VERMONT COBRE: PROJECT 1: INKT CELL ACTIVATION BY CD ID & ITS LIGANDS

佛蒙特州 COBRE：项目 1：通过 CD ID 激活 INKT 细胞

• 批准号: 7720915
• 负责人: JONATHAN E BOYSON
• 金额: $ 17.2万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720915
• 关键词: Acute; Address; Affect; Affinity; Amino Acids; Anabolism; Area; Asthma; Autoimmunity; Binding; Biological Assay; CD8B1 gene; Carditis; Cell Count; Cell Wall; Cell physiology; Cells; Centers of Research Excellence; Ceramide glucosyltransferase; Chemicals; Class; Collaborations; Communicable Diseases; Complement; Computer Retrieval of Information on Scientific Projects Database; Consult; Data; Dimerization; Enzymes; Event; Faculty; Fluorescence; Fostering; Frequencies; Funding; Glycolipids; Glycosphingolipids; Goals; Grant; Heterodimerization; Heterogeneity; Human; Immune response; Immunity; Immunology; Inclusion Bodies; Individual; Injury; Institution; Interferon Type II; Laboratories; Leukocytes; Ligands; Liver; Lung; Lymphocytic choriomeningitis virus; MHC Class II Genes; Major Histocompatibility Complex; Mammals; Maps; Maternal-Fetal Exchange; Mediating; Mentors; Methodology; Mus; Mutagenesis; Pathway interactions; Personal Satisfaction; Phase; Physiological; Play; Predisposition; Pregnancy loss; Preparation; Production; Progress Reports; Proteins; Publications; RNA Interference; Reagent; Recombinants; Regulation; Relative (related person); Reporting; Research; Research Personnel; Resistance; Resources; Role; Science; Series; Shapes; Site-Directed Mutagenesis; Sorting - Cell Movement; Source; Specificity; Standards of Weights and Measures; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Time; United States National Institutes of Health; Variant; Vermont; Virus Diseases; Work; Writing; airway hyperresponsiveness; base; career; congenic; cytokine; design; expression vector; interest; killer T cell; novel; pathogen; research study; response; tumor immunology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Invariant natural killer T (iNKT) cells comprise a novel subset of T cells that has a profound effect on infectious disease, autoimmunity, tolerance induction, and tumor immunology. Their relatively high frequency in the gut, liver, and lung, together with their ability to rapidly secrete large amounts of a wide variety of cytokines and to activate other leukocyte subsets suggests that they may play an important role in early events of a developing immune response. All human iNKT cells express an unusual invariant T cell receptor that recognizes glycolipids derived from bacterial cell walls. In addition to these exogenous glycolipid ligands, it has been clear for some time that iNKT cells can recognize mammalian endogenous CD1d ligands. The identity, however, of natural activating ligands in mammals has remained elusive. Preliminary data from our laboratory suggests that human iNKT cells may be able to differentiate endogenous CD1d ligands from different cells. Our hypothesis is that this specificity is mediated by the iNKT TCR Vb, since the TCRa chain is invariant. This was supported by site-directed mutagenesis studies in a single amino acid change in the CDR3b loop significantly affected TCR recognition of CD1d loaded with endogenous ligand. To directly assess the contribution of iNKT TCRs to CD1d/endogenous ligand recognition, we have been developing a recombinant soluble iNKT TCR that can be multimerized to produce a high-affinity reagent with which we can directly assess relative TCR binding affinity to CD1d and its ligands using a fluorescence based approach. This "TCR