Functional cloning of S. mansoni egg-specific Th2 cells

曼氏沙门氏菌卵特异性 Th2 细胞的功能克隆

• 批准号: 7476341
• 负责人: MARKUS MOHRS
• 金额: $ 21.83万
• 依托单位: TRUDEAU INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476341
• 关键词: Acute; Adjuvant; Adoptive Transfer; Allergic; Amino Acids; Animals; Antigens; Asthma; Back; Bacteria; Basophils; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Line; Cell Separation; Cells; Cloning; Condition; Data; Deposition; Development; Disease; Employee Strikes; Engineering; Epitopes; Evolution; Funding; Generations; Goals; Green Fluorescent Proteins; Health; Helminth Antigens; Helminths; Human; Hybridomas; Hypersensitivity; Immune response; Immunity; Immunization; In Vitro; Individual; Infection; Injection of therapeutic agent; Interferons; Interleukin-4; Interleukins; Link; MHC Class I Genes; MHC Class II Genes; Methods; Molecular Profiling; Mus; Ovalbumin; Parasites; Population; Production; Protein S; Proteins; Protozoa; Reagent; Recombinants; Reporter; Research; Role; Schistosoma mansoni; Schistosomiasis; Shapes; Specificity; System; T-Cell Receptor; T-Lymphocyte; Testing; Th2 Cells; Toxoplasma gondii; Transgenic Mice; Transgenic Organisms; Translating; Virus; Week; Work; World Health Organization; aluminum sulfate; base; cytokine; egg; immunopathology; in vivo; insight; lymph nodes; men; novel strategies; pathogen; response; skills; tool

DESCRIPTION (provided by applicant): Schistosomiasis is a serious health problem and more than 200 million people are infected world wide. Like immunity to many helminth parasites, the immune response to Schistosoma mansoni is tightly linked to a robust Th2 response characterized by the expression of interleukin (IL)-4 by CD4+ T cells. The abrupt emergence of Th2 cells weeks after infection of the human or murine host coincides with the deposition of S. mansoni eggs. Intriguingly, injection of eggs alone induces a robust Th2 response even in the absence of infection or adjuvants. Despite the obvious importance of Th2 cells in the immune response to helminthic infections, research has been held back by the inability to follow parasite-specific CD4+ T cell responses. This is due to the fact that helminth-specific T cell receptors (TCR) have not been isolated to generate TCR transgenic mice. This situation is compounded by the fact that as of to date it is not possible to engineer antigen-transgenic helminth parasites that can be used in conjunction with existing TCR-transgenic mice. The availability of both pathogen- specific TCR transgenic mice and recombinant pathogens has been instrumental in studying immunity to many pathogens associated with Th1 immunity. Additionally, in contrast to many bacteria, protozoa and viruses, helminthic antigens are generally not known. One exception is IPSE/alpha-1, a basophil activating protein of S. mansoni eggs, whose function is conserved between men and mice. To follow helminth-specific Th2 responses by their signature, the expression of IL-4, we have previously developed IL-4 reporter mice (4get and KN2). We have demonstrated that expression of the IL-4 reporter is only induced when CD4+ T cells are exposed to both their cognate antigen and Th2 polarizing conditions. Consequently IL-4 expressing CD4+ T cells in helminth-infected mice are highly enriched for cells bearing TCRs specific for parasitic antigens. Here we will use IL-4 reporter mice to isolate helminth-specific Th2 cells generated in response to S. mansoni eggs and their antigens to establish Th2 cell lines and CD4+ T cell hybridomas. We have two independent aims. In Aim 1 we will functionally clone S. mansoni egg-specific Th2 cell hybridomas. We will isolate egg-specific Th2 cells either directly from immunized mice or use an adoptive transfer system to expand and reselect such cells in vivo. In Aim 2 we will functionally clone Th2 cells specific for the S. mansoni egg antigen IPSE/alpha-1. We will use a combination of antigen-specific immunization and selection to expand and reselect IPSE/alpha-1-specific Th2 cells for the generation of hybridomas. Our preliminary data with S. mansoni eggs, IPSE/alpha-1 reagents, IL-4 reporter mice and Th2 cell hybridomas demonstrate that we have acquired all required reagents and technical skill to pursue the proposed research. The identification of S. mansoni egg specific TCRs from cloned Th2 hybridomas is the first critical step in the generation of TCR transgenic mice. The generation of S. mansoni-specific hybridomas will be proof of principle for this new approach that can subsequently be used for other pathogens. Schistosomiasis is a serious health problem and more than 200 million people are infected world wide. Schistosoma mansoni is a multicellular helminth parasite that chronically infects men and mice. The adaptive immune response is in both men and mice tightly linked to a vigorous Th2 response that is initiated by and extensively directed against S. mansoni eggs. Despite the obvious importance of this disease, S. mansoni egg-specific Th2 cells and their T cell receptors (TCR) of have not been cloned. The isolation of pathogen-specific TCRs is a critical first step in the generation of antigen-specific TCR transgenic mice. Such pathogen- specific TCR transgenic mice have been instrumental in studying the immune response to many type 1 associated diseases caused by bacteria, viruses and protozoa. Here we will use a novel approach in which we employ interleukin (IL)-4 reporter mice to functionally isolate S. mansoni egg-specific Th2 cells by their signature cytokine, IL-4. Subsequently we will generate and clone T cell hybridomas to isolate the S. mansoni egg-specific TCRs. The work outlined in this application will be a critical step towards the generation of tools urgently needed to study schistosomiasis, other helminthic diseases and fundamental principles of Th2 immunity.

