Flow cytometric analysis of CEM and Jurkat T cells transduced with HTLV Tax. Jurkat or CEM cells (10⁶) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h, 2,500 rpm) in the presence of 8 μg of Polybrene/ml. CEM and Jurkat cells (10⁶) were mock infected as a negative control for apoptosis. Cells were resuspended in IMDM with 10% FBS, stained with Annexin V-PE (AV-PE) and 7-AAD, and analyzed by flow cytometry at 48 and 72 h postinfection. (A) Representative flow cytometric analysis of LV-transduced CEM and Jurkat cells at 72 h postinfection. GFP⁺ cells were gated and analyzed for AV (horizontal axis) and 7-AAD staining (vertical axis). Representative panels of transduced CEM (left column) and Jurkat (right column) cells. The percentage of individual subpopulations in each quadrant is indicated. Cells that are AV-PE single positive (lower right quadrant) and AV-PE⁺ 7-AAD⁺ positive (upper right quadrant) are early- and late-stage apoptotic cells, respectively. (B) Annexin V and 7-AAD staining of Jurkat and CEM cells transduced with LVs. Jurkat and CEM cells were transduced with LVs and GFP⁺ cells were analyzed for AV and 7-AAD staining by flow cytometry. CEM cells (subpanels A and B) and Jurkat cells (subpanels C and D) were analyzed at 48 h (blue bars) or 72 h (purple bars) postinfection. The percentage of GFP⁺ cells that were positive for AV staining (subpanels A and C) or which stained for 7-AAD (subpanels B and D), after infection with HR′CMV-Tax1/GFP transduction (Tax1), HR′CMV-Tax1(−)/GFP [Tax1(−)], HR′CMV-Tax2/GFP (Tax2), or HR′CMV-GFP (GFP) are indicated. Values of AV and 7-AAD expression with HR′CMV-Vpr/GFP (VPR) were significantly higher than those obtained with the other vectors, which in turn were higher than those obtained with medium alone (Mock). The data were compiled from three independent experiments. Statistical analysis was performed by ANOVA (P < 0.005).

Clonogenic colony-forming activity of lentiviral vector- transduced CD34⁺ cells. GFP⁺ CD34⁺ cells were purified by FACS, and isolated cells (10³) were plated in 10-mm dishes containing 2 ml of MethoCult H4433. Mature colony types were identified by morphology as granulocyte-macrophage CFU (CFU-GM), burst forming unit-erythroid (BFU-E), or highly proliferative pluripotent type (HPP-CFC), as previously described (31). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34⁺ GFP⁺ cells (10³) plated was determined at 14 days postplating. Purified CD34⁺ GFP⁺ cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34⁺ cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34⁺ cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10³ purified CD34⁺ GFP⁺ cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10³ CD34⁺ GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10³ CD34⁺ GFP⁺ cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA (P < 0.005).

Schematic representation of multigene lentiviral vectors. Multigene vectors based on the use of an internal ribosomal entry site sequence from the encephalomyocarditis virus (80). The target gene and reporter gene are driven by an immediate-early CMV promoter (IE CMV prom). pHR′CMV-Tax1(−)/GFP contains the HTLV-1 tax gene inserted in the antisense orientation and does not encode for functional Tax1. pHR′CMV-Vpr/GFP encodes the HIV-1 vpr gene (Vicente Planelles). SA, splice acceptor; SD, splice donor; RRE, HIV-1 Rev response element; Ψ, psi packaging signal for the transfer vector.

FACS isolation of CD34⁺ cells infected with lentiviral vectors. Purified CD34⁺ cells (3 × 10⁶) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34⁺ GFP⁺ cells. Note that cells transduced with LVs encoding a bicistronic mRNA display approximately 5- to 10-fold-lower levels of GFP in comparison with cells transduced with HR′CMV-GFP, as has previously been reported (103). The percentage of CD34⁺ GFP⁺ cells is displayed in the upper right panel. Mock-transduced CD34⁺ cells were isolated only on the basis of CD34⁺ expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.

PCR analysis of clonogenic colonies. Clonogenic colonies were cultivated from LV-transduced CD34⁺ cells isolated by FACS on the basis of CD34 and GFP coexpression, as described in Fig. 3. Colonies were randomly isolated by aspiration from the methycellulose medium under an inverted microscope (Leica DMIL). (A) Q-PCR analysis of high-molecular-weight DNA from clonogenic colonies arising from lentiviral vector- transduced CD34⁺ cells. Clonogenic colonies were identified by morphology after 12 to 14 days in culture, and DNA was processed for PCR analysis as previously described (31). DNA from cells was assayed for the presence of HTLV-1 tax/rex (nucleotides 7336 to 7495) and human β-globin sequences. The numbers of cells in each colony varied and were reflected by the β-globin signal obtained. The amplified HTLV-1 and β-globin products were 159 and 110 bp, respectively. The hematopoietic colonies were identified as follows: lanes 1 to 3, 6, 8, and 12, CFU-GM; lanes 4, 9, 10, and 13, BFU-E; and lanes 7 and 14, HPP-CFC. Colonies 1, 6, 8, and 12 were from FACS experiment 1; colonies 2, 4, 11, and 13 were from FACS experiment 2; colonies 3, 5, and 9 were from FACS experiment 3; and colonies 7, 10, and 14 were from FACS experiment 4. “Mock” represents clonogenic colonies derived from sorted CD34⁺ cells that were mock infected and sorted only on the basis of CD34 expression. The colony analyzed in lane 6 was scored as negative for tax/rex sequences, suggesting that this colony arose from maturation of a nontransduced CD34⁺ cell, which presumably contaminated the FACS-purified cell population. (B) RT-PCR analysis of RNA from clonogenic colonies. RNA was isolated from clonogenic colonies by using Trizol, and a 10-μl aliquot of RNA representing 25% of the total sample was analyzed by RT-PCR with primers 670 and 671 and a OneStep RT-PCR kit (Qiagen). “Tax1-RT” represents the sample from lane 4 assayed with heat-inactivated reverse transcriptase and was used as a negative control for this experiment. RNA extracted from 10⁴ SLB-1 cells, an HTLV-1 transformed T-cell line, was used as a positive control (SLB). Colonies 1, 2, and 4 to 8 were CFU-GM; colonies 3, 9, and 10 were BFU-E; and colonies 11 and 12 were from HPP-CFC. Colonies 1, 4, 7, and 10 were from FACS experiment 2; colonies 2, 5, 8, and 11 were from FACS experiment 3; and colonies 3, 6, 9, and 12 were from FACS experiment 4. (C) Low-field (×20) light and fluorescent micrographs were taken of a CFU-GM arising from CD34⁺ cells infected with HR′CMV-GFP (GFP, top panel), HR′CMV-Tax1/GFP (Tax1, middle panel), or HR′CMV-Tax2/GFP (Tax2, bottom panel) at 14 days postplating. Note that GFP expression is generally 5- to 10-fold lower in cells infected with the bicistronic LVs than in cells infected with HR′CMV-GFP, as has previously been reported (102).