MHC restriction and antigen characterization of mucosal CD8aa TCRab IEL

描述（由申请人提供）：虽然肠上皮内淋巴细胞（IEL）是人体最大的T细胞区室之一，但我们对其发育和功能的了解明显有限。本研究的目的是深入了解IEL生物学的最基本方面，解决其抗原主要组织相容性复合体（MHC）限制的问题，以及可能的抗原特异性问题。我们的研究将集中在CD8aa TCRa¿IEL人群上。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，该杂交瘤随后将用于许多功能分析。最初，它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，这些抗原制剂由各种细胞（包括各种树突状细胞和上皮细胞）呈递。在阳性激活的情况下，将使用从各种MHC缺陷小鼠品系中分离的抗原提呈细胞来研究限制性MHC分子的鉴定。抗原鉴定将尝试通过高效液相色谱序列分离和质谱。在第二个目标中，我们将对先前产生的杂交瘤的TCRa链和-¿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，并将其注射到各种致命辐照的MHC宿主中。对不同BM嵌合体的分析应使我们能够确定CD8aa TCRa¿IELs在体内的MHC限制。这两个目标是互补的，并将导致CD8aa TCRa¿IELs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¿IELs生物学的新的重要信息。更好地了解肠道免疫系统有望为许多肠道炎症性疾病开发新的治疗方式。

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.