Treg cell diversity and homeostatic control

Treg 细胞多样性和稳态控制

• 批准号: 10333377
• 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/10333377
• 关键词: Ablation; Acute; Affect; Antibodies; Arthritis; Autoimmune; Autoimmune Diseases; Autoimmunity; Back; Bar Codes; Biological Markers; Biology; Blocking Antibodies; CD4 Positive T Lymphocytes; Cell Separation; Cell surface; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; 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; Inflammation; Inflammatory; Joints; Knowledge; Lesion; Locales; Location; Lymphoid Tissue; Maintenance; Malignant Neoplasms; Mediator of activation protein; Membrane Proteins; Mining; Mus; Pathway interactions; Patients; Peripheral; Phenotype; Phosphorylation; Play; Population; Process; Proteome; Regulation; Regulatory T-Lymphocyte; Rest; Role; Signal Transduction; Sorting - Cell Movement; Structure; Surface; TSLP gene; Testing; Therapeutic; Thymus Gland; Tissues; Tumor Necrosis Factor Receptor; Variant; Work; antagonist; base; biomarker identification; biomarker panel; combinatorial; cytokine; immune function; immunoregulation; in vivo; interest; loss of function; lymphoid organ; member; morphogens; novel; overexpression; receptor; single-cell RNA sequencing; stem cells; therapeutic biomarker; 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.

表达转录因子FoxP3细胞的CD4+T调节(Treg)起关键和非冗余作用 在维持免疫耐受和控制炎症方面的作用。CD4+T细胞中Treg比例的变化 处于严格的动态平衡控制之下。Treg细胞具有相当大的多样性，具有分化的能力 控制某些免疫功能，甚至是具有额外免疫功能的组织驻留树。我们最近 最近的单细胞RNAseq研究已经精细地绘制了这些图表，表明Treg亚群表达不同的效应器 分子。在小鼠和人类中，Treg亚集结构似乎非常相似。我们假设特雷格 淋巴组织中的亚群对不同的动态平衡信号做出反应，这些不同的输入条件 特定的Treg函数。(1)我们的单细胞转录组数据，突出了Treg的多样性 亚型以不偏不倚的方式确定了一些用于鉴定和分类的细胞表面标记，但 我们将通过执行组合转录组/蛋白质组分析(scRNAseq组合)来扩展这个基础 用DNA标记抗体(CITE-SEQ)对表面蛋白进行定量以鉴定一组流式细胞术 准确区分Treg亚型的标记，在老鼠和人类中都是如此。这将与这部小说有关 了解Treg亚群的先验知识，提供生物标记物，并支持功能细胞分选 实验。我们将评估Treg亚群在调节其他免疫细胞(增殖和 分化)，评估它们的发育起源和稳定性(胸腺或外周，可塑性？)，以及它们如何 自身免疫性病变或肿瘤病变各有不同。(2)IL_2是Treg细胞的典型控制者，而其他常见的 细胞因子(IL7、15、21和TSLP)和IL33也会影响Treg。我们的结果显示了一个明显不同的分布 细胞因子受体在Treg亚群中的分布，表明不同的生长因子支持它们。我们将使用 获得(体内细胞因子注射)或功能丧失(阻断抗体)策略来修改Treg 亚群平衡，通过流式细胞术读出(STAT磷酸化、Treg亚群数量和激活)和 ScRNAseq.(3)大肿瘤坏死因子受体(TNFR)超家族的大多数成员在Treg细胞中高表达， 一些已经被证明会影响Treg的维持和生存，但缺乏一个全面的图景。 我们的数据显示了TNFR在Treg子集之间的差异表示。我们假设TNFR分子 与细胞因子受体网络，为不同的Treg亚群提供特定的营养信号。我们已经开发出 一种基于CRISPR的强大的活体筛查，基于条码干细胞，以评估一组基因如何 参与Treg动态平衡。我们将分析整个TNFR家族在维护不同 Treg亚群，在不同淋巴组织中的基线和在营养细胞因子刺激后。我们还将 组合测试几个TNFR，以寻找冗余、互补或拮抗。这些结果 将提供对Treg种群的多样性的澄清和更深入的理解，它们由 细胞因子或表面受体，以及如何为治疗目的而特定地操纵它们。