Tracking anti-fungal CD4+ T cells in vivo

描述(由申请人提供)：我们提出了一个探索性项目，以创造多肽MHC II四聚体，将促进对地方性真菌病的免疫力和产生IL-17的CD4+T细胞的长期记忆生物学的深入了解。进入长寿记忆细胞池的Ag特异性T细胞代表了免疫记忆的细胞基础，而免疫记忆是疫苗免疫和保护性免疫的基础。对于疫苗的基本设计来说，需要了解这个池中持续存在的T细胞。对于辅助性T细胞17的寿命有不同的看法。在这场辩论中，人们对接种真菌疫苗后进入记忆池的CD4+T细胞以及这些细胞是否保持产生IL-17的能力存在认识差距。这一知识差距是疾病预防策略的障碍，因为Th17细胞促进疫苗对真菌的免疫。我们假设，抗二型真菌--芽囊霉菌、组织胞浆和球孢子虫--的抗原特异性的CD4+T细胞进入记忆池，并在再次暴露于病原体时长期保持产生IL-17的能力。我们将使用一种新型的TCR转基因小鼠来验证我们的假设，以破译保存在二相性真菌中的显性、保护性抗原的身份，并将该抗原的T细胞表位引入多肽-MHC(PMHC)四聚体中，以跟踪体内内源性抗原特异性CD4+T细胞在接种真菌疫苗和再次暴露于肺部后在生理条件下的进化、持久性、表型和功能。我们的目标是：1)鉴定共有的保护性真菌抗原和表位，并创建和验证多肽MHC四聚体。我们将鉴定、克隆和表达疫苗诱导的CD4+T细胞识别的真菌抗原，这些细胞介导对地方性真菌病的保护性免疫。我们的初步数据强烈指向Calnexin。我们将推导出BL/6小鼠第II类MHC识别的抗原表位，并合成和验证pMHC四聚体，使我们能够监测和跟踪生理条件下内源性表位特异性的CD4+T细胞。2)确定pMHC四聚体疫苗诱导的抗真菌T17细胞的持久性和表型。我们将使用pMHC四聚体来确定NAVE BL/6小鼠内源性表位特异性CD4+T细胞池的大小。在接种的小鼠中，我们将使用pMHC四聚体来追踪介导对真菌病耐药的记忆性CD4+T细胞的长期命运、持久性和表型，重点是阐明Th17细胞在接种后对芽生菌病、组织胞浆菌病和球孢子菌病的持续程度。我们的工作将对CD4+T细胞的表型和功能产生新的见解，这些细胞对地方性真菌病具有保护性免疫，特别是IL-17产生细胞在较长时期内的持久性。这一结果将对从事真菌学和免疫学工作的人产生广泛的意义和影响。

DESCRIPTION (provided by applicant): We propose an exploratory project to create peptide MHC II tetramers that will advance insight into the immunity to endemic mycoses and the biology of long-lived memory among IL-17 producing CD4+ T cells. Ag- specific T cells that enter the pool of long-lived memory cells represent the cellular basis of immunological memory, which is the underpinning of vaccine- and protective-immunity. An understanding of T cells that persist in this pool is needed for the rationale design of vaccines. There are differing views about the longevity of T helper 17 cells. Amid this debate, there's a knowledge gap about CD4+ T cells that enter the memory pool after fungal vaccination, and whether the cells retain the ability to produce IL-17. This knowledge gap is a barrier to disease prevention strategies since Th17 cells promote vaccine immunity to fungi. We hypothesize that Ag-specific CD4+ T cells that protect against dimorphic fungi - Blastomyces, Histoplasma, and Coccidioides - enter the memory pool and retain the ability long-term to produce IL-17 on re-exposure to the pathogen. We'll test our hypothesis by using a novel TCR transgenic mouse to decipher the identity of a dominant, protective Ag conserved in dimorphic fungi, and introduce the antigen's T cell epitope into peptide-MHC (pMHC) tetramers to track the evolution, persistence, phenotype and function of endogenous Ag-specific CD4+ T cells in vivo under physiological conditions after fungal vaccination and re-exposure in the lung. Our aims are to: 1) Identify the shared protective fungal Ag and epitope and create and validate peptide MHC tetramers. We will identify, clone and express the fungal Ag recognized by vaccine induced CD4+ T cells that mediate protective immunity to endemic mycoses. Our preliminary data point strongly toward calnexin. We'll deduce the peptide epitope of the Ag recognized by the class II MHC of BL/6 mice and synthesize and validate pMHC tetramers to let us monitor and track endogenous epitope specific CD4+ T cells under physiological conditions. 2) Define the persistence and phenotype of vaccine-induced anti-fungal T17 cells using pMHC tetramers. We will use pMHC tetramers to define the size of the endogenous pool of epitope-specific CD4+ T cells in na¿ve BL/6 mice. In vaccinated mice, we will use pMHC tetramers to track the long-term fate, persistence and phenotype of memory CD4+ T cells that mediate resistance to the mycoses, with an emphasis on clarifying the extent to which Th17 cells persist after vaccination to blastomycosis, histoplasmosis, and coccidioidomycosis. Our work will yield new insight into the phenotype and function of CD4+ T cells that confer protective immunity to the endemic mycoses, and especially the persistence of IL-17 producing cells over an extended period. The results will be broadly significant and impactful for those working in Mycology & Immunology.