Transcription factor GATA-1 potently represses the expression of the HIV-1 coreceptor CCR5 in human T cells and dendritic cells
Blood Sundrud et al. 106: 3440

Supplemental materials for: Sundrud et al, Vol 106, Issue 10, 3440-3448

Files in this Data Supplement:

• Figure S1. Expression of GATA-3 and GATA-1 in transduced cells (JPG, 51.1 KB) -

  

  Purified CD4⁺ T cells were activated and transduced with either HDV, HDV.GATA-3 or HDV.GATA-1 viruses. Cells were expanded and sorted for mCD24 expression. Sorted cells were lysed and Western blot analyses were performed to determine GATA-3 (top panel), GATA-1 (middle panel) and -actin (bottom panel) expression using specific antibodies. The membranes were stripped following each blot and reprobed in the order indicated (see “Materials and methods”). These data are representative of two separate Western blot experiments using transduced cells obtained from different donors.

• Figure S2. Phenotypic analysis of T_N, T_CM and T_EM cells transduced with HDV, HDV.GATA-3 and HDV.GATA-1 following IL-2-dependent expansion (JPG, 344 KB) -

  

  (A) CD4⁺-naive (T_N – CD45RO^–CCR7⁺), central memory (T_CM – CD45RO⁺CCR7⁺) and effector memory (T_EM – CD45RO⁺CCR7^–) T-cell subsets were transduced with HDV, HDV.GATA-3 or HDV.GATA-1 and expanded in IL-2-supplemented media for 8 to 10 days. Unsorted cells were phenotypically analyzed for the expression of CD45RO and CCR7 (left panel) or CD4 (right panel) via FACS analysis. Cells were costained with an anti-mCD24 antibody to gate on transduced populations. (B) Primary human NKT cells were activated (see “Materials and methods”) and transduced with HDV, HDV.GATA-3 or HDV.GATA-1. The cells were expanded in IL-2–containing media for 8 to 10 days, and expression of the invariant / chains of the NKT cell TCR (V24 and V11) were evaluated via flow cytometric analyses. Transduced NKT cells were identified and gated on by costaining unsorted cells with an anti-mCD24 antibody.

• Figure S3. Expression of GATA-1 in human T cells does not induce GATA-3 expression (JPG, 139 KB) -

  

  Human T_N cells purified from adult blood or neonatal umbilical cord blood were activated through the TCR in Th1 or Th2 cytokine conditions (see “Materials and methods”). Additionally, T_N cells were activated without polarizing cytokines and transduced with the control HDV or HDV.GATA-1. All cells were expanded, and transduced cells were sorted based on mCD24 expression. Cells were then either left un-stimulated (resting) or restimulated with anti-CD3 and anti-CD28 antibodies (restimulated) for 8 hours. Resting and restimulated T cells were harvested, and GATA-3 expression was evaluated by quantitative real-time PCR. These data were analyzed by normalizing GATA-3 expression levels to GAPDH levels; copy numbers were determined by comparing the normalized C_t values to a standard curve of GATA-3 (see “Materials and methods”). The data are displayed as GATA-3 transcript numbers plus or minus SD. Raw GATA-3 expression values with C_t values greater than 37 cycles were not considered as significant GATA-3 expression. These data represent three experiments using T_N cells from three independent adult or umbilical cord blood donors.