Monocyte programmed death ligand-1 expression after 3–4 days of sepsis is associated with risk stratification and mortality in septic patients: a prospective cohort study

The patients in this study were from the emergency department (ED) of Beijing Chao-yang Hospital. There are about 250,000 ED admissions per year in this university teaching hospital. Patients admitted to the ED at days 1–2 after the onset of signs of systemic inflammatory response syndrome (SIRS) were evaluated for possible enrollment according to the inclusion and exclusion criteria. The eligible patients were treated according to the international guidelines for management of septic shock [2]. The beginning of vasopressive therapy was defined as the onset of the septic shock. Considering the investigation’s focus on the second phase of sepsis, blood samples were obtained at days 3–4 after the onset of SIRS [5, 14].

According to the diagnostic criteria of the 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference [15], sepsis was defined by an identifiable site of infection, which was evidence of a systemic inflammatory response (SIRS) manifested by at least two of the following criteria: (a) body temperature >38.5 °C or <36 °C; (b) heart rate >90 beats per minute; (c) respiratory rate >20 breaths per minute or PaCO₂ <32 mmHg; and (d) white cell count >12,000/mm³ or <4000/mm³, or percentage of immature neutrophils >10 %. Septic shock was defined as sepsis-induced hypotension despite adequate fluid resuscitation. Sepsis-induced hypotension was defined as a systolic blood pressure (SBP) <90 mmHg or mean arterial pressure (MAP) <70 mmHg or SBP decrease >40 mmHg or less than two standard deviations below normal for age in the absence of other causes of hypotension. According to the criteria of the International Sepsis Forum Consensus Conference on Definitions of Infection [16], the infection was defined on the basis of clinical features, laboratory findings, and an imaging test. The exclusion criteria were: (a) age less than 18 years; (b) patients with HIV infection or cancer if they were treated with a high dose of corticoids or if they presented with an aplasia (polymorphonuclear neutrophil count of less than 0.5 G/L); (c) patients who died within 2 days of the onset of septic shock; (d) the signs of SIRS occurred more than 3 days prior to admission, or patients were transferred from another hospital; and (e) patients who declined to consent.

Clinical characteristics of patients, including age, gender, past medical history, vital signs, and results of correlative laboratory examinations, were recorded at days 3–4 after the onset of septic shock. Sepsis-related organ failure assessment (SOFA) score and simplified acute physiology score II (SAPS II) were calculated according to related clinical and demographic data. This study was approved by the Beijing Chao-yang Hospital Ethics Committee. Written informed consent was obtained from the patients and volunteers. Consents for patients who were unable to provide consent were provided by first-degree relatives. During follow-up the following data were collected: comorbidities (chronic obstructive pulmonary diseases, congestive heart failure, cerebrovascular disease, diabetes), and the outcome after 28 days (survival or death).

Samples of peripheral blood were collected in ethylenediamine tetraacetic acid (EDTA) anticoagulant tubes. The samples were transported to the laboratory at 4 °C within 2 h. Erythrocytes were lysed and cells were stained by a researcher who was blinded to the clinical data. Antibodies were purchased from BD Bioscience (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). According to the manufacturer’s recommendations monoclonal antibodies and their isotype controls were used: BV421-labeled anti-PD1 (5 μl, clone EH12.1), PE-labeled anti-PD-L1 (20 μl, clone M1H1), APC-H7 labeled anti-CD3 (5 μl, clone SK7), FITC-labeled anti-CD4 (5 μl clone OKT4), FITC-labeled anti-CD8 (20 μl, clone RPA-T8), and APC-H7-labeled anti-CD14 (5 μl, clone MφP9) per 100 μl of whole blood. Samples were run on a Gallios™ Flow Cytometer (Beckman Coulter, Inc.) and analyzed using Gallios Software Version 1.0 (Beckman Coulter, Inc.). Lymphocytes were gated by forward scatter (FSC) and side scatter (SSC), and T-cells subsets were further identified by CD3⁺ and CD4⁺ staining. Monocytes were identified by CD14⁺ staining (Fig. 1). At least 3000 cells were analyzed from each sample. The threshold was defined using an isotype control. Results are expressed as percentages and the mean of fluorescence intensities (MFI).

The baseline characteristics were described as frequencies, percentages, median and inter-quartile ranges. Comparisons between groups were made using the Pearson χ ² test for categorical data and the Mann-Whitney test for continuous variables. For multi-group comparisons, the Kruskal-Wallis test was applied. Age, white blood cell (WBC), lymphocytes, SOFA score, and SAPS II were stratified using cutoff values based on the population median [5]. PD-1 and PD-L1 expressions were stratified using the optimal threshold indicated by the receiver operating characteristic (ROC) curve. Using cutoff values determined by ROC curves, Kaplan-Meier survival curves were established, and the log-rank test was applied for the comparisons of survival distributions. Binary logistic regression was used to identify the variables associated with 28-day mortality in patients with septic shock. Variables with P < 0.15 in univariate analysis were conserved in the model. All statistical tests were two-tailed, and P < 0.05 was considered statistically significant. All data were analyzed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).