Molecular Characterization of Myelin-Specific T cells from MS Patients

对MS易感基因的全基因组分析强调了MHC II类基因的重要性， 强烈暗示向CD4T细胞提呈抗原是MS发病机制中的一个关键过程。 然而，在体外研究MS患者的髓鞘特异性CD4T细胞一直是非常困难的，因为 T细胞受体对相关多肽-MHC复合体的亲和力。因此，有两个基本问题 仍未解决：第一，MS患者中髓鞘特异性T细胞的哪些分子特性 从健康个体中分离出来的？第二，髓鞘特异性和病毒特异性有区别吗？ 可以被利用来获得治疗收益的T细胞？在目标1中，我们将通过以下方式研究TCR识别 多发性硬化症患者的髓鞘特异性T细胞。成像实验确定了免疫学上的变化 髓鞘特异性T细胞与病毒特异性T细胞形成突触的比较和结构研究表明 从MS患者中分离的一些髓鞘特异性TCR的异常结合拓扑结构。加西亚实验室 最近发展了一种酵母肽-MHC展示方法来鉴定一种全新的多肽配体 给出了TCR，并表明其中一个肽被非常规TCR拓扑识别，并且 未能诱导信号。这意味着TCR结合拓扑可以对信令产生深远的影响。 这一方法将被用来研究TCR拓扑结构对髓鞘特异性T细胞功能的影响 细胞，并确定服用这种多肽是否可以预防人类自发性疾病 TCR/MHC转基因小鼠。在目标2中，我们将与Love博士和Hafler博士合作研究髓鞘和 来自多发性硬化症患者和对照受试者的病毒特异性T细胞通过纳米电池设备进行了鉴定。抗原性 T细胞将用机器人设备从纳米细胞中分离出来，这使得详细的分子 髓鞘特异性T细胞群的特征。多发性硬化症髓鞘特异性T细胞的转录组 患者和对照受试者将被检查，重点是与以下相关的基因的表达 对MS、转录因子和细胞因子信号分子的易感性。髓鞘特异性克隆性扩增 将通过TCR链的测序和髓鞘反应性TCR的频率来评估 外周血和脑脊液T细胞的序列将通过TCR的光照度测序来确定 曲目。这些研究将确定髓鞘特异性T细胞是否具有特定的细胞因子活性 和/或与健康受试者相比，MS患者的基因表达程序优先扩展。

Genome-wide analyses of MS susceptibility loci have emphasized the importance of MHC class II genes, strongly implicating antigen presentation to CD4 T cells as a key process in the pathogenesis of MS. However, it has been very difficult to study myelin-specific CD4 T cells from MS patients ex vivo, due to low T cell receptor affinities for the relevant peptide-MHC complexes. Therefore, two fundamental questions remain unresolved: First, which molecular properties distinguish myelin-specific T cells in patients with MS from those in healthy individuals? Second, are there distinguishing properties of myelin-specific and virusspecific T cells that could be exploited for therapeutic gain? In Aim 1, we will study TCR recognition by myelin-specific T cells from MS patients. Imaging experiments identified alterations in immunological synapse formation by myelin-specific compared to virus-specific T cells, and structural studies demonstrated unusual binding topologies by some myelin-specific TCRs isolated from MS patients. The Garcia lab recently developed a yeast peptide-MHC display approach to identify entirely novel peptide ligands for a given TCR and showed that one ofthe peptides was recognized with a non-conventional TCR topology and failed to induce signaling. This means that TCR binding topology can have a profound effect on signaling. This approach will be used to examine the irnpact of TCR topology on the function of myelin-specific T cells and determine whether administration of such peptides can prevent spontaneous disease in human TCR/MHC transgenic mice. In Aim 2, we will collaborate with Drs. Love and Hafler to study myelin and virus-specific T cells from MS patients and control subjects identified with the nanowell device. Antigenresponsive T cells will be isolated from nanowells with a robotic device, which enables detailed molecular characterization of myelin-specific T cell populations. The transcriptome of myelin-specific T cells from MS patients and control subjects will be examined, with an emphasis on the expression of genes associated with susceptibility to MS, transcription factors and cytokine signaling molecules. Clonal expansion of myelinspecific will be assessed by sequencing of TCR chains, and the frequency of myelin-reactive TCR sequences in peripheral blood and CSF T cells will be determined by lllumina sequencing ofthe TCR repertoire. These studies will determine whether myelin-specific T cells with particular cytokine proflles and/or gene expression programs are preferentially expanded in MS patients compared to healthy subjects.