Treg cell diversity and homeostatic control

Treg 细胞多样性和稳态控制

基本信息

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
项目类别：
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

项目摘要

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单元的CD4+ T调节（Treg）播放键和非冗余维持免疫学耐受性和控制炎症的作用。 CD4+ T细胞中的Treg比例处于严格的体内控制之下。 Treg细胞具有相当大的多样性，具有差异的能力控制某些免疫功能，甚至控制具有免疫力外功能的组织居民Treg。我们的最新消息最近的单细胞RNASEQ工作已将这些作品绘制得很细，表明Treg子集表达了不同的效应器分子。 Treg子集结构在小鼠和人类中似乎非常相似。我们假设那个treg 淋巴组织中的子集对不同的稳态提示反应，并且这些不同的输入条件特定的Treg功能。（1）我们的单细胞转录组学数据，强调了Treg的多样性以公正的方式进行亚表格型，确定了一些细胞表面标记以识别和分类，但是我们将通过执行组合的转录组/蛋白质组分析来扩展此基础（Scrnaseq合并通过DNA标记抗体的表面蛋白质定量 - cite-seq），以识别一组流式细胞仪在小鼠和人类中都准确区分Treg亚表格的标记。这将与这本小说有关在Treg子集上的先验知识，提供生物标志物并启用功能性分类的细胞分类实验。我们将评估Treg子集在调节其他免疫细胞（增殖和分化），评估其发育起源和稳定性（胸腺或外围，可塑性？），以及它们如何自身免疫性或肿瘤病变有所不同。（2）IL2是Treg细胞的规范控制器但是细胞因子（IL7、15、21和TSLP）和IL33也影响Tregs。我们的结果表明分布明显不同 Treg子集中的细胞因子受体的，这表明不同的生长因子支持它们。我们将使用增益 - （体内细胞因子给药）或功能丧失（阻止抗体）策略以修改Treg 子集平衡，通过流式细胞仪（Stat磷酸化，Treg子集数和激活）读取 scrnaseq。（3）大型TNF受体（TNFR）超家族的大多数成员在Treg细胞中过度表达，有些人已被证明会影响Treg的维护和生存，但缺乏全面的图片。我们的数据显示了TNFR跨Treg子集的差异表示。我们假设TNFR分子具有细胞因子接收器的网络，向不同的Treg子集提供特定的营养信号。我们已经发展了基于条形码干细胞的强大基于CRISPR的体内屏幕，以评估基因集的方式参与Treg稳态。我们将分析整个TNFR家族在维护不同的角色上的作用 Treg子集，在不同淋巴组织中的基线和营养细胞因子刺激后。我们也会通过组合测试几个TNFR，以寻找冗余，完整性或对抗。这些结果将提供对Treg人群多样性的澄清和更深入的了解，他们的控制细胞因子或表面受体，以及如何专门操作治疗意图。