Figure 4.

Mixed Lymphocyte Reaction Assays with PBMCs from the TLHM6 Monkey plus Transplanted iPSC-derived RPE Cells

(A) To evaluate the PBMC-RPE MLR assay using allogeneic iPS-RPE cells, fresh PBMCs (2 × 10⁶ cells/well) prepared from a TLHM6 monkey at pre-operation (pre-OPE) or at 8 weeks post-OPE were cultured with 46a iPS-RPE cells for 96 hr. Before the assay, the RPE cells were irradiated (20 Gy), and 1 × 10⁴ cells were cultured in a 24-well plate. Following the assay, the PBMCs were stained with anti-CD4, anti-CD8, anti-CD11b, anti-CD20, anti-CD56, or anti-Ki-67 antibody (plus each isotype control antibody) at 4°C for 30 min and then analyzed using flow cytometry. Compared with the PBMCs pre-OPE, all cell types in the PBMCs at post-OPE, except for CD11b⁺ cells, had greatly proliferated after co-culture with allogeneic iPS-RPE cells. Numbers (%) in the histogram indicate double-positive cells (e.g., CD4⁺/Ki-67⁺).

(B–D) Expression of MHC class II (MHC-II) and CD86 (B7-2) co-stimulatory molecules on B cells exposed to iPS-RPE cells. PBMCs or LN cells in the presence of iPS-RPE cells were stained with anti-MHC-II, anti-CD86, and anti-CD20 antibodies and analyzed using flow cytometry. (B) TLHM6 PBMCs (post-OPE, 8W), (C) TLHM6 LN cells (post-OPE, 16W), and (D) K-254 PBMCs (normal control). Numbers (%) in the histogram indicate double-positive cells (e.g., CD20⁺/MHC-II⁺).