Cytokine Production Analysis by Intracellular Staining

Cytokine production was analyzed by intracellular staining (ICS) assay. PBMCs were incubated with or without the relevant peptides (20 μg/ml) plus anti-CD28 mAb (4 μg/ml) (BD Biosciences) and the Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; eBioscience), or with the Cell Stimulation Cocktail (phorbol 12-myristate 13-acetate [PMA], ionomycin, brefeldin A, and monensin; eBioscience) as a positive control, for 18 h at 37°C [16 (link),22 (link)]. Cells were washed and then stained with APC-labeled—HLA-A*0201 dextramers complexed to corresponding peptides, PeCy7-labeled mAb to CD8 (BioLegend), and the dump channel reagents. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) at 4°C for 20 min, rewashed with the BD Perm/Wash buffer (BD Biosciences), and stained with different combinations of AlexaFluor700-labeled IL-17A (BioLegend) and FITC-labeled anti-IFN-γ (BioLegend) for 20 min at 4°C. Cells were washed, acquired with the LSRFortessa cytometer, and analyzed with FlowJo software. IL-17, IFN-γ or IL-17/IFN-γ producing cells were analyzed in CD8⁺dextramer⁺ cells after exclusion of B cells, monocytes, NKT cells, NK cells, and CD4⁺ T cells (dump channel).