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. 2013 Jan;36(1):3-10.
doi: 10.1097/CJI.0b013e3182791234.

Evaluation of long-term transgene expression in piggyBac-modified human T lymphocytes

Yozo Nakazawa  1 , Sunandan Saha, Daniel L Galvan, Leslie Huye, Lisa Rollins, Cliona M Rooney, Matthew H Wilson
Affiliations

Evaluation of long-term transgene expression in piggyBac-modified human T lymphocytes

Yozo Nakazawa et al. J Immunother. 2013 Jan.

Abstract

The piggyBac transposon system is a promising nonviral method to genetically modify T cells for immunotherapeutic applications. To evaluate the regulation and stability of transgene expression in human T cells modified with piggyBac-transposons, peripheral blood mononuclear cells were nucleofected with transposase and an enhanced green fluorescence protein (eGFP)-expressing transposon. Single-cell clones that were subsequently stimulated and expanded exhibited homogenous eGFP expression for >26 weeks in culture. CD3 stimulation of the T-cell receptor together with CD28-mediated costimulation resulted in an approximate 10-fold transient increase in eGFP expression, but immunomodulatory cytokines, including interferon-γ, interleukin-12, interleukin-4, and transforming growth factor-β, did not alter transgene expression in actively dividing, activated, or resting T cells. Epigenetic modification with 5-azacytidine or trichostatin-A increased transgene expression indicating that piggyBac-mediated transgene expression could be modulated by methylation or histone acetylation, respectively. We performed transposon copy number analysis of populations of stably transfected T cells, comparing transposon plasmids of 5.6 and 3.5 kb. The smaller vector achieved an average of 22 transposon copies per cell, whereas the larger vector achieved 1.6 copies/cell, implying that transposon copy number can be engineered to be low or high depending on the vector used. Our results provide important insight into the ability of piggyBac to achieve stable genetic modification of T cells for immunotherapy applications and how transgene expression might be regulated by TCR activation, cytokines, and epigenetic mechanisms.

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Figures

FIGURE 1
FIGURE 1. Generation of piggyBac-modified T-cell clones
5 × 106 IL-15-treated peripheral blood mononuclear cells (PBMCs) from a healthy donor were nucleofected with pIRII-eGFP (5 μg) and pCMV-piggyBac (5 μg). Two days later, the nucleofected cells were stained with anti-CD28 antibody, and then ~500 cells of dual GFP- and CD28-positive cells were single-sorted into five 96-well plates. Each cell was stimulated with OKT3 and co-cultured with aK562 cells in the presence of IL-15 for 3 weeks. Well-growing clones were repeatedly stimulated with OKT3 in the presence of aK562 cells in the presence of IL-15 every 3 weeks after nucleofection. GFP expression was determined by flow cytometry at 12 weeks after eGFP-gene transfer. The percentage of GFP expression and mean fluorescence intensity (MFI) are indicated in each clone.
FIGURE 2
FIGURE 2. Stable and long-term expression of piggyBac-transgenes
T-cell clones were stimulated with OKT3 and co-cultured with aK562 cells at a ratio of 2:1 in IL-15-containing media 3 weeks after transfection, and every 3 weeks thereafter until 24 weeks after transfection. GFP expression was determined by flow cytometry just before each stimulation (i.e. every 3 weeks). The percentage of GFP expression and mean fluorescence intensity (MFI) for each is indicated.
FIGURE 3
FIGURE 3. T cell receptor stimulation increased expression of piggyBac-transgenes
Five representative clones were stimulated in OKT3/CD28 mAb-coated wells for 4 days and then cultured for 35 days. Activated clones were analyzed for GFP expression at the indicated time points (continuous line). On day 12 after activation, a small portion of each activated clone was reactivated with an OKT3/CD28 mAbs-coated plate for 4 days and thereafter analyzed for GFP expression as the same time points (dashed line).
FIGURE 4
FIGURE 4. Immunomodulatory cytokines do not affect transgene expression in piggyBac-transposed human T cells
Five representative clones (C1, C2, C3, C8, and C19) were stimulated on anti-CD3/CD28 antibody coated plates for 4 days and then cultured for 35 days. Stimulated clones were classified into 3 different groups based on the activation state such as “actively dividing”, “activated”, and “resting”. Those in “actively dividing” received cytokines on day 1, “activated” received cytokines on day 5, and “resting” received cytokines on day 21. Clones were treated with interferon-gamma (IFN-γ) (10 ng/mL), IL-12 (10 ngmL), IL-4 (1,000 IU/mL) or transforming growth factor-beta (TGF-β) (5 ng/mL) for 4 days. Four days after the treatment, the clones were analyzed for GFP expression by flow cytometry.
FIGURE 5
FIGURE 5. PiggyBac-transgenes are partially regulated by epigenetic mechanisms
For epigenetic analysis, two agents were used. (A) For methylation analysis, 5 representative clones were stimulated with OKT3/CD28 mAbs, in the presence of the methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-AzaC) at the indicated concentration. Four days after treatment, clones were analyzed for GFP expression by flow cytometry. (B) For histone deacetylation analysis, 5 representative clones were stimulated on OKT3/28 mAb-coated plates for 4 days. Six days after treatment, the histone deacetylase inhibitor, trichostatin A (TSA) was added into the cultures at the indicated concentration. Twenty-four hours after treatment, TSA-treated clones were analyzed for GFP expression by flow cytometry.
FIGURE 5
FIGURE 5. PiggyBac-transgenes are partially regulated by epigenetic mechanisms
For epigenetic analysis, two agents were used. (A) For methylation analysis, 5 representative clones were stimulated with OKT3/CD28 mAbs, in the presence of the methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-AzaC) at the indicated concentration. Four days after treatment, clones were analyzed for GFP expression by flow cytometry. (B) For histone deacetylation analysis, 5 representative clones were stimulated on OKT3/28 mAb-coated plates for 4 days. Six days after treatment, the histone deacetylase inhibitor, trichostatin A (TSA) was added into the cultures at the indicated concentration. Twenty-four hours after treatment, TSA-treated clones were analyzed for GFP expression by flow cytometry.
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