Abstract

BACKGROUND: Dendritic cells (DCs) play a critical role in the immune response and are a candidate for immunotherapy in cancer. Since gibbon ape leukemia virus (GALV) transduction of CD34+ cells is reasonably efficacious, we assessed the efficacy of GALV transduction of CD34+ derived DCs as a possible approach to creating genetically modified DCs for immunotherapy.
METHODS: Peripheral blood CD34+ cells were transduced with retroviruses obtained from the PG13/LN C8 cell line, with the neomycin gene as a marker gene. After prestimulation of hematopoietic cells for 24 hours with 10 ng/mL interleukin (IL)-3, 10 ng/mL IL-6, 100 ng/mL stem cell factor, 100 ng/mL granulocyte-macrophage colony stimulating factor and 8 µg/mL protamine sulfate, the cells were cultured in a transforming media prior to differentiating into DCs by GM-CSF, TNF-a and IL-4. Immunophenotyping analyses for confirmation of the generated DCs, colony formation assay and PCR were done for the expression of neomycin gene in the transduced cells.
RESULTS: Titration of viral vectors indicated a transduction efficiency of 1X10⁵ CFU/mL. Transduction efficiency for the CD34+ cells transformed to DCs was 45% and 38% before and after DC differentiation, respectively. Additionally, a mean (SEM) of 26.9% (11.4%) and 41.4% (11.8%) of the genetically modified DCs were positive for CD86+ HLA-DR and CD1a+CD14, respectively
CONCLUSION: This study showed that the majority of transduced CD34+ cells were successfully differentiated into cells identical to DCs according to morphology and immunophenotyping features, which could be a potential application in immunotherapy.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a critical role in initiating the immune response. They develop in peripheral organs and are unique in their ability to prime naive T cells.¹ As a result, they represent a particularly attractive cell component for immunotherapy.² Recent advances in the isolation and culture of DCs from CD34+ cells make it possible to generate ex vivo DCs that are quantitatively suitable for clinical trials and retain their function as APCs.³ A potentially powerful strategy in cancer gene therapy involves the use of genetically engineered DCs with a defined gene that results in long-term transgene expression, which is the ambition of much research today.^4,5 Various strategies (including viral and nonviral approaches) have been used to investigate genetically modified DCs.² Among the viral strategies, retrovirus-based vectors have the capacity of infecting the dividing cells. Given this, they can be employed as transducing agents for CD34+ cells because genetic modification in primitive stem cells deliver their progeny such as DCs and results in long-term expression of antigen.^6,7 However, a low transduction efficacy due to a low titer of virus is the main drawback of retroviral transduction.⁸ Various transduction efficacies of CD34+-derived DCs using retroviral vectors have been reported in previous studies.^8,9 Similarly, it has been shown that Moloney murine leukemia virus (MoMLV)-based vectors carrying the env protein of gibbon ape leukemia virus (GALV) can produce high-titer retroviral vectors with a wide host range,^1011,12 Accordingly, we aimed to evaluate the transduction of CD34+-derived DCs by this viral vector. resulting in significant transduction efficacy in primate and human cells.

Consequently, owing to the importance of retroviral construct as one of the main factors affecting the persistent expression of transgene, and also the promising results obtained following the transduction of CD34+ cells with GALV produced from TE-FLY MOSALF cells and their derivation into DCs,¹³ we were encouraged to examine GALV produced from another packaging cell line, PG13, which showed good efficiency in CD34+ cell transduction in previous reports,¹¹ although there is no evidence on the efficacy of transmission of the defined gene to their cell progenies such as DCs. In addition, to establish a clinically applicable method, a cell free supernatant of the viral vector was used for transduction. To evaluate transduction and derivation efficacy of DCs, gene expression and immunophenotype analysis were carried out on the genetically modified DCs.

Methods

CD34+ cell purification
Apheresis samples (n=5) obtained from donors were stimulated with recombinant granulocyte-colony stimulating factor (G-CSF). Mononuclear cells were isolated by density gradient centrifugation on Ficoll 1.077 and depleted from the adherent cells by 2-hour incubation on flat bottom flasks in DMEM +10% FBS medium at 37ºC in a 5% CO2 atmosphere. The CD34+ cells were isolated by positive immunoselection using Dynal magnetic bead (GIBCO-BRLGrand Island, NY, USA) and their purity was enumerated by flowcytometery using phycoerythrin (PE)-conjugated anti CD34+ (R7125 Dako) monoclonal antibody.

