Interleukin 2 (il 2)

Suspension culture flasks (Sumitomo Bakelite, Tokyo, Japan) were pre-coated with 5 μg/ml anti-CD3 mAb and 5 μg/ml anti-CD28 mAb by incubation at room temperature for 45 min. PBMCs (1 × 10⁵–1 × 10⁶/ml) were suspended in AlyS505N-7 medium (Cell Science & Technology) containing 700 IU/ml IL-2, 5% FBS, and 1% penicillin-streptomycin-amphotericin B (Fujifilm Wako). The cells were then seeded into the flacks pre-coated with anti-CD3 and anti-CD28 mAbs. For short-term stimulation of CD8⁺ T cells derived from PBMCs, Dynabead human T-activator CD3/CD28 (Thermo Fisher Scientific) was used.

After removal of subcutaneous fat, skin specimens were minced and digested for 2 hours with 3 mg/mL of collagenase type III in RPMI 1640 medium (Wako, Osaka, Japan). The isolated cells were washed and incubated overnight in Iscove’s modified medium (Wako) supplemented with 10% Fetal Bovine Serum, L-Alanyl-L-Glutamine, penicillin/streptomycin, and 3.5 μL/L β-mercaptoethanol before analysis. In Figure 4, cells were isolated by short-term explant culture in the presence of 100 IU/mL of IL-2 (Wako) and 20 ng/mL of IL-15 (Wako). A hundred thousand of the isolated cells were incubated with or without 10 μg/mL GD3 (Adipogen AG, Liestal, Switzerland) for 15 hours before flow cytometry analysis. Concentration of GD3 was determined according to previous reports on its plasma concentration, functional assays (10 (link), 17 (link), 18 (link)), and our titration results using control blood T cells (Supplementary Figure S2C)

Peripheral mononuclear cells were isolated by Ficoll density gradient centrifugation (density 1.077; GE Healthcare Bio-Sciences, IL, USA). As for isolation of epidermal and dermal T cells, whole-skin specimens were incubated in 5 U/ml dispase (Life Technologies, CA, USA) overnight at 4 °C, and the epidermis was separated from the dermis. The epidermis was cut with scissors and incubated in collagenase III (3 mg/ml; Worthington Biochemical Corporation, NJ, USA) for 90 minutes with deoxyribonuclease (5 jig/ml; Sigma-Aldrich, MO, USA) in RPMI 1640 medium with 10% fetal bovine serum. A single-cell suspension was prepared by pipetting. The dermis was digested in the same way in collagenase III with deoxyribonuclease and further processed by a Medicon tissue disruptor (BD Biosciences, CA, USA). Short-term expansion culture was also applied to collect skin T cells from the whole-skin specimens or the epidermal/dermal specimens separated by dispase (Life Technologies) in the presence of 100 IU/ml of IL-2 (Wako, Osaka, Japan) and 20 ng/ml of IL-15 (Wako). The consistency of the T-cell phenotypes isolated from different body sites or different surgical techniques was also investigated in the subanalyses of our results (Supplementary Fig. 2).