Transcription factor GATA-1 potently represses the expression of the HIV-1 coreceptor CCR5 in human T cells and dendritic cells by Mark S. Sundrud, Scott E. VanCompernolle, Karla A. Eger, Tullia C. Bruno, Arun Subramaniam, Srinivas Mummidi, Sunil K. Ahuja, and Derya Unutmaz Blood Volume 106(10):3440-3448 November 15, 2005 ©2005 by American Society of Hematology

Endogenous GATA-1 expression is inversely related to CCR5 expression in human CD34+ hematopoietic stem cells during DC differentiation. Endogenous GATA-1 expression is inversely related to CCR5 expression in human CD34+ hematopoietic stem cells during DC differentiation. Purified and expanded primary human HSCs were cultured in stem cell media or DC-differentiating media and 2 endpoints were analyzed: (A) cell-surface CCR5 expression was assessed at the indicated time points via flow cytometry and (B) endogenous GATA-1 mRNA levels were determined via quantitative real-time PCR (see “Materials and methods”). The findings represent 2 experiments performed using stem cells isolated from independent umbilical cord blood samples Mark S. Sundrud et al. Blood 2005;106:3440-3448 ©2005 by American Society of Hematology

Ectopic expression of GATA-1 potently reduces cell-surface expression of CCR5 in primary human cell targets of HIV-1. Ectopic expression of GATA-1 potently reduces cell-surface expression of CCR5 in primary human cell targets of HIV-1. (A) Primary human HSC-derived DCs were transduced with HDV, HDV.GATA-1, or HDV.GATA-3 and cultured for an additional 4 days. CCR5 expression levels on HSC-derived DCs were determined via flow cytometric analyses by costaining cells with anti-CCR5, anti-CD1c (Miltenyi Biotech) to monitor DC differentiation and anti-mCD24 to resolve DCs expressing the lentiviral transgene. (B) Primary human conventional T-cell subsets (top 3 panels) or NKT cells (bottom) were TCR activated and transduced to express GATA-1, GATA-3, or the control HDV (see “Materials and methods”). Cells were expanded in the presence of IL-2, and cell-surface CCR5 expression was determined on unsorted populations of transduced cells and costained with anti-mCD24 antibodies. Additionally, NKT cells were costained with a fluorescein isothiocyanate (FITC)–conjugated anti–human Vβ11 antibody (Coulter), to exclude any contaminating non-NKT cells. These results are representative of at least 3 experiments performed on each cell type from unique adult or umbilical cord blood preparations. Mark S. Sundrud et al. Blood 2005;106:3440-3448 ©2005 by American Society of Hematology

GATA-1–induced CCR5 down-regulation inhibits R5-tropic HIV infection. GATA-1–induced CCR5 down-regulation inhibits R5-tropic HIV infection. (A) T cells expressing HDV (▵), HDV.GATA-3 (□), or HDV.GATA-1 (•) were prepared as described in the caption of Figure 2 and superinfected with replication-competent CCR5-tropic HIV-1 (R5.HIV), at an MOI of 1 either in the absence (top left) or presence (top right) of HIV protease inhibitors and AZT, which were added at day 2 after infection. Infections were determined based on GFP expression as indicated. T cells expressing GATA-3 or GATA-1 were also infected with either VSV-G.HIV (bottom left) or X4.HIV (bottom right) at 1 MOI. All infection data are shown as the percentage of GFP+ T cells at the indicated times. (B) Culture supernatants were also collected from T cells infected with R5.HIV at the indicated times after infection, and viral replication was quantified via p24 ELISA. These data are representative of 3 independent experiments performed using T cells isolated from individual donors. Error bars indicate standard deviation of duplicate samples. Mark S. Sundrud et al. Blood 2005;106:3440-3448 ©2005 by American Society of Hematology

GATA-1 expression in primary human T cells suppresses CCR5 promoter activity. 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 characterized14,15 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. Mark S. Sundrud et al. Blood 2005;106:3440-3448 ©2005 by American Society of Hematology

Ectopic GATA-1 expression in human TN,TCM, and TEM cells induces a Th2 cytokine profile. Ectopic GATA-1 expression in human TN,TCM, and TEM cells induces a Th2 cytokine profile. CD45RO–RA+ naive and CD45RO+RA– memory T cells were purified from adult blood PBMCs of healthy donors. Memory T cells were further sorted by FACS for expression of CCR7 into TCM (CCR7+) and TCM (CCR7–) cells. The T-cell populations were TCR activated and transduced with HDV, HDV.GATA-1, or HDV.GATA-3 at the time of activation (see “Materials and methods”). Following 8 to 10 days of expansion, intracellular expression of the cytokines IFNγ, IL-4, IL-5, and IL-13 were determined by flow cytometry. For these experiments transduced cells were left unsorted, but mCD24+ cells were identified and gated on by costaining with an anti-mCD24 antibody. These data are representative of 3 separate experiments using T-cell subsets purified from different adult donors. Mark S. Sundrud et al. Blood 2005;106:3440-3448 ©2005 by American Society of Hematology

Expression of GATA-1 in human TN,TCM, and TEM cell subsets induces expression of Th2-associated chemokine receptors. Expression of GATA-1 in human TN,TCM, and TEM cell subsets induces expression of Th2-associated chemokine receptors. Human TN, TCM, and TEM cells were purified, activated, and transduced with HDV alone, HDV.GATA-1, or HDV.GATA-3 as described in caption for Figure 5. Following expansion, unsorted cells were stained for chemotactic receptors as indicated or an isotype control in conjunction with an anti-mCD24 antibody to gate on transduced populations. The percentage of cells positive for the appropriate chemokine receptor is shown, with the gray, tinted peak representing isotype control staining. Results are representative of 3 experiments using T-cell subsets from distinct adult donors. Mark S. Sundrud et al. Blood 2005;106:3440-3448 ©2005 by American Society of Hematology