iPS-HPCs are not susceptible to allogenic CTLs in vitro. (A) In vitro–generated HLA-A2–specific CTLs were mostly CD8⁺. (B) To determine the susceptibility of the iPS-HPCs to CTL cytotoxicity, a CTL cytotoxicity assay was performed. HLA-A2–specific CTLs were used as effector cells. iPS-HPCs (HLA-A2), immortalized B cells (HLA-A2–expressing positive control), and non–HLA-A2 immortalized B cells served as target cells. iPS-HPCs show very low susceptibility to killing by allogenic cytotoxic T cells. ***P < .001. As expected, HLA-A2 immortalized B cells were lysed. (C) To further study the interaction of HPCs and HLA-A2–specific CTLs, IL-2 release by CTLs was measured by the ELISPOT assay following stimulation of the CTLs with iPS-HPCs (HLA-A2), immortalized B-cells (HLA-A2–expressing positive control), and non–HLA-A2 cells (negative control). iPS-HPCs failed to activate CTLs in a time-dependent manner (mean ± SD). (D) To upregulate MHC-I expression by iPS-HPCs, HPCs were stimulated with IFN-γ for 48 hours. iPS-HPCs were harvested, and the expression of MHC-I molecules was analyzed. IFN-γ (empty overlay) upregulated expression of MHC-I molecules by HPCs compared with untreated iPS-HPCs (dark). Gray represents the isotype control. (E) After IFN-γ treatment, iPS-HPCs showed significantly increased susceptibility to CTL killing compared with untreated cells (mean ± SD).