Fitc mouse igg1 κ isotype control

1 × 10⁵ epithelial target cells were washed and re-suspended in the cell staining buffer (Biolegend) containing different dye-conjugated primary antibodies at 1 μg/ml or 5 μg/ml for 30 min at 4°C. Cells were then washed and stained with the Zombie NIR (cell death marker, Biolegend). The stained cells were analyzed by flow cytometry with a FACSCanto II cytometer (BD Biosciences). Primary antibodies targeting the various surface ligands and receptors as well as the corresponding control include: FITC anti-human HLA-A,B,C (Clone W6/32, Biolegend), PE anti-human HLA-E (Clone 3D12, Biolegend), FITC anti-human ICAM-1/CD54 (Clone DX2, Biolegend), FITC anti-human Fas/CD95 (Clone DX2, Biolegend), APC anti-human Nectin-2/CD112 (Clone TX31, Biolegend), APC anti-human PVR/CD155 (Clone SKII.4, Biolegend), PE anti-human MICA (Clone 159227, R&D Systems), PE anti-human MICB (Clone 236511, R&D Systems), PE anti-human ULBP-2/5/6 (Clone 165903, R&D Systems), FITC Mouse IgG1 κ Isotype Control (Clone MOPC-21, Biolegend), APC Mouse IgG1 κ Isotype Control (Clone MOPC-21, Biolegend), and PE Mouse IgG2b κ Isotype Control (Clone MPC-11, Biolegend). Cell death analysis by Annexin V staining was performed using the FITC Annexin V detection kit (Biolegned) following the manufacturer’s protocol.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation. For surface staining, PBMCs were stained for 30 min in the dark at 4°C with the monoclonal antibodies PE-CF594 mouse anti-human CD4 (BD Biosciences, San Diego, CA, USA), PE anti-human CD25 (BioLegend, San Diego, CA, USA), FITC anti-human CD161 (BioLegend), 7AAD (BD Pharmingen, San Diego, CA, USA), and CD45RO PE-Cy7 (BD Biosciences) or with FITC Mouse IgG1 κ isotype control (BioLegend) and Mouse IgG1 κ isotype control PE (eBioscience, San Diego, CA, USA). For intracellular staining of cytokines, incubate PBMCs in RPMI 1640 medium (Gibco, Life Technologies, Shanghai, China) in 5% CO₂ at 37°C, then stimulate these cells for 5 h, PBMCs were stimulated for 5 h with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Steinheim, Germany) and 1 μg/mL ionomycin (BD Pharmingen) in the presence of 10 μg/mL Brefeldin-A (BFA; BioLegend) and subsequently fixed and permeabilized by Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Then the cells were stained for 30 min away from the light at 4°C with anti-human IL-17A APC (eBioscience) or Mouse IgG1 κ Isotype Control APC (eBioscience).

TGF-β1 (PEPRO TECH, China, 100-21-10 μg), PC-MSC (Provided by Shenzhen 150 Biomedical Co.,Ltd.), ESC (Provided by ATCC), FITC Mouse IgG1 κ Isotype control (Biolegend, 7313762), PE Mouse IgG1 κ Isotype control (Biolegend, 9217868), APC Mouse IgG1 κ Isotype control (Biolegend, 9282592), PE Mouse Anti-Human CD73 (Biolegend, 8151901), APC Mouse Anti-Human CD105 (Biolegend, 9277133), FITC Mouse IgG2a κ Isotype control (Biolegend, 8241935), FITC Mouse Anti-Human CD14 (Biolegend, 8299630), FITC Mouse Anti-Human CD45 (Biolegend, 8206704), DMEM/F12 medium (Peiyuan, Shanghai, L310 kJ), Fetal bovine serum (Gemini, 900-108), Trypsin digestive fluid (Beyo, C0203), DPBS (Beyo, C0221G), CCK8 (Beyotime, C0039), EDU staining kit (Beyotime, C0081s), Hematoxylin-eosin staining kit (Solarbio, G1120), DEPC-treated water (Solarbio, R1600), trichloromethane (Sinopharm Chemical Reagent Co. LTD., 10006818), GelRed dye (Biotium, #41003), RNA Loading Buffer (TaKaRa, 9168), Tris-Borate-EDTA Buffer (TBE) 10× Powder, pH8.3 (TaKaRa, T9122), RNAsimple Total RNA Kit (TIANGEN, DP419), All-in-One First-Strand Synthesis Master Mix (Yugong Biolabs, EG15133S), SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205-02), and Methacrylate anhydride (MAA, 0.1 mL/1 g gelatin), ELISA Kit (Solarbio, SEKH-0052), MateRegen^® Gel (2104006, BioRegen Biomedical (Changzhou) Co., Ltd.).