Immunofluorescence labeling and flow cytometry analysis
Peripheral blood (50 μl) was collected from each subject, and the following fluorescently labeled monoclonal antibodies were added: FITC-conjugated anti-human CD19, FITC-conjugated anti-human 14, FITC-conjugated anti-human OX40, PE-conjugated anti-human OX40L, and PC5-conjugated anti-human CD4 (all purchased from BioLegend, San Diego, CA, USA), followed by an incubation in the dark at room temperature for 30 minutes. Next, 200 μl of red blood cell lysis buffer (Beckman Coulter, Brea, CA, USA) were added, and the mixtures were incubated at 37 °C for 10 minutes. Finally, each specimen was washed with 1 ml of PBS, centrifuged at 2000 rpm for 5 minutes, resuspended in 500 μl of PBS, and detected using a flow cytometer (Beckman Coulter, Brea, CA, USA). FlowJo version 10.4 software was used to analyze the raw flow cytometry data.