Fixation permeabilisation kit

Cells were stained with Zombie Red and then with antibodies against CD3 (OK3), CD8 (SK1), PD-1 (EH12.2H7), CD45RA (HI100), and CCR7 (G043H7). For nuclear staining of Ki67 (Ki67), we used the eBioscience Fixation/Permeabilisation kit. For IFNγ (clone 4S.B3) staining, the Fixation and Permeabilization buffer from Invitrogen was used. Cells were analyzed on a CytoflexS cytometer (Beckman Coulter).

Cells from spleens and lymph nodes were depleted of erythrocytes by hypotonic lysis. The cells were washed with FACS washing buffer (2% FBS, 0.1% NaN₃ in PBS) twice and were then incubated with fluorescence-conjugated antibodies against cell surface molecules for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. Isotype antibodies were included as negative controls. For intracellular cytokine staining, single-cell suspensions were stimulated with 50 ng/ml PMA and 1 μM ionomycin in the presence of brefeldin A solution for 4 h. After stimulation, cells were stained with fluorescence-conjugated antibodies against CD4 and CD25, fixed and permeabilised using a fixation/permeabilisation kit (eBioscience, San Diego, CA, USA) and stained with fluorescence-conjugated specific antibodies against IFN-γ, IL-17A, and Foxp3 in accordance with the manufacturer’s instructions. Flow cytometry was performed using a Becton Dickinson FACSCalibur machine.

For surface staining, single-cell suspensions were washed once with fluorescence-activated cell sorting (FACS) washing buffer (2% FBS, 0.1% NaN3 in PBS) and blocked with Fc receptor blocking solution. Cells were then incubated with fluorescence-conjugated antibodies against cell surface molecules for 30 min at 4 °C. After washing with FACS buffer, the cells were fixed and permeabilized using a fixation/permeabilisation kit (eBioscience) and stained with fluorescence-conjugated specific antibodies against cytokines following the manufacturer’s instructions. Flow cytometry was performed using a Becton Dickinson FACS Canto (BD Biosciences, San Jose, CA, USA), and the data were analysed with the FlowJo software (Flowjo, Ashland, OR, USA). The following anti-human monoclonal antibodies were used in the staining assay: BV510-labelled anti-CD3 (300448), APC-labelled anti-CD4 (357408), PE-labelled anti-CD8a (300908), PE-Cy7-labelled anti-CD45RA (304126), BV421-labelled anti-CCR7 (353208), PerCP-Cy5.5-labelled anti-TNF-α (502926), BV421-labelled anti-GzmB (396414), and FITC-labelled anti-IFN-γ (11-7319-82); all antibodies were from eBioscience or BioLegend.