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

项目摘要： 长期以来，主要组织相容性复合体(MHC)分子一直被认为是 先天免疫细胞群和适应性免疫细胞群。超越呈现多肽的经典MHC I和II类 分子，在人类中存在着各种结构上相关的蛋白质，它们被发现存在于 不同的抗原类型到各种T细胞亚型。其中包括单态的MHC相关蛋白1。 (MR1)，历史上以将微生物衍生的核黄素代谢物呈递给Mucosal- 相关不变T(MAIT)细胞。在这个初步观察的基础上，我们的团队和其他人已经表明 MR1的配基组比以前认识的要多样化得多。MR1能够呈递配体 除了最初描述的利必特代谢物之外，还有各种大小和化学结构的 携带完全重排的αβTCR的MR1限制性T细胞(MR1T)。最近，有新的报道称 已显示出MR1依赖的细胞毒活性的MR1T克隆对完整的 没有任何微生物感染或微生物配体。由于这些观测的性质是前所未有的 和基于MR1的免疫疗法的潜在影响，重要的是由 MR1在癌症诱导的细胞功能障碍的背景下被识别。考虑到我们自己 研究和这些新的报告，我们认为MR1作为细胞代谢组的传感器在两个 微生物感染和内源性功能障碍，如癌症，呈现一系列不同的代谢物 抗原。由于我们在研究MR1生物化学方面的既定记录，以及我们现有的合作 作为MR1的配基发现流水线，亚当斯实验室是唯一准备调查抗原身份的机构 MR1是在癌症的背景下提出的。我们的调查将集中在浆细胞癌，多个 骨髓瘤，作为这种癌症的细胞系，已经被证明在该细胞上有异常高表达的MR1 表面，表明可能有较高的抗原量。我们认为我们已有的微生物配基发现 MR1的流水线可以用来研究肿瘤相关抗原(TAAs) 通过在骨髓瘤细胞系中直接表达我们的MR1结构或通过在这些细胞系中添加其 将裂解物放入转基因生产细胞的培养液中；这两种技术我们以前都使用过。身份识别 通过我们的合作，将通过LC-MS/MS确定从这些实验中负载到MR1中的配体 俄克拉何马大学健康科学中心的希尔德布兰德实验室。根据这一决心，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MR1口袋。最后， 我们将使用MR1/TAA四聚体对供者血液样本中的MR1T克隆进行染色，并研究它们的 转录表型阐明对这些抗原有反应的同源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.