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Endogenous ligand of the NK activating receptor NKp46

NK 激活受体 NKp46 的内源性配体

基本信息

批准号：
10116279
负责人：
Judy Lieberman
金额：
$ 22.13万
依托单位：
BOSTON CHILDREN'S HOSPITAL
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：
2020-03-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10116279
关键词：
Affect Aftercare Allogenic Antineoplastic Agents Apoptosis Autologous Binding Binding Proteins Blocking Antibodies CD8-Positive T-Lymphocytes Cell Communication Cell Line Cell surface Cells Cellular Stress Chemotherapy-Oncologic Procedure Chimeric Proteins Cross-Priming Data Dendritic Cells Dependence Down-Regulation Eating Endoplasmic Reticulum Equilibrium Flavivirus Fusobacterium Infections Genes Human Immune Immune response Immunity Immunologic Surveillance Impairment Incubated Infection Infiltration Influenza A virus Influenza Hemagglutinin Knock-out Knowledge Ligands Link Lymphocyte Malignant Neoplasms Mammals Manuscripts Mediating Mediator of activation protein Metapneumovirus Mus NK Cell Activation Natural Killer Cells Phagocytes Pharmaceutical Preparations Play Pregnancy Preparation Production Proteins Radiation therapy Receptor Cell Recombinants Reovirus Infections Role Sialic Acids Signal Transduction Stress Surface Plasmon Resonance Testing Therapeutic Tumor Cell Line Viral Work ZIKV infection Zika Virus base biophysical techniques calreticulin cancer cell cell killing chemotherapeutic agent chemotherapy cytokine cytotoxicity endoplasmic reticulum stress fetal fetus cell first responder genetic manipulation graft vs host disease immunogenic cell death immunological synapse immunological synapse formation in vivo inhibitor/antagonist knock-down mouse model neoplastic cell overexpression receptor response tool trophoblast tumor tumor growth virtual

项目摘要

Natural killer cells (NK) act as a first line of defense against infection and cancer. NK activation is controlled by a balance of signals transmitted by activating and inhibitory NK receptors. The activating receptor NKp46 is considered the major activating receptor in natural cytotoxicity against autologous, allogeneic and xenogeneic target cells. NKp46 is conserved in mammals and virtually ubiquitously expressed on NK, suggesting it is functionally important. Mice deficient in Ncr1, the gene encoding NKp46, are impaired in tumor immune surveillance, have more severe influenza A, metapneumovirus, reovirus and fusobacterium infections and graft versus host disease. The endogenous ligand of this important activating NK receptor is not known, despite decades of searching. Our preliminary work suggests that NKp46 recognizes calreticulin (CRT), which is normally found inside the endoplasmic reticulum (ER) but gets transferred to the cell surface in ER-stressed cells or tumor cells treated with some cancer drugs. An NKp46-Ig fusion protein specifically pulls down cell-surface CRT (ecto-CRT) and preliminary data using surface plasmon resonance indicate specific binding. Knocking down CALR, the gene encoding for CRT, or adding blocking antibodies to CRT inhibits NKp46-mediated NK killing. Some chemotherapeutic agents and radiotherapy induce an immunostimulatory type of programmed cell death in cancer cells, known as immunogenic cell death (ICD), which removes surviving tumor cells after treatment. The mechanism behind ICD has been linked to ecto-CRT, which serves as an “eat me”/phagocytic signal for dendritic cells, which then activate anti-tumor CD8+ T cells by cross-priming. Here we hypothesize that NKp46 recognizes ecto-CRT, and that NK recognition of ER-stressed cells via NKp46 interaction with ecto-CRT plays an important role in ICD and NK-mediated immune defense more generally. In preliminary data, infection with ZIKV, which replicates in the ER and causes ER stress, and treatment of tumor cell lines with ICD-inducing chemotherapy drugs induces ecto-CRT and NKp46-dependent NK killing. Moreover, CRT-coated tumor cells become NK cell targets, and NCR1 knockout in an NK cell line strongly inhibits its killing of targets with exposed CRT. To test our hypotheses, we will first confirm that ecto-CRT is a ligand for NKp46 by using biophysical methods to characterize ecto-CRT and NKp46 binding and identifying the regions of CRT responsible for the interaction. Genetic manipulation or blocking of NKp46 on NK and of ecto-CRT on tumor targets will examine the role of this receptor-ligand interaction in NK recognition, immune synapse formation and functional responses to infected and tumor cells. NK killing of untreated tumor cells and tumor cells treated with ICD-inducing and noninducing drugs will be compared. The in vivo role of NKp46 in controlling ZIKV infection and mouse tumors (without and with chemotherapy) will be assessed by comparing viral levels and tumor growth, NK cell infiltration of tumors and survival in wild-type and Ncr1-/- mice.

自然杀伤细胞（NK）是抵抗感染和癌症的第一道防线。通过活化和抑制NK受体传递的信号的平衡。活化受体NKp 46是被认为是针对自体、同种异体和异种的天然细胞毒性中的主要活化受体。 NKp 46在哺乳动物中是保守的，并且实际上在NK细胞上普遍表达，这表明它是靶细胞。 Ncr 1（编码NKp 46的基因）缺陷的小鼠在肿瘤免疫中受损，监测，有较严重的甲型流感，偏肺病毒，呼肠孤病毒和梭杆菌感染和移植这种重要的活化NK受体的内源性配体是未知的，尽管我们的初步工作表明，NKp 46识别钙网蛋白（CRT），这是一种通常存在于内质网（ER）内，但在ER-1应激细胞中转移到细胞表面一种NKp 46-β IG融合蛋白特异性地拉低细胞表面的蛋白质， CRT（体外CRT）和使用表面等离子体共振的初步数据表明特异性结合。下调CRT编码基因CALR或在CRT上添加阻断抗体可抑制NKp 46介导的NK细胞一些化疗药物和放疗诱导免疫刺激型的程序化细胞癌细胞死亡，称为免疫原性细胞死亡（ICD），它会在癌细胞死亡后清除存活的肿瘤细胞。 ICD背后的机制与体外循环CRT有关，其充当“吃我”/吞噬细胞，树突状细胞的信号，然后通过交叉免疫激活抗肿瘤CD 8 + T细胞。 NKp 46识别外膜CRT，并且NK通过NKp 46与外膜CRT的相互作用识别ER-CRT应激细胞，在ICD和NK细胞介导的免疫防御中起重要作用。用ZIKV（其在ER中复制并引起ER应激）处理肿瘤细胞系，以及用ICD-GFP诱导的肿瘤细胞系处理肿瘤细胞系。化疗药物可诱导体外CRT和NKp 46-NK依赖性NK杀伤，而且，CRT-CRT包被的肿瘤细胞成为NK细胞靶标，并且NK细胞系中的NCR 1敲除强烈抑制其对暴露于NCR 1的靶标的杀伤。为了验证我们的假设，我们将首先通过使用生物物理方法证实外膜CRT是NKp 46的配体。表征外膜CRT和NKp 46结合的方法，以及鉴定CRT中负责NKp 46结合的区域的方法。基因操作或阻断NK细胞上的NKp 46和肿瘤靶点上的外显CRT将检查这种受体-配体相互作用在NK识别、免疫突触形成和功能反应中作用未处理的肿瘤细胞和用ICD-10诱导和治疗的肿瘤细胞的NK杀伤 NKp 46在控制ZIKV感染和小鼠肿瘤中的体内作用将通过比较病毒水平和肿瘤生长、NK细胞浸润（无和有化疗）来评估在野生型和Ncr 1-nc/-nc小鼠中的肿瘤和存活率。