tetramer" can be used in relatively straightforward binding assays directly on CD1d-expressing cells to calculate the relative Kd of different NKT TCR:CD1d ligand combinations. It would also complement and enhance approaches proposed to define biosynthetic pathways and the heterogeneity of glycosylceramides used in the formation of natural CD1d ligands and/or their precursors. For example, one potential strategy would be to use an iNKT TCR tetramer to FACS-sort CD1d+ cells that have been rendered deficient in endogenous ligand production, either through RNAi or through chemical mutagenesis. In the last year, we successfully expressed both the TCRa and b chains from two iNKT TCRs for use in these experiments. However, we were unable to successfully refold these TCRs using standard methodology. We have, therefore, now modified our design which now employs fos/jun dimerization on the C-terminus of the TCR chains to increase the level of heterodimerization. This approach has been successfully used for years in the facilitation of MHC class II heterodimer formation. We have successfully cloned these new constructs into expression vectors, expressed the individual recombinant chains as inclusion bodies, and are awaiting our first results from protein refolds. We have also made substantial headway on our second Aim, which is to dissect the biosynthetic pathways necessary for endogenous glycolipid formation. Since it is well known that mouse endogenous ligands are dependent on glucosylceramide synthase, we have been developing a targeting strategy in this species as a positive control. We have been able to successfully use an RNAi strategy to target this enzyme (the first in the GSL biosynthesis series). Furthermore, we have interesting preliminary data to suggest that, in contrast to the mouse, the human endogenous CD1d ligand may not be a glycosphingolipid. The third area of interest is our observation that there are dramatic strain differences in the iNKT cell number and function. These strain-dependent differences are correlated with significant physiological effects such as shared susceptibility to aGalCer-induced airway hyperreactivity (a cardinal feature of asthma), aGalCer-induced liver injury, and aGalCer-induced pregnancy loss. Given the ability of NKT cells to dramatically alter and shape developing immune responses, we hypothesize that variability in NKT cell number and function could be a major determinant in strain-dependent variability in resistance to certain pathogens. These observations are now in press. We have extended this work by identifying the 129/SvImJ strain as one of a few strains with severely impaired responses to aGalCer, which should prove valuable in identifying the determinants underlying these strain-dependent differences. We are now completing a thorough characterization of this strain in anticipation of its use in a congenic mapping strategy to identify loci involved in strain-dependent NKT cell function variability. Publications 2007: Rymarchyk, S.L., H. Lowenstein, J. Mayette, S.R. Foster, D. Damby, I.W. Howe, I. Aktan, R.E. Meyer, M.E. Poynter, and J.E. Boyson. Widespread natural variation in NKT cell number and function. Immunology (in press). J.E. Boyson, I. Aktan, D. Barkhuff, and A. Chant. NKT cells at the maternal-fetal interface. (submitted). Olson Jr., C.M., Bates, T.C., Radolf, J.D., Huber, S.A., Boyson, J.E., and J. Anguita. Local regulation of interferon gamma by invariant natural killer T cells modulates acute lyme carditis. (submitted). Mentoring Summaries: Elizabeth