Viral vector preparation and titration on Hela cell line
PG13/LN C8 cells (ATCC-CRL-10686), which had already been cultured in HAT/HT medium and 10-7 M methotrexate for 5/2/5 days, respectively, were trypsinized and plated at 9X10⁵/60mm dishes (n=6). After 72 hours, the medium was replaced. It was then aspirated every 12 hours for 3-4 times and filtered through 0.45 µm filters. This viral condition media (VCM) was then used for G418 resistance tittering on Hela cells; 5X10⁴ cells/well were cultivated in a 6-well culture plate in DMEM containing 10% FBS (GIBCO-BRL, Grand Island, NY, USA), 1% protamine sulfate (Sigma, St. Louis, MO, USA). After 24 hours, they were refed with the medium and infected with 100 µL diluted VCM (10-1-10-3). The virus-containing medium was aspirated and the cells were trypsinized. Cell suspension (containing the medium and 500 µg/ml G418 (GIBCO-BRL, Grand Island, NY, USA) with the dilutions ranging from 1/10 to 1/500 were then prepared. Finally, the colonies were counted on each plate by day.^10-14

Gene transduction of CD34+ cells
Retroviral transduction was performed as follows: 2X10⁵ CD34+ cells (n=4) were incubated for 24 hours in a RetroNectin-coated plate in serum free media supplemented with 10 ng/mL IL3, 10 ng/mL IL6, 100 ng/mL SCF, 100 ng/mL GM-SCF and 8µg/mL protamine sulfate and VCM with the viral titer of 10⁴-10⁵

Differentiation of CD34+ cells to DCs
The DCs were generated using 2X10⁴ cells/mL (4X10⁴ cells/well) in the presence of 100 ng/mL GM-CSF (R&D), 10 ng/mL TNF-a (R&D), 100 ng/mL SCF (R&D) and 200 u/mL IL-4 (R&D) in serum free media at 37ºC in a 5% CO2 atmosphere. The cultures were then split every 4-5 days with medium containing fresh growth factors. SCF was added only for the first four days. Both cells in supernatant and adherent cells were recovered using a 5mM EDTA solution (Sigma, St. Louis, MO, USA) and counted on days indicated.

Enumerating immunophenotype of the generated DCs
The immunophenotypes were identified by flowcytometry analysis using monoclonal antibodies conjugated to PE or FITC:CD1_-FITC (Dako F7141), CD14-PE (Dako R0864), CD3-FITC (Dako F0818), CD19-FITC (Dako F0768), CD86-FITC (Dako F7205), CD56-PE (Dako R7251), HLA-DR-FITC and PE (DakoR7267 and F7266 ) (Dako, Denmark). After incubation with 2% mouse serum at 4ºC, the cells were incubated with conjugated monoclonal antibodies for 30 minutes at 4ºC at a final concentration of 10⁶ cells resuspended in PBS 1X per each reaction and analysed by flowcytometer.

G418-resistance colony formation assay
To assess G418 resistance of the neomycin gene-transduced cells, colony formation assay was performed. The transduced and non-transduced CD34+ cells and derived DCs were cultivated in DMEM containing 10% FBS, 1% protamine sulfate and 500 µg/mL G418. After 14 days of cultivation, colony formation was evaluated by microscopic and PCR analyses. CFU/mL. They were refed after 24 and 72 hours. The cells were then harvested from the plates, seeded into new dishes and cultivated in liquid culture with GM-CSF (100 ng/mL), TNF-a (10 ng/mL) and IL-4 (200 U/mL) (R&D) in serum free media for DC generation.

PCR analysis for the expression of neomycin gene
To ascertain whether the transduced cells expressed the neomycin gene, DNA was extracted following the standard procedures from equal numbers of infected and mock-infected cells. The extracted DNA was amplified in a 25 µL reaction with the primers specific for STR or the neomycin gene in an Eppendorf Mastercycler gradient. The sequences of the primers were as follows: (STR F) GATCCCAAGCTCTTCCTCTT-3', (STR R) 5'-ACGTTTGTGTGTGCATCTGT-3', (neo F) 5'-CTGAAGCGGGAAGGGACT-3' and (neo R) 5'-GGCCACAGTCGATGAATC-3'. The amplification profiles were 1 minutes 94ºC, 1 minutes 55ºC and 1 minute 72ºC for 40 cycles. The expected size of the amplified products -neo and STR- was 359 bp and 14 bp for STR, respectively. Amplified products were analyzed in a 2% agarose gel run in TBE buffer and stained with ethidium bromide.

Results

Determination of multiplicity of infection of VCM on Hela cells
Hela cells were used for the detection of VCM titer in a G418 containing media. After plating of Hela cells for 24 hours, they were infected by VCM. 24 hours later, they were trypsinized and the cell suspension was diluted from 1/10 to 1/500. The concentration of 500 µg/mL G418 (GIBCO) was used to select the transduced cells. Then, the produced colonies were counted on each plate by day.10-14 The results indicated that multiplicity of infection was: 1.0_105 CFU/mL (Figure 1).

Retroviral transduction of CD34+ cells and expression in DCs
Retroviral transduction of the CD34+ cell population was done by culturing of the prestimulated CD34+ cells in a serum free media containing SCF, GM-CSF, IL-3 and IL-6 in the presence of VCM. Refeeding was done by fresh media after 24 and 72 hours. Then, TNF-a was added by day 4. The generation of DCs was estimated by analyzing the morphology and phenotype of the cell populations.
Derivation of CD34+ cells to DCs
The purified CD34+ cells (often more than 90%) were cultured for up to 10 days in the serum free media containing SCF, GM-CSF, TNF-a and IL4. As a result, the total cell number, including the adherent and non-adherent cells were considerably increased. On day 10, a heterogeneous cell population was observed, which included non-adherent cells with round morphology or cytoplasmic spikes peculiar to DCs along with a monolayer of large adherent flat macrophage-like cells (Figure 2).

