Extended Data Fig. 9: T_reg cells regulate astrogliosis by suppressing the IL-6–STAT3 signalling pathway.

a, DEREG or wild-type littermates were administered diphtheria toxin intraperitoneally on days 6, 9 and 12, and administered PBS or AREG protein intraventricularly on days 7, 9 and 12 after stroke onset. The GFAP⁺ areas and neurological scores were determined on day 14 (n = 7 PBS-treated wild type, n = 9 AREG-treated wild type, n = 10 PBS-treated DEREG, n = 8 AREG-treated DEREG). Data were pooled from two independent experiments. b, Ischaemic DEREG or wild-type littermates were administered diphtheria toxin intraperitoneally on days 6, 9 and 12 after stroke onset, and administered PBS or AREG protein (50 μg kg⁻¹) intraventricularly on days 7, 9 and 12 after stroke onset. TUNEL⁺ cells in the ischaemic brains were measured on day 14 (n = 7 PBS-treated wild-type mice, n = 7 PBS-treated DEREG mice, n = 3 AREG-treated DEREG mice). Data were obtained from a single experiment. c, Ischaemic DEREG or wild-type littermates were administered diphtheria toxin intraperitoneally on day 0 after stroke onset, and administered PBS or AREG protein (50 μg kg⁻¹) intraventricularly on day 1 after stroke onset. Neurological score and infarct region were evaluated on day 3 (n = 3 PBS-treated wild-type mice, n = 5 others). Data were obtained from a single experiment. d, Heat map of the qPCR analysis of neurotoxic markers of astrocytes on day-14 post-ischaemic brains of Cd3e^−/− mice injected with PBS, wild-type T_reg cells, or Areg^−/− T_reg cells on day 5 after stroke onset (n = 3). Data are representative of two independent experiments. e, KEGG pathway analysis of day-14 post-ischaemic brains of diphtheria-toxin-treated DEREG or wild-type littermates. Data were obtained from a single experiment. f, Microglia, CD45^highCD11b^high, CD45⁻ and astrocytes were isolated from the day-14 post-ischaemic brains of diphtheria-toxin-treated DEREG mice (n = 4). The relative expression levels of Il6 were evaluated by qPCR (relative to Hprt1). Data were obtained from a single experiment. g, ELISA for IL-6 of serum from ischaemic DEREG or wild-type littermates (n = 14 wild-type mice, n = 13 DEREG mice), Cd3e^−/− (CD3KO) mice transferred with wild-type or Areg^−/− T_reg cells (n = 2 intact mice, n = 6 PBS-treated mice, n = 6 wild-type T_reg-cell-transferred mice, n = 5 Areg^−/− T_reg-cell-transferred mice), and Rag2^−/− (Rag2KO) mice transferred with wild-type or Il1rl1^−/− T_reg cells (n = 3 PBS-treated mice, n = 6 wild-type T_reg-cell-transferred mice, n = 6 Il1rl1^−/− T_reg-cell-transferred mice). Data were pooled from two independent experiments. h, Relative Gfap and Stat3 mRNA expression levels (relative to Hprt1) in primary astrocytes stimulated with the indicated cytokines for 24 h (n = 3). Data are representative of two independent experiments. i, Microglia and astrocytes isolated from 12 adult mice were stimulated for 24 h with brain lysates from control or ischaemic mice in the presence or absence of AREG. Relative Il6 mRNA expression levels (relative to Hprt1) were evaluated by qPCR (n = 3). Data are representative of three independent experiments. j, GFAP⁺pSTAT3⁺ cells in ischaemic brains were measured on day 14 after stroke onset and representative photos are shown (n = 8 PBS-treated wild type, n = 5 PBS-treated DEREG, n = 6 AREG-treated DEREG). Data are representative of two independent experiments. k, Wild-type and Areg^−/− T_reg cells were transferred into Cd3e^−/− mice on day 5 after stroke onset. Representative photographs of Fig. 4h of GFAP⁺pSTAT3⁺ cells in the day-14 post-ischaemic brains are shown. Data are representative of two independent experiments and shown as mean ± s.e.m. P values were determined by one-way ANOVA.

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