Induction of TNF-α in macrophages by αβ T cell–derived by IFN-γ. (A) Immunohistochemical staining of macrophages/microglia (Iba1) and astrocytes (GFAP) in wt, Rag1^−/−, Tcrd^−/−, and sham-operated wild type wt mice after MCAO (scale bar = 50 μm). (B) Frequency of TNF-α–positive brain macrophages, microglia, and peripheral (spleen) macrophages analyzed 3 days after MCAO. Brain macrophages were identified as CD45^high, CD11b⁺, or CD11c⁻ and were distinguished from microglia by the higher expression of CD45. Frequency of TNF-α–positive brain macrophages (C) and microglia (D) in wt, Rag1^−/−, and Tcrd^−/− mice analyzed by flow cytometry 3 days after stroke. Representative dot plots show CD11b⁺ CD45^high and CD11b⁺ CD45^intermediate-gated populations, identifying macrophages and microglia, respectively. (E) In this experiment, Rag1^−/− mice were reconstituted 1 hour before stroke induction with 1 × 10⁷ CD3⁺ cells isolated from wt, Tcrd^−/−, or Ifng^−/− mice. The production of TNF-α by macrophages was analyzed 3 days after stroke by flow cytometry. In all experiments, data show the means ± SDs of 9-12 animals per group, analyzed in 3-4 independent experiments, and the statistical analysis with the use of 1-way ANOVA with Bonferroni posthoc test. **P < .01, ***P < .001.