Immunophenotypic analysis of the generated DCs
As can be seen in (Figure 3), flowcytometric analysis of cell surface markers of the derived DCs revealed lack of CD3, CD19 and CD56 expression on the generated DCs. Also, we found that 26.9% (11.4) and 41.4% (11.8) of the genetically modified DCs were positive for CD86+ HLA-DR and CD1a+CD14, respectively.

Colony formation assay for the transduced G418-resistance gene
For determining G418 resistance of the neomycin gene transduced cells, colony assay was performed. The transduced CD34+ cells in DMEM containing 10% FBS, 1% protamine sulfate and 500mg/mL G418 survived well, while the control CD34+ cells viability declined an much as 90% after a week. Similarly, the transduced derived DCs tolerated the medium well, while CD34+ control cell derived DCs were entirely destroyed.

PCR results
To verify the genetic transfer of the neomycin gene in CD34+ cells and their derived DCs, PCR analysis was done for detecting Neomycine gene in addition to STR, as internal control.

As can be seen in (Fig 4A and B), our findings indicated that 45% to 49% of CD34+ cells and 29% to 38% of DCs were positive for this selectable marker, Neomycine, in agarose and polyachrylamide gel electrophoreses.

Discussion

Transduction of DCs, which is a key approach for the induction of immune responses (including antitumoral responses), is currently considered a promising strategy for cellular immunotherapy.¹⁴ The potential of ex vivo generation of DCs enables the development of genetic modification for stimulating immune responses against the desired antigen. Additionally, retroviral transduction enables gene transduction into the stem cells and consequently, into their progenies such as DCs.^15,16 However, this optimism is hampered by some limitations, such as low transduction efficacy in the genetic modification of DCs.²
 Accordingly, we aimed to optimize the retroviral transduction of CD34-derived DCs, using the GALV vector. For this purpose, mPBCD34+ cells were used as target cells, cultured in serum-free medium (containing a defined cytokine cocktail) and transduced on CH296-coated plates preloaded with VCM. Further, in the present study, we used retronectins, an extracellular matrix protein, and protamin sulfate for improving the transduction efficacy.^17,18

The results of viral vector titration on Hela cells gave a reasonable infective titer for CD34+ transduction compared with the results obtained by previous reports; 5X10^{5 19} and 7X10⁵.¹² Higher titers obtained in other studies are the results of different methods of vector preparation, mostly using ultracentrifugation for concentrating the supernatants containing the viral vector. However, so that maximal biological activity of the viral vector could be preserved, we did not concentrate the supernatant.

We then determined the immunophenotype of the generated transduced DCs by flowcytometry. The results showed that the expression of CD86+ HLA-DR and CD1a+CD14 was positive among the generated DCs. For evaluating the transduction efficacy of CD34- derived DCs, PCR analysis was carried out. The results showed that a large proportion (45%-49%) of CD34+ cells that were transduced by GALV expressed the neomycin gene. Also, 29%-38% of the generated DCs, following CD34+ cells differentiation, represented the neomycin gene, as well. Furthermore, the existence of a G418-resistance colony in the transduced CD34+ and DCs confirmed the PCR results. The diverse range of transduction efficacy obtained in the previous reports ranged from 11.5% to 86%. For example, the average transduction efficiency for the cord blood and bone marrow CD34+-derived DCs was about 10% to 20%.8 Heemskerk et al reported 40%-60% cell transduction following amphotropic retroviral transduction.⁹ Using GALV produced by TE-FLY MOSALF cells¹³ it has been shown that more than 70% of DCs derived from cord blood or mobilized progenitors have been transduced. Various sources of DCs, vectors and methods of transduction resulted in these differences.²⁰

The higher efficiency observed in some experiments seems to be mainly due to cocultivation on viral vector producer cells, which is not desirable in clinical approaches8 or following the combination of centrifugal and liposomal methods.13 In the transduction process, however, owing to the maintenance of maximum biological activity of the viral vector, viral concentration was not performed in our experiment.

One of the main advantages of our experiment is the use of a modified version of the amphotropic retroviral vectors. Its envelope has been replaced by the GALV env gene and produced by packaging the cell line, PG13, resulting in tropism for a wide range of cell types and higher viral titers.12 Also, its receptors on human mPBCD34+ cells have been shown previously.12 Therefore, optimization of this viral vector source for genetic modification will be achievable, which was preliminarily assessed in the present study. Although we did not reach a high transduction efficiency, especially in the genetically modified DCs, we believe that additional optimization, especially in the transduction procedure of CD34+ cells and their derivation into DCs, will be beneficial.

In conclusion, as a result of DC resistance to retroviral transduction, we attempted GALV transduction of CD34+ cells for genetic manipulation of DCs GALV transduction might substitute for other vectors, which have been unsuccessful. However, further studies are required to optimize this GALV vector to improve gene transduction efficacy, and to improve the functional behavior of the genetically modified DCs.

Acknowledgment
This project is granted by the Hematology, Oncology and