Conventional and unconventional T cells infiltrate the ischemic hemisphere and produce high amounts of cytokines at the site of the injury. (A) Absolute numbers of T lymphocytes in ischemic hemispheres at days 1, 3, and 7 after MCAO. Cell counts were determined by flow cytometric analysis of CNS-infiltrating cells after Percoll density centrifugation and staining for CD45 and CD3. For absolute quantification, TrueCount tubes were used. (B) Frequency of CD4⁺, CD8⁺, and γδ T lymphocytes in ischemic hemispheres at days 1, 3, and 7 after stroke. Data were obtained after flow cytometric analysis of CNS-infiltrating cells stained for CD45, CD3, NK1.1, CD4, CD8, and γδ T-cell receptor. (C) GFP-positive γδ T cells were visualized in the penumbra area of the ischemic hemisphere in Tcrd-H2BEGFP mice 3 days after MCAO (red indicates GFAP-positive astrocytes; blue, DAPI nuclear staining; scale bar = 20 μm). Flow cytometric analysis of IFN-γ (D) and IL-17A (E) produced by CD4⁺, CD8⁺ and γδ T cells isolated from ischemic hemispheres at different days after stroke induction. Right panels show intracellular stainings for IFN-γ and IL-17A of gated CD4⁺, CD8⁺, and γδ T cells of 1 representative experiment at day 3. (F) Comparison of IFN-γ and IL-17A expression by T cells isolated from brain, peripheral blood, and spleen 3 days after stroke. (A,B,D,F) The graphs show mean ± SD of 12-14 animals per group, in 3-4 independent experiments for each time point. Statistical significances analyzed by 1-way ANOVA with Bonferroni posthoc test (*P < .05) in all cases.