Molecular and functional investigation of the role of CD1 in gamma delta T cell surveillance

γδ T细胞构成针对感染因子和癌性免疫应答的重要组分。 转化，但生化机制，他们检测抗原通过其体细胞 重组T细胞受体（TCR）的作用尚不清楚。与αβ TCR不同，α β TCR仅限于识别抗原 在主要组织相容性复合物（MHC）分子的背景下，γδ TCR可以识别多种配体， 从自身MHC到完整的、未加工的病毒糖蛋白。我们最近的工作建立了CD 1 分子作为人Vδ1 γδ T细胞亚群的配体，产生强大的功能性、生物化学和生物学特性。 结构性证据。我们试图将我们的研究扩展到人类肠道，在那里γδ T细胞，特别是Vδ1+， T细胞占优势。我们的初步数据表明，CD 1识别是强大的，并存在于所有个人 研究表明，肿瘤中的CD 1反应性γδ T细胞与 健康的邻近组织。因此，该提案的长期目标是充分表征这种CD 1反应性的细胞因子。 在肿瘤与健康组织中，检测其功能性效应子表型、TCR库和 免疫调节信号，除了TCR，形成募集，激活和潜在的扩张 这些细胞与一种高度相关的人类疾病结直肠癌的关系。我们的第一个目标， “Characterization of CD1-specific γδ T cells in normal and diseased tissue."，试图利用经典的细胞 通过直接离体功能和转录分析补充扩增，以分析CD 1反应性和非反应性 来源于肿瘤和邻近健康组织的反应性T细胞群。这些数据将提供洞察力， 与健康组织相比，肿瘤微环境中调节γδ T细胞的信号。我们的第二 目的，“阐明γδ TCR与CD 1/脂质复合物结合的分子机制"，将 重点在于表征这些细胞表达的γδ TCR与CD 1/脂质抗原之间的相互作用。 我们将使用蛋白质生物化学、生物物理学和x射线晶体学来阐明分子机制， 其中γδ TCR识别CD 1/脂质。我们的努力将大大扩大我们对具体的 在人类健康和疾病中调节γδ T细胞活性的信号。我们的第三个目标，“确定 以及配体、共刺激和/或共受体分子在CD 1特异性γδ T细胞活化中的作用， “结肠中的表型”将表征可以调节结肠的配体和免疫调节信号。 人结直肠癌中CD 1反应性γδ T细胞的活性。我们将联合收割机RNAseq和 使用脐带血来源的幼稚Vδ1细胞进行分化测定以测试候选信号的相关性。这 将通过0 P9/DL 1系统由体外衍生的天然VSlT细胞补充。γδ T细胞可以是 促炎或调节，因此我们试图了解这些细胞在这种疾病中发挥的作用，如果有的话， 状态总之，这些目标将开始揭开γδ T细胞在人类免疫生物学中的神秘面纱，无论是在 细胞和分子水平。

γδ T cells constitute an important component of the immune response against infectious agents and cancerous transformations, yet the biochemical mechanisms by which they detect antigen through their somatically recombined T cell receptor (TCR) remain unclear. Unlike αβTCRs, which are restricted to recognizing antigens in the context of Major Histocompatibility Complex (MHC) molecules, γδTCRs can recognize a diversity of ligands ranging from self MHC to intact, unprocessed, viral glycoproteins. Our recent work has established CD1 molecules as ligands for a subpopulation of human Vδ1 γδ T cells, producing robust functional, biochemical and structural evidence. We seek to extend our studies to the human gut, where γδ T cells, and in particular, Vδ1+ T cells, predominate. Our preliminary data suggests that CD1 recognition is robust and present in all individuals examined, and that there exist important functional differences between CD1 reactive γδ T cells in tumors versus healthy adjoining tissue. Thus, the long-term goal of this proposal is to fully characterize this CD1 reactive population in tumors versus healthy tissue, examining their functional effector phenotypes, TCR repertoire and immunomodulatory signals, in addition to the TCR, that shape the recruitment, activation and potential expansion of these cells in the context of a highly relevant human disease, colorectal cancer. Our first aim, “Characterization of CD1-specific γδ T cells in normal and diseased tissue.”, seeks to use classical cellular expansions complemented by direct ex vivo functional and transcript analysis to profile CD1 reactive and non- reactive T cell populations derived from tumor and adjoining healthy tissue. These data will provide insight into the signals that regulate γδ T cells within the tumor microenvironment compared to healthy tissue. Our second aim, “Elucidation of the molecular mechanisms by which γδ TCRs bind to CD1/lipid complexes.”, will focus on characterizing the interaction between the γδ TCRs expressed by these cells and CD1/lipid antigen. We will use protein biochemistry, biophysics and x-ray crystallography to elucide the molecular mechanisms by which the γδ TCR recognizes CD1/lipid. Our effort will significantly expand our understanding of the specific signals that regulate γδ T cell activity in human health and disease. Our third aim, “Determine the presence and role of ligand, co-stimulatory and/or co-receptor molecules in CD1 specific γδ T cell activation and phenotype in the colon” will characterize the ligand and immunomodulatory signals that may regulate the activity of CD1 reactive γδ T cells in the context of human colorectal cancer. We will combine RNAseq and differentiation assays using cord blood derived, naïve Vδ1 cells to test the relevance of candidate signals. This will be complemented by in vitro derived native Vδ1 T cells through the OP9/DL1 system. γδ T cells can be either pro-inflammatory or regulatory, therefore we seek to understand which role these cells play, if any, in this disease state. Together, these aims will begin to unravel the mystery of γδ T cells in human immunobiology, both at the cellular and molecular levels.