Molecular Characterization of Myelin-Specific T cells from MS Patients

MS易感基因座的全基因组分析强调了MHC II类基因的重要性， 强烈暗示抗原提呈至CD 4 T细胞是MS发病机制的关键过程。 然而，由于低的细胞毒性，离体研究来自MS患者的髓鞘特异性CD 4 T细胞非常困难。 T细胞受体对相关肽-MHC复合物的亲和力。因此，两个基本问题 仍然没有解决：首先，哪些分子特性区分MS患者的髓鞘特异性T细胞 与健康人的差异第二，是否存在髓鞘特异性和病毒特异性的区别特性 T细胞可以用于治疗？在目标1中，我们将通过以下方式研究TCR识别： 来自MS患者的髓鞘特异性T细胞成像实验确定了免疫学上的改变， 与病毒特异性T细胞相比，髓鞘特异性T细胞形成突触，结构研究表明 从MS患者中分离的一些髓鞘特异性TCR的不寻常的结合拓扑结构。加西亚实验室 最近开发了一种酵母肽-MHC展示方法，以鉴定一种全新的肽配体， 并显示其中一种肽被非常规TCR拓扑结构识别， 无法诱导信号。这意味着TCR结合拓扑结构可以对信号传导具有深远的影响。 这种方法将用于检查TCR拓扑结构对髓鞘特异性T细胞功能的影响。 细胞并确定施用此类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博士合作研究髓磷脂， 来自MS患者和对照受试者的病毒特异性T细胞，其用Mellell装置鉴定。抗原应答 T细胞将用机器人装置从T细胞中分离出来， 髓鞘特异性T细胞群的表征。MS髓鞘特异性T细胞的转录组 将对患者和对照受试者进行检查，重点是与以下相关的基因表达： MS易感性、转录因子和细胞因子信号分子。髓鞘特异性克隆性扩增 将通过TCR链的测序和髓磷脂反应性TCR的频率来评估 外周血和CSF T细胞中的序列将通过TCR的Illumina测序来确定 保留曲目。这些研究将确定是否髓鞘特异性T细胞与特定的细胞因子proflets 和/或基因表达程序优先在MS患者中扩展。

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.