Bonney I currently meet periodically with all trainees, and discuss science as well as help with reports, presentations, and publications. I moreover participate in general career advising. Jon Boyson, a COBRE Junior Faculty is a co-investigator on my grant, "Placental immunity to LCMV" HD047224. We are also collaborating on production of Tetramers to detect NKT cells and LCMV-specific CD8 T cells. In addition, I am a consulting collaborator on his recent application: CD1D-Restricted T cells and Pregnancy Loss R01AI067897 My goal this year is to help Jon receive R21 level funding for his COBRE project, and then late help with preparation of the related R01 Sally Huber During the last year, I met with Jon twice to discuss a potential collaboration to determine cross-talk between gamma-delta T cells and Natural Killer T cells, both of which recognize CD1d, a non-classical major histocompatibility complex class I like molecule. Both NKT and gamma-delta T cells are innate effectors and modulate developing adaptive immunity in virus infection. The question to be addressed is whether they synergize or antagonize each other. I also provided help on how to write his first Progress Report on his NIH RO1 grant. Jon is currently in his second year of his RO1 and is on track to be phased out of the COBRE this year. Presumably, I would then be assigned a new mentee.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 不变的天然杀伤T（INKT）细胞包括一个新的T细胞子集，该细胞对传染病，自身免疫性，耐受性诱导和肿瘤免疫学具有深远影响。它们在肠道，肝脏和肺中相对较高的频率，以及它们能够快速分泌大量各种细胞因子并激活其他白细胞细胞亚群的能力，这表明它们可能在发生免疫反应的早期事件中起重要作用。 所有人类Inkt细胞表达了一种不寻常的T细胞受体，该受体识别源自细菌细胞壁的糖脂。除了这些外源性糖脂配体外，一段时间以来很明显inkt细胞可以识别哺乳动物内源性CD1D配体。然而，哺乳动物中自然激活配体的身份仍然难以捉摸。与我们实验室的初步数据表明，人Inkt细胞可能能够区分内源性CD1D配体与不同细胞。我们的假设是，由于TCRA链是不变的，因此该特异性是由Inkt TCR VB介导的。在CDR3B环的单个氨基酸变化中，位于定向的诱变研究支持了这一点，从而显着影响了带有内源配体的CD1D的TCR识别。 为了直接评估inkt TCR对CD1D/内源配体识别的贡献，我们一直在开发一个重组可溶性Inkt TCR，可以进行多种二合中以产生高亲和力试剂，我们可以直接评估与CD1D及其配体的相对TCR结合亲和力使用基于荧光的方法。该“ TCR四聚体”可以直接在表达CD1D的细胞上的相对直接的结合测定中使用，以计算不同NKT TCR的相对KD：CD1D配体组合。它还将补充和增强方法，以定义生物合成途径以及用于形成天然CD1D配体和/或其前体的形成中的糖基辛酰胺的异质性。例如，一种潜在的策略是使用Inkt TCR四聚体来通过RNAi或通过化学诱变使内源性配体产生不足的FACS-SORT CD1D+细胞。去年，我们成功地表达了来自两个Inkt TCR的TCRA和B链，以用于这些实验。但是，我们无法使用标准方法成功地重新分配这些TCR。因此，我们现在已经修改了我们的设计，该设计现在在TCR链的C末端采用FOS/JUN二聚化，以提高异二聚化水平。多年来，这种方法已成功地用于促进MHC II类异二聚体形成。我们成功地将这些新构建体克隆到表达载体中，将单个重组链作为包容物体表示，并正在等待我们从蛋白质再生中的首次结果。 我们还取得了第二个目标，即剖析内源性糖脂形成所需的生物合成途径。由于众所周知，小鼠内源性配体取决于葡萄糖基酶合酶，因此我们一直在该物种中制定靶向策略作为阳性对照。我们已经能够成功使用RNAi策略来靶向这种酶（GSL生物合成系列中的第一个酶）。此外，我们还有有趣的初步数据，可以表明与小鼠相比，人内源性CD1D配体可能不是糖磷脂。 感兴趣的第三个领域是我们观察到的inkt细胞数和功能存在巨大的应变差异。这些依赖性差异与显着的生理作用相关，例如对agalcer诱导的气道高反应性（哮喘的基本特征），agalcer诱导的肝损伤和agalcer诱发的妊娠丧失的共同敏感性。鉴于NKT细胞显着改变和塑造了产生免疫反应的能力，我们假设NKT细胞数和功能的变异性可能是抗菌株依赖性变异性的主要决定因素。这些观察现在正在印刷中。我们通过将129/SVIMJ菌株作为对Agalcer响应严重受损的少数菌株之一来扩展这项工作，这对于确定这些菌株依赖性差异的决定因素应该很有价值。现在，我们正在完成对这种菌株的彻底表征，以期在先天映射策略中使用它，以识别涉及应变依赖性NKT细胞功能变异性的基因座。 出版物2007： Rymarchyk，S.L.，H。Lowenstein，J。Mayette，S.R。 Foster，D。Damby，I.W。Howe，I。Aktan，R.E。 Meyer，M.E。Poynter和J.E. Boyston。 NKT单元格数和功能的广泛自然变化。免疫学（印刷中）。 J.E. Boyston，I。Aktan，D。Barkhuff和A. Chant。母体界面处的NKT细胞。 （提交）。 小奥尔森（C.M. Olson Jr.），贝茨（T.C.不变天然杀伤细胞对干扰素伽马的局部调节调节急性莱姆心炎。 （提交）。 指导摘要： 伊丽莎白·邦尼 我目前会定期与所有学员会面，并讨论科学以及有关报告，演讲和出版物的帮助。 我还参加一般职业建议。 乔恩·博伊森（Jon Boyson）是一名柯布尔初级教师，是我赠款“胎盘免疫对LCMV” HD047224的共同投资者。 我们还正在协作四聚体的生产，以检测NKT细胞和LCMV特异性CD8 T细胞。 此外，我是他最近申请的咨询合作者：CD1D限制的T细胞和妊娠损失R01AI067897 我今年的目标是帮助乔恩（Jon 莎莉·休伯（Sally Huber） 在过去的一年中，我两次与乔恩（Jon）会面，讨论了一个潜在的合作，以确定伽马 - 戴尔塔T细胞和天然杀伤T细胞之间的串扰，这两种细胞都识别CD1D是一种非经典的主要组织相容性复合物I类喜欢的分子。 NKT和伽马 - 戴尔塔T细胞都是先天效应子，并调节病毒感染中的适应性免疫。要解决的问题是它们是否协同或彼此对抗。我还为如何在他的NIH RO1赠款上撰写他的第一个进度报告提供了帮助。 乔恩（Jon）目前正处于RO1的第二年，并有望从今年的鞋垫中逐步淘汰。据推测，我将被分配一个新的受训者。