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，以创建肽MHC II四聚体，这将有助于深入了解对地方性真菌病的免疫和产生IL-17的CD4+ T细胞的长期记忆生物学。进入长寿命记忆细胞池的抗原特异性T细胞代表了免疫记忆的细胞基础，这是疫苗和保护性免疫的基础。了解存留在这个池中的T细胞是设计疫苗的基本原理所必需的。关于辅助性T - 17细胞的寿命存在不同的观点。在这场争论中，关于真菌接种后进入记忆池的CD4+ T细胞，以及这些细胞是否保留产生IL-17的能力，存在知识空白。由于Th17细胞促进疫苗对真菌的免疫，这种知识差距是疾病预防策略的障碍。我们假设ag特异性CD4+ T细胞能够抵抗二态真菌——芽孢菌、组织浆菌和球虫——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，以解读二态真菌中保守的显性保护性Ag的身份，并将抗原的T细胞表位引入肽- mhc （pMHC）四聚体中，以跟踪内源性Ag特异性CD4+ T细胞在真菌接种和再次暴露于肺部后的生理条件下的体内进化、持久性、表型和功能。我们的目标是：1)鉴定共享的保护性真菌Ag和表位，并创建和验证肽MHC四聚体。我们将鉴定、克隆和表达由疫苗诱导的介导地方性真菌病保护性免疫的CD4+ T细胞识别的真菌Ag。我们的初步数据强烈指向钙连蛋白。我们将推断BL/6小鼠ⅱ类MHC识别的Ag的肽表位，并合成和验证pMHC四聚体，以便在生理条件下监测和跟踪内源性表位特异性CD4+ T细胞。2)利用pMHC四聚体确定疫苗诱导的抗真菌T17细胞的持久性和表型。我们将使用pMHC四聚体来确定na - ve BL/6小鼠表位特异性CD4+ T细胞内源性库的大小。在接种疫苗的小鼠中，我们将使用pMHC四聚体来追踪介导真菌病抗性的记忆CD4+ T细胞的长期命运、持久性和表型，重点是阐明Th17细胞在接种芽孢菌病、组织浆菌病和球虫菌病后的持续程度。我们的工作将为CD4+ T细胞的表型和功能提供新的见解，这些细胞赋予对地方性真菌病的保护性免疫，特别是IL-17产生细胞在较长时间内的持久性。这些结果将对真菌学和免疫学工作者具有广泛的意义和影响。

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.