DataSheet_1_Increased Expression of Tim-3 Is Associated With Depletion of NKT Cells In SARS-CoV-2 Infection.docx

In the ongoing coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), natural killer T (NKT) cells act as primary initiators of immune responses. However, a decrease of circulating NKT cells has been observed in COVID-19 different stages, of which the underlying mechanism remains to be elucidated. Here, by performing single-cell RNA sequencing analysis in three large cohorts of COVID-19 patients, we found that increased expression of Tim-3 promotes depletion of NKT cells during the progression stage of COVID-19, which is associated with disease severity and outcome of patients with COVID-19. Tim-3+ NKT cells also expressed high levels of CD147 and CD26, which are potential SARS-CoV-2 spike binding receptors. In the study, Tim-3+ NKT cells showed high enrichment of apoptosis, higher expression levels of mitochondrial genes and caspase genes, with a larger pseudo time value. In addition, Tim-3+ NKT cells in COVID-19 presented a stronger capacity to secrete IFN-γ, IL-4 and IL-10 compared with healthy individuals, they also demonstrated high expression of co-inhibitory receptors such as PD-1, CTLA-4, and LAG-3. Moreover, we found that IL-12 secreted by dendritic cells (DCs) was positively correlated with up-regulated expression of Tim-3 in NKT cells in COVID-19 patients. Overall, this study describes a novel mechanism by which up-regulated Tim-3 expression induced the depletion and dysfunction of NKT cells in COVID-19 patients. These findings not only have possible implications for the prediction of severity and prognosis in COVID-19 but also provide a link between NKT cells and future new therapeutic strategies in SARS-CoV-2 infection.

在由严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）引发的新型冠状病毒肺炎（coronavirus disease 2019，COVID-19）疫情中，自然杀伤T细胞（natural killer T，NKT）是免疫应答的核心启动因子。然而，在COVID-19的不同病程阶段均已观测到循环NKT细胞数量减少，但其潜在调控机制仍有待阐明。本研究针对三大型COVID-19患者队列开展单细胞RNA测序（single-cell RNA sequencing）分析，结果显示在COVID-19进展期，T细胞免疫球蛋白黏蛋白分子3（Tim-3）的表达上调可促进NKT细胞耗竭，该现象与患者的疾病严重程度及预后密切相关。Tim-3阳性（Tim-3+）的NKT细胞还高表达CD147与CD26，二者均为潜在的SARS-CoV-2刺突蛋白结合受体。本研究中，Tim-3+ NKT细胞呈现出凋亡通路高度富集的特征，线粒体基因与半胱天冬酶（caspase）基因的表达水平显著升高，且拟时间（pseudo time）值更大。此外，相较于健康个体，COVID-19患者体内的Tim-3+ NKT细胞分泌干扰素-γ（IFN-γ）、白细胞介素-4（IL-4）及白细胞介素-10（IL-10）的能力更强，同时还高表达程序性死亡受体1（PD-1）、细胞毒性T淋巴细胞相关抗原4（CTLA-4）、淋巴细胞活化基因3（LAG-3）等共抑制受体。进一步分析发现，COVID-19患者体内树突状细胞（dendritic cells，DCs）分泌的IL-12与NKT细胞中Tim-3的表达上调呈正相关关系。总体而言，本研究揭示了一种全新的调控机制：COVID-19患者体内Tim-3表达上调可诱导NKT细胞耗竭与功能失调。上述研究结果不仅可为COVID-19的疾病严重程度与预后预测提供潜在参考依据，还为NKT细胞与未来SARS-CoV-2感染的新型治疗策略建立了关联纽带。