Figure 5
Figure 5. iPS-HPCs induce CD8 T-cell anergy due to poor MHC-I expression and lack of costimulatory molecules. (A) iPS-HPCs do not express the costimulatory molecules CD80 or CD86, respectively, as determined by flow cytometry, suggesting their inability to present antigen to T cells. Furthermore, iPS-HPCs express the T-cell inhibitory ligand PD-L1 but not PD-L2. (B) Expression levels of costimulatory molecules were unchanged following treatment with IFN-γ. (C) IFN-γ secretion by isotype control–treated CTLs cocultured with IFN-γ–pretreated iPS-HPCs was increased by 50-fold compared with isotype control or after treatment with an anti-CD28 antibody. IFN-γ served to upregulate the expression of MHC-I. However, anti-CD28 antibody–treated CTLs cocultured with IFN-γ–pretreated iPS-HPCs secreted 120 times more IFN-γ compared with isotype control or anti-CD28 antibody–treated CTLs cultured with untreated HPCs (mean ± SD). **P < .01; *P < .05. This result showed that delivery of costimulation through the anti-CD28 antibody rescued the stimulatory capacity of HPCs. (D) Anti-CD28 antibody–treated CTLs cocultured with IFN-γ–pretreated HPCs highly release IL-2 compared with isotype controls or anti-CD28 antibody–treated CTLs cocultured with untreated iPS-HPCs (mean ± SD). **P < .01; *P < .05. (E) Alloreactive T cells stimulated with iPS-HPCs did not appear to be stimulated, as their growth remained slow and in single-cell suspension. However, T cells stimulated with immortalized B cells formed clusters as a sign of stimulation. (F) T cells restimulated with iPS-HPCs failed to respond to stimulation with immortalized B cells in the absence of IL-2 as determined by IFN-γ secretion. After addition of IL-2, T-cell proliferation recovered suggesting that IL-2 stimulation reactivated anergic T cells (mean ± SD). **P < .01. In contrast, T cells stimulated with immortalized B cells in the first and second stimulations did not show significantly increased secretion of IFN-γ in the presence of IL-2. Thus, iPS-HPCs induce T-cell anergy that was rescued by IL-2. (G) iPS-HPCs were pretreated with a PD-L1–blocking antibody or isotype control antibody and then cocultured with CTLs for 3 days. The CTLs were then plated into an IFN-γ ELISPOT plate with irradiated M23 cells and treated with recombinant IL-2. IFN-γ production by IL-2–treated CTLs cocultured with iPS-HPCs pretreated with the PD-L1–blocking antibody was significantly higher than that of IL-2–treated CTLs cocultured with untreated iPS-HPCs (mean ± SD). *P < .05. This experiment definitively suggests that PD-L1 has a role in the induction of T-cell anergy by iPS-HPCs. These experiments were repeated 3 times.

iPS-HPCs induce CD8 T-cell anergy due to poor MHC-I expression and lack of costimulatory molecules. (A) iPS-HPCs do not express the costimulatory molecules CD80 or CD86, respectively, as determined by flow cytometry, suggesting their inability to present antigen to T cells. Furthermore, iPS-HPCs express the T-cell inhibitory ligand PD-L1 but not PD-L2. (B) Expression levels of costimulatory molecules were unchanged following treatment with IFN-γ. (C) IFN-γ secretion by isotype control–treated CTLs cocultured with IFN-γ–pretreated iPS-HPCs was increased by 50-fold compared with isotype control or after treatment with an anti-CD28 antibody. IFN-γ served to upregulate the expression of MHC-I. However, anti-CD28 antibody–treated CTLs cocultured with IFN-γ–pretreated iPS-HPCs secreted 120 times more IFN-γ compared with isotype control or anti-CD28 antibody–treated CTLs cultured with untreated HPCs (mean ± SD). **P < .01; *P < .05. This result showed that delivery of costimulation through the anti-CD28 antibody rescued the stimulatory capacity of HPCs. (D) Anti-CD28 antibody–treated CTLs cocultured with IFN-γ–pretreated HPCs highly release IL-2 compared with isotype controls or anti-CD28 antibody–treated CTLs cocultured with untreated iPS-HPCs (mean ± SD). **P < .01; *P < .05. (E) Alloreactive T cells stimulated with iPS-HPCs did not appear to be stimulated, as their growth remained slow and in single-cell suspension. However, T cells stimulated with immortalized B cells formed clusters as a sign of stimulation. (F) T cells restimulated with iPS-HPCs failed to respond to stimulation with immortalized B cells in the absence of IL-2 as determined by IFN-γ secretion. After addition of IL-2, T-cell proliferation recovered suggesting that IL-2 stimulation reactivated anergic T cells (mean ± SD). **P < .01. In contrast, T cells stimulated with immortalized B cells in the first and second stimulations did not show significantly increased secretion of IFN-γ in the presence of IL-2. Thus, iPS-HPCs induce T-cell anergy that was rescued by IL-2. (G) iPS-HPCs were pretreated with a PD-L1–blocking antibody or isotype control antibody and then cocultured with CTLs for 3 days. The CTLs were then plated into an IFN-γ ELISPOT plate with irradiated M23 cells and treated with recombinant IL-2. IFN-γ production by IL-2–treated CTLs cocultured with iPS-HPCs pretreated with the PD-L1–blocking antibody was significantly higher than that of IL-2–treated CTLs cocultured with untreated iPS-HPCs (mean ± SD). *P < .05. This experiment definitively suggests that PD-L1 has a role in the induction of T-cell anergy by iPS-HPCs. These experiments were repeated 3 times.

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