(A) Schematic of culturing system. Naive CD4⁺ T cells were stimulated with α-CD3/CD28 and cultured under Th1-polarizing conditions (IL-12, α–IL-4). On day 3, cells were removed from stimulation and given IFN-γ, α–IL-4, and IL-2 for an additional 2 days. (B) At day 5, transcript analysis was performed via qRT-PCR. Transcript was normalized to Rps18 and presented as fold-change compared with WT control (n = 4 biological replicates from 4 independent experiments, mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-tailed unpaired Student’s t test). (C) Representative flow cytometric analysis for CXCR3 on IFN-γ–treated Th1 cells at day 5. Data are displayed as MFI fold-change compared with WT controls (n = 3 biological replicates from 3 independent experiments, mean ± SEM; **P < 0.01, 2-tailed unpaired Student’s t test). (D) An immunoblot was performed to assess the relative abundance of the indicated proteins. β-Actin serves as a loading control (n = 4 independent experiments, mean ± SEM; *P < 0.05, ***P < 0.001, 2-tailed unpaired Student’s t test). (E) ChIP assays were performed to assess STAT1 association with Cxcr3 in WT and Ikzf3^–/– Th1 cells. Publicly available ChIP-Seq data for STAT1 (GSM994528) were examined to identify potential regions of STAT1 enrichment. Sequencing tracks were viewed using IGV, and regulatory regions of interest are indicated by blue boxes. Approximate ChIP primer locations at the Cxcr3 promoter (prom.) and 3′ enhancer (enhc.) are indicated with gray arrows. (F) The indicated regions were analyzed for STAT1 enrichment. Data were normalized to total input. Percentage enrichment relative to input was divided by IgG, and data are presented as fold-change relative to IgG. (n = 4 biological replicates from 4 independent experiments, mean ± SEM; **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey’s multiple comparisons test.)