Determining the role of AIRE and AIRE-expressing tumor associated macrophages in tumor growth and immunity

项目摘要/摘要 自身免疫调节基因(AIRE)通过促进胸腺自身反应性T细胞的缺失来预防自身免疫。 虽然大多数研究是在胸腺进行的，但AIRE也在次级淋巴器官中表达，在那里它被认为是 通过与自身反应性的CD4+T细胞相互作用和删除来促进外周免疫耐受。然而， 与胸腺相比，AIRE在外周的作用可能更多方面。我们最近确认了艾尔 在肿瘤相关巨噬细胞(TAM)中表达，已知的是抑制抗肿瘤免疫反应。 虽然表达Aire的肿瘤相关巨噬细胞(ATAM)是我们小组最近才发现的， 先前的工作表明，AIRE与抑制抗肿瘤免疫有关。最近在小鼠身上研究AIRE的工具有 促进了外周AIRE表达和aTAM的研究。我们已经在几个常见的地方找到了TAM 共同表达巨噬细胞标志物(CD11b、F4/80和CD64)的实体瘤模型 和质量细胞术(CyTOF)。此外，对几个公开的人类RNA-seq数据集的询问支持 AIRE在肿瘤常驻髓系细胞类型中的表达。这一初步数据与治疗 靶向aTAM以改善癌症免疫治疗的潜力使这一人群值得彻底 功能调查。这项提议将检验aTAM是一种免疫抑制物质的假设， 亲肿瘤细胞群。本提案的目标1将定义表型、转录和表观遗传学 ATAM的配置文件。目的2确定ATAM诱导AIRE表达的机制(S)。目标 3将确定aTAM在抗肿瘤免疫过程中调节局部和系统免疫反应的作用。 该研究方法将使用多种方法进行，包括通过RNA-SEQ的单细胞分析， 利用新的遗传小鼠进行的体外共培养、流式细胞术和ATAC-SEQ、体内分析 模特们。这些拟议的研究将是AIRE在任何肿瘤中表达的第一个特征 并将进一步建立我们对外周血细胞功能的理解。 表达细胞类型。这可能导致发现与治疗耐药相关的新途径 进一步加深了我们对全球AIRE功能的理解。从翻译上讲，这项工作可能会识别新的扰动 以改善患者对免疫治疗的反应。这项研究项目和奖学金培训将是 由加州大学旧金山分校(UCSF)资助的顶级研究机构在 马修·斯皮策博士、刘易斯·拉尼尔博士和詹姆斯·加德纳博士的实验室，以及来自马修·斯皮策博士的专家指导。 马修(麦克斯)克鲁梅尔。拉尼尔博士在免疫细胞生物学方面取得了广泛的基础性发现。Dr。 斯皮策在系统免疫学和单细胞方法方面拥有专业知识。詹姆斯·加德纳博士在 外周AIRE和小鼠模型生成的研究。马克斯·克鲁梅尔博士在肿瘤髓系细胞方面拥有专业知识 生物学和癌症免疫疗法。总的来说，这个设施和团队为以下人员提供了丰富的训练环境 完成这项研究并培养从事学术研究事业所需的专业技能。

PROJECT SUMMARY/ABSTRACT AutoImmune Regulator gene (AIRE) prevents autoimmunity by promoting thymic deletion of self-reactive T cells. While most studied in the thymus, AIRE is also expressed in secondary lymphoid organs, where it is thought to contribute to peripheral tolerance through interaction with and deletion of self-reactive CD4+ T cells. However, AIRE’s role in the periphery is likely more multi-faceted than in the thymus. We recently identified AIRE expression in tumor associated macrophages (TAMs), which are known to inhibit anti-tumor immune responses. While Aire-expressing tumor associated macrophages (aTAMs) were only recently discovered by our group, prior work implicates AIRE in inhibiting anti-tumor immunity. Recently available tools to study AIRE in mice have facilitated investigation of peripheral AIRE expression and aTAMs. We have found aTAMs in several common solid tumor models expressing common macrophage markers (CD11b, F4/80, and CD64) by both flow cytometry and mass cytometry (CyTOF). Additionally, interrogation of several public human RNA-seq datasets supports AIRE expression in tumor resident myeloid cell types. This preliminary data together with the therapeutic potential for targeting aTAMs to improve cancer immunotherapy make this a population deserving of thorough functional investigation. This proposal will test the hypothesis that aTAMs are an immunosuppressive, pro-tumoral cell population. Aim 1 of this proposal will define the phenotypic, transcriptional, and epigenetic profiles of aTAMs. Aim 2 will determine the mechanism(s) driving induction of AIRE expression in aTAMs. Aim 3 will identify the role of aTAMs in regulating local and systemic immune responses during anti-tumor immunity. This research approach will be carried out using a variety of methods including single cell analysis via RNA-seq, CyTOF, flow cytometry, and ATAC-seq, ex vivo co-cultures, and in vivo assays utilizing novel genetic mouse models. These proposed studies will be the first characterization of AIRE expression in any tumor resident immune cells and will further establish our understanding of the function of peripheral AIRE- expressing cell types. This could result in the discovery of novel pathways relevant to therapeutic resistance and further our understanding of global AIRE function. Translationally, this work may identify novel perturbations to improve patient responses to immunotherapy. This research project and fellowship training will be conducted at a top-funded research institution, the University of California, San Francisco (UCSF), in the laboratories of Dr. Matthew Spitzer, Dr. Lewis Lanier, and Dr. James Gardner with expert mentorship from Dr. Matthew (Max) Krummel. Dr. Lanier has made extensive foundational discoveries in immune cell biology. Dr. Spitzer has expertise in systems immunology and single cell methods. Dr. James Gardner has expertise in the study of peripheral AIRE and mouse model generation. Dr. Max Krummel has expertise in tumor myeloid cell biology and cancer immunotherapies. Overall, this facility and team provide a rich training environment for completion of this research and development of professional skills necessary for a career in academic research.