Treg cell diversity and homeostatic control

Treg 细胞多样性和稳态控制

• 批准号: 10551213
• 负责人: CHRISTOPHE O. BENOIST
• 金额: $ 51.25万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-11 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10551213
• 关键词: Ablation; Acute; Affect; Antibodies; Arthritis; Autoimmune; Autoimmune Diseases; Autoimmunity; Back; Bar Codes; Biological Markers; Blocking Antibodies; CD4 Positive T Lymphocytes; Cell Count; Cell Maintenance; Cell Separation; Cell Survival; Cell physiology; Cell surface; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cues; Cytokine Receptors; DNA; Data; Development; Dissection; Equilibrium; FOXP3 gene; Family; Family member; Feedback; Flow Cytometry; Genes; Goals; Grant; Growth Factor; Heterogeneity; Homeostasis; Human; IL2 gene; IL7 gene; Immune Tolerance; Immunologic Surveillance; Immunooncology; Immunotherapy; In Vitro; Inbred Strains Mice; Individual; Infiltration; Inflammation; Inflammatory; Joints; Knowledge; Lesion; Locales; Location; Lymphoid Tissue; Maintenance; Malignant Neoplasms; Mediator; Membrane Proteins; Mining; Mus; Organ; Pathway interactions; Patients; Peripheral; Phenotype; Phosphorylation; Play; Population; Process; Proliferating; Proteome; Regulation; Regulatory T-Lymphocyte; Rest; Role; Signal Transduction; Sorting; Structure; Surface; T cell differentiation; T-Lymphocyte Subsets; TSLP gene; Testing; Therapeutic; Thymus Gland; Tissues; Tumor Necrosis Factor Receptor; Variant; Work; antagonist; base; biomarker panel; combinatorial; cytokine; immune function; immunoregulation; in vivo; interest; loss of function; lymphoid organ; member; morphogens; novel; overexpression; receptor; segregation; single-cell RNA sequencing; stem cells; therapeutic target; transcription factor; transcriptome; transcriptomics; tumor

CD4+ T regulatory (Treg) that express the transcription factor FoxP3 cells play key and non-redundant roles in maintaining immunologic tolerance and controlling inflammation. Treg proportions within CD4+ T cells are under tight homeostatic control. There is considerable diversity to Treg cells, with differential ability to control certain immune functions, and even tissue-resident Tregs with extra-immunologic functions. Our recent recent single-cell RNAseq work has charted these finely, showing that Treg subsets express different effector molecules. Treg subset structure appears very similar in mouse and humans. We hypothesize that Treg subsets in lymphoid tissues respond to different homeostatic cues, and that these different inputs condition specific Treg functions. (1) Our single-cell transcriptomics data, which highlighted the diversity of Treg subphenotypes in an unbiased fashion, identified some cell surface markers for identification and sorting, but we will expand this base by performing a combined transcriptome/proteome analysis (scRNAseq combined with surface protein quantitation by DNA-tagged antibodies - CITE-seq) to identify a panel of flow cytometry markers that accurately distinguish Treg subphenotypes, in both mouse and human. This will relate this novel landscape to prior knowledge on Treg subsets, provide biomarkers, and enable cell sorting for functional experimentation. We will assess the function of Treg subsets in regulating other immunocytes (proliferation and differentiation), assess their developmental origin and stability (thymic or peripheral, plasticity?), and how they vary in autoimmune or tumor lesions. (2) IL2 is the canonical controller of Treg cellsbut other common- cytokines (IL7, 15, 21 and TSLP) and IL33 also affect Tregs. Our results show a strikingly divergent distribution of cytokine receptors among Treg subsets, suggesting that different growth factors support them. We will use both gain- (in vivo cytokines administration) or loss-of-function (blocking antibodies) strategies to modify Treg subset balance, read out by flow cytometry (STAT phosphorylation, Treg subset number and activation) and scRNAseq. (3) Most members of the large TNF Receptor (TNFR) superfamily are over-expressed in Treg cells, and some have been shown to affect Treg maintenance and survival, but a comprehensive picture is lacking. Our data show differential representation of TNFR across Treg subsets. We hypothesize that TNFR molecules network with cytokine receptors to provide specific trophic signals to distinct Treg subsets. We have developed a powerful CRISPR-based in vivo screen, based on barcoding stem cells, to evaluate how sets of genes partake in Treg homeostasis. We will analyze the role of the entire TNFR family on maintenance of different Treg subsets, at baseline in different lymphoid tissues and after stimulation by trophic cytokines. We will also test several TNFRs combinatorially, to search for redundancy, complementarity or antagonism. These results will provide a clarification and deeper understanding of the diversity of Treg populations, their control by cytokines or surface receptors, and how they might be specifically manipulated for therapeutic intent.

CD 4+调节性T细胞（Treg）即表达转录因子FoxP 3的细胞发挥关键和非冗余作用 在维持免疫耐受和控制炎症中的作用。CD 4 + T细胞内的Treg比例 都处于严格的自我平衡控制之下Treg细胞具有相当大的多样性，具有分化能力， 控制某些免疫功能，甚至是具有免疫外功能的组织驻留型T细胞。我们最近 最近的单细胞RNAseq工作已经很好地描绘了这些，表明Treg亚群表达不同的效应子， 分子。Treg亚群结构在小鼠和人类中似乎非常相似。我们假设Treg 淋巴组织中的亚群对不同的稳态线索做出反应，这些不同的输入条件 特定Treg功能。(1)我们的单细胞转录组学数据突出了Treg的多样性， 亚表型，确定了一些细胞表面标志物的鉴定和分选，但 我们将通过联合转录组/蛋白质组分析（scRNAseq联合 通过DNA标记的抗体进行表面蛋白定量- CITE-seq）以鉴定流式细胞术的一组 在小鼠和人类中，准确区分Treg亚表型的标记物。这将关系到这本小说 横向于关于Treg亚群的先前知识，提供生物标志物，并使得能够进行细胞分选以用于功能检测。 实验我们将评估Treg亚群在调节其他免疫细胞（增殖和免疫应答）中的功能。 分化），评估其发育起源和稳定性（胸腺或外周，可塑性？），以及它们如何 在自身免疫性或肿瘤病变中变化。(2)IL 2是Treg细胞的典型控制者，但其他常见的调节因子 细胞因子（IL 7、15、21和TSLP）和IL 33也影响TcR。我们的结果显示了一个显著的发散分布 Treg亚群之间的细胞因子受体，表明不同的生长因子支持他们。我们将使用 修饰Treg的获得（体内细胞因子施用）或功能丧失（阻断抗体）策略 亚群平衡，通过流式细胞术读出（STAT磷酸化、Treg亚群数量和活化），以及 scRNAseq. (3)大TNF受体（TNFR）超家族的大多数成员在Treg细胞中过表达， 有些已经被证明会影响Treg的维持和存活，但缺乏全面的了解。 我们的数据显示TNFR在Treg亚群中的差异表达。我们假设TNFR分子 与细胞因子受体的网络，提供特定的营养信号，以不同的Treg亚群。我们已经开发 一个强大的基于CRISPR的体内筛选，基于条形码干细胞，以评估基因组如何 参与调节性T细胞稳态。我们将分析整个TNFR家族在维持不同的 Treg亚群，在不同淋巴组织中的基线和营养细胞因子刺激后。我们还将 组合测试几种TNFR，以寻找冗余、互补或拮抗。这些结果 将提供一个澄清和更深入的了解Treg群体的多样性，他们的控制， 细胞因子或表面受体，以及它们如何被特异性地操纵用于治疗目的。