Dendritic cell heterogeneity and its role in pulmonary fungal infection

项目摘要 机会性真菌病原体烟曲霉是免疫功能低下和 危重病人，表现为从急性到慢性的各种肺部疾病。在这些 临床症状，烟曲霉菌引起不同的适应性CD4T细胞反应，但其机制 这些不同的反应是由什么引起的，目前尚不清楚。传统的树突状细胞(CDC)对 感知和启动对这种真菌病原体的免疫反应，并可能是不同T细胞的介质 对烟曲霉菌的反应。最近的研究已经建立了CDC异质性的转录基础 通过单细胞分析，特别是证明了cDC2群体的分裂，这被认为是 规范的抗原提呈细胞，表达MHC-II类，并启动CD4T细胞。我们现在分开 基于转录因子差异表达的cDC2s分化为cDC2a和cDC2b T-bet和RoRγt。此外，众所周知，cDC2的解剖定位有助于空间定位。 与病原体衍生产品共存，从而实现有效的病原体感知、抗原摄取和 随后的CD4T细胞活化。根据烟曲霉菌的致病特性和最新发现的 CDC2异质性，我们的特定假设是cDC2A和cDC2B亚集在 肺和引流淋巴结，促进不同类型的适应性CD4T细胞对烟曲霉菌的反应。 在这项研究中，我们研究了cDC2亚群在临床相关的侵袭性小鼠模型中的功能。 肺曲霉菌病。在具体目标1中，我们将定义cDC2子集的时间和空间动力学 应用高含量免疫荧光法和流式细胞术检测急性烟曲霉菌感染过程中的变化。 我们还将在体外评估cDC2A和cDC2B的功能特性。在具体目标2中，我们将阐明 CDC2亚群对烟曲霉菌感染获得性免疫的直接影响 特异性地消融cDC2A和cDC2B亚群，并建立它们在体内的功能意义。此外，由 研究cDC2A和cDC2a亚群特异性消融中其他免疫细胞亚群的空间重组 CDC2B，我们可以确定它们在调节肺免疫的细胞回路中的意义。这项研究采用了和 开发新的遗传工具、显微镜方法和计算方法，以产生系统水平 了解肺部免疫生物学，研究宿主与病原体的相互作用。此外，这项建议是 为正在接受培训的内科科学家量身定做，因为它调查了 CDC2亚群诱导对临床相关病原体烟曲霉菌的适应性免疫 抗真菌治疗策略和疫苗开发。

Project Summary The opportunistic fungal pathogen Aspergillus fumigatus is a major health concern in immunocompromised and critically ill patients, manifesting as a variety of pulmonary conditions ranging from acute to chronic. In these clinical syndromes, A. fumigatus elicits a diverse adaptive CD4 T cell response, but the mechanisms by which these varied responses are induced remains unknown. Conventional dendritic cells (cDCs) are crucial for sensing and initiating immune responses to this fungal pathogen and are the likely mediators of diverse T cell responses to A. fumigatus. Recent studies have established the transcriptional basis for cDC heterogeneity through single cell analyses, specifically demonstrating a division in the cDC2 population, which are considered the canonical antigen presenting cells, expressing MHC Class II and priming CD4 T cells. We now separate cDC2s into two novel subsets—cDC2A and cDC2B—based on differential expression of the transcription factors T-bet and RORγt, respectively. In addition, the anatomic positioning of cDC2s is known to facilitate spatial colocalization with pathogen-derived products allowing for efficient pathogen sensing, antigen uptake, and subsequent CD4 T cell activation. Based on the pathogenic properties of A. fumigatus and recent discoveries of cDC2 heterogeneity, our specific hypothesis is that cDC2A and cDC2B subsets will localize differently in lung and draining lymph node and facilitate different adaptive CD4 T cell response types to A. fumigatus. In this study, we investigate cDC2 subsets’ functionality in a clinically relevant murine model of invasive pulmonary aspergillosis. In Specific Aim 1, we will define the temporal and spatial dynamics of cDC2 subsets during acute A. fumigatus infection by applying high-content immunofluorescence methods and flow cytometry. We will also assess cDC2A and cDC2B functional properties in vitro. In Specific Aim 2, we will elucidate the direct impact of cDC2 subsets on adaptive immunity in A. fumigatus infection by implementing genetic tools to specifically ablate cDC2A and cDC2B subsets and establish their functional significance in vivo. In addition, by investigating spatial reorganization of other immune cell subsets in subset-specific ablation of cDC2A and cDC2B, we can identify their significance in cellular circuits governing lung immunity. This study employs and develops novel genetic tools, microscopy methods, and computational approaches to generate a systems level understanding of lung immunobiology and study host-pathogen interactions. Furthermore, this proposal is tailored for a physician-scientist in training, as it investigates the basic features of and mechanisms by which cDC2 subsets induce adaptive immunity to the clinically relevant pathogen A. fumigatus, with implications for anti-fungal therapeutic strategies and vaccine development.