Extended Data Fig. 2: Mouse brain T cells are effector memory cells and secrete IFNγ.
From: The subfornical organ is a nucleus for gut-derived T cells that regulate behaviour
a, Intravascular labelling strategy of circulating leukocytes via retro-orbital (R.O.) injection of a fluorophore-conjugated CD45 antibody. Representative FACS strategy to isolate extravascular tissue CD4 T cells from the mouse brain. Gating strategy was kept the same across all organs used for the experiment. b, Quantification of absolute numbers of extravascular CD4 αβ T cells in the brain with different incubation times of CD45 intravascular labelling. c, Representative FACS strategy of CD45 IV-negative extravascular CD4 T cells in the mouse brain. d, Representative FACS expression of T-bet in the steady-state brain, lung, liver and inguinal lymph node. e, FACS analysis of brain T cell population in Il15ra KO mice. **P < 0.01, ***P < 0.001 by unpaired parametric t-test (n = 5 per group). Grubbs’ outlier test (P < 0.05) was used to determine outliers, which were excluded. f, IFNγ reporter with endogenous polyA transcript” (GREAT) mice were used to look at IFNγ-YFP expression in various immune cells in steady-state tissue. g, Comparison of IFNγ-YFP expression in CD4 T cells (CD45 IV-, CD11b-, CD3+, CD4+, TCRβ+), CD8 T cells (CD45 IV-, CD11b-, CD3+, CD8+, TCRβ+), NK cells (CD45 IV-, CD11b-, CD3-, NK1.1+), γδ T cells (CD45 IV-, CD11b-, CD3+, TCRγδ+), and T and NK cell negative lymphocytes (CD45 IV-, CD11b-, CD3-, NK1.1-). (n = 6; repeated twice). h, Comparison of IFNγ-YFP expression across various immune cells subsets in the brain. i, Quantification of IFNγ-YFP+ cells in CD4 T cells, CD8 T cells and NK cells in the brain with or without 4-hour cell stimulation (PMA–ionomycin with Brefeldin A). *P < 0.05, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Bonferroni’s multiple comparisons test (n = 2 per group).
