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)谱系的组成是个人免疫状态的关键决定因素。已有研究表明，T细胞库中的“空洞”可导致对某些抗原无反应，而T细胞逃避负选择过程可导致自身反应性TCR表达和自身免疫性疾病。因此，了解用于产生T细胞库的机制是至关重要的，并代表了这项建议的主要长期目标。目前的理论认为，只有那些通过胸腺两个发育检查点(正选择和负选择)的T细胞才能为成熟的T细胞谱系做出贡献，并且这些选择过程使用相同的TCR配体(自体肽+自体MHC)，相对亲和力是唯一决定结果的因素。最近发现的蛋白酶体的胸腺特异性成分，即产生与MHC分子结合的自体肽的蛋白酶，挑战了这一教条，并强烈表明，一组独特的多肽配体用于正向选择。然而，由这样一组独特的配体激活T细胞从来没有被直接证明过，这是这一提议的主要具体目的。具体地说，胸腺特异的蛋白酶体亚单位(？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能够被自然选择的T细胞库识别的新的多肽/MHC配体，我们将在体外混合正常的脾或淋巴T细胞，并测量选定的T细胞的反应，包括最近的事件，如细胞内钙水平的变化和ZAP70和ERK激酶的激活，以及下游事件，包括增殖、细胞因子的产生和向效应细胞的分化。在过继转移和肿瘤移植模型中，将观察体内对表达5t的细胞的反应，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变化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5t。这些实验将验证或反驳胸腺特异性多肽配体在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.