Essential role of program death 1-Ligand 1 in regulatory T-cell- Afforded protection against blood-brain barrier damage after stroke

Abstract

Background and Purpose-Our recent research revealed that adoptively transferred regulatory T cells (Tregs) reduced acute ischemic brain injury by inhibiting neutrophil-derived matrix metalloproteinase-9 (MMP-9) and protecting against blood-brain barrier damage. The mechanisms underlying Treg interactions with neutrophils remain elusive. This study evaluates the contribution of program death 1-ligand 1 (PD-L1) to Treg-mediated neutrophil inhibition and neuroprotection after cerebral ischemia. Methods-In vitro experiments were performed using a transwell system or a coculture system allowing cell- To-cell contact. Focal cerebral ischemia was induced in mice for 60 minutes. Tregs (2×106) isolated from donor animals (wild- Type or PD-L1"'") were intravenously injected into ischemic recipients 2 hours after middle cerebral artery occlusion (MCAO). MMP-9 production, blood-brain barrier permeability, and brain infarct were assessed at 1 or 3 days after MCAO. Results-In vitro experiments reveal that Treg-mediated inhibition of neutrophil MMP-9 required direct cell- To-cell contact. The suppression of MMP-9 was abolished when Tregs were pretreated with PD-L1 neutralizing antibodies or when neutrophils were pretreated with PD-1 antibodies. In vivo studies confirmed that intravenous administration of Tregs pretreated with PD-L1 antibodies or Tregs isolated from PD-L1-deficient mice failed to inhibit MMP-9 production by blood neutrophils 1 day after 60 minutes MCAO. Furthermore, the blood-brain barrier damage after MCAO was greatly ameliorated in PD-Ll-competent Treg- Treated mice but not in PD-Ll-compromised Treg- Treated mice. Consequently, PD-L1 dysfunction abolished Treg-mediated brain protection and neurological improvements 3 days after MCAO. Conclusions-PD-L1 plays an essential role in the neuroprotection afforded by Tregs against cerebral ischemia by mediating the suppressive effect of Tregs on neutrophil-derived MMP-9.