A search for genes that regulate allogeneic stem cell competition

描述（由申请人提供）：免疫活性脊椎动物的移植物排斥可利用T和NK细胞对造血细胞移植物[HCT]和组织移植物的应答。移植物抗宿主病（GVHD）是一种T细胞介导的综合征，在对免疫抑制的宿主进行HCT后发生，即使供体和受体之间的MHC [人类HLA，H2小鼠]相同。虽然GVHD归因于T细胞对非HLA次要抗原的应答，但先天免疫系统的可能贡献尚未得到充分研究。我们将研究先天组织相容性系统在原索动物、小鼠和人类同种异体HCT中的作用。在Botryllus schlosseri（一种与脊椎动物密切相关的原脊索动物）中，一个单一的基因位点FuHC控制着移植物的接受或排斥。共享一个等位基因的葡萄球菌菌落形成嵌合体，不共享等位基因的菌落排斥。葡萄球菌嵌合体形成利用类似于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，并且种群携带数千个等位基因。我们假设，研究干细胞排斥或诱导耐受的机制，在葡萄球菌将提供新的见解的途径，导致耐受诱导哺乳动物。我们已经确定，映射和克隆的FuHC。FuHC与在哺乳动物干细胞上高度表达的基因（Tie 1和Tek）共享结构域同源性，并且与涉及NK细胞信号传导和细胞粘附的基因（CD 155和Igsf 4）共享序列和结构同源性，暗示NK样细胞作为该原始识别系统的介体。在葡萄球菌嵌合体中，一个伴侣的循环细胞可以竞争并取代另一个伴侣的生殖细胞和/或索马。宿主组织的替换遵循“赢家”与“输家”品系的体细胞和生殖系干细胞竞争潜力（SCP）的遗传等级。我们的研究已经将这些掠夺性细胞谱系定义为可前瞻性分离的干细胞，并表明FuHC基因座和种系竞争遗传在移植的纯化干细胞水平上起作用。利用葡萄孢SCP性状，我们建议研究调节宿主中异基因干细胞竞争的进化分子机制。我们计划：（i）鉴定可能调节葡萄球菌中干细胞竞争的基因，（ii）研究所选基因在干细胞移植植入和组织竞争性再增殖中的作用，和（iii）研究与改变葡萄球菌中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。将通过qRT-PCR和原位杂交验证选定基因的表达模式，并通过敲除和体内植入研究评价其功能。将分析小鼠和人类基因的等位基因多态性，与改变葡萄球菌中干细胞竞争潜力的基因同源;如果发现，我们将确定这些是否与移植结果相关。

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.