Single Cell definition of pathogenic T cells in Drug-induced Stevens-Johnson-Syndrome/Toxic epidermal necrolysis

项目摘要 药物引起的Stevens-Johnson综合征（SJS）和中毒性表皮坏死松解症（TEN）是最常见的 免疫介导的药物不良反应（IM-ADR）与水疱、粘膜脱落 和表皮坏死。SJS/TEN的死亡率、个人残疾和医疗费用不成比例 在美国，其发病率为5例/1，000，000/年。药物特异性HLA基因筛查 风险等位基因具有100%的阴性预测值，并且在某些情况下具有成本效益，然而，只有2-8%的 携带药物特异性HLA风险等位基因的个体在暴露于相关药物时发生SJS/TEN。强 HLA I类相关性，SJS/TEN水疱液中存在产生颗粒溶解素的CD 8 + T细胞， 以及迫切需要了解除了HLA风险等位基因之外， SJS/TEN的发展构成了本研究的科学前提和意义。利用我们广泛的 现有的生物样本库中，冷冻保存的水疱液样本来自于具有严格表型的急性药物依赖的患者， 诱导的SJS/TEN和来自火烧伤和深Ⅱ度烧伤患者的水疱细胞作为非抗原驱动的 对照组，我们将使用整合的多组学方法在单细胞水平上定义抗原驱动的CD 8 + T细胞。 SJS/TEN病理部位的细胞及其转录组学特征。 在具体目标1中，我们将使用批量和单细胞T细胞受体（TCR）测序方法， 定量急性SJS/TEN患者水疱液中活化的CD 8 + T细胞的TCR克隆性。我们 假设药物抗原驱动活化的CD 8 + T细胞的募集和/或克隆扩增 具有在急性疾病期间将在水疱液中富集的特异性T细胞受体（TCR）。在一些 在某些情况下，显性TCR克隆型可能在具有相同HLA风险等位基因的无关个体之间共享。 将根据活化（CD 3 + CD 8 + CD 137+）和非活化（CD 137+）对来自水疱液的单个活T细胞进行分选。 (CD3+ CD 8 + CD 137-）谱，并进行TCR库测序。在具体目标2中，我们将定义 急性SJS/TEN患者水疱液中活化CD 8 + T细胞的转录组学特征。 来自疱液的单细胞的RNA-seq分析将与来自多参数的索引分选数据相结合 流式细胞仪面板，这将使我们能够识别与潜在的 扩增的抗原驱动的TCR克隆型。我们的假设是，水疱液中的CD 8 + T细胞将具有 表达表型，包括颗粒溶解素和其他细胞溶解介质，以及早期激活 如CD 137。 我们将使用新的方法将致病性T细胞受体克隆型和它们的表型 签名.这将为HLA I类限制性、药物性和非药物性的免疫发病机制提供重要的见解。 诱导的SJS/TEN有助于预防、早期诊断和确定合理的治疗靶点。它还将 为适用于其他严重免疫介导疾病的单细胞研究提供路线图。

PROJECT SUMMARY Drug-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are the severest of immunologically-mediated adverse drug reactions (IM-ADR) associated with blistering, mucosal sloughing and epidermal necrosis. The mortality, personal disability and healthcare costs of SJS/TEN are out of proportion to its incidence of 5 cases per 1,000,000/year in the United States. Genetic screening with drug-specific HLA risk alleles has 100% negative predictive value and is cost-effective in some settings, however, only 2-8% of individuals carrying a drug-specific HLA risk allele when exposed to the relevant drug develop SJS/TEN. Strong HLA class I associations, the presence of granulysin-producing CD8+ T cells in the SJS/TEN blister fluid, and the critical need to understand what factors in addition to the HLA risk allele are necessary for development of SJS/TEN form the scientific premise and significance of this study. Using our extensive existing biobank of cryopreserved blister fluid samples from patients with rigorously phenotyped acute drug- induced SJS/TEN, and blister cells from patients with fire and scald partial thickness burns as non-antigen driven controls we will use an integrated multi-omic approach to define at a single-cell level the antigen-driven CD8+ T cells at the site of pathology of SJS/TEN and their transcriptomic signatures. In Specific Aim 1 we will use bulk and single cell T-cell receptor (TCR) sequencing approaches to quantify the TCR clonality of activated CD8+ T cells in the blister fluid of acute SJS/TEN patients. Our hypothesis is that the drug antigen drives recruitment and/or clonal expansion of activated CD8+ T cells with specific T-cell receptor(s) (TCR) that will be enriched in blister fluid during acute disease. In some cases the dominant TCR clonotype, may be shared amongst unrelated individuals with the same HLA-risk allele. Single live T cells from blister fluid will be sorted according to activated (CD3+CD8+CD137+) and non-activated (CD3+CD8+CD137-) profile and subjected to TCR repertoire sequencing. In Specific Aim 2 we will define the transcriptomic signatures of activated CD8+ T cells in the blister fluid of patients with acute SJS/TEN. RNA-seq profiling of single cells from blister fluid will be combined with index sorting data from multi-parameter flow cytometry panels that will enable us to identify transcriptomic signatures associated with potentially expanded antigen-driven TCR clonotypes. Our hypothesis is that the CD8+ T cells in blister fluid will have an expression phenotype that includes granulysin and other cytolytic mediators, and early activation markers such as CD137. We will use novel approaches to link the pathogenic T-cell receptor clonotypes and their phenotypic signatures. This will provide important insights into the immunopathogenesis of HLA class I restricted, drug- induced SJS/TEN to fuel prevention, early diagnosis and identification of rationale therapeutic targets. It will also provide a roadmap for single cell studies applicable to other severe immunologically mediated diseases.