Figure 3.

Accumulation of IL-17⁺ γδ T cells at the meninges is associated with increased infarct size. (a) Flow cytometric gating strategy of brain microglia (CD45^intCD11b^int) and infiltrating leukocytes (CD45^high) including monocytes/macrophages (Mono/MΦ; CD11b⁺Ly6G⁻CD11c⁻), neutrophils (PMN; CD11b⁺Ly6G⁺CD11c⁻), CD4⁺ T cells (CD11b⁻CD4⁺CD8⁻), CD8⁺ T cells (CD11b⁻CD8⁺CD4⁻), B cells (CD11b⁻CD4⁻CD8⁻CD19⁺) and natural killer cells (NK; CD11b⁻NK1.1⁺) in AC Res and AC Sens naïve mice and days 1, 2 and 3 after MCAO. Graphs represent absolute number of cells in the ischemic hemisphere (n = 5–9 per group, Supplementary Table 3). Bottom right graph, percentage of leukocyte reduction 2 days after reperfusion (n = 8–9 per group, Supplementary Table 3). Values are mean ± s.e.m. **P < 0.01 versus AC Res; ^#P < 0.05 and ^##P < 0.01 versus neutrophils (Student’s t-test). (b) Cxcl1 and Cxcl2 mRNA expression in brains of AC Res and AC Sens mice 1 d after MCAO (n = 10 per group). Values are fold change over brain from naïve mice. (c) Flow cytometric gating strategy of meningeal PMN in AC Res and AC Sens mice 1 d after MCAO. Graph represents PMN cell number quantification in the meninges (n = 9 per group). (d) Flow cytometry analysis of meningeal IL-17⁺ γδ T cells (CD45^highCD11b⁻TCR-β⁻TCR-γδ⁺IL-17⁺-GFP) of AC Res and AC Sens IL-17-eGFP mice 16 h after MCAO. Left graph represents the total number of γδ T cells and right graph, total number of IL-17⁺ γδ T cells in the meninges of AC Res and AC Sens mice subjected to sham surgery (n = 7 per group) and after MCAO (AC Res, n = 7; AC Sens, n = 6). Each data point (n) is derived by pooling 2 hemispheres. (e) Representative photomicrographs of meningeal γδ T cells (green; TCR-γδ-GFP⁺), laminin (red) and cell nuclei (blue, To-Pro-3) 16 h after MCAO. Coronal section is taken at Bregma + 3 mm. Columns and line plots represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; n.s., not significant (Student’s t-test)