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Instructions: The crosstalk between T lymphocytes and microglia plays an important role in modulating neuroinflammation after ischemic stroke. We have demonstrated FasL mutant (gld) mice attenuated brain inflammation after ischemic injury, with a dramatic accumulation of double negative (DN) T cells in brain. However, the effects of DN T lymphocytes in modulating pro-inflammatory (M1) or anti-inflammatory (M2) microglial polarization are not fully understood.Methods: Middle cerebral artery occlusion (MCAO) was induced for 60 min followed by 24 h and 72 h reperfusion in both gld and C57BL/6J mice. DN T lymphocytes isolated from gld and C57BL/6J mice after MCAO were cocultured with primary microglia for 24 h. The subtypes of T lymphocytes and microglial polarizations in ischemic brain were measured by flow cytometry, real-time PCR and immunofluorescence.Oxygen glucose deprivation (OGD)-induced neurons were treated by the coculture supernatant for 24 h, followed by propidium iodide and calcein AM staining. In inhibition study, DN T cells were pretreated with ethyl-3,4-dephostatin, a inhibitor of protein tyrosine phosphatases nonreceptor type 2 (PTPN2), for 24 h.Results:In vivo, compared to C57BL/6J mice, DN T cells were significantly accumulated in ischemic cerebral hemisphere in gld mice 72 h after MCAO, accompanied by increased M2 microglial polarization. In vitro, microglia underwent M1 polarization when cocultured with DN T cells from C57BL/6J mice, whereas shifted to M2 phenotype when cocultured with gld-derived DN T cells. In addition, the coculture supernatant from gld mice attenuated OGD-induced neuronal death and neurotoxicity. Furthermore, DN T lymphocytes from gld mice exhibited a profound downregulation of PTPN2 in protein levels, which was accompanied by an enhanced protein level of TNF-α. In contrast, inhibition of PTPN2 abolished the decreased expression of TNF-α in DN T cells from gld mice, and simultaneously reduced the M2-microlgial polarization.Conclusions: DN T cells with FasL mutation drives M2-microglial polarization and attenuated neuroinflammation in experimental stroke. The underlying mechanisms may involve the abolishment of FasL/PTPN2 pathway in inhibiting TNF-α expression in DN T cells with FasL mutation.