompatibility system in allogeneic HCT in protochordates mice and humans. In Botryllus schlosseri (a protochordate closely related to vertebrates), a single gene locus, the FuHC, controls graft acceptance or rejection. Botryllus colonies that share one allele form chimeras, colonies that do not, reject. Botryllus chimera formation utilizes a system analogous to NK cells. However, like the mammalian MHC, the FuHC gene is highly polymorphic and populations carry thousands of alleles. We hypothesize that studying the mechanism of stem cell rejection or induced tolerance in Botryllus will provide novel insights into the pathways that lead to tolerance induction in mammals. We have identified, mapped, and cloned the FuHC. The FuHC shares domain homology to genes highly expressed on mammalian stem cells (Tie1 and Tek), and sequence and structural homology to genes involved in NK cell signaling and cell adhesion (CD155 and Igsf4) implicating NK-like cells as mediators of this primitive recognition system. In Botryllus chimeras, circulating cells of one partner can compete and replace the germline and/or soma of the other partner. The host tissues' replacement follows genetic hierarchies for somatic and germline stem cell competitive potential (SCP) of "winner" vs. "loser" strains. Our studies have defined these predatory cell lineages as prospectively isolatable stem cells and showed that the FuHC locus and germline competition inheritance operate at the level of transplanted, purified stem cells. Utilizing the Botryllus SCP trait we propose to investigate the evolutionary molecular mechanisms that regulate allogeneic stem cell competition in a host. We plan to: (i) identify genes that may regulate stem cell competition in Botryllus, (ii) investigate the role of selected genes in stem cell transplantation engraftment and competitive repopulation of tissues, and (iii) investigate the potential role of genes homologous to genes altering stem cell competition potential in Botryllus in mammalian transplantation immunity. Using high throughput sequencing platforms we plan to study the molecular regulation of SCP by comparing differential expression of genes between "winner" and "loser" strains. Boolean analysis will be carried out on this data to identify candidate SCP genes. The expression patterns of selected genes will be validated via qRT-PCR and in situ hybridization, and their function evaluated by knockdown and in vivo engraftment studies. Allelic polymorphism of mouse and human genes, with homology to genes that alter stem cell competition potential in Botryllus will be analyzed; if found, we will determine whether these are related to transplantation outcomes.