Using CRISPR/Cas9 Pro-code technology to dissect the migratory dendritic cell signature

使用 CRISPR/Cas9 Pro-code 技术剖析迁移树突状细胞特征

• 批准号: 9976980
• 负责人: Steven Tiwen Chen
• 金额: $ 4.39万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-08 至 2023-07-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976980
• 关键词: Ablation; Address; Affect; Antigen-Presenting Cells; Antigens; Antitumor Response; Architecture; Autoantigens; Bar Codes; Biological Assay; Bone Marrow; Bone Marrow Transplantation; CD 200; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CRISPR/Cas technology; Cancer Model; Candidate Disease Gene; Cell Communication; Cell Shape; Cells; Cellular biology; Clinical Trials; Coculture Techniques; Confocal Microscopy; Cues; Cytometry; Data; Dendritic Cells; Development; Environment; Flow Cytometry; Gene Expression Profile; Genes; Genetic Transcription; Genomics; Heterogeneity; Histology; Homeostasis; Human; Immune; Immune response; Immune system; Immunosuppression; In Vitro; Incubated; Knock-out; Lead; Lesion; Lung Neoplasms; Malignant neoplasm of lung; Measures; Molecular; Molecular Profiling; Mus; Myeloid Cells; Non-Small-Cell Lung Carcinoma; Outcome Study; Ovalbumin; Patients; Peptides; Phenotype; Play; Proteins; Regulatory Pathway; Regulatory T-Lymphocyte; Resistance; Role; Shapes; T cell response; T-Lymphocyte; Technology; Testing; Time; Tumor Antigens; Tumor Immunity; Work; cancer cell; cancer immunotherapy; cancer therapy; cancer type; central tolerance; chemokine; cytokine; cytotoxic; effector T cell; experimental study; gene function; genetic signature; high dimensionality; immune checkpoint; immune checkpoint blockade; immunogenic; immunoregulation; in vivo; insight; lymph nodes; migration; molecular phenotype; novel; novel strategies; novel therapeutics; peripheral tolerance; progenitor; programmed cell death ligand 1; programs; recruit; response; single-cell RNA sequencing; treatment response; tumor; tumor growth; tumor microenvironment; tumor progression; uptake

PROJECT SUMMARY Immune checkpoint blockade (ICB) has revolutionized the treatment of cancer but only about 20% of patients respond to treatment. The immune composition of the tumor microenvironment (TME) has been implicated in determining response to ICB. In particular, myeloid cells (MC) have been associated with both tumor progression and anti-tumor immunity. Among MC, classical type I dendritic cells (cDC1s) are uniquely capable of incorporating microenvironmental cues in the context of antigen (Ag) uptake and migrating to the lymph node (LN) where they present Ag and shape effector T cell responses. At the same time, cDC1s are also important in central and peripheral tolerance and the induction of T regulatory cells (Tregs). Using bulk and single cell RNA sequencing (scRNAseq) of mouse and human non-small cell lung cancer (NSCLC) lesions, we have identified a common transcriptional program in cDC1s that is upregulated upon migration to the LN. This migratory signature includes many immunoregulatory genes (e.g. PD-L1, CD200, ITGB8, SOCS2), therefore suggesting that tumors may hijack cDC1 tolerogenic programs to evade the immune system. Though these data provide a comprehensive transcriptional profile of migratory cDC1s, the function and relevance of many of the genes remain unknown. Our central hypothesis is that the upregulated genes in this signature control cDC1 homeostasis and phenotype. In Aim1, we will be using CRISPR/Cas9 Protein Barcode (Pro-code) technology to interrogate multiple KO to the migratory signature in steady state and in tumors, and performing high dimensional phenotyping in vivo to identify and characterize the genes that regulate the unique phenotype and molecular profile of cDC1s. In Aim 2, we will use in vitro OT-I/OT-II assays and tumor killing assays to determine how the migratory signature influences cDC1-T cell priming and activation. And in Aim 3, we will perform single KOs to the migratory cDC1 signature and determine changes to immune composition in the TME and tumor progression. The outcome of these studies will be a mechanistic, functional, and contextual understanding of the cDC1 transcriptional signature in the TME. This project may yield novel insights into DC biology and identify new ways to modulate or target this compartment for anti-tumor immunity.

项目摘要 免疫检查点阻断（ICB）彻底改变了癌症的治疗，但只有约20%的患者 对治疗有反应。肿瘤微环境（TME）的免疫组成与肿瘤的发生有关。 确定对ICB的响应。特别是，髓样细胞（MC）与肿瘤进展 和抗肿瘤免疫。在MC中，经典的I型树突状细胞（cDC 1）具有独特的能力， 在抗原（Ag）摄取和迁移到淋巴结的背景下结合微环境线索 (LN)在那里它们呈现Ag和形状效应T细胞应答。与此同时，cDC 1也在 中枢和外周耐受性以及调节性T细胞（T细胞）的诱导。使用散装和单细胞RNA 通过对小鼠和人非小细胞肺癌（NSCLC）病变的scRNAseq测序，我们已经确定了 cDC 1中一种常见的转录程序，在迁移到LN时上调。这种迁徙 特征包括许多免疫调节基因（例如PD-L1、CD 200、ITGB 8、SOCS 2），因此表明 肿瘤可能劫持cDC 1耐受原性程序以逃避免疫系统。虽然这些数据提供了一个 迁移性cDC 1的全面转录谱，许多基因的功能和相关性 仍然未知。我们的中心假设是，在这个标签中上调的基因控制cDC 1 稳态和表型。在Aim 1中，我们将使用CRISPR/Cas9蛋白质条形码（Pro-code）技术 在稳定状态和肿瘤中询问多个KO的迁移特征，并在高水平上 体内三维表型分析，以鉴定和表征调节独特表型的基因， cDC 1 s的分子特征。在目标2中，我们将使用体外OT-I/OT-II测定和肿瘤杀伤测定来确定 迁移信号如何影响cDC 1-T细胞的启动和激活。在目标3中，我们将表演单曲 科斯与迁移性cDC 1特征的结合，并确定TME和肿瘤中免疫组成的变化 进展这些研究的结果将是一个机制，功能，和上下文的理解， TME中的cDC 1转录特征。该项目可能会产生对DC生物学的新见解， 新的方法来调节或靶向抗肿瘤免疫的这个隔室。