IFN-γ and STAT1 are required for efficient induction of CXCR3 on CD4⁺ but not CD8⁺ T cells and T-bet, but not Eomes, controls CXCR3 induction. After stimulation with anti-CD3 and anti-CD28 antibody, cells were rested 24 hours without stimulation. CXCR3 surface protein expression was measured by flow cytometric staining of wild-type CD4 (A) and CD8 (B) cells in the presence of 50 μg/mL IFN-γ–neutralizing antibody or an isotype control. Stained cells treated with anti–IFN-γ are represented by hollow gray peaks, cells stimulated in the presence of control antibody are depicted by solid peaks, and dotted lines represent a PE-labeled isotype control. Surface CXCR3 was also analyzed on STAT1^−/− CD4 (C) and CD8 (D) T cells. STAT1^+/+ cells are represented by solid peaks, STAT1^−/− cells are represented by hollow gray peaks, and isotype control staining is repressed by a dotted line. Levels of CXCR3 (E,F), T-bet (G,H), and Eomes (I,J) mRNA in activated CD4 and CD8 T cells from WT (□) as well as STAT1^−/− mice (■) were measured by real-time RT-PCR. Data were normalized to the housekeeping gene GAPDH, and the results are presented as fold-induction of gene expression over nonactivated cells. Histograms in panels A and B are representative of 3 independent experiments, while panels C and D represent 1 of 5 independent experiments. Mean fluorescence intensity (MFI) data represent the mean (± SEM) for all experiments. Resting WT CD4⁺ and CD8⁺ T cells in these experiments yielded average MFIs of 39.8 (± 10.0) and 29.5 (± 2.6), respectively, while STAT1^−/− CD4⁺ and CD8⁺ T cells resulted in 32.6 (± 10.7) and 34.1 (± 11.4), respectively, prior to activation. (E-J) Averaged results (± SEM) of 5 independent experiments. Statistical analysis was performed using the Mann-Whitney rank-sum test (SigmaStat; Systat Software, San Diego, CA). *P < .05 was considered significant.