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

项目摘要 在过去的R01资助期内，我们已将MHC区域内的因果关系映射为 特定的人类白细胞抗原-DR结合槽氨基酸位点，已确定&gt；100非类风湿性关节炎(RA) MHC风险等位基因，并已证明这些等位基因在很大程度上是 在CD4+T细胞调节元件内。如果我们能定义遗传机制 作为RA易感性的基础，那么就有可能定义治疗策略 废除或预防风湿性关节炎。这一点的核心是确定参与介导疾病的关键T细胞 易感性--就其独特的TCR序列特征和致病细胞而言 各州。在这里，我们假设人类白细胞抗原-DR风险等位基因在胸腺内起作用，有利于选择 “前哨TCR”，当自身抗原被呈递给前哨TCR时， T细胞增强剂改变T细胞特异性基因调控，使幼稚T细胞表达 哨兵TCRs转变为致病状态。我们将“哨兵TCR”定义为 与瓜氨酸多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关键T细胞基因的调节，这些基因调节 T细胞向致病状态的转变。因此，具有罕见的“前哨TCR”的T细胞可以在 合适的条件，触发最初的自身免疫反应，推动免疫扩散 反应，并引发持续性的关节炎症。但是，关键的致病T细胞状态， 前哨T细胞受体，以及特定的因果调节性T细胞等位基因的作用还没有完全确定。 确定血液和组织中TCR序列的全套测序，单细胞分析 解决T细胞状态和基因工程以询问因果T细胞等位基因和基因 代表了令人兴奋的方法，我们的实验室已经在其中开发了专业知识。在这份提案中，我们 寻求为这一模式建立支持。首先，我们将证明人类白细胞抗原II类人类白细胞抗原RA的风险 等位基因改变了TCR谱系，以容纳“前哨TCR”，使用了300个TCR和基因数据 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 单细胞数据对RA风险等位基因在细胞表面标记和转录本上的影响 同样的300个人。最后，我们将确定非编码等位基因的分子机制 在CD4+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和 了解ATAC测序数据的影响，并确认非编辑功能 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.