Specific Contribution of a New IG Isotype (IGT) in Teleost Fish Immune Responses

描述（由申请人提供）：虹鳟鱼（我们的鱼模型），已知含有至少三种免疫球蛋白同型，IgM， IgD和最近发现的IgT。到目前为止，人们对IgT的蛋白质结构和分布以及它在免疫中的特殊作用一无所知。我们已经产生了抗IgT的抗体，这使我们发现了一种新的只表达表面IgT的B细胞亚群。值得注意的是，IgT+ B细胞代表了鳟鱼肠道中最普遍的B细胞类型。经肠道寄生虫C. shasta感染鳟鱼后，存活鱼的肠道上皮和固有层中出现大量IgT+ B细胞浸润。最值得注意的是，我们发现针对寄生虫的igt特异性滴度仅限于肠道粘液，而IgM是唯一参与血清反应的同型。我们发现大多数肠道细菌被IgT包裹，进一步支持了IgT在肠道稳态中的作用。总的来说，这些数据表明，IgT代表了哺乳动物IgA的功能等效。本提案的总体目标是使用我们的鱼-寄生虫模型来获得粘膜免疫的原始见解，这将有助于分析人类粘膜免疫学的现有范式。迄今为止，用于研究肠道黏膜免疫的动物模型仅限于哺乳动物。因此，我们对鱼类的研究将为该领域提供一个新的系统发育维度。由于鱼类在肠道中缺乏表皮斑块和淋巴结，因此预期局部粘膜反应的空间和功能复杂性较低，从而有助于分析滤泡外的IgT，进而分析IgA反应。因此，本提案中产生的数据可能阐明肠道IgT/ iga介导的免疫的新机制，这些机制不依赖于卵泡结构的存在。寻找IgT/IgA生产的滤泡外途径将是开发新的粘膜疫苗接种策略的基础。因此，本提案的目标是：AIM 1：鳟鱼肠道中IgT的生化表征、产生和运输。在这里，我们将完成肠道IgT和IgT分泌细胞的表征，我们将通过新发现的虹鳟鱼Ig聚合受体（TIgPr）研究IgT转运到肠道区域的机制。目的2：参与肠IgT免疫应答发展的机制和途径。在这里，我们将研究IgT和IgT+ B细胞对角鼻虫反应的滤泡外机制和途径。为此，我们将对感染寄生虫的鱼类的IgT反应进行时空分析，并研究IgT+ B细胞归巢到感染鱼类肠道的途径。目标3。目的探讨肠道IgT和IgT+B细胞对沙斯塔梭菌的主要作用。本研究的主要目的是确定寄生虫诱导的IgT特异性反应是否具有保护作用，并阐明IgT和IgT+ B细胞保护鱼类免受寄生虫侵袭的作用机制。

DESCRIPTION (provided by applicant): Rainbow trout (our fish model), is known to contain at least three immunoglobulin isotypes, IgM, IgD and the recently discovered IgT. Thus far nothing is known about the protein structure and distribution of IgT, and its specific role in immunity. We have generated antibodies against IgT that has led us to the finding of a novel B cell subset expressing exclusively surface IgT. Significantly, IgT+ B cells represent the most prevalent B cell type in the gut of trout. Infection of trout with C. shasta, a gut parasite, revealed a massive infiltration of IgT+ B cells in the gut epithelium and lamina propria of survivor fish. Most notably, we found that IgT-specific titers against the parasite were confined to gut mucus, whereas IgM was the only isotype involved in serum responses. Supporting further the role of IgT in gut homeostasis, we found that a majority of intestinal bacteria were coated with IgT. Collectively, these data indicate that IgT represents the functional equivalent of mammalian IgA. The overarching goal of this proposal is to use our fish-parasite model to gain primordial insights of mucosal immunity that will facilitate the analysis of existing paradigms in human mucosal immunology. To date, the animal models being used to study gut mucosal immunity are limited to mammalian species. Thus, our studies with fish will provide a new phylogenetic dimension into the field. Since fish lack peyer patches and lymph nodes in the gut, a lower spatial and functional complexity of the local mucosal responses should be expected, thereby facilitating the analysis of extrafollicular IgT, and by extension, IgA, responses. Accordingly, data generated in this proposal is likely to elucidate novel mechanisms of intestinal IgT/IgA-mediated immunity that are independent of the presence of follicular structures. Finding extrafollicular pathways of IgT/IgA production will be fundamental to developing novel mucosal vaccination strategies. Thus, the goals of this proposal are: AIM 1: Biochemical characterization, production and transport of IgT in the gut of trout. Here we will complete the characterization of gut IgT and IgT-secreting cells, and we will investigate the mechanisms involved in the transport of IgT into the gut luminal area, through a newly discovered rainbow trout Ig polymeric receptor (TIgPr). AIM 2: Mechanisms and pathways involved in the development