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

项目总结/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中进行的，但AIRE也在次级淋巴器官中表达， 通过与自身反应性CD 4 + T细胞的相互作用和自身反应性CD 4 + T细胞的缺失来促进外周耐受。然而，在这方面， AIRE在外周中的作用可能比在胸腺中的作用更多方面。我们最近发现了AIRE 在肿瘤相关巨噬细胞（TAM）中的表达，其已知抑制抗肿瘤免疫应答。 虽然表达Aire的肿瘤相关巨噬细胞（aTAMs）最近才被我们的小组发现， 先前的工作涉及AIRE抑制抗肿瘤免疫。最近可用的研究小鼠AIRE的工具有 有助于研究外周AIRE表达和aTAM。我们在几个常见的 通过流式细胞术和流式细胞术检测表达常见巨噬细胞标志物（CD 11b、F4/80和CD 64）的实体瘤模型 和质谱流式细胞术（CyTOF）。此外，对几个公共人类RNA-seq数据集的询问支持 AIRE在肿瘤驻留骨髓细胞类型中的表达。这一初步数据与治疗 靶向aTAMs以改善癌症免疫治疗的潜力使这一人群值得进行彻底的研究。 功能调查。该提议将检验aTAM是免疫抑制剂的假设， 促肿瘤细胞群。本提案的目标1将定义表型、转录和表观遗传 aTAM的概况。目的2将确定驱动aTAM中AIRE表达的诱导的机制。目的 3将鉴定aTAM在抗肿瘤免疫期间调节局部和全身免疫应答中的作用。 该研究方法将使用多种方法进行，包括通过RNA-seq进行单细胞分析， CyTOF、流式细胞术和ATAC-seq、离体共培养和利用新型遗传小鼠的体内测定 模型这些拟议的研究将是AIRE在任何肿瘤中表达的第一个特征 常驻免疫细胞，并将进一步建立我们对外周AIRE功能的理解- 表达细胞类型。这可能导致发现与治疗抗性相关的新途径 并进一步了解全球AIRE功能。翻译，这项工作可能会发现新的扰动 来提高病人对免疫疗法的反应该研究项目和奖学金培训将 在一个顶级资助的研究机构，加州大学弗朗西斯科分校（UCSF）进行，在 Matthew Spitzer博士、刘易斯Lanier博士和James Gardner博士的实验室， 马修·克鲁梅尔。拉尼尔博士在免疫细胞生物学方面有着广泛的基础性发现。博士 斯皮策在系统免疫学和单细胞方法方面具有专长。詹姆斯·加德纳博士在 外周AIRE研究及小鼠模型建立。Max Krummel博士在肿瘤骨髓细胞方面具有专业知识 生物学和癌症免疫疗法。总的来说，这个设施和团队提供了一个丰富的培训环境， 完成这项研究和发展必要的专业技能的职业生涯中的学术研究。

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.