Figure 4.
Figure 4. The HIF1 complex regulates the CD8+ T cell transcriptional program but is not essential for T cell proliferation. (A and B) Proliferation analysis of WT and HIF1−/− CTLs (A) and P14-LCMV CTLs treated with and without rapamycin (B). Cells were seeded at 0.3 × 106/ml, and CTL numbers were counted after 24 h. Data are mean ± SEM of five experiments. (C) Heat map showing the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1−/− CTLs, as determined by microarray. (D) Real-time PCR analysis of IFN-γ expression in WT and HIF1−/− CTLs. Data are mean ± SEM of three experiments in triplicate. (E) Immunoblot analysis of T-bet and Blimp1 expression in WT and HIF1−/− CTLs. Data are representative of two experiments. (F) Real-time PCR analysis of Perforin mRNA expression in WT and HIF1−/− CTLs. Data are mean ± SEM of three experiments in triplicate (**, P < 0.01). (G) Immunoblot analysis of Perforin protein expression in WT and HIF1−/− CTLs (top) and P14-LCMV CTLs treated ± rapamycin for 20 h (bottom). Data are representative of at least three experiments. (H) IL-2–maintained CTLs were placed in either hypoxic (1%) or normoxic (20%) oxygen for 24 h before being subjected to immunoblot analysis for HIF1α, Glut1, and perforin expression. Data are representative of three experiments. (I) ChIP was performed with anti–Pol II, and the changes in Pol II binding to the Perforin transcription start site and the second exon were quantified by real-time PCR. Data were normalized to input DNA amounts and plotted as fold over the values for Pol II binding to the HPRT proximal promoter. Data are mean ± SEM of three experiments performed in duplicate. (J) P14-LCMV T cells were activated for 2 d with gp33-41 and then cultured for a further 4 d with IL-2 in different glucose concentrations. Cells were then subjected to immunoblot analysis for perforin expression. Data are representative of two experiments. Molecular mass is indicated in kilodaltons. Black lines (solid or dotted) indicate that intervening lanes have been spliced out.

The HIF1 complex regulates the CD8+ T cell transcriptional program but is not essential for T cell proliferation. (A and B) Proliferation analysis of WT and HIF1−/− CTLs (A) and P14-LCMV CTLs treated with and without rapamycin (B). Cells were seeded at 0.3 × 106/ml, and CTL numbers were counted after 24 h. Data are mean ± SEM of five experiments. (C) Heat map showing the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1−/− CTLs, as determined by microarray. (D) Real-time PCR analysis of IFN-γ expression in WT and HIF1−/− CTLs. Data are mean ± SEM of three experiments in triplicate. (E) Immunoblot analysis of T-bet and Blimp1 expression in WT and HIF1−/− CTLs. Data are representative of two experiments. (F) Real-time PCR analysis of Perforin mRNA expression in WT and HIF1−/− CTLs. Data are mean ± SEM of three experiments in triplicate (**, P < 0.01). (G) Immunoblot analysis of Perforin protein expression in WT and HIF1−/− CTLs (top) and P14-LCMV CTLs treated ± rapamycin for 20 h (bottom). Data are representative of at least three experiments. (H) IL-2–maintained CTLs were placed in either hypoxic (1%) or normoxic (20%) oxygen for 24 h before being subjected to immunoblot analysis for HIF1α, Glut1, and perforin expression. Data are representative of three experiments. (I) ChIP was performed with anti–Pol II, and the changes in Pol II binding to the Perforin transcription start site and the second exon were quantified by real-time PCR. Data were normalized to input DNA amounts and plotted as fold over the values for Pol II binding to the HPRT proximal promoter. Data are mean ± SEM of three experiments performed in duplicate. (J) P14-LCMV T cells were activated for 2 d with gp33-41 and then cultured for a further 4 d with IL-2 in different glucose concentrations. Cells were then subjected to immunoblot analysis for perforin expression. Data are representative of two experiments. Molecular mass is indicated in kilodaltons. Black lines (solid or dotted) indicate that intervening lanes have been spliced out.

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