A search for genes that regulate allogeneic stem cell competition

描述（由申请人提供）：免疫能力强的脊椎动物的移植排斥反应可以利用T和NK细胞对造血细胞移植和组织移植的反应。移植物抗宿主病（GVHD）是一种t细胞介导的综合征，即使供体和受体之间的MHC [HLA人，H2小鼠]相同，也会在免疫抑制宿主的HCT后发生。虽然GVHD归因于t细胞对非hla次要抗原的反应，但先天免疫系统的可能贡献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HCT中的作用。在schlosserbotryllus（一种与脊椎动物密切相关的原脊索动物）中，单个基因位点FuHC控制移植物的接受或排斥。共享一个等位基因的菌落形成嵌合体，没有等位基因的菌落则排斥。Botryllus嵌合体的形成利用类似NK细胞的系统。然而，像哺乳动物MHC一样，FuHC基因是高度多态性的，种群携带数千个等位基因。我们假设，研究Botryllus干细胞排斥或诱导耐受性的机制将为哺乳动物诱导耐受性的途径提供新的见解。我们已经识别、绘制并克隆了富强子对撞机。FuHC与哺乳动物干细胞上高表达的基因（Tie1和Tek）具有结构域同源性，与NK细胞信号传导和细胞粘附相关基因（CD155和Igsf4）具有序列和结构同源性，暗示NK样细胞是这一原始识别系统的介质。在Botryllus嵌合体中，一个伴侣的循环细胞可以竞争并取代另一个伴侣的种系和/或体细胞。宿主组织的替换遵循体细胞和种系干细胞竞争潜力（SCP）的“赢家”vs.的遗传等级。“失败者”压力。我们的研究将这些掠食性细胞系定义为可分离的干细胞，并表明FuHC位点和种系竞争遗传在移植纯化的干细胞水平上起作用。利用Botryllus的SCP特性，我们打算研究宿主体内调节异体干细胞竞争的进化分子机制。我们计划：(i)鉴定可能调节Botryllus干细胞竞争的基因，（ii）研究选定基因在干细胞移植植入和组织竞争性繁殖中的作用，以及（iii）研究改变Botryllus干细胞竞争潜力的基因同源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”菌株之间基因的差异表达来研究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，并通过敲除和体内植入研究来评估其功能。分析了鼠源性和人源性基因的等位基因多态性，这些等位基因与改变肉芽孢杆菌干细胞竞争潜能的基因具有同源性；如果发现，我们将确定这些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. PUBLIC HEALTH RELEVANCE: For future use of hematopoietic chimerism in therapies, many fundamental issues related to the molecular regulation of cellular chimerism need to be elucidated. Here we plan and suggest to search for genes that regulate stem cell activity in a protochordate model organism that demonstrate genetic hierarchies for somatic and germline stem cell competition in a chimera. We will then seek to determine if mouse and human genes with homology to genes that prevent stem cell competition in this protochordate, can be related to mammalian hematopoietc stem cell transplantation immunity. Since protochordates are believed to be the link between vertebrates and invertebrates, this study has the potential to discover an ancestral recognition system regulating blood based chimerism that may still be present in humans.