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）构成人体最大的T细胞区室之一，但我们对其发育和功能的理解明显有限。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）限制性问题，并可能解决其抗原特异性问题。我们的研究将集中在CD 8aa TCR α IEL人群。在本项目的第一部分，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，所述T细胞杂交瘤随后将用于许多功能测定。最初，它们将用不同的抗原制剂（例如上皮内细胞裂解物、细菌裂解物、食物抗原制剂）刺激，所述抗原制剂由包括各种树突细胞和上皮细胞群体的各种细胞呈递。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呈递细胞研究限制性MHC分子的鉴定。然后尝试通过HPLC连续分馏和质谱法进行抗原鉴定。在第二个目标中，我们将测序和克隆先前产生的杂交瘤的TCR α和TCR β链。然后，我们将逆转录病毒介导这些TCR进入骨髓细胞（BM），并将它们注射到各种致死辐射的MHC宿主中。分析不同的BM嵌合体应该使我们能够定义体内CD 8aa TCR α IEL的MHC限制。这两个目标是互补的，并将导致识别抗原和CD 8aa TCR α IEL的MHC限制。这项研究的结果将为我们提供大量关于CD 8aa TCR α IELS生物学的新的重要信息。对肠道免疫系统的更好理解有望为许多肠道炎症性疾病开发新的治疗方法。 公共卫生相关性：鉴于肠道免疫系统在产生耐受性和保护性免疫中发挥的重要作用，令人惊讶的是，该系统在临床实践中的潜力很少。非常规的上皮内淋巴细胞构成了肠道中大量的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靶抗原是什么以及它们的识别机制，这些信息对于理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。

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.