The study was approved by the Ethics Committee of the 1st Affiliated Hospital of Harbin Medical University. Written informed consent was regularly obtained from all patients upon admission to department Intensive Care Medicine, the 1st Affiliated Hospital of Harbin Medical University (the intensive care center for severe COVID-19 patients in Harbin, Heilongjiang Province). The 63 confirmed COVID-19 patients in the intensive care unit (ICU) were severe/critical according to the Guidelines of the Diagnosis and Treatment of New Coronavirus Pneumonia (version 6) published by the National Health Commission of China or to the Sequential Organ Failure Assessment（SOFA）score. All the patients enrolled in this retrospective single-center study were admitted to the ICU from Feb. 12th to Mar. 26th, 2020.
Severe COVID-19 patients were defined as those satisfying any of the following criteria: 1. Patients who had hypoxemia at rest; hypoxemia was defined as arterial oxygen tension (PaO2) over inspiratory oxygen fraction (FIO2) of less than 300 mmHg or arterial oxygen saturation of 93% or lower. 2. Patients whose breathing rates were greater than 30 breaths per minute.
Critical patients were defined as COVID-19 patients who required high-ﬂow nasal cannula or higher-level oxygen support measures to correct hypoxemia.
All medical record information, including epidemiological, demographic, clinical manifestation, and laboratory data, were obtained. All data were checked by a team of trained physicians.
Laboratory confirmation of COVID-19 was performed by the local CDC according to the Chinese CDC protocol. Throat swab specimens were collected from all patients, and the samples were maintained in viral transport medium for laboratory testing. Specimens, including sputum and blood, were cultured to identify pathogenic bacteria or fungi that may be associated with COVID-19 infection. A lymphocyte test kit (Beckman Coulter Inc., FL, USA) was used for lymphocyte subset analysis. The concentrations of serum cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were quantitatively determined by a Human Th1/Th2 Cytokine Kit (Saiji Biotec, Hangzhou, China) as described in the instruction manual.
The expression of PD-1 was analyzed using a BD FACSCalibur (BD Biosciences, CA, USA) ﬂow cytometer. Peripheral blood cells were surface stained with mouse anti-human CD8-FITC (BD Biosciences, CA, USA), CD4-PE (BD Biosciences, CA, USA) and CD279 (PD-1)-APC (Biolegend, CA, USA) monoclonal antibodies for 15 minutes in the dark at room temperature. After lysing with 1X BD FACS Lysing Solution(BD Biosciences, CA, USA), cells were then washed two times with PBS. Data were analyzed using Kaluza software (Version 2.1)( Beckman Coulter, IN, USA). The following monoclonal antibody panel was used for surface staining: CD8-FITC (BD Biosciences, CA, USA), CD4-PE (BD Biosciences, CA, USA) and CD279 (PD-1)-APC (Biolegend, CA, USA) for PD-1 expression on lymphocytes; CD14-FITC (BD Biosciences, CA, USA) and HLA-DR-PerCP (Miltenyi, Bergisch Gladbach, Germany) for HLA-DR expression on monocytes; CD4-FITC (Quanto Bio, Beijing, China), CD25-PE (Quanto Bio, Beijing, China) and CD127-APC (Quanto Bio, Beijing, China) for Treg cells. The stained samples were further lysed, washed, and resuspended in 200 µl of PBS before acquisition.
Apoptosis was determined by a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, CA, USA) after lysing samples with a lysing solution that did not contain a fixative agent. Then, the cells were washed and stained with Annexin V-FITC and PI as described in the instruction manual.
Acquisitions of samples were carried on FACSCalibur ﬂow cytometer (BD Biosciences, CA, USA). Cytokines data were analyzed using FCAP Array™ software (Version 3.0) (BD Biosciences, CA, USA), other data were analyzed using Kaluza software (Version 2.1)( Beckman Coulter, IN, USA).
Figure 1 shows the patient flowchart in this prospective and observational study.
The quantized variables of parameters are expressed as the mean ± standard deviation, and the significance was tested by t-test. Nonparametric variables are expressed as median and quartile intervals [M (P25, P75)], and significance was tested by Wilcoxon rank-sum test. P < 0.05 was considered statistically significant in all statistical analyses. The Spearman rank correlation coefficient was used to analyze correlations. All statistical analyses were performed by Statistical Analysis System (SAS) (version 9.1.3, SAS Institute Inc., Cary, NC, USA).