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. 2012 Feb 15;188(4):1726-33.
doi: 10.4049/jimmunol.1102879. Epub 2012 Jan 13.

Diverse functions of distal regulatory elements at the IFNG locus

Patrick L Collins  1 , Melodie A Henderson, Thomas M Aune
Affiliations

Diverse functions of distal regulatory elements at the IFNG locus

Patrick L Collins et al. J Immunol. .

Abstract

Previous studies have identified multiple conserved noncoding sequences (CNS) at the mouse Ifng locus sufficient for enhancer activity in cell-based assays. These studies do not directly address biology of the human IFNG locus in a genomic setting. IFNG enhancers may be functionally redundant or each may be functionally unique. We test the hypothesis that each IFNG enhancer has a unique necessary function using a bacterial artificial chromosome transgenic model. We find that CNS-30, CNS-4, and CNS+20 are required at distinct stages of Th1 differentiation, whereas CNS-16 has a repressive role in Th1 and Th2 cells. CNS+20 is required for IFN-γ expression by memory Th1 cells and NKT cells. CNS-4 is required for IFN-γ expression by effector Th1 cells. In contrast, CNS-16, CNS-4, and CNS+20 are each partially required for human IFN-γ expression by NK cells. Thus, IFNG CNS enhancers have redundant necessary functions in NK cells but unique necessary functions in Th cells. These results also demonstrate that distinct CNSs are required to transcribe IFNG at each stage of the Th1 differentiation pathway.

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Figures

FIGURE 1
FIGURE 1
BAC transgenes used in this study. Top: Locations of 190 kb and 210 kb BAC transgenes, Th1 and Th2 DNase I hypersensitivity and conserved sequences between humans and mice or between humans and platypus. Grey bars represent deletion locations. For conservation analysis, green bars are conserved sequences and solid lines represent a fully aligned genome. Bottom: Integration check of transgenes. Shown are human-specific PCR integration checks for BAC transgenic mice.
FIGURE 2
FIGURE 2
CNS-4 is required for human IFN-γ production. A, Transgenic CD4+ cells were cultured under Th1 and Th2 polarizing conditions for three days. Human IFN-γ (left) or mouse IFN-γ (right) concentrations were determined from cultures. B, Day five CD4+ Th1 cultures were restimulated with anti-CD3, or IL-12 and IL-18, or IL-12 and IL-2. Human and mouse IFN-γ concentrations were determined by ELISA. Results are representative of independent replicates. Error bars are standard deviations. * P < 0.05.
FIGURE 3
FIGURE 3
CNS-16 represses human IFN-γ production. A, Transgenic CD4+ cells were cultured under Th1 and Th2 polarizing culture conditions for three days. Human IFN-γ (left) or mouse IFN-γ (right) concentrations were determined in cultures. B, Transgenic CD8+ cells were cultured under Th1 polarizing conditions for three days. IFN-γ was measured in culture by ELSIA. Results are representative of independent replicates. C, Transgenic CD4+ cells were cultured under Th1 conditions for three days and mRNA levels were determined by quantitative PCR. Error bars are standard deviation. D, Transgenic CD8+ T cells were cultured under Th1 polarizing conditions for seven days and restimulated with PMA/Ionomycin. Interferon gamma was determined by intracellular cytokine staining. Quantification of the percentages of human IFN-γ+ cells from three independent replicates is shown. * P < 0.05.
FIGURE 4
FIGURE 4
CNS+20 is required for antigen specific recall responses. Transgenic mice were immunized with OVA in CFA, and boosted at day 10 with OVA in IFA. At day 35 mice splenocytes were restimulated with HEL protein, OVA, or OVA 257-264 peptide. IFN-γ concentrations were determined by ELISA. Results are means of three independent replicates. Error bars are standard error of the mean. * P < 0.05.
FIGURE 5
FIGURE 5
CNS+20 is required for in vivo generated Th1 cells. A, Splenocytes from day 35 immunized transgenic mice were restimulated with IL-12 and IL-18 and IFN-γ was assayed by intracellular cytokine staining. B, Quantification of the percentages of human IFN-γ positive cells in mouse IFN-γ+, CD4+ or mouse IFN-γ+, CD8+ populations or MFI of human IFN-γ positive cells. Results are means of three independent replicates and error bars are standard error of the mean. * P < 0.05.
FIGURE 6
FIGURE 6
CNS+20 is required for IFN-γ production by antigen-stimulated NKT cells. A, Splenocytes were isolated from transgenic mice and stimulated with a mock treatment or with alpha GalCer. Human and mouse IFN-γ concentrations in culture supernatants were determined by ELISA. Results are representative of three independent experiments. Error bars are standard error of the mean. B, Splenocytes from transgenic mice were stimulated with IL-12 and IL-18 and IFN-γ was assayed by intracellular cytokine staining. NKT cells were gated on MHC class II−, CD1d+ populations. * P < 0.05.
FIGURE 7
FIGURE 7
CNS-16, CNS-4 and CNS+20 are required for IFN-γ expression by NK cells. A, NK cells were purified from transgenic splenocytes and stimulated with IL-12 and IL-18, or given a mock stimulus. IFN-γ concentrations were determined by ELISA. Results are representative of four independent experiments and error bars represent standard deviation. B, Representative cell purity of purified NK cells. C, Splenocytes from day 35 immunized transgenic mice were restimulated with IL-12 and IL-18 and IFN-γ was determined by intracellular cytokine staining. NK cells were determined as CD8−, CD4−, DX5+. D, Quantification of the percentages of human IFN-γ positive cells in mouse IFN-γ+ CD4−, CD8-populations. Results are means of three independent replicates and error bars represent standard error of the mean.
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