Regulatory T Cell Transplantation Protects Against Cerebral Ischemic Injury Via Modulating Peripheral Immune Response

OBJECTIVES:Ischemic stroke elicits a profound inflammatory response involving both innate and adaptive immunity. The reciprocal interaction between the immune system and the ischemic brain helps determine stroke pathology and represents a promising target for stroke treatment. Recent evidence suggests that peripheral regulatory T cells (Tregs), an innate immuno-modulator, might be an endogenous protective mechanism that counterbalances cerebral inflammation following ischemic stroke. However, the therapeutic potential of Treg transplantation in stroke has not been evaluated and the underlying mechanism for Treg-afforded neuroprotection is still poorly understood.METHODS:Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for60minutes in mice or120minutes in rats. Tregs were isolated from donor animals by CD4and CD25double selection and systemically transferred to ischemic recipients at2,6or24hours after MCAO. Brain infarction and neurological performance was assessed up to28days post-stroke. The mechanisms of Treg-afforded neuroprotection were investigated using cell-specific depletion, gene knockout mice, bone marrow chimeras and in vitro Treg-neutrophil co-cultures.RESULTS:Using both mouse and rat models of transient focal cerebral ischemia, we demonstrate that adoptively transferred Tregs markedly reduced brain damage and improved long-term neurological outcomes with a wide temporal window of efficacy (24h). Maximal protection occurred upon early Treg transfer with a2h delay after MCAO, which resulted in approximately50%reduction of infarct volume. The protective effects of Tregs were associated with decreased cerebral inflammation and reduced infiltration of peripheral inflammatory cells into the lesioned brain. Treg treatment also attenuated blood-brain barrier (BBB) disruption as early as1day after ischemia, as manifested by reduced extravasation of plasma-derived IgG and exogenous tracers, preserved continuity of tight junction protein ZO-1and protected ultrastructure of tight junctions by electron microscopy. Surprisingly, using Tregs with the CD45.1congenic marker or cell tracker labeling, we noticed that exogenous Tregs exerted early neuroprotection without penetrating into the brain parenchyma. Rather, Tregs suppressed peripheral neutrophil-derived matrix metallopeptidase-9(MMP-9), thereby preventing proteolytic damage of the BBB. Treg-afforded neuroprotection after MCAO was abolished by1) gene knockout of MMP-9,2) transplantation of bone marrow from MMP-9knockout mice into irradiated wide type mice, or3) depletion of neutrophils with anti-Grl antibody, In vitro studies using Treg-neutrophil co-cultures further confirmed that Tregs suppressed neutrophil-derived MMP-9in a cell-cell contact dependent manner, In additions to its potent central neuroprotection, Treg treatment was shown to ameliorate post-stroke lymphopenia, suggesting an additional beneficial effect on systemic immune status.CONCLUSIONS:Our study demonstrates that Treg adoptive therapy is a novel and potent cell-based therapy targeting post-stroke inflammatory dysregulation and neurovascular disruption.