Collagenase 1 and 2

Eight-week-old C57BL/6J male mice (Central Laboratory of Animals; Korea) were maintained under specific pathogen-free conditions with a temperature of 22-24°C, humidity of 50-60%, and a lighting regimen of 12 h light and 12 h dark. All animal experiments were performed under an experimental protocol approved by the Ethics Review Committee for Animal Experimentation of Dong-eui University (A2018-001).
After sacrificing the mice, the skin was extracted and cut to about 5 × 5 mm, and then skin tissue was immersed in phosphate-buffered saline (PBS, pH 8.0) containing dispase (Sigma Aldrich, USA) solution (2.5 unit/ml) for O/N at 4°C. Dermis and epidermis were chopped finely for separation and treated with collagenase I and II (Sigma Aldrich) solution (2.5 unit/ml each) at 37°C for 3 h, followed by treatment with 0.05% trypsin at 37°C for 20 min. The reaction was stopped by adding the same amount of DMEM containing 20% FBS and antibiotics/antimycotics and filtering with a cell strainer (70 μm pore), followed by collection through centrifugation at 12,000 ×g for 20 min.
Extracted epidermal cells were incubated under 37°C and 5% CO₂ using DMEM containing 20% FBS and antibiotics/antimycotics, and 1× human keratinocyte growth supplement (HKGS; GibcoBRL, USA). Extracted dermal cells were incubated at 37°C with 5% CO using DMEM containing 20% FBS and antibiotics/antimycotics.

Mice were infected intradermally in the right ear with 2x10⁶ stationary-phase promastigotes of L. amazonensis in 20 µl PBS. After 18 h, 15 days and 60 days post-infection, the ears were collected. Control was performed with ears from naïve mice. The dermal sheets were opened and added to the wells of a 24-well plate in DMEM (Sigma) with 1% penicillin/streptomycin and 100 µg/ml each of collagenase I and II (Sigma), followed by 90 min incubation at 37°C. The tissue was then macerated with a tissue mixer for 3 min, filtered through a 70 µm filter and washed with PBS at 400 g for 5 min at 15°C. Cells were resuspended in RPMI/10% FBS for further use.

Tumor samples from six patients with RCC were digested in RPMI-1640 containing collagenase I and II (0.1 mg ml⁻¹, Sigma-Aldrich) and DNAse I, minced, and digested for 1 h using the GentleMACS system (Miltenyi Biotec). Live γδ, CD4 and CD8 T cells were isolated from each digest using the BD FACSAria Fusion flow cytometer. Bulk RNA libraries (used for demultiplexing) were prepared according to the Smart-seq2 protocol^{75 (link)} and sequenced on an Illumina HiSeq 4000. Samples were then pooled by cell type (into γδ, CD4 and CD8 T-cell groups) before library preparation. Libraries were constructed using 10x Genomics Chromium 5′ (v1) and 10x Genomics Chromium V(D)J kit (PN-1000005) and sequenced on an Illumina NovaSeq 6000 with paired-end 100 base pair read lengths. For γδ TCR amplification, custom oligonucleotide sequences displayed in Supplementary Table 2 were used.