Suppression of Bystander T Helper 1 Cells by Iris Pigment Epithelium-Inducing Regulatory T Cells via Negative Costimulatory Signals

Abstract

To determine whether iris pigment epithelium (IPE)-induced T regulatory (Treg) cells can suppress the activation of bystander T cells with cell contact via costimulatory interactions. CD8(+) T cells were cocultured with IPE, x-irradiated, and then used as regulators (IPE-induced Treg cells). The target CD4(+) T cells from wild-type control or knockout donors were used for the assay. T-cell activation was assessed for proliferation by examining both [(3)H]-thymidine incorporation and cytokine production. Expression of costimulatory molecules on IPE-induced Treg cells was evaluated using RT-PCR, immunostaining, and flow cytometry. Expression of costimulatory receptors on target T cells or Treg cells was evaluated by flow cytometry. Neutralizing antibodies were then used to abolish regulatory function. CD8(+) IPE-induced Treg cells significantly suppressed the activation of effector target T cells, e.g., T-cell proliferation and cytokine production such as Th1, Th2, and Th17 cytokines. Although IPE-induced Treg cells expressed various costimulatory molecules, including programmed cell death 1 ligand 1 (PD-L1), only PD-L1 on the Treg cells was actually delivered to target Th1 cells using cell-to-cell interaction (T-T interaction). If neutralizing antibodies for PD-L1 were cocultured with Treg cells, Th1 suppression was impaired. Moreover, Treg cells failed to suppress IFNgamma production by target CD4(+) T cells from programmed cell death 1 (PD-1) knockout donors. Th1-specific inhibition was exclusively achieved with direct cell contact. T cells exposed to IPE in the eye that acquires full regulatory capacity express negative costimulators and suppress bystander Th1-type effector cells.