Il 15

Frozen HMNCs were recovered and rested in complete RPMI supplemented with 2ng/mL IL-15 (Miltenyl Biotec) and 25ug/mL DNAse-I (Sigma-Aldrich) for 45 mins at 37°C in 5% CO₂. CD56⁺ NK cells were magnetically isolated from HMNCs using a negative selection kit (Stemcell, Vancouver, Canada) and from peripheral blood samples using a RosetteSep™ kit (Stemcell). Whole PBMCs were isolated by density centrifugation using Ficoll-Paque™ PLUS and CD3⁺ T cells were magnetically isolated from PBMCs using a negative selection kit (Stemcell). PBMCs or CD3⁺ T Cells were stimulated with anti-CD3 (5µg/mL; Tonbo Biosciences, California, USA) and anti-CD28 (2µg/mL; Tonbo Biosciences) and co-cultured with either hepatic or peripheral blood NK cells at a 1:1 (PBMC : NK) ratio in cRPMI supplemented with 2ng/mL IL-15, unless otherwise indicated. A monoclonal antibody (mAb) against CD160 (MBL, Massachusetts, USA) or an IgG isotype control (Biolegend, California, USA) was added at 10µg/mL to assess its role in NK cell killing. After 24 hr the percentage of dead T cells was assessed using the coculture flow cytometry panel (Supplementary Table 2).

Human skin specimens were collected from healthy patients undergoing plastic surgery at Duke University Medical Center and used anonymously. All human samples for this study were obtained according to the protocols approved by the Institutional Review Board at Duke University. Samples of normal human skin obtained were cultured in 24-well plates. The human skin samples were incubated in skin explant media modified from Clark et al. (32 (link)) (DMEM; 10% FBS; 0.1 mM non- essential amino acids (Thermo Fisher Scientific, Waltham, MA); 1 mM sodium pyruvate; 2 mM L-Glutamine; 1% Pen/Strep (Thermo Fisher Scientific); IL-2 (5 unit/ml, PromoCell, Heidelberg, Germany); and IL-15 (7.5 ng/ml, Tonbo Biosciences, San Diego, CA). For other experiments, cells were then cultured in skin explant media without IL-2 and IL-15 for 24 hours before being collected. Cells that migrated into the culture media were harvested and utilized for further FACS sorting. FACS-sorted T cells were treated with recombinant 2 nM IL-15 or 3.1 nM IL-27 (BioLegend, San Diego, CA) or vehicle control for 24 hours and then collected for flow analysis.