Figure 2. Foxp3 activation of MHC class I promoter activity in Jurkat cells maps to the IRE and core promoter.

A. Jurkat T cells were cotransfected with a 1.0 µg of the MHC class I PD1 promoter construct containing DNA sequences from −416 to +32 bp (WT), ligated to a luciferase reporter gene and 2.0 µg of a Foxp3 expression vector or control vector. Results are expressed relative to class I promoter activity of the control co-transfection. Results are the average ± SEM of 6 experiments. *** p<0.0004.

B. Jurkat T cells were cotransfected with 2.0 µg of Foxp3 or control vector (no Foxp3) and 1.0µg of a series of 5’ truncation deletion mutants of the class I promoter, as diagrammed. The results for each construct are normalized to the control transfection with empty vector. Although the absolute activity of the constructs differed, all of them were transcriptionally active (12).The results are the average ± SEM of 4 separate experiments. *, p<0.2; ** p=0.016.

C. Jurkat T cells were cotransfected with 2.0 µg Foxp3 or control vector (no Foxp3) and either WT (1.0 µg) or a class I promoter construct containing a mutation in the interferon response element (IRE). The results for each construct are normalized to the control transfection with empty vector. Although the absolute activity of the two constructs differed, both were transcriptionally active. The results are the average ± SEM of 4 separate experiments. ** p=0.019.

D. Jurkat T cells were cotransfected with 2.0 µg Foxp3 or control vector and 1 µg of a series of 3’ truncation deletion mutants of the class I promoter. The results for each construct are normalized to the control transfection with empty vector. Although the absolute activity of the constructs differed, all of them were transcriptionally active (16). The results are the average ± SEM of 3 separate experiments.

E. Jurkat T cells were cotransfected with 2.0 µg Foxp3 or control vector and 1.0 µg of either WT or a MHC class I promoter construct containing a −50 to +3 deletion (drop out). The results are average ± SEM of 4 separate experiments. ** p=0.018.

F. Jurkat T cells were cotransfected with 2.0 µg Foxp3 or control vector and 1.0 µg of either WT, IRE mutant, the drop-out or a mutant containing both the IREmut and the drop-out (IREm/drop-out). The results for each construct are normalized to the control transfection with empty vector. Although the absolute activity of the constructs differed, all of them were transcriptionally active (16).The results are the average ± SEM of 5 separate experiments.**, p<0.01; ***,p<0.004