Results Treg Treatment Elevates the Number of Functional Tregs in the Blood and Spleen After MCAO

Cerebral ischemia induces prompt and robust local inflammation in the brain, which is characterized by the activation of glial cells and infiltration of leukocytes. Mounting evidence has demonstrated that overactivation of these inflammatory cells releases a large number of cytotoxic molecules and exacerbates brain injury. Interestingly, the immune responses elicited by focal cerebral ischemia are not restricted to the brain but extend into the periphery. Activation of cytokines, chemokines, and chemokine receptors has been observed in peripheral blood after cerebral ischemia and is associated with rapid neurological deterioration and poor functional outcomes in patients with stroke. In response to such widespread and pervasive inflammation, multiple anti-inflammatory mechanisms are launched. However, these anti-inflammatory mechanisms negatively affect the function and composition of the systemic innate and adaptive immune systems. As a consequence, there is poststroke immunosuppression, predisposing stroke victims to infections and associated complications. Although it is well accepted that inflammation amplifies brain damage and represents a promising target for stroke management, the application of anti-inflammatory agents in patients with stroke must be carefully titrated to avoid catastrophic immunosuppression. Regulatory T cells (Tregs) are a specialized population of T cells that play essential roles in suppressing inflammatory responses and maintaining immune homeostasis. As a result of their potent immunomodulatory properties, much of the stroke literature has focused its recent attention on Tregs. Clinical and animal studies demonstrate that the number of circulating Tregs increases after stroke and remains elevated for several weeks. Some of these Tregs exit the peripheral circulation, infiltrate into the ischemic brain, and continue Background and Purpose—Cerebral ischemia has been shown to result in peripheral inflammatory responses followed by long-lasting immunosuppression. Our recent study demonstrated that intravenous delivery of regulatory T cells (Tregs) markedly protected against transient cerebral ischemia by suppressing neutrophil-derived matrix metallopeptidase 9 production in the periphery. However, the effect of Tregs on systemic inflammatory responses and immune status has not been fully characterized. Methods—Cerebral ischemia was induced by middle cerebral artery occlusion for 60 minutes in mice or 120 minutes in rats. Tregs were isolated from donor animals by CD4 and CD25 double selection and transferred intravenously to ischemic recipients at 2 hours after middle cerebral artery occlusion. Animals were euthanized on different days after reperfusion. The effects of Tregs on systemic inflammation and immune status were evaluated using flow cytometry, ELISAs, and immunohistochemistry. Results—Systemic administration of purified Tregs raises functional Tregs in the blood and peripheral organs, including spleen and lymph nodes. These exogenous Tregs remain in the blood and peripheral organs for ≥12 days. Functionally, Treg adoptive transfer markedly inhibits middle cerebral artery occlusion–induced elevation of inflammatory cytokines (interleukin-6 and tumor necrosis factor α) in the blood. Furthermore, Treg treatment corrects long-term lymphopenia and improves cellular immune functions after ischemic brain injury. As a result, Treg-treated animals exhibit decreased bacterial loads in the blood during recovery from cerebral ischemic attack. Conclusions—Treg treatment did not exacerbate poststroke immunosuppression. On the contrary, Treg-treated animals displayed improved immune status after focal cerebral ischemia. (Stroke. 2013;44:00-00.)