MHC restriction and antigen characterization of mucosal CD8aa TCRab IEL

粘膜 CD8aa TCRab IEL 的 MHC 限制和抗原表征

• 批准号: 8114949
• 负责人: Florence Lambolez
• 金额: $ 27.27万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8114949
• 关键词: Address; Agonist; Antigen Targeting; Antigen-Presenting Cells; Antigens; Apoptosis; Biological Assay; Biology; Bone Marrow Cells; CD8B1 gene; Cell Line; Cells; Cellular Stress; Cellular biology; Chimera organism; Cloning; Consensus; Data; Dendritic Cells; Development; Epithelial Cells; Fractionation; Generations; High Pressure Liquid Chromatography; Histocompatibility Antigens Class II; Hour; Hybridomas; Immune; Immune response; Immune system; Immunity; In Vitro; Inflammatory Bowel Diseases; Intestines; Knock-out; Lead; Link; Lymphocyte; Lymphocyte Biology; Lymphocyte Subset; Lymphocyte antigen; Major Histocompatibility Complex; Mass Spectrum Analysis; Methods; Modality; Modeling; Mouse Strains; Mus; Pathway interactions; Peptides; Phenotype; Play; Population; Population Heterogeneity; Preparation; Process; Research; Retroviridae; Role; Signal Transduction; Small Intestines; Specificity; System; T-Cell Antigen Receptor Specificity; T-Cell Receptor; T-Lymphocyte; Thymoma; Thymus Gland; Transgenic Mice; Transgenic Model; bacterial lysate; base; clinical practice; experience; food antigen; in vivo; insight; intraepithelial; novel therapeutics; pathogen; thymocyte

DESCRIPTION (provided by applicant): Although intestinal intraepithelial lymphocytes (IEL) constitute one of the largest T cell compartments of the body our understanding of their development and function is conspicuously limited. The objective of the presented study is to provide insight into the most fundamental aspects of IEL biology, addressing the question of their antigen-major histocompatibility complex (MHC) restriction and potentially also their antigen specificity. Our research will focus on CD8aa TCRa¿ IEL populations. In the first part of this project, we will immortalize IEL by fusing them with a thymoma cell line to generate T-cell hybridomas that will be subsequently used in a number of functional assays. Initially, they will be stimulated with different antigen preparations (such as intraepithelial cell lysates, bacterial lysates, food antigen preparations) presented by a variety of cells including various populations of dendritic cells and epithelial cells. In case of positive activation, the identification of the restricting MHC molecules will be investigated using antigen-presenting cells isolated from various MHC deficient mice strains. Antigen identification will then be attempted by HPLC sequential fractionations and mass spectrometry. In the second aim, we will sequence and clone the TCRa- and -¿ chains of the hybridomas generated previously. We will then retrovirally transfect these TCRs into bone marrow cells (BM) and inject them into various lethally irradiated MHC hosts. Analysis of the different BM chimeras should allow us to define the MHC restriction of CD8aa TCRa¿ IELs in vivo. Both aims are complementary and will lead to the identification of the antigen and MHC restriction of CD8aa TCRa¿ IELs. The results of this study will provide us with a tremendous amount of new and important information regarding the biology of CD8aa TCRa¿ IELs. Better understanding of the intestinal immune system holds promise of the development of novel therapeutic modalities for a number of intestinal inflammatory disorders. PUBLIC HEALTH RELEVANCE: Given the profound role intestinal immune system plays in generation of tolerance and protective immunity, it is surprising how little the potential of this system has been utilized in clinical practice. Unconventional intraepithelial lymphocytes constitute a substantial population of T cells in the intestine, yet their role remains elusive. Here we set out to explain what are the target antigens for these cells and the mechanisms of their recognition, information that is critical in understanding how the intestinal immune lymphocytes are capable of eliciting an immune response against pathogens.

描述(由申请人提供)：尽管肠道上皮内淋巴细胞(IEL)是人体最大的T细胞隔间之一，但我们对其发育和功能的了解明显有限。这项研究的目的是深入了解IEL生物学的最基本方面，解决它们的抗原主要组织相容性复合体(MHC)限制的问题，并潜在地解决它们的抗原特异性。我们的研究将集中在CD8aa TCRA？IEL人群。在这个项目的第一部分，我们将通过将IEL与胸腺瘤细胞系融合来产生T细胞杂交瘤，从而使IEL永生化，该杂交瘤将随后用于一些功能分析。最初，他们将被不同的抗原制剂(如上皮内细胞裂解物、细菌裂解物、食物抗原制剂)刺激，这些抗原制剂由包括不同群体的树突状细胞和上皮细胞在内的各种细胞呈递。在阳性激活的情况下，将使用从各种MHC缺陷小鼠品系分离的抗原提呈细胞来研究限制MHC分子的鉴定。然后，将尝试通过高效液相色谱顺序分离和质谱学进行抗原鉴定。在第二个目标中，我们将对先前产生的杂交瘤细胞的TCRA链进行测序和克隆。然后，我们将这些TCR逆转录病毒转染到骨髓细胞(BM)中，并将它们注射到各种致死照射的MHC宿主中。对不同的BM嵌合体的分析应该允许我们确定CD8aa TCRA？IEL在体内的MHC限制。这两个目的是相辅相成的，并将导致CD8aa TCRA？IEL的抗原鉴定和MHC限制。这项研究的结果将为我们提供大量关于CD8aa TCRA？IEL生物学的新的重要信息。对肠道免疫系统的更好了解有望开发出一些肠道炎症性疾病的新治疗方法。 与公共卫生相关：鉴于肠道免疫系统在产生耐受和保护性免疫中发挥的深远作用，令人惊讶的是，该系统在临床实践中的潜力是如此之少。非常规上皮内淋巴细胞在肠道中构成了大量的T细胞，但它们的作用仍然难以捉摸。在这里，我们开始解释这些细胞的目标抗原是什么，以及它们的识别机制，这些信息对于了解肠道免疫淋巴细胞如何能够引发针对病原体的免疫反应至关重要。