Supplymetary Figure 1. Capacity of iPS-RPE cells to suppress activation of bystander T cells. (A) Cultured iPS-RPE cells (454E2: gray bars) and control RPE cells (ARPE-19: black bars) were used for the assay. In the presence of anti-human CD3 abs and rIL-2, purified CD4+ T cells were co-cultured with from 5 x 103 to 5 x 105 (confluent) iPS-RPE cells or control RPE cells for 48 h. Results indicate the IFN-g production by T cells exposed to RPE cells. (B) After separation of CD4+ T cells from a TLHD1 donor, the autologous iPS-RPE cells (TLHD1) and allogeneic iPS-RPE cells (454E2) were prepared and co-cultured. After 48 h, supernatants of the T-RPE cell culture were collected to measure IFN-g production. Data are the mean ± SEM of three ELISA determinations. * P <0.05, ** P <0.005, *** P <0.0005, as compared to the positive control (T cells alone). (C) Mixed lymphocyte reactions (MLRs) were prepared with CFSE-labeled PBMCs (n=1) plus mitomycin C (MMC)-treated allogeneic PBMCs (n=2). The iPS-RPE (454E2) and ARPE-19 cells were cultured with the MLRs. After 96 h, the PBMCs were harvested for flow cytometric analysis. The Y-axis in the graph indicates the number of CFSE-positive cells. * P <0.05, as compared to the positive control (open bar).
Supplymentary Figure 2. Capacity of iPS-RPE cell–derived soluble factors to suppress activation of T cells. CD4+ T cells in the presence of anti-CD3 abs and rIL-2 were co-cultured with human recombinant proteins such as IL-1RA, IL-6, IL-8/CXCL8, IL-21, IL-22, MIF, TSP-1, TNF-a, TNFRI, MIG/CXCL9, MCP-1/CCL2, MIP-3a/CCL20, TGFb1, and TGFb2 and evaluated with IFN-g production by the T cells. Data are the mean ± SEM of three ELISA determinations. * P <0.05, ** P <0.005, *** P <0.0005, as compared to the positive control (CD4+ T cells without recombinant proteins, open bar). ND, not detected.