Expression of human IFN-γ from a 190-kb IFNG-BAC reporter transgene in CD4⁺ T cells is Stat4 and T-bet dependent. Transgenic CD4⁺ T cells from C57BL/6 (Wt), C57BL/6.Tbx21^−/− (T-bet^−/−), or C57BL/6.Stat4^−/− mice were cultured under Th1 or Th2 conditions for 3 d (primary stimulation) or 5 d. After 5 d, cultures were harvested and restimulated with plate-bound anti-CD3 only for 2 d (secondary stimulation). Levels of human IFN-γ (left panel) and of murine IFN-γ (right panel) were determined by ELISA.

Regulation of IFNG-BAC transgene activity in CD8⁺ T cells. A, CD8⁺ T cells were purified from IFNG-BAC⁺ Wt, Stat4^−/−, or T-bet^−/− mice and were cultured under Th1 or Th2 polarizing conditions as in Fig. 1. Cultures were harvested after 3 d (primary responses) or 2 d after restimulation with anti-CD3 (effector responses) and were analyzed for levels of human or mouse IFN-γ by ELISA. B, CD8⁺ T cells were also purified from IFNG-BAC⁺ Wt or T-bet^−/− OT-I TCR transgene-positive mice and stimulated with peptide Ag, APCs, and IL-2. After 5 d, cultures were harvested and restimulated with peptide and APCs. After an additional 2 d, cultures were harvested and IFN-γ ELISA assays performed.

Deletions made in this study. Positions of 40-kb deletions from the IFNG-BAC transgene are shown: red, Δ1; green, Δ2; blue, Δ3; black, Δ4 and small 1-kb CNS deletions; brown, Δ-77; and purple, Δ-30. Relative positions of human IFNG, Il26, and Il22 in the IFNG-BAC transgene are shown below. Evolutionary mouse-human sequence conservation, locations of CNS (red, >70% sequence conservation between mouse and human spanning ≥200 bp), and locations of transposons (green) within the 190-kb human IFNG-BAC transgene are as identified using the DCODE Web site (www.dcode.org).

Functional consequences of large 40-kb deletions from the 190-kb human IFNG-BAC transgene. A–D, Transgenic CD4⁺ T cells were cultured under Th1 or Th2 conditions as in Fig. 1. Human IFN-γ levels were determined for (A) IFNG-BAC, Δ1, and Δ2 CD4⁺ T cells, and for (B) IFNG-BAC, Δ3/4, and Δ4 CD4⁺ T cells. C, Murine IFN-γ levels were determined for IFNG-BAC, Δ3/4, and Δ4 CD4⁺ T cells. D, CD8+ T cells were cultured under Tc1 or Tc2 conditions as in Fig. 2, and human IFN-γ levels were determined. E, IFNG-BAC, Δ3/4 and Δ4 CD4⁺ T cells were cultured under Th1 conditions for 3 d, and mRNA levels were determined by quantitative PCR. Data points represent results of independent experiments. F, Freshly isolated NK cells were stimulated with nothing or with IL-12 plus IL-18. After 48 h of culture, IFN-γ levels were determined. G, IFNG-BAC or Δ3/4 BAC transgenic splenocytes were stimulated with αGalCer for 2 d. Supernatant IFN-γ levels were determined by ELISA (all except E), and results are averages of at least three independent experiments. Error bars are SEM.

CNS-30 is necessary for human IFN-γ production by T cells. Cells were cultured from IFNG-BAC, Δ3/4 BAC, or Δ-30 BAC transgenic mice. A, CD4⁺ T cells were cultured for 3 d under Th1 or Th2 conditions and processed for ChIP assays with Abs specific for Runx3 or IgG. Values for IgG controls were subtracted from means. B, CD4⁺ T cells were cultured under Th1 or Th2 polarizing conditions. Levels of human and mouse IFN-γ were determined by ELISA. C, Effector Th1 cells were stimulated with nothing, IL-12, IL-18, or with IL-12 plus IL-18. After 48 h, culture IFN-γ levels were determined by ELISA. Results are averages of at least three independent experiments. D, CD4⁺ and CD8⁺ T cells were cultured for 4 d under Th1 conditions, restimulated with PMA/ionomyocin, and analyzed for intracellular cytokine staining. Cells are gated on CD4⁺ or CD8⁺ populations. Results of individual experiments are shown.

CNS-30 is not necessary in NK cells, macrophages, and dendritic cells. A, Freshly isolated NK cells were stimulated with nothing, IL-12, IL-18, or with IL-12 plus IL-18. After 48 h, culture IFN-γ levels were determined by ELISA. B, IFNG-BAC and Δ-30 CD4⁺ T cells were cultured under Th1 conditions for 3 d (left panel) or NK cells were cultured for 2 d with IL-12 and IL-18 (right panel) and mRNA levels were determined by quantitative PCR. Data points represent results of individual experiments. C, Freshly isolated macrophages or dendritic cells were cultured for 2 d and stimulated with nothing or with IL-12 and IL-18, and culture fluids were harvested and analyzed for IFN-γ levels by ELISA. Results are averages of at least three independent experiments. Error bars are SEM. D, Whole spleen was stimulated with PMA/ionomyocin and analyzed for intracellular cytokine staining. Cell gating is presented in Supplemental Fig. 1.

Copy number-dependent expression. Relative copy numbers of three different Δ-77 lines were determined by quantitative PCR analysis of genomic DNA. CD8⁺ T cells were cultured for 4 d under Th1 polarizing conditions and were analyzed for IFN-γ levels by intracellular cytokine staining. Cells were gated on CD8⁺ cell populations. Replicates were subjected to a linear regression analysis.

CNS-30 is necessary for RNAP II recruitment to the IFNG locus. Δ3/4, Δ-30, Δ4, or IFNG-BAC CD4⁺ T cells were cultured for 3 d under Th1 conditions and processed for ChIP assays with Abs specific for normal rabbit IgG or (A) H4Ac marks, (B) H3K9Me2 marks, or (C) RNAP II. Results are presented as fraction of input of DNA as determined from a standard curve and are the average of three independent experiments. Values for IgG controls were <0.001 and were subtracted from means. Error bars are SD. Significance was determined by a control t test. p < 0.025.