Clonal CD8+ T cell expansion in CAVD

Background: Calcific aortic valve disease (CAVD) is a complex cardiovascular pathology, culminating in aortic stenosis, heart failure and premature mortality, with no comprehensive treatment strategy, except invasive valve replacement. While inflammation may contribute to valvular fibro-calcific pathology and T cells have been identified within the leaflets, their contribution to CAVD pathogenesis remains unclear. Methods: To elucidate the heterogenous phenotype of the immune populations present in CAVD patients, deep phenotypic screens of paired valve and peripheral blood cells were conducted via flow cytometry (n=20) and immunohistochemistry (n=10). Following identification of a significant population of memory T cells; specifically, CD8+ T cells within the valve, single cell RNA sequencing and paired single T cell receptor sequencing was conducted on an additional 4 patients on CD45+ CD3+, CD4+ or CD8+ T cells. Results: Through unsupervised clustering, six T cell clusters were identified within the patient. Tissue resident memory (TRM) T cells were detected for the first time within the valve, exhibiting a highly cytotoxic, activated, and terminally differentiated phenotype. This pan-pro-inflammatory signal was differentially identified in T cells originating from the valve, and not observed in the blood, indicative of an adaptive, local not-systemic inflammatory signature in CAVD patients. T cell receptor analysis identified hyperexpanded clones within the CD8+ T cell central memory (TCM) population, with TRM cells comprising most of the large and medium clonal expansion within the T cell population. Clonal expansion occurred within the valve more than the blood, with clonally expanded receptors remaining within their tissue of origin. Clonal interaction network analysis demonstrated the greatest proportion of clones originating from CD8+ TCM and TRM populations, exhibiting a tissue resident, cytotoxic environment within the valve. Conclusion: Taken together, the data identified T cell sub-populations within the context of CAVD and further predicted possible epitopes responsible for the clonal expansion of the valvular T cells, which may be important for propagating autoimmune driven inflammation in CAVD.研究背景：钙化性主动脉瓣疾病（Calcific aortic valve disease, CAVD）是一种复杂的心血管病理状态，最终可进展为主动脉瓣狭窄、心力衰竭及过早死亡，目前除侵入性瓣膜置换术外，尚无全面的治疗方案。尽管炎症可能参与瓣膜纤维钙化病理过程，且瓣膜瓣叶内已检测到T细胞，但T细胞在CAVD发病机制中的具体作用仍不明确。 研究方法：为阐明CAVD患者体内免疫细胞群的异质性表型，本研究通过流式细胞术（flow cytometry, n=20）与免疫组化（immunohistochemistry, n=10）对配对的瓣膜组织与外周血细胞进行了深度表型筛选。在鉴定出瓣膜内存在大量记忆T细胞（尤其是CD8+ T细胞）后，我们又对另外4例患者的CD45+ CD3+、CD4+或CD8+ T细胞开展了单细胞RNA测序（single cell RNA sequencing）与配对单细胞T细胞受体测序（paired single T cell receptor sequencing）。 研究结果：通过无监督聚类分析，我们在患者体内鉴定出6个T细胞亚群。本研究首次在瓣膜组织中检测到组织驻留记忆T细胞（Tissue resident memory T cells, TRM），其表型呈现高度细胞毒性、活化状态及终末分化特征。该泛促炎信号仅在瓣膜来源的T细胞中呈差异性表达，外周血T细胞中未检测到此特征，提示CAVD患者体内存在适应性的局部而非全身性炎症特征。T细胞受体测序分析显示，CD8+中央记忆T细胞（CD8+ T cell central memory T cells, TCM）群体中存在过度扩增的克隆，且TRM细胞构成了T细胞群体中绝大多数大、中型克隆扩增的组分。克隆扩增在瓣膜组织中的发生频率高于外周血，且克隆扩增的受体均保留在其起源组织内。克隆相互作用网络分析表明，大部分克隆源自CD8+ TCM与TRM群体，这提示瓣膜内存在组织驻留性的细胞毒性微环境。 研究结论：综上，本研究明确了CAVD患者体内的T细胞亚群，并进一步预测了可能驱动瓣膜T细胞克隆扩增的表位，这些表位或在推动CAVD自身免疫性炎症进展中发挥重要作用。