描述（由申请人提供）：血吸虫病是一种严重的健康问题，全世界有超过 2 亿人受到感染。与对许多蠕虫寄生虫的免疫一样，对曼氏血吸虫的免疫反应与强大的 Th2 反应密切相关，其特征是 CD4+ T 细胞表达白细胞介素 (IL)-4。人类或鼠类宿主感染后几周，Th2 细胞突然出现，与曼氏沙门氏菌卵的沉积同时发生。有趣的是，即使在没有感染或佐剂的情况下，单独注射鸡蛋也会诱导强烈的 Th2 反应。尽管 Th2 细胞在蠕虫感染的免疫反应中具有明显的重要性，但由于无法跟踪寄生虫特异性 CD4+ T 细胞反应，研究一直受到阻碍。这是因为尚未分离出蠕虫特异性 T 细胞受体 (TCR) 来产生 TCR 转基因小鼠。迄今为止，还不可能设计出可与现有 TCR 转基因小鼠结合使用的抗原转基因蠕虫寄生虫，这一事实使情况变得更加复杂。病原体特异性 TCR 转基因小鼠和重组病原体的可用性有助于研究与 Th1 免疫相关的许多病原体的免疫力。此外，与许多细菌、原生动物和病毒不同，蠕虫抗原通常是未知的。一个例外是 IPSE/alpha-1，它是曼氏沙门氏菌卵的一种嗜碱性粒细胞激活蛋白，其功能在男性和小鼠之间是保守的。为了通过其特征（IL-4 的表达）跟踪蠕虫特异性 Th2 反应，我们之前开发了 IL-4 报告小鼠（4get 和 KN2）。我们已经证明，只有当 CD4+ T 细胞暴露于其同源抗原和 Th2 极化条件时，IL-4 报告基因的表达才会被诱导。因此，感染蠕虫的小鼠中表达 IL-4 的 CD4+ T 细胞高度富集，其中含有对寄生虫抗原具有特异性的 TCR。在这里，我们将使用 IL-4 报告小鼠分离响应曼氏沙门氏菌卵及其抗原而产生的蠕虫特异性 Th2 细胞，以建立 Th2 细胞系和 CD4+ T 细胞杂交瘤。我们有两个独立的目标。在目标 1 中，我们将功能性克隆曼氏曼氏酵母卵特异性 Th2 细胞杂交瘤。我们将直接从免疫小鼠中分离卵特异性 Th2 细胞，或使用过继转移系统在体内扩增和重新选择此类细胞。在目标 2 中，我们将功能性克隆对曼氏沙门氏菌卵抗原 IPSE/alpha-1 具有特异性的 Th2 细胞。我们将结合使用抗原特异性免疫和选择来扩展和重新选择 IPSE/α-1 特异性 Th2 细胞以产生杂交瘤。我们对曼氏酵母卵、IPSE/alpha-1 试剂、IL-4 报告小鼠和 Th2 细胞杂交瘤的初步数据表明，我们已经获得了开展拟议研究所需的所有试剂和技术技能。从克隆的 Th2 杂交瘤中鉴定曼氏沙门氏菌卵特异性 TCR 是产生 TCR 转基因小鼠的第一个关键步骤。曼氏沙门氏菌特异性杂交瘤的产生将证明这种新方法的原理，随后可用于其他病原体。血吸虫病是一种严重的健康问题，全世界有超过 2 亿人受到感染。曼氏血吸虫是一种多细胞蠕虫寄生虫，长期感染人类和小鼠。在人和小鼠中，适应性免疫反应与强烈的 Th2 反应密切相关，该反应由曼氏沙门氏菌卵发起并广泛针对曼氏沙门氏菌卵。尽管这种疾病的重要性显而易见，但曼氏沙门氏菌卵特异性 Th2 细胞及其 T 细胞受体 (TCR) 尚未被克隆。病原体特异性 TCR 的分离是产生抗原特异性 TCR 转基因小鼠的关键的第一步。这种病原体特异性 TCR 转基因小鼠在研究对由细菌、病毒和原生动物引起的许多 1 型相关疾病的免疫反应方面发挥了重要作用。在这里，我们将使用一种新方法，其中我们使用白细胞介素 (IL)-4 报告小鼠通过其标志性细胞因子 IL-4 功能性分离曼氏沙门氏菌卵特异性 Th2 细胞。随后，我们将生成并克隆 T 细胞杂交瘤，以分离曼氏曼氏酵母卵特异性 TCR。本申请中概述的工作将是朝着生成研究血吸虫病、其他蠕虫病和 Th2 免疫基本原理急需的工具迈出的关键一步。

项目成果

Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses.

• DOI: 10.1084/jem.20082460
• 发表时间: 2009-08-03
• 期刊: The Journal of experimental medicine
• 作者: Everts B;Perona-Wright G;Smits HH;Hokke CH;van der Ham AJ;Fitzsimmons CM;Doenhoff MJ;van der Bosch J;Mohrs K;Haas H;Mohrs M;Yazdanbakhsh M;Schramm G
• 通讯作者: Schramm G