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Brain. 2017 Jul 1;140(7):1914-1931. doi: 10.1093/brain/awx111.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

Mao L1,2,3, Li P1,4, Zhu W1, Cai W1, Liu Z5, Wang Y5, Luo W6, Stetler RA1,2, Leak RK7, Yu W4, Gao Y2, Chen J1,2, Chen G8, Hu X1,2,5.

Author information

Pittsburgh Institute of Brain Disorders and Recovery and Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
State Key Laboratory of Medical Neurobiology and Institute of Brain Sciences, Fudan University, Shanghai 200032, China.
Life Science Research Centre of Taishan Medical University, Taishan 271016, Shandong, China.
Department of Anesthesiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China.
China-America Institute of Neuroscience, Beijing Luhe Hospital, Capital Medical University, Beijing 100010, China.
AstraZeneca Pharmaceutical Company, Waltham, Massachusetts 02452, USA.
Division of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA.
Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, China.


Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thrombolytic treatment for ischaemic stroke.


CCL2; blood–brain barrier; haemorrhagic transformation; matrix metalloproteinase 9; regulatory T cell

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