Studies of Antigen Stimulation

描述（由申请人提供）：我们研究的目的是解释具有强大生化、定量和机制基础的II类MHC分子的抗原提呈，以避免主导这个复杂领域的许多经验主义。我们研究了模式蛋白抗原鸡卵清溶菌酶（HEL）是如何被抗原呈递细胞（APC）加工的，并建立了其各种肽与I-Ak分子的加工、选择和结合的化学基础。这使我们处于这样一种情况，即这些参数可以与T细胞反应的生物学密切相关，即与HEL克隆的特异性和频率密切相关。 该更新的第一个目的是检查当APC被细胞因子激活时诱导的HEL肽的翻译后修饰。有证据表明，活化的APC可以修饰HEL，以产生针对修饰肽的特异性T细胞。检查的最初焦点是HEL肽，其中它们的酪氨酸和色氨酸被硝化（或氧化）。这些计划是表征T细胞的特异性，诱导变化的APC的生物学，以及使用质谱方法的变化的生物化学性质。将在携带T细胞受体作为转基因的小鼠中检查T细胞对修饰肽的生物学;我们打算鉴定T细胞在炎症和组织病理学中的可能作用，重点是表达HEL的β细胞。 在第二个目标中，寻求解释为什么对各种HEL表位的应答与APC呈递的肽-MHC复合物的数量无关。我们可以定量来自HEL的各种肽-MHC复合物的密度：高水平的那些诱导的T细胞数量与低约一至两百倍的那些诱导的T细胞数量大致相同。我们考虑了一些可能的解释，例如APC表面上复合物的分子竞争、竞争或协同性，复合物的持续时间，最后调节鼠溶菌酶的存在，该溶菌酶是一种通常在APC上表达的强交叉反应蛋白。通过了解这些重要的变量，我们应该能够对CD 4 T细胞反应有更全面的了解。 实验是基于; i）使用具有改变APC中表位表达或表位持续性的相关生物化学变化的HEL分子，ii）用TCR受体转基因小鼠追踪对多于一个表位的细胞应答，iii）在转移系统中使用APC;和iv）遗传性地消除鼠溶菌酶基因。

DESCRIPTION (provided by applicant): The purpose of our research is to explain antigen presentation by class II MHC molecules having a strong biochemical, quantitative and mechanistic foundation in order to avoid the many empiricisms that dominate this complex area. We investigated how the model protein antigen hen egg-white lysozyme (HEL) was processed by antigen presenting cells (APC); and established the chemical basis for the processing, selection by, and binding of, its various peptides to I-Ak molecules. This now places us in a situation where these parameters can be strictly related to the biology of the T cell response, i.e. to the specificities and frequencies of HEL clones. The first aim of this renewal is to examine post translational modification of the HEL peptides induced when APC are activated by cytokines. Evidence is presented that activated APC can modify HEL to generate specific T cells to the modified peptides. The initial focus of examination is on HEL peptides in which their tyrosines and tryptophans are nitrated (or oxidized). The plans are to characterize the specificities of the T cells, the biology of the APC that induces the changes, and the biochemical nature of the changes using mass spectrometry approaches. The biology of the T cells to modified peptides will be examined in mice bearing a T cell receptor as a transgene; we intend to identify the possible role of the T cells in inflammation and tissue pathology focusing on beta cells expressing HEL. In the second aim, an explanation is sought for why the response to the various HEL epitopes does not relate to the number of peptide-MHC complex presented by APC. We can quantitate the density of the various peptide-MHC complexes from HEL: those represented at a high level induce about the same number of T cells as those presented at about one- or two-hundred fold less. We consider a possible number of explanations such as molecular competition, competition or cooperativity of the complexes on APC surface, the time of persistence of the complexes, and finally the modulating presence of the murine lysozyme, a strong cross-reactive protein expressed normally on APC. By knowing these important variables, we should be able to have a much fuller understanding of the CD4 T cell responses. The experiments are based on; i) using HEL molecules with relevant biochemical changes that alter either epitope expressions or the persistence of an epitope in APC, ii) tracing the cellular response with TCR receptor transgenic mice to more than one epitope, iii) using APC in transfer systems; and, iv) genetically ablating the murine lysozyme gene.