Cx3cr1 2a9 1

T cell phenotype was assessed by flow cytometry (LSRFortessa, BD) using fluorochrome-conjugated antibodies specific for CD3 (clone UCHT1; BD), CD4 (SK3; BD), CD8 (SK1; BD), CD45RO (UCHL1; BD), CX3CR1 (2A9-1; BioLegend), CD57 (HNK-1; BioLegend), CD27 (M-T271; BD), CD28 (CD28.2; BioLegend), CD69 (FN50; BD), and CCR7 (3D12; BD). Viable cells were gated using Live/Dead Aqua viability dye (Invitrogen). For induction and detection of intracellular cytokines, cells were cultured overnight with 20ng/ml recombinant human IL-15 (247-ILB; R&D Systems) or medium control (RPMI 1640 [Gibco], supplemented with 10% fetal bovine serum [Gemini Bio-Products], 1% L-glutamine [Gibco], and 1% penicillin/streptomycin [Gibco]), then treated with brefeldin A (GolgiPlug, BD) for 6h prior to harvest. After Live/Dead and surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD) for 20 min on ice and stained for 40 minutes on ice with anti-IFNγ (B27, BD) and anti-TNF (MAb11, BD). For intracellular accumulation of cytolytic molecules, cells were treated with IL-15-supplemented or control medium for 48 hours, then harvested, stained with Live/Dead and surface antibodies, treated with Cytofix/Cytoperm, stained with anti-granzyme B (GB11; BD) and anti-perforin (B-D48; BioLegend).