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

项目总结 免疫检查点阻断(ICB)使癌症的治疗发生了革命性的变化，但只有大约20%的患者 对治疗有反应。肿瘤微环境的免疫成分(TME)已被认为与 确定对ICB的反应。特别是，髓系细胞(MC)与这两种肿瘤的进展有关 和抗肿瘤免疫。在MC中，经典的I型树突状细胞(CDC1s)是唯一能够 在抗原(Ag)摄取和向淋巴结迁移的背景下结合微环境线索 (Ln)它们呈现抗原和形状效应T细胞反应。同时，cDC1s也在 中枢和外周耐受与T调节细胞(Tregs)的诱导。使用散装和单细胞RNA 小鼠和人非小细胞肺癌(NSCLC)病变的测序(ScRNAseq)，我们已经确定 在cDC1s中的一种常见的转录程序，在迁移到LN时上调。这种迁徙 Signature包括许多免疫调节基因(例如PD-L1、CD200、ITGB8、SOCS2)，因此提示 肿瘤可能会劫持cDC1耐受程序来逃避免疫系统。尽管这些数据提供了一个 迁移性cDC1s的全面转录谱，许多基因的功能和相关性 仍然不为人所知。我们的中心假设是这个信号中上调的基因控制cDc1 动态平衡和表型。在Aim1，我们将使用CRISPR/Cas9蛋白质条形码(Pro-CODE)技术 在稳态和肿瘤中询问多个KO的移行特征，并执行高性能 体内的空间表型分析，以确定和表征调节独特表型和 CDC1s的分子图谱。在目标2中，我们将使用体外OT-I/OT-II试验和肿瘤杀伤试验来确定 迁移特征如何影响CDC1-T细胞的启动和激活。在目标3中，我们将表演单人 KOS对移行性cDC1信号的影响，并确定TME和肿瘤中免疫成分的变化 进步。这些研究的结果将是对以下问题的机械性、功能性和语境的理解 TME中的cDc1转录签名。该项目可能会对DC生物学产生新的见解，并确定 调节或靶向该隔室以获得抗肿瘤免疫的新方法。

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.