Intermolecular Interactions in the Immunological Synapse

描述（由申请人提供）：T细胞表面蛋白的表达 与荧光蛋白融合，使得能够研究 活细胞中免疫突触。使用巧光共振能量 这些蛋白质之间的转移（FRET），分子之间的相互作用， 由于FRET要求蛋白质 彼此之间的距离在10 nm以内。与青色荧光蛋白（CFP）融合的CD 3 ζ， 将使用绿色（G）FP的衍生物和与黄色（Y）FP融合的CD 4， 分析II类限制性T细胞上的免疫突触。CFP和YFP 形成用于FRET的合适对，这使得能够测量TCR-CD 4 活细胞中的相互作用。初步数据显示， 识别抗原的T细胞分子。除了复制基因 转化为T细胞系，将制造表达这些蛋白质的转基因小鼠， 为许多实验提供了幼稚细胞的来源。最早的时候 在成熟突触形成之前， 研究，比较CD 4和TCR募集到接触区域，以及它们的 Ca 2+信号传导。突触形成对拮抗剂的反应 以及在对抗原应答的拮抗作用期间。的 脂筏的作用将使用筏相关的FP探测。T的能力 细胞与树突细胞上自然加工的抗原形成突触， 追究结晶学证据表明CD 4会二聚化。 1 过敏和免疫学研究部分 3 R01GM65230-01 October 2001 尼古拉斯·加斯科因 这将使用CD 4-YFP和CD 4-CFP之间的FRET进行测试。罚款 已经观察到突触相关的“系链”结构，并且它们与 将研究T细胞活化。人们对免疫学知之甚少 I类限制性T细胞中的突触。CD 3 zeta-CFP和CD 8-YFP将用于 研究突触的形成和TCR与 I类限制系统中的辅助受体。T细胞受体（TCR）系统，其中 激动剂和拮抗剂肽特别好地表征， 对突触中TCR-共受体相互作用的影响可能与 将使用TCR-MHCp复合物的结合常数。“TCR”的作用 突触中的“α-链连接肽基序”（α-CPM）将是 研究了使用来自CD 3-CFP，共受体-YFP， 和TCR，幼稚，激活和记忆T细胞之间突触形成的差异 细胞将被研究。双光子显微镜将用于T细胞成像 与淋巴结或胸腺内的组织细胞相互作用。免疫 在淋巴结中免疫应答期间和在阳性免疫应答期间 胸腺中的阴性选择将被可视化。

DESCRIPTION (provided by the applicant): Expression of T cell surface proteins fused to fluorescent proteins enables investigation of the formation of the immunological synapse in living cells. Using fluorescence resonance energy transfer (FRET) between these proteins, interactions between molecules during recognition of antigen can be visualized, since FRET requires that the proteins be within 10nm of each other. CD3zeta fused to cyan fluorescent protein (CFP), a derivative of green (G)FP, and CD4 fused to yellow (Y)FP will be used to analyze the immunological synapse on class II-restricted T cells. CFP and YFP form a suitable pair for FRET, which enables measurement of TCR-CD4 interactions in living cells. Preliminary data show FRET between these molecules on T cells recognizing antigen. In addition to transfecting the genes into T cell lines, transgenic mice will be made that express these proteins, to provide a source of naive cells useful for many experiments. The earliest time points in antigen recognition, before formation of the mature synapse, will be studied, to compare CD4 and TCR recruitment to the contact region, and their interaction, to Ca2+ signaling. Synapse formation in response to antagonists and during antagonism of a response to antigen will also be investigated. The role of lipid rafts will be probed using raft-associated FPs. The ability of T cells to make synapses with naturally processed antigen on dendritic cells will be investigated. There is evidence from crystallography that CD4 dimerizes. 1 ALLERGY AND IMMUNOLOGY STUDY SECTION 3 R01GM65230-01 OCTOBER 2001 GASCOIGNE, NICHOLAS R. This will be tested using FRET between CD4-YFP and CD4-CFP. Fine synapse-related "tether" structures have been observed, and their relevance to T cell activation will be studied. Little is known about the immunological synapse in class I-restricted T cells. CD3zeta-CFP and CD8-YFP will be used to investigate formation of the synapse and interactions between the TCR and coreceptor in a class I-restricted system. A T cell receptor (TCR) system where agonist and antagonist peptides are particularly well characterized and where effects on TCR-co-receptor interaction in the synapse can be related to the binding constants for the TCR-MHCp complex will be used. The role of the "TCR alpha-chain connecting peptide motif" (alpha-CPM) in the synapse will be investigated. Using T cells from mice transgenic for CD3-CFP, co-receptor-YFP, and TCR, differences in synapse formation between naive, activated and memory T cells will be investigated. Two-photon microscopy will be used to image T cells interacting with tissue cells within lymph node or thymus. Immunological synapse formation during an immune response in lymph node and during positive and negative selection in the thymus will be visualized.