Hril 15

Citrate‐treated peripheral blood samples were obtained from healthy blood donors recruited at the Institute for Transfusion Medicine of the UKE and the Healthy Blood Donor Cohort at the Leibniz Institute for Experimental Virology in Hamburg, Germany. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of healthy donors by density‐gradient centrifugation, washed, and resuspended in complete medium (RPMI‐1640 medium [Sigma] supplemented with 10 % [v/v] fetal bovine serum, Sigma). Primary NK cells were isolated and enriched from freshly isolated PBMC through negative‐selection strategy using the EasySep^TM Human NK cell Enrichment Kit (StemCell Technologies) according to the manufacturer's protocol. NK cells were cultivated in complete media at 2 × 10⁶ cells/ml and stimulated with human recombinant IL‐15 (5 ng/ml, hrIL‐15; R&D Systems).

Frozen PBMCs were thawed and cultured at 2 × 10⁶ cells/mL overnight in RPMI supplemented with 5% autologous plasma, 40 U/mL of human recombinant (hr) interleukin (IL)-2 (Roche), 20 ng/mL of hrIL-7 (R&D Systems), and 10 ng/mL of hrIL-15 (R&D Systems) to obtain armed-CD8⁺ T lymphocytes. Then, CD3⁺ T cells were purified by negative selection (CD3⁺CD56⁺ NKT Cell Isolation Kit, human; Miltenyi Biotech), and 4 × 10⁵ CD3⁺ lymphocytes were stained with APC anti-CD107a/LAMP-1 (BD Biosciences, clone H4A3) and stimulated in the presence or not of anti-CD3/CD28 (1 bead/cell) for 4 hours. Brefeldin A (Sigma-Aldrich) was added at 10 μg/mL for the last 3 hours of cell incubation. Then, CD3⁺ T cells were stained with a combination of the following fluorochrome-conjugated antibodies FITC anti-human CD8 (BD Biosciences, Clone RPAT8), V500 anti-human CD45RA (BD Biosciences, Clone HI100), BB700 anti-human CCR7, PE anti-human IFN-γ (BD Biosciences, Clone B27-RUO), and BV421 anti-human Granzyme B to measure cytotoxicity and cytokine production. Intracytoplasmic molecules were evaluated by using the fixation/permeabilization solution kit BD Cytofix-Cytoperm (BD Biosciences), according to the manufacturer's instructions. For the evaluation of CD107a/LAMP-1 and IFN-γ positive events, the medium sample was used for setting the gate. The gating strategy used is shown in eFigure 2.