Ficoll hypaque solution

Human fresh peripheral blood samples of all individuals were obtained from the children with acute HSP and HCs. Peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation with Ficoll-Hypaque solution (CL5020, CEDARLANE, Netherlands) were transferred to sterile tubes and washed twice with phosphate-buffered saline (PBS). Subsequently, human CD4⁺T cells were isolated by human CD4⁺T cell isolation kit (number 130-096-533) (Miltenyi Biotec GmbH, Germany). Additionally, human PBMCs were stained with FITC-Mouse Anti-Human CD4 (BD Biosciences, San Diego, CA, USA), clone name: RPA-T4, isotype control: FITC Mouse IgG2b, κ, clone name: eBMG2b; PE-Mouse Anti-Human CXCR5 Biotinylated Monoclonal Antibody (BioLegend, San Diego, CA), clone name: J252D4, isotype control: PE Mouse IgG2a, κ, clone name: eBM2a; APC-Anti-Human ICOS (CD278) (BD Biosciences, San Diego, CA, USA), clone name: ISA-3, isotype control: APC Mouse IgG2a, κ, clone name: MOPC-173; PerCP-Cy5.5 Mouse Anti-Human CD279 (PD-1) (BD Biosciences, San Diego, CA, USA), clone name: EH12.1, isotype control: PerCP-Cy5.5 Mouse IgG1, κ, clone name: MOPC-21. All the staining procedures were performed according to the manufacturers' protocols, and the stained cells were analyzed by a flow cytometer of FACSCalibur and CELLQUEST software (Becton Dickinson, Sparks, MD, USA).

Peripheral whole blood specimens were collected from the HC and patients during the acute and convalescence stages of EV71-associated HFMD, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Hypaque solution (CL5020, CEDARLANE, The Netherlands). Human CD4⁺T cells were isolated from PBMCs using the human CD4⁺T Cell Isolation Kit (130-096-533) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's protocol.