Functional cloning of S. mansoni egg-specific Th2 cells

描述(申请人提供)：血吸虫病是一个严重的健康问题，全世界有超过2亿人感染。像对许多寄生虫的免疫一样，对曼氏血吸虫的免疫反应与以CD4T细胞表达白细胞介素4为特征的强大的Th2反应紧密相连。在感染人类或小鼠宿主数周后，Th2细胞突然出现，这与曼氏血吸虫虫卵的沉积相吻合。有趣的是，即使在没有感染或佐剂的情况下，单独注射鸡蛋也能诱导强大的Th2反应。尽管Th2细胞在寄生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的CD4T细胞反应，研究一直受到阻碍。这是由于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。到目前为止，还不可能设计出可以与现有的TCR转基因小鼠结合使用的抗原转基因蠕虫寄生虫，这一事实加剧了这种情况。病原体特异性TCR转基因小鼠和重组病原体的可获得性有助于研究对许多与Th1免疫相关的病原体的免疫。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/α-1，这是曼氏血吸虫卵子中的一种嗜碱性细胞激活蛋白，其功能在人和小鼠之间是保守的。为了跟踪蠕虫特异性Th2反应的签名和IL-4的表达，我们以前培育了IL-4报告小鼠(4get和Kn2)。我们已经证明，只有当CD4T细胞同时暴露于其同源抗原和Th2极化条件下时，才能诱导IL-4报告基因的表达。因此，在蠕虫感染的小鼠中，表达IL-4的CD4T细胞高度富含携带寄生虫抗原特异性TCR的细胞。在这里，我们将使用IL-4报告小鼠分离虫卵及其抗原所产生的蠕虫特异性Th2细胞，以建立Th2细胞系和CD4T细胞杂交瘤。我们有两个独立的目标。在目标1中，我们将功能性地克隆曼氏血吸虫卵特异性Th2细胞杂交瘤。我们将直接从免疫小鼠中分离卵子特异性Th2细胞，或者使用过继转移系统在体内扩增和重新选择这种细胞。在目标2中，我们将功能性地克隆针对曼氏血吸虫虫卵抗原IPSE/α-1的Th2细胞。我们将使用抗原特异性免疫和选择相结合的方法来扩增和重新选择IPSE/α-1特异性Th2细胞，以产生杂交瘤。我们用曼氏血吸虫卵、IPSE/α-1试剂、IL-4报告小鼠和Th2细胞杂交瘤的初步数据表明，我们已经获得了进行拟议研究所需的所有试剂和技术技能。从克隆的Th2杂交瘤中鉴定曼氏血吸虫卵特异性TCRs是制备TCR转基因小鼠的第一个关键步骤。曼氏杆菌特异性杂交瘤的产生将证明这一新方法的原理，该方法随后可用于其他病原体。血吸虫病是一个严重的健康问题，全世界有超过2亿人感染。曼氏血吸虫是一种多细胞寄生虫，慢性感染人和老鼠。人和小鼠的获得性免疫反应都与强烈的Th2反应密切相关，Th2反应是由曼氏血吸虫卵启动的，并广泛针对曼氏血吸虫卵。尽管这种疾病具有明显的重要性，但曼氏血吸虫卵特异性Th2细胞及其T细胞受体(TCR)尚未被克隆。病原体特异性TCR的分离是制备抗原特异性TCR转基因小鼠的关键第一步。这种病原体特异性TCR转基因小鼠在研究由细菌、病毒和原虫引起的许多1型相关疾病的免疫反应方面发挥了重要作用。在这里，我们将使用一种新的方法，即我们使用白介素4报告小鼠来通过其标志性细胞因子IL-4来功能性地分离曼氏血吸虫卵特异性Th2细胞。随后，我们将产生并克隆T细胞杂交瘤，以分离曼氏血吸虫卵特异性TCRs。这项申请中概述的工作将是朝着研究血吸虫病、其他蠕虫疾病和Th2免疫的基本原理迫切需要的工具的产生迈出关键的一步。

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.