Immunospot s6 ultra analyzer

PPD-specific IFN-γ release was determined by using an enzyme-linked immunospot (ELISpot) assay according to the manufacturer’s instructions (U-CyTech, Utrecht, Netherlands). Briefly, 96-well PVDF plates (Millipore, Bedford, MA, USA) were coated with anti-human IFN-γ coating antibody overnight at 4°C. The wells were blocked for 1 hr at 37°C, then 2.5 × 10⁵ PBMCs in 100 μL RPMI-1640 complete medium was inoculated into each well in the presence of PPD (20 μg/mL). RPMI-1640 complete medium served as a negative control and 2.5 μg/mL phytohemagglutinin (PHA) (Sigma-Aldrich) treatment as a positive control. After 20 h incubation at 37°C, the plates were incubated with a biotin-labeled detection antibody at 37°C for 1 hr and subsequently horseradish peroxidase (HRP)-conjugated streptavidin working solution for another 1 h. AEC substrate solution was added to each well for 30 min in the dark at room temperature. Color development was stopped by thoroughly rinsing both sides of the PVDF membrane with demineralized water. The plates were dried in the dark at room temperature. Spots were counted using a C.T.L. ImmunoSpot^® S6 Ultra Analyzer (Cellular Technology Limited, Shaker Heights, OH, USA). The number of BCG-specific IFN-γ-producing cells was calculated based on the spot-forming units (SFUs) per 2.5 × 10⁵ PBMCs after deducting the background SFUs of the paired negative control wells.

The ELISPOT assays were performed by mixing engineered T cells or control effector CD8⁺ T cells (10⁵ cells) with target cells at a 4:1 effector-target ratio, and then added to the IFN-γ antibodies-precoated plates (Dakewe, China). Effector CD8⁺ T cells alone were treated as negative control, while phytohemagglutinin (PHA) stimulated CD8⁺ T cells were treated as positive control. Plates were incubated for 18 to 24 h at 37 °C and 5% CO₂. For allogeneic reaction, TCR⁻/HLA-I⁻ T cells were co-cultured with irradiated allogenic PBMCs (60 Gy) at a concentration of 5 × 10⁴ cells per well in IFN-γ ELISPOT plates. In a parallel experiment, allogeneic PBMCs were co-cultured with TCR⁻/HLA⁻ T cells that had been irradiated. Two groups of cells were incubated in a 1:1 ratio for 18 h. The experiment was subsequently performed according to the manufacturer’s instructions. Briefly, cells were removed from the plates and plates were washed for 6 times with washing buffer. Then, biotinylated IFN-γ antibodies were added and incubated for 1 h at 37 °C. After washing for 6 times, streptavidin-HRP was added and incubated for 1 h at 37 °C. Subsequently, AEC buffer was added to each well and incubated for 10 min at room temperature in the dark. Plates were scanned and the number of positive spots was calculated using the ImmunoSpot S6 ultra analyzer (Cellular Technology Ltd., USA).