Proteasome subunit beta5t and thymus-specific peptides in T cell selection

T 细胞选择中的蛋白酶体亚基 beta5t 和胸腺特异性肽

• 批准号: 8701462
• 负责人: John J. Monaco
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701462
• 关键词: Adoptive Transfer; Affinity; Aminopeptidase; Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens; Autoimmune Diseases; Autoimmune Process; Autoimmune Responses; Autoimmunity; Bacteria; Binding; CD8B1 gene; Calcium; Cell physiology; Cells; Cleaved cell; Complex; Cytosol; Cytotoxic T-Lymphocytes; Development; Diagnosis; Disease; Ectopic Expression; Effector Cell; Epithelial Cells; Epitopes; Etiology; Eukaryotic Cell; Event; Exopeptidase; Generations; Graft Rejection; Immune; Immune response; Immunologic Deficiency Syndromes; In Vitro; Individual; Interferons; Knowledge; Label; Lead; Life; Ligands; Major Histocompatibility Complex; Mature T-Lymphocyte; Measurable; Measures; Methods; Modeling; Monitor; Mouse Cell Line; Mus; Neoplasm Transplantation; Outcome; Pathogenesis; Pathway interactions; Peptide Hydrolases; Peptides; Peripheral; Phase; Phosphotransferases; Process; Production; Proteasome Inhibitor; Proteins; Relative (related person); Role; Shapes; Site; Specificity; T cell response; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; Therapeutic; Thymus Gland; Tissues; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Ubiquitin; Virus; antigen processing; cytokine; design; human disease; in vivo; lymph nodes; multicatalytic endopeptidase complex; neoplastic cell; promoter; public health relevance; receptor expression; research study; response

DESCRIPTION (provided by applicant): The composition of the antigen-specific T cell receptor (TcR) repertoire is a critical determinant of the immune status of an individual. It has been shown that 'holes' in the T cell repertoire can lead to unresponsiveness to certain antigens, while escape of T cells from the process of negative selection can lead to self-reactive TcR expression and autoimmune disease. Thus, understanding the mechanisms used to generate the T cell repertoire are critical, and represent the major long term objective of this proposal. Current dogma dictates that only those T cells passing two developmental checkpoints in the thymus, positive selection and negative selection, can contribute to the mature T cell repertoire, and that these selective processes operate with the same TcR ligands (self peptides + self MHC), with relative affinity being the sole determinant of the outcome. The recent discovery of a thymus-specific component of proteasomes, the proteases that produce the self-peptides that bind to MHC molecules to create these ligands, challenges this dogma, and strongly suggests that a unique set of peptide ligands is used for positive selection. However, T cell activation by such a unique set of ligands has never been directly demonstrated, and is the major specific aim of this proposal. Specifically, the thymus-specific proteasome subunit (¿5t) will be ectopically expressed in non-thymic antigen presenting cells. In order to determine whether these cells express new peptide/MHC ligands that can be recognized by the naturally selected T cell repertoire, normal splenic or lymph node T cells will be mixed in vitro, and selected T cell responses will be measured, including proximal events such as changes in intracellular calcium levels and ZAP70 and erk kinase acticvation, as well as downstream events including proliferation, cytokine production and differentiation into effector cells. In vivo responses to ¿5t-expressing cells will be observed in adoptive-transfer and tumor transplantation models to determine whether in vitro responses correlate with in vivo recognition. To determine whether alterations in protease activity in otherwise normal host cells can lead to autoimmune responses, ¿5t will be expressed in transgenic animals under the control of regulatable, tissue-specific promoters. These experiments will either validate or contradict the proposed role of thymic-specific peptide ligands during T cell selection and development.

描述（由申请方提供）：抗原特异性T细胞受体（TcR）库的组成是个体免疫状态的关键决定因素。已经表明，T细胞库中的“孔”可导致对某些抗原的无反应性，而T细胞从阴性选择过程中逃逸可导致自身反应性TcR表达和自身免疫性疾病。因此，理解用于产生T细胞库的机制是至关重要的，并且代表了该提议的主要长期目标。目前的教条规定，只有那些T细胞通过胸腺中的两个发育检查点，阳性选择和阴性选择，可以有助于成熟的T细胞库，这些选择性过程与相同的TcR配体（自身肽+自身MHC），相对亲和力是唯一的决定因素的结果。最近发现的胸腺特异性组分的蛋白酶体，蛋白酶产生的自我肽，结合到MHC分子，以创建这些配体，挑战这一教条，并强烈建议，一套独特的肽配体用于积极的选择。然而，这种独特的配体组的T细胞活化从未被直接证明，并且是该提议的主要具体目标。特别是，胸腺特异性蛋白酶体亚单位（<$5t）将异位表达在非胸腺抗原呈递细胞。为了确定这些细胞是否表达可被天然选择的T细胞库识别的新的肽/MHC配体，将在体外混合正常的脾或淋巴结T细胞，并测量选择的T细胞应答，包括近端事件，如细胞内钙水平的变化和ZAP 70和erk激酶活化，以及下游事件，包括增殖，细胞因子产生和分化成效应细胞。体内反应将在过继转移和肿瘤移植模型中观察表达5 t的细胞，以确定体外应答是否与体内识别相关。为了确定蛋白酶活性在其他正常宿主细胞中的改变是否会导致自身免疫应答，将在可调控的组织特异性启动子的控制下在转基因动物中表达p5 t。这些实验将验证或反驳胸腺特异性肽配体在T细胞选择和发育过程中的作用。