Uncover the new subsets of epidermal Langerhans cells

项目总结 朗格汉斯细胞(LCS)是表达C型凝集素朗格林(CD207)的皮肤树突状细胞(DC) 在皮肤中调节适应性免疫和免疫耐受，并参与各种类型的皮肤 疾病。成年LCS来源于胚胎卵黄囊来源的巨噬细胞和胎肝单核细胞。 稳定状态。有趣的是，LCS也可以来源于骨髓或外周单核细胞，并且 在发炎的情况下重新填充皮肤。然而，由于缺乏个体的分子图谱 LC水平，在我们对单个CD207表皮LC群体如何 诱导免疫力和耐受性。幸运的是，单细胞RNA测序等新技术 (scRNA-seq)可以评估细胞间的转录变异，而转座酶的单细胞检测- 可及染色质测序(scatac-seq)可以评估单细胞的表观基因组异质性。 以不偏不倚的方式解决问题。最近，我们在小鼠中发现了两个主要的LC亚群，ATF3Bal2a1b-(MLc1) 和ATF3-Bal2a1b(MLC2)亚集，以及包括ATF3(HLC1)亚集在内的三个主要LC亚集 ScRNA-seq.我们还发现，在ATF3基因敲除的小鼠中，缺乏ATF3可以促进LC的成熟并促进 LCS诱导Th1和Th17细胞分化，提示ATF3LC1诱导的免疫抑制功能。 因此，这些初步数据支持我们的假设，即LC是由不同子集组成的异类 具有不同的免疫功能。我们的目标是使用单细胞分析平台和LC命运图 和突变小鼠模型来进一步验证这一点。我们将在R61阶段实现两个具体目标：目标1) 通过分析胚胎期间的LC来表征MLC1和MLC2的基因特征和调控元件， 使用scRNA-seq和rsRNA-seq研究稳定状态和炎症状态的LCS的年轻和衰老发育 目的2)建立ATF3NegEGFP报告小鼠，以命运图ATF3 LC1胚胎发育和 ATF3Lc1和ATF3-Lc2亚群在成人和衰老发育过程中稳态的动态变化 并在体外通过分选ATF3EGFP LC1和ATF3-来表征LC亚群 Lc2细胞和再衍生的ATF3.loxp小鼠，与hLangerin-Cre小鼠杂交，产生LC-3loxp小鼠。 特异性/时间诱导的ATF3KO用于体内功能研究。在R33阶段，我们将进行以下具体工作 目的：目的3)利用LC特异性的ATF3缺失hLCcre.ATF3KO在体内对ATF3Lc1亚群进行功能鉴定 为评价ATF3Lc1亚群在不同疾病模型中的潜在免疫调节作用， 包括自身免疫性白癜风、黑色素瘤和真菌感染模型。我们的工作将揭开LC的神秘面纱 子集及其特定功能，这将为LCS的生物学提供新的见解，并导致 发展以LC为基础的皮肤病干预策略。

PROJECT SUMMARY Langerhans cells (LCs) are skin-resident dendritic cells (DCs) expressing the C-type lectin Langerin (CD207) that mediate both adaptive immunity and immune tolerance in skin and are involved in various types of skin diseases. Adult LCs are originated from embryonic yolk-sac-derived macrophages and fetal liver monocytes in the steady state. Interestingly, LCs could also be derived from the bone marrow or peripheral monocytes and repopulate the skin under inflammatory conditions. However, due to the lack of molecular profiles at individual LC level, a significant gap remains in our understanding on how a single CD207+ epidermal LC population can induce both immunity and tolerance. Fortunately, new technologies such as the single-cell RNA-sequencing (scRNA-seq) can evaluate cell-to-cell transcriptomic variation, while the single-cell assay for transposase- accessible chromatin using sequencing (scATAC-seq) can assess the epigenomic heterogeneity at single-cell resolution in an unbiased manner. Recently, we identified two major LC subsets in mice, ATF3+Bal2a1b- (mLC1) and ATF3-Bal2a1b+(mLC2) subsets, and three major LC subsets in human including ATF3+ (hLC1) subset using scRNA-seq. We also found in ATF3 knockout mice that lack of ATF3 enhances LC maturation and promotes LCs-induced Th1 and Th17 cell differentiation suggesting immune suppressive function induced by ATF3+LC1. Hence, these preliminary data support our hypothesis that LCs are heterogeneous consisting of distinct subsets with different immune functions. Our objective is to use single-cell analysis platforms plus the LC fate-mapping and mutation mouse models to further validate this. We will pursue two Specific Aims in the R61 phase: Aim 1) Characterize the gene signatures and regulatory elements of mLC1 and mLC2 by profiling LCs during embryonic, young, and aging development at steady-state and repopulated LCs at inflamed state using scRNA-seq and scATAC-seq; Aim 2) Generate ATF3negEGFP reporter mice to fate-map ATF3+LC1 embryonic development and the dynamic change of ATF3+LC1 and ATF3-LC2 subset at steady state during adult and aging development and at inflammatory state and functionally characterize LC subsets in vitro by sorting ATF3EGFP+ LC1 and ATF3- LC2 cells and rederiving ATF3.loxp mice, which will be crossed with hLangerin-Cre mice to generate LC- specific/time-induced ATF3KO for in vivo functional study. In the R33 phase, we will pursue the following Specific Aim: Aim 3) Functionally characterize ATF3+LC1 subset in vivo using LC-specific ATF3 deletion hLCcre.ATF3KO mice to evaluate the potential immune regulation function of ATF3+LC1 subset in the different disease models, including autoimmune vitiligo, melanoma, and fungi infection models. Our work will uncover the mystery of LC subsets with their specific functions, which will provide new insights into the biology of LCs and lead to the development of LC-based intervention strategies for skin diseases.