Microglial activation in immunotherapy recipients. (A) CD11c expression was quantified by Q-PCR using total brain RNA from untreated, day 6, day 10, and day 13 immunotherapy recipients. β-Actin was used as a reference gene to calculate normalized fold expression. The bar graph shows the mean ± SD for each group (n = 3 mice per group; representative of two independent experiments). Asterisks denote a statistically significant increase (*, P < 0.01) relative to untreated LCMV carrier mice. (B) Representative flow cytometric plots show the frequency of microglia (CD45^lo/int Thy1.2⁻ Ly6C⁻ Ly6G⁻ CD11b⁺) expressing CD11c-YFP in the brains of naive, carrier, and day 12 immunotherapy mice. Boxes and associated percentages denote the frequency of CD11c-YFP⁺ and CD11c-YFP⁻ microglia. (C) Bar graph shows the frequency of CD11c-YFP⁺ microglia (mean ± SD) in naive, carrier, and immunotherapy (IT) recipients at days 12 and 13. All data are shown from a combination of 19 independent experiments (n = 1-4 mice per group per experiment; *, P < 0.05). (D and E) Representative histograms (D) and bar graphs (E) show the expression of MHC I (H-D^bK^b) and MHC II (I-A^b) on CD11c-YFP⁺ and CD11c-YFP⁻ microglia at day 12 after immunotherapy. Isotype control antibody staining is shown in gray on the histograms. Microglia were gated as described in B. Bar graphs show the geometric mean fluorescent intensity (GMFI) represented as mean ± SD (n = 5 mice per group). Data are representative of three independent experiments, and asterisks denote statistical significance (*, P < 0.05). (F and G) Representative histograms and bar graphs depict the expression of CXCL9 (F) and CCL5 (G) by microglia from carriers and day 12 immunotherapy recipients. Microglia were gated as described in B. Boxes denote the frequency of chemokine-expressing cells. Bar graphs show the mean ± SD. GMFI is shown (n = 4–5 mice per group; three independent experiments; *, P < 0.05).