Ultralow attachment u bottom plates

H1-derived islet-like cells were transfected with siCTL or siNL#2 and were then left untreated or treated with IFNα (2000 U/ml) for 24 hours. The cells were detached using Accutase for 5 min at 37°C, and then the HLA-ABC expression at the cell surface was evaluated by flow cytometry and the NLRC5 KD efficiency by Western blotting. For the coculture, 100,000 H1-derived islet-like cells were cultured with 50,000 HLA-A2 PPI-specific CD8⁺ T cells (42 (link)) in human islet medium supplemented with IL-2 (25 U/ml; R&D Systems), IL-15 (5 ng/ml; PeproTech), and anti-human CD107a–fluorescein isothiocyanate (Thermo Fisher Scientific) in ultralow attachment U-bottom plates (Corning). The cocultured cells were then incubated at 37°C for 4 hours. Cells were washed with flow cytometry buffer (2% BSA in PBS with 5 mM EDTA) and then incubated with mouse anti-human CD8 allophycocyanin (APC) (BD Biosciences) at 4°C for 30 min. Last, the cells were fixed with 4% PFA at 4°C for 20 min and analyzed using a BD LSRFortessa X-20 flow cytometer (San Jose, CA, USA).
The percentage of CD107a⁺ of the CD8⁺ T cells was calculated using FlowJo software version v10. Briefly, the single cells were identified as described above, and then CD8⁺ T cells were first selected to allow the calculation of the percentage of CD107a⁺ (fig. S10B).