Fig. 4

Immunogenicity of RPE and LbL-RPE cells in vitro. a Expression of HLA class I and HLA class II on RPE and LbL-RPE cells. RPE and LbL-RPE cells in the presence of recombinant IFN-γ (100 ng/mL) were cultured for 48 h then stained with anti-HLA-I or HLA-II antibodies. The isotype control is shown in red. Numbers in the histogram indicate that the percentage of positive cells was less than 0.5% when determined by isotype-specific controls. b Detection of co-stimulatory molecules using anti-CD80, CD86, or B7-H3 (blue) in RPE and LbL-RPE cell cultures with or without IFN-γ. Isotype control is shown in red. Numbers in the histogram indicate that the percentage of positive cells was less than 0.5% when determined by isotype-specific controls. c Production of IFN-γ in CD4⁺ T cells exposed to RPE or LbL-RPE cells. Purified CD4⁺ T cells (5 × 10⁵, from human PBMCs) were co-cultured with RPE or LbL-RPE cells for 48 h, and the levels of IFN-γ in the supernatants measured (data represent the mean ± SEM of three independent experiments; ND, not detected; ****p < 0.0001; **p < 0.01). d Detection of inflammatory cytokine IFN-γ by human PBMCs when co-cultured with RPE or LbL-RPE cells. PBMCs (5 × 10⁵) were cultured with RPE or LbL-RPE cells for 120 h, and the levels of IFN-γ in the supernatants measured (data represent the mean ± SEM of three independent experiments; ***p < 0.001; *p < 0.05)