Investigation into the Endogenous Ligand Repertoire of the Non-Classical MHC-Related Protein, MR1 in Multiple Myeloma Cell Lines

项目摘要： 主要组织相容性复合物（MHC）分子长期以来一直被认为是 先天性和适应性免疫细胞群。除了经典的肽呈递MHC I类和II类 分子，在人类中存在多种其他结构相关的蛋白质， 不同的抗原类型到各种T细胞亚型。其中包括单态MHC相关蛋白1 (MR1)其在历史上以将微生物来源的核黄素代谢物呈递给粘液菌而闻名， 相关不变T（MAIT）细胞。在这一初步观察的基础上，我们的小组和其他人已经证明， MR 1的配体组比以前认识到的更加多样。MR 1能够呈递配体 除了最初描述的核糖基代谢物之外，还有各种大小和化学结构， 携带完全重排的αβ TCR的MR 1限制性T细胞（MR 1 Ts）。最近有报道称 的MR 1 T克隆，这些克隆已经显示出对完整的肿瘤组织的MR 1依赖性细胞毒活性。 不存在任何微生物感染或微生物配体。由于这些观察的前所未有的性质 以及基于MR 1的免疫疗法的潜在影响，重要的是， MR 1在癌症诱导的细胞功能障碍的背景下被确定。考虑到我们自己的 研究和这些新的报告，我们提出，MR 1作为一个传感器的细胞代谢组在这两个 在微生物感染和内源性功能障碍（如癌症）的背景下， 抗原由于我们建立了研究MR 1生物化学的跟踪记录， 作为MR 1的配体发现管道，亚当斯实验室是唯一准备研究抗原的身份 MR 1在癌症中的表现。我们的调查将集中在浆细胞癌，多个 骨髓瘤，因为这种癌症的细胞系已经显示在细胞中具有异常高的MR 1表达 表面，表明更高的可能的抗原负荷。我们认为我们已经发现的微生物配体 MR 1的管道可以适用于研究肿瘤相关抗原（TAA）， 通过在这些细胞系中直接表达我们的MR 1构建体或通过加入它们的 裂解物进入转染的生产细胞的培养基中;这两种技术我们以前都用过。鉴定 通过我们的合作，将通过LC-MS/MS测定从这些实验中加载到MR 1中的配体 与俄克拉荷马州大学健康科学中心的希尔德布兰德实验室合作。根据这一决定，我们 将利用我们的结构生物学专业知识来确定潜在的配体如何结合MR 1口袋。最后， 我们将使用MR 1/TAA四聚体对来自供体血液样品的MR 1 T克隆进行染色，并研究它们的生物学特性。 转录表型，以阐明对这些抗原有反应性的同源T细胞群体。

Project Abstract: Major Histocompatibility Complex (MHC) molecules have been long been appreciated as the bridge between the innate and adaptive immune cell populations. Beyond the peptide-presenting classical MHC class I and II molecules, there exists a variety of other structurally related proteins in humans that have been found to present diverse antigen types to a variety of T cell sub-types. Among these is the monomorphic MHC-related protein 1 (MR1) which is historically known for the presentation of microbially-derived riboflavin metabolites to Mucosal- Associated Invariant T (MAIT) cells. Expanding on this initial observation, our group and others have shown that the ligandome of MR1 is much more diverse than previously appreciated. MR1 is capable of presenting ligands of various sizes and chemical structures beyond the originally described ribityl metabolites to a vast array of MR1-restricted T cells (MR1Ts) carrying fully rearranged αβ TCRs. Recently, there have been emerging reports of MR1T clones that have shown MR1-dependent cytotoxic activity against cancerous tissues in the complete absence of any microbial infection or microbial ligands. Due to the unprecedented nature of these observations and the potential impact of an MR1-based immunotherapy, it is important that the ligands being presented by MR1 in the context of cancer-induced cellular disfunction be identified. Taking into consideration our own research and these novel reports, we propose that MR1 acts as a sensor of the cellular metabolome in both the context of microbial infection and endogenous disfunction like cancer, presenting a diverse array of metabolite antigens. Due to our established track record investigating the biochemistry of MR1 and our existing collaborative ligand discovery pipeline for MR1, The Adams Lab is uniquely poised to investigate the identity of the antigens that MR1 is presenting in the context of cancer. Our investigation will focus on the plasma cell cancer, multiple myeloma, as cell lines of this cancer have been shown to have abnormally high expression of MR1 at the cell surface, indicating a higher possible antigenic load. We propose that our established microbial-ligand discovery pipeline for MR1 can be adapted to investigate the tumor associated antigens (TAAs) being presented on the surface of myeloma cells by either expressing our MR1 construct directly in these cell lines or by adding their lysate into the media of transfected producing cells; both techniques that we have used before. Identification of the ligands loaded into MR1 from these experiments will be determined by LC-MS/MS through our collaboration with the Hildebrand Lab at the Oklahoma University Health Sciences Center. Following this determination, we will capitalize on our structural biology expertise to determine how potential ligands bind the MR1 pocket. Finally, we will use MR1/TAA tetramers to stain MR1T clones from donor blood samples and investigate their transcriptional phenotype to shed light on the cognate T cell population that is reactive to these antigens.