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

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

展开

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.

天然杀伤细胞（NK）是针对感染和癌症的第一道防线。 NK激活由通过激活和抑制性NK受体传播的信号平衡。激活受体NKP46是认为自体，同种异体和异种的自然细胞毒性中的主要激活接收器靶细胞。 NKP46在哺乳动物中保守，几乎普遍于NK表达，这表明它是功能很重要。缺乏NCR1的小鼠，编码NKP46的基因，在肿瘤免疫中受损监视，具有更严重的流感，元蛋白病毒，依孢病毒和梭形病毒感染以及移植物相对于宿主疾病。尽管数十年的搜索。我们的初步工作表明NKP46识别钙网蛋白（CRT），通常在内质网（ER）内发现，但在ER压力细胞中转移到细胞表面或用某些癌症药物治疗的肿瘤细胞。 NKP46-Ig融合蛋白专门拉下细胞表面使用表面等离子体共振的CRT（ECTO-CRT）和初步数据表明特定的结合。敲门在CALR下，编码CRT的基因或添加阻断抗体以抑制NKP46介导的NK 杀人。一些化学疗法剂和放射疗法会诱导一种免疫刺激类型的编程细胞癌细胞中的死亡，称为免疫原性细胞死亡（ICD），该死亡可去除生存肿瘤细胞治疗。 ICD背后的机制与Ecto-Crt有关，该机制用作“吃我”/吞噬树突状细胞的信号，然后通过交叉染色激活抗肿瘤CD8+ T细胞。在这里我们假设 NKP46识别Ecto-CRT，并且NK通过NKP46与Ecto-Crt的相互作用识别ER应激细胞在ICD和NK介导的免疫防御中起着重要作用。在初步数据中，感染 ZIKV在ER中复制并引起ER应力，并用ICD诱导的肿瘤细胞系处理化学疗法药物诱导ECTO-CRT和NKP46依赖性NK杀戮。此外，CRT涂层的肿瘤细胞成为NK细胞靶标，NK细胞系中的NCR1敲除强烈抑制其对靶标的杀死 CRT。为了检验我们的假设，我们将首先确认Ecto-Crt是使用生物物理的NKP46的配体表征Ecto-CRT和NKP46结合并识别负责CRT区域的方法相互作用。 NK和ECTO-CRT在肿瘤靶标上的NKP46的遗传操纵或阻塞将检查这种接受者 - 配体相互作用在NK识别，免疫突触形成和功能反应中的作用感染和肿瘤细胞。 NK杀死未处理的肿瘤细胞和用ICD诱导和将比较非诱导药物。 NKP46在对照ZIKV感染和小鼠肿瘤中的体内作用（没有化学疗法）将通过比较病毒水平和肿瘤生长，NK细胞浸润来评估野生型和NCR1 - / - 小鼠的肿瘤和生存。