目的:严重急性呼吸综合征冠状病毒-2(SARS-CoV-2)通过T细胞调节抗病毒免疫,但这些细胞在2019年冠状病毒病(COVID-19)患者中是否活跃或丰富尚不清楚。本研究旨在研究T细胞群及其亚群的时间偏移,COVID-19患者的T辅助细胞(Th)细胞(CD4)和T细胞毒性(Tc)细胞(CD8)。
方法:纳入30例确诊(鼻拭子逆转录-聚合酶链反应(RT-PCR)确诊)的COVID-19患者。根据氧饱和度(SpO2)水平,患者分为两类:(i)轻度(n=11)发热且SpO2水平>95%,和(ii)呼吸机严重(n=19),并按照印度医学研究理事会(ICMR)指南在重症监护病房(ICU)中。该研究还招募了30名年龄性别匹配的对照组,这些对照组没有与COVID-19无关的传染病。炎症性疾病和严重合并症损害免疫力的患者被排除在研究之外。免疫表型流式细胞术用于评估T细胞活力,Th,在第二次COVID-19波(变体:B.1.61)的第1天(入院时)和第4天(降低感染负荷),轻度和重度COVID-19患者的Tc细胞群。分类变量表示为频率和百分比,p值通过卡方检验计算。所有变量均以中位数和Q1(25百分位数)和Q3(75百分位数)表示。Mann-Whitney检验用于比较研究组。通过使用配对样品t检验计算Δ平均差。统计学上显著的水平被认为是p<0.05。
结果:血红蛋白,总白细胞计数(TLC),淋巴细胞,单核细胞,患者嗜酸性粒细胞明显减少(p<0.05)。严重COVID-19患者的CD4和CD8细胞显着降低与对照组(CD4,中位数49;CD8,40.12;p>0.05)。Th-EM(效应记忆)-Tim-3(T细胞免疫球蛋白结构域和粘蛋白结构域3)显着升高(p=0.002),Tc-EMRA(效应记忆细胞再表达)-Tim-3+,Tc-Naive-Tim-3+,轻度COVID-19患者的Tc-EM-PD1+和Tc-CM(中枢记忆)-Tim-3+显著低于对照组(p<0.05)。同样,在严重的COVID-19患者中,Th-EMRA-Tim-3+,Th-Naive-PD1+,Th-EM-PD1+,Th-EM-Tim3+和Th-CM-Tim-3+显示显著降低(p<0.05),Tc-EMRA-Tim-3+,Tc-Naive-Tim-3+,Tc-EM-PD1+,和Tc-CM-Tim-3+显示相似的结果。在温和的vs.严重组,T细胞减少(p=0.001),Th-EMRA-Tim-3+(p=0.024),和Th-Navie-Tim-3+(p=0.005),并且显着增加(p<0.05)Tc-Naive-Tim3(p=0.001),Tc-EM-Tim-3+(p=0.031),观察到Tc-CM-Tim-3+(p=0.08)。重症COVID-19患者Th-Naive-Tim3+显著升高(第4天-第1天;δ43,p=0.019),Th-EM-Tim3+(δ16.24,p=0.033),和Th-CM-Tim3+(δ13.57,p=0.041)。
结论:T细胞群和CD8亚群有助于区分轻度和重度COVID-19患者。监测T细胞,尤其是CD8亚群的变化,对于诊断和治疗轻度和重度危重患者具有重要意义。
OBJECTIVE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) modulates antiviral immunity via T cells, but whether these cells are active or abundant in coronavirus disease 2019 (COVID-19) patients is unknown. The present study aimed to investigate the temporal shifting in the T-cell population and their subsets, T-Helper (Th) cell (CD4) and T-Cytotoxic (Tc) cell (CD8) in COVID-19 patients.
METHODS: Thirty confirmed COVID-19 patients (nasal swab reverse transcription-polymerase chain reaction (RT-PCR) confirmed) were enrolled. On the basis of oxygen saturation (SpO2) levels, patients were stratified into two categories: (i) mild (n=11) having fever and SpO2 level >95%, and (ii) severe (n=19) on the ventilator, and in the intensive care unit (ICU) as per the Indian Council of Medical Research (ICMR) guidelines. Thirty age-sex-matched controls without infectious diseases unrelated to COVID-19 were also enrolled in the study. Patients with inflammatory diseases and severe comorbidities that compromise immunity were excluded from the study. Immunophenotyping flow cytometry assay was used to evaluate T-cell viability, Th, and Tc cells population in mild and severe COVID-19 patients on day 1 (at admission) and day 4 (decreasing the infection load) in the second COVID-19 wave (variant: B.1.61). Categorical variables were expressed as frequency and percentage and p-values were calculated by Chi-square test. All the variables were represented in median and Q1 (25 percentile) and Q3 (75 percentile). The Mann-Whitney test was used to compare the study groups. The Δ mean differences were calculated by using the Paired samples t-test. The statistically significant level was taken as p<0.05.
RESULTS: Hemoglobin, total leukocyte count (TLC), lymphocytes, monocytes, and eosinophils were significantly reduced in patients (p<0.05). A significant decrease of CD4 and CD8 cells in severe COVID-19 patients vs. controls (CD4, median 49; CD8, 40.12; p>0.05) was seen. Th-EM (effector memory)-Tim-3 (T-cell immunoglobulin domain and mucin domain 3)+ was significantly higher (p=0.002) however, Tc-EMRA (effector memory cells re-expressing)-Tim-3+, Tc-Naive-Tim-3+, Tc-EM-PD1+ and Tc-CM (central memory)-Tim-3+ significantly reduced (p<0.05) in mild COVID-19 patients than controls. Similarly, in severe COVID-19 patients, Th-EMRA-Tim-3+, Th-Naive-PD1+, Th-EM-PD1+, Th-EM-Tim 3+ and Th-CM-Tim-3+ showed a significant reduction (p<0.05) and Tc-EMRA-Tim-3+, Tc-Naive-Tim-3+, Tc-EM-PD1+, and Tc-CM-Tim-3+ showed similar results. In mild vs. severe group, decreased T-cells (p=0.001), Th-EMRA-Tim-3+ (p=0.024), and Th-Navie-Tim-3+ (p=0.005), and significantly increased (p<0.05) Tc-Naive-Tim3+ (p=0.001), Tc-EM-Tim-3+ (p=0.031), and Tc-CM-Tim-3+ (p=0.08) were observed. Severe COVID-19 patients showed a significant increase in Th-Naive-Tim3+ (day 4-day 1; δ43, p=0.019), Th-EM-Tim3+ (δ 16.24, p=0.033), and Th-CM-Tim3+ (δ 13.57, p=0.041).
CONCLUSIONS: T-cell populations and CD8 subset help to differentiate the mild and severe COVID-19 patients. Monitoring T cells, especially CD8 subset changes, has important implications for diagnosing and treating mild and severe patients being critically ill.