Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy

Abstract

Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols.

FIG. 1.

The effect of cytokine stimulation on lentiviral transduction efficiency. Freshly isolated primary natural killer (NK) cells were cultured in CellGro SCGM medium with 10% human serum in the presence of cytokines as indicated in the figure. After 24 hr of stimulation, NK cells were transduced at an multiplicity of infection (MOI) of 20 using the VSV-G pseudotyped lentiviral “gene ontology” (LeGO)-G2 vector. Seventy-two hours post-transduction, acquisition of enhanced green fluorescent protein (eGFP) expression was carried out by flow cytometry. Data from five independent donors. *p<0.05; **p<0.01; one-way ANOVA repeated measures.

FIG. 2.

Inhibition of innate immune signaling pathways for enhancement of lentiviral transduction efficiency. NK cells stimulated with interleukin (IL)-2/IL-21 for 2 days were transduced in the presence of various inhibitors. (a) 2-Aminopurine (5 mM), an inhibitor of double-stranded RNA (dsRNA)-activated protein kinase (PKR). BAY11-7082 (5 μM), an inhibitor of IκB-α. BX795 (2 μM), an inhibitor of TBK1/IKKɛ complex. The proteasome inhibitor bortezomib (15 ng/ml) was also included in this experiment. Results are from one representative donor. (b) Results from two independent donors where IRAK1/4 inhibitor was present at various concentrations during the transductions. None of the samples showed any significant improvement of transduction efficiency.

FIG. 3.

Dose response, nontoxicity, and reversibility of BX795 treatment. (A) NK cells stimulated with IL-2/IL-21 for 2 days were transduced in the presence of various concentrations of BX795. Percentage of eGFP-expressing NK cells after transduction in the presence of BX795 was assessed by flow cytometry. Enhancement of transduction efficiency by BX795 is dose dependent and a concentration of 6 μM is sufficient to get maximum response. Data from three independent donors. *p<0.05; ns=not significant, paired t-test. (B) Analysis of Annexin V and propidium iodide (PI) staining after treatment with BX795 in the presence of IL-2 and IL-21 revealed no toxic effect on NK cells up to 10 μM. Data from one representative donor. (C) NK cells were transduced after overnight pretreatment with 6 μM BX795 and subsequent washing away of the inhibitor. Transduction efficiency at various time points after washing away of the inhibitor (white bars: 0, 2, 4, 6 hr) were similar to those that were not pretreated with BX795, and further treatment during the transduction protocol still displayed identical levels of transduction improvement. Data from three independent donors.

FIG. 4.

Effect of stimulation time on genetic modification efficiency. Freshly isolated NK cells were either directly transduced (day 0) or stimulated in CellGro SCGM medium with 10% human serum in the presence of IL-2 (1000 U/ml) and IL-21 (20 ng/ml) for 1, 2, 3, or 4 days and subjected to lentiviral transduction in the presence and absence of BX795. For each transduction, eGFP expression was acquired 72 hr later. The efficiency of transduction peaked after 2 days of stimulation and remained more or less stable until day 4. Data from four donors. n.s., not significant; **p<0.01; ***p<0.001; two-way ANOVA repeated measures.

FIG. 5.

Function of genetically modified NK cells is not altered. (a) Results of cytotoxicity assays against K562 cells with transduced and nontransduced cells treated with or without BX795. The treatment did not alter the cytotoxic activity of NK cells. Data from four donors. (b) Analysis of degranulation on GFP+ and GFP− cells within the same sample as assessed by flow cytometry reveals no difference in the activity of genetically modified and nonmodified cells. Data from one representative donor. (c) Degranulation against no target (negative control). (d) Degranulation against PMA/ionomycin stimulation (positive control). (e) Degranulation against K562 cells. White bars indicate GFP− cells, and gray bars indicate GFP+ cells. Data from four independent donors.

FIG. 6.

Phenotypic analysis of NK cells. Phenotype of nontransduced NK cells that were cultured (a) without BX795 exposure and (b) with BX795 exposure as well as NK cells transduced (c) in the absence of BX795 and (d) in the presence of BX795 were analyzed by flow cytometry. Distribution of NKG2A and KIR expressing populations comparatively analyzed in GFP+ and GFP− cells presents the polyclonality of transduced cells. In the lower panels, histograms show expression of various cell surface receptors (light gray shaded: isotype control; black line: GFP− cells; dark gray shaded: GFP+ cells). In the histograms for transduced cells, GFP+ and GFP− cells are overlaid. No difference in the expression level of analyzed receptors is observed.

FIG. 7.

The use of BX795 treatment for enhancing transduction efficiency in other cell types. Human cells lines of hematopoietic origin (NK92, U266, Jurkat, CEM, RPMI 8226) as well as the mouse macrophage cell line B10R were transduced in the absence and presence of 6 μM BX795. An enhancement in transduction efficiency either as elevated number of GFP+ cells (a–f) or as increase in GFP fluorescence (g–i) was observed for all cell types tested except U266. Data from one representative experiment for each cell line, run in triplicate.