M5273

To assess cytokines, perforin (PRF) and granzyme B (GZMB) production, CD8⁺CCR4⁺ T cells were sorted and left overnight in T cell medium (Roswell Park Memorial Institute-1640 medium (11875093) supplemented with 1% (v/v) glutaMAX-I (35050061), 1% (v/v) non-essential amino acids (11140068), 1 mM sodium pyruvate (11360088), 50 μM β-mercaptoethanol (31350010), penicillin (50 U/mL), streptomycin (50 µg/mL) (15070063), 1% (v/v) kanamycin (15160047) (all from Gibco, Life Technologies) and 5% (v/v) human serum (Swiss Blood Centre, Basel)). Cells were then stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (P1585, Sigma-Aldrich) and 1 μg/mL ionomycin (I0634, Sigma-Adrich), resuspended in T cell medium, for 5 hours at 37°C in a 5% carbon dioxide-humidified atmosphere. After 2.5 hours, 10 μg/mL brefeldin A (B5936, Sigma-Aldrich) and 2 mM monensin (M5273, Sigma-Aldrich) were supplemented. Where indicated, directly conjugated anti-CX3CR1 antibody (online supplemental table 1) was added to the cells 30 min prior to fixation. Intracellular staining of cytokines, PRF and GZMB, was performed using fluorochrome-conjugated antibodies (online supplemental table 1) and Cytofix/Cytoperm kit (554714, BD Biosciences), according to the manufacturer’s instructions. All samples were acquired on BD LSRFortessa (BD Biosciences), and the results were analysed with FlowJo software V.10.7.1 (Tree Star).