Defining the influence of RA genetic susceptibility factors on T cell antigen specificity and functional state

项目摘要 在过去的R 01资助期间，我们绘制了MHC区域内的因果效应， 特异性HLA-DR结合沟氨基酸位点，鉴定>100例类风湿关节炎（RA）非 MHC风险等位基因在整个基因组中，并已证明这些等位基因在很大程度上是 在CD 4 + T细胞调节元件内。如果我们能明确基因机制 支持RA易感性，那么就有可能定义治疗策略， 消除或预防RA。其核心是定义参与介导疾病的关键T细胞 易感性-就其独特TCR序列特征和其致病细胞而言 states.在此，我们假设HLA-DR风险等位基因在胸腺内起作用，以利于选择 因此，当自身抗原呈递给前哨TCR时， T细胞增强子改变T细胞特异性基因调控，这使得幼稚T细胞能够表达 使前哨TCR转变为致病状态。我们将“哨兵TCR”定义为 与瓜氨酸肽结合并触发初始自身免疫反应的受体。风险 T细胞启动子和增强子中的等位基因改变了调节T细胞增殖的关键T细胞基因的调节。 T细胞向致病状态的转变。因此，具有罕见的“哨兵TCR”的T细胞可以在 在适当的条件下，触发最初的自身免疫反应， 反应，并引发持续的关节炎症。但是，关键的致病性T细胞状态， 前哨TCR和特异性因果调节T细胞等位基因的作用尚未完全确定。 用于确定血液和组织中TCR序列的全套测序， 解决T细胞状态，以及基因工程来询问因果T细胞等位基因和基因 代表了我们实验室已经开发出的令人兴奋的专业方法。在本提案中，我们 寻求对这一模式的支持。首先，我们将证明HLA II类HLA RA风险 等位基因改变TCR库以窝藏“哨兵TCR”，使用来自300名受试者的TCR和基因型数据， 健康的个体。接下来，我们将使用多基因RA风险模型来定义关键的T细胞状态 RA风险等位基因影响单细胞数据的表面标志物和转录本， 同样的300人。最后，我们将定义非编码等位基因的分子机制 在CD 4 + T细胞中使用基因组编辑;为此，我们将开发和应用编辑策略， 原代T细胞，测序DNA以确认所需编辑的存在，并获得RNA， ATAC测序数据，以了解编辑的影响，并确认非功能性 编码等位基因

Project Summary During this past R01 funding period, we have mapped causal effects within the MHC region to specific HLA-DR binding groove amino acid sites, identified >100 rheumatoid arthritis (RA) non- MHC risk alleles across the genome, and have demonstrated that these alleles are largely within CD4+ T cell regulatory elements. If we could define the genetic mechanisms underpinning RA susceptibility, then it may be possible to define therapeutic strategies to abrogate or prevent RA. Central to this is defining the key T cells involved in mediating disease susceptibility – both in terms of their unique TCR sequence features and their pathogenic cell states. Here, we hypothesize that HLA-DR risk alleles act within the thymus to favor selection of “sentinel TCRs”, and that when autoantigens are presented to sentinel TCRs, risk alleles within T cell enhancers alter T cell specific gene regulation, which enables naïve T cells expressing sentinel TCRs to transition into a pathogenic state. We define “sentinel TCRs” as those receptors that bind to citrullinated peptides and trigger the initial autoimmune response. Risk alleles in T cell promoters and enhancers alter regulation of critical T cell genes that regulate the transition of T cells into pathogenic states. Hence, a T cell with a rare “sentinel TCR” can, under the right conditions, trigger the initial autoimmune response, drive a spreading immune response, and initiate persistent joint inflammation. But, the key pathogenic T cell states, sentinel TCRs, and the action of specific causal regulatory T cell alleles are not yet fully defined. Repertoire sequencing to define TCR sequences in blood and tissue, single cell analyses to resolve T cell states, and genetic engineering to interrogate causal T cell alleles and genes represent exciting methodologies that our lab has developed expertise in. In this proposal we seek to build support for this model. First, we will demonstrate that HLA class II HLA RA risk alleles alter TCR repertoire to harbor “sentinel TCRs”, using TCR and genotype data from 300 healthy individuals. Next, we will use polygenic RA risk models to define the key T cell states that RA risk alleles influence with single cell data on surface markers and transcripts in the same 300 individuals. Finally, we will define the molecular mechanisms of non-coding alleles using genomic editing in CD4+ T cells; to this end we will develop and apply strategies to edit primary T cells, sequence DNA to confirm the presence of the desired edit, and obtain RNA and ATAC sequencing data to understand the impact of the edit and confirm the functionality of non- coding alleles.