of gut IgT immune responses to Ceratomyxa shasta. Here we will investigate the extrafollicular mechanisms and pathways by which IgT and IgT+ B cells respond to Ceratomyxa shasta. To this end, we will perform a temporal-spatial analysis of the IgT response in fish infected with the parasite, and we will investigate pathways of IgT+ B cell homing into the gut of infected fish. AIM 3. To investigate the primary effector functions of gut IgT and IgT+B cells against C. shasta. The main goals of this aim are to establish whether the generated IgT-specific responses induced by the parasite are protective, and to elucidate the effector mechanisms by which IgT and IgT+ B cells contribute to protecting fish from parasite invasion. . PUBLIC HEALTH RELEVANCE: We have demonstrated that IgT, a newly discovered teleost immunoglobulin, is the functional homolog of mammalian IgA. We have established a model system that uses a gut parasite to induce strong IgT-specific responses in the gut of rainbow trout. Thus, the overarching goal of this proposal is to use our fish-parasite model to gain primordial insights of mucosal immunity immunity that will facilitate the analysis of existing paradigms in human mucosal immunology. To date, the animal models being used to study mucosal immunity are limited to mammalian species. Thus, our studies with fish will provide a novel phylogenetic dimension into the field. It should be stressed that the selective forces (i.e. host-microbial pathogenic or mutualistic interactions) that have shaped fish and mammalian mucosal immune systems are similar. Thus, some of the novel immunological solutions driven by these selective forces in fish and mammals are likely to share fundamental structural and mechanistic aspects. In fact, the mammalian intestinal IgA is considered a "primitive form of adaptive immunity that regulates microbial communities in the gut". Indeed, the features of mucosal immunity in mammals may have remained "primitive" precisely because the selective forces and physical constraints of mucosal surfaces of fish and mammals are basically very similar. Since fish lack peyer patches and lymph nodes in the gut, a lower spatial and functional complexity of the local mucosal responses should be expected, thereby facilitating the analysis of extrafollicular IgT, and by extension, IgA, responses. Accordingly, data generated in this proposal is likely to elucidate novel mechanisms of intestinal IgT/IgA-mediated immunity that are independent of the presence of follicular structures. Finding extrafollicular pathways of IgT/IgA production will be fundamental to developing novel mucosal vaccination strategies. In addition, this knowledge will be instrumental to help treating diseases that develop as result of the dysregulation of mucosal immune responses (i.e., inflammatory bowel disease, Crohn's disease). It is worth noting that the beneficial effects of fish diets are well-known (i.e., low fat, low cholesterol). Thus, the consumed of fish diets is growing at a very fast pace. Our studies on the fish immune system will also benefit society by contributing to the fish farming industry in the short and long-term. Among the major problems in fish farming are those related to health and disease issues. Therefore, a better knowledge of the fish immune system is essential to the development of new therapeutic agents and vaccines to prevent fish diseases.