Adoptive Regulatory T Cell Therapy Protects Against TPA-induced Lethal Hemorrhagic Transformation After Stroke

Objective:Blood brain barrier (BBB) damage is one of the most disabling consequences of stroke in the clinic. Thrombolytic treatment of ischemic stroke with recombinant tissue plasminogen activator (tPA), the only effective treatment for stroke, may exacerbate BBB breakdown and lead to lethal hemorrhagic transformation (HT). Identifying novel BBB protective agents may therefore reduce ischemia/reperfusion (I/R) injury and improve the efficacy and safety of tPA treatment. Our recent research suggests that adoptive regulatory T cell (Tregs) therapy could protect against cerebral ischemia via BBB protection. The present study aimed to further investigate the effect of Tregs on tPA-enhanced BBB damage and HT, and explore the mechanisms underlying Tregs-mediated protection of BBB.Methods:Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). tPA (10mg/kg) was continuously infused into the femoral vein over 20 min at 2h after MCAO. Immediately after tPA, Tregs, whichwere isolated from donor mice by CD4 and CD25 double selection were transferred to the MCAO mice through femoral vein. Cerebral hemorrhage was quantified by spectrophotometric hemoglobin assay of brain homogenates. BBB permeability was quantified at 24h after MCAO. Brain infarction and neurological performance was assessed up to 21 days post-stroke. To explore the involvement of PD-L1 in Treg-afforded BBB protection, Tregs were prepared from wild-type or PD-L1-/- mice and infused to the recipients at 2h after MCAO. BBB permeability and brain infarct were assessed at 1 or 3 days post-MCAO. In vitro experiments were carried out using a transwell system or a co-culture system allowing cell-to-cell contact.Results:Intraventricular injection of tPA at 2h after MCAO reproducibly results in HT in the ischemic territory at 24h after MCAO. Administration of Tregs dramatically reduced the tPA-induced cerebral hemorrhage in stroke mice. Further studies demonstrated that Treg treatment attenuated BBB damage after tPA-induced reperfusion, as manifested by reduced extravasation of plasma-derived IgG and exogenous evans blue dye and preserved continuity of tight junction protein ZO-1 and claudin. As a result, Treg-treated animals exhibited markedly reduced brain damage and improved long-term neurological outcomes lasting out to 21 days after MCAO and tPA treatment. Further studies suggest that Tregs inhibited tPA-potentiated MMP-9 and CCL2 expression in the ischemic brain. Deficiency of either one of these two factors will partially abolish the BBB protective effect of Tregs. 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 Abs or when neutrophils were pre-treated with PD-1 Abs. In vivo studies confirmed that intravenous administration of Tregs pre-treated with PD-L1 Abs or Tregs isolated from PD-L1-deficient mice failed to inhibit MMP-9 production by blood neutrophils 1 day after 60 min middle cerebral artery occlusion (MCAO). Furthermore, the BBB damage after MCAO was greatly ameliorated in PD-L1-competent Treg-treated mice, but not in PD-L1-compromised Treg-treated mice. Consequently, PD-L1 dysfunction abolished Treg-mediated brain protection and neurological improvements 3 days after MCAO.Conclusions:Our study suggests that Treg adoptive therapy reduces tPA induced BBB damage and HT after brain ischemia/reperfusion. Tregs may represent a potent cell-based therapy to improve the efficacy and increase the safety of thrombolytic treatment for stroke victims. PD-L1 plays an essential role in the BBB protection afforded by Tregs against cerebral ischemia by mediating the suppressive effect of Tregs on neutrophil-derived MMP-9.