GATA-1 expression in primary human T cells suppresses CCR5 promoter activity. (A) Total resting CD4⁺ T cells TCR activated and transduced with HDV, HDV.GATA-3, or HDV.GATA-1 were expanded, and cell-surface expression of CCR5 was assessed on unsorted cells by staining with either isotype control (gray peaks) or anti-CCR5 (white peaks) antibodies in conjunction with an anti-mCD24 antibody. The histograms are gated on the mCD24⁺ populations, and the percentage of CCR5-positive T cells are displayed. The histograms are representative of 5 independent stainings performed on cells isolated from different blood donors. (B) Total CD4⁺ human T cells expressing HDV, HDV.GATA-1, or HDV.GATA-3 as above were sorted based on mCD24 expression (see “Materials and methods”) and either left unstimulated (resting) or restimulated via anti-CD3 and anti-CD28 crosslinking for 18 hours (restimulated). Cells were lysed, cDNA was generated, and quantitative real-time PCR was performed using gene-specific primers. CCR5 mRNA expression levels are normalized to GAPDH levels and are displayed as fold differences in relative expression. These data represent 3 independent real-time PCR reactions run from cDNA generated from separate sets of cells from different donors. (C, left) Genomic organization of human CCR5 and the GATA-1 cis-binding sites. Open boxes are exons, dashed lines connecting exons are introns. Exons and introns are numbered. Nucleotide numbering and gene structure are according to Mummidi et al.³⁹  GATA-1–binding positions are indicated by diamonds, and the GATA binding sites previously characterized^14,15  are marked with asterisks. The luciferase reporter construct is schematically shown below the gene structure (see “Materials and methods”). (C, right) Transduced human T cells positively sorted for mCD24 expression were restimulated through the TCR (see “Materials and methods”) and are transfected with either the full-length CCR5 promoter driving firefly luciferase expression or a promoterless firefly luciferase plasmid. Cells were cotransfected to normalize for transfection efficiency. Transfected cells were lysed, and luciferase expression was quantified. Firefly luciferase activity was normalized to Renilla activity within samples, and the data are displayed as the relative fold induction of firefly luciferase driven by CCR5 promoter over the promoterless control vector. These data represent 3 independent sets of transfections performed on cells from separate donors. Error bars indicate standard deviation of duplicate samples.