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

描述（由申请人提供）：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。研究表明，T细胞库中的“孔”可导致对某些抗原的无反应，而T细胞从负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，了解用于生成T细胞库的机制至关重要，并且代表了本提案的主要长期目标。目前的理论认为，只有那些通过胸腺中两个发育检查点（阳性选择和阴性选择）的T细胞才能形成成熟的T细胞库，并且这些选择过程与相同的TcR配体（自身肽+自身MHC）一起操作，相对亲和力是结果的唯一决定因素。最近发现的一种胸腺特异性的蛋白酶体成分，这种蛋白酶能产生与MHC分子结合产生这些配体的自肽，挑战了这一教条，并强烈表明一组独特的肽配体用于阳性选择。然而，这样一组独特的配体对T细胞的激活从未被直接证明，这是本提案的主要具体目标。具体来说，胸腺特异性蛋白酶体亚基（¿5t）将在非胸腺抗原提呈细胞中异位表达。为了确定这些细胞是否表达可被自然选择的T细胞库识别的新肽/MHC配体，将在体外混合正常脾或淋巴结T细胞，并测量选定的T细胞反应，包括近端事件，如细胞内钙水平的变化和ZAP70和erk激酶激活，以及下游事件，包括增殖，细胞因子产生和分化为效应细胞。将在过继转移和肿瘤移植模型中观察对表达¿5t细胞的体内反应，以确定体外反应是否与体内识别相关。为了确定正常宿主细胞中蛋白酶活性的改变是否会导致自身免疫反应，将在可调节的组织特异性启动子的控制下在转基因动物中表达。这些实验将验证或反驳胸腺特异性肽配体在T细胞选择和发育中的作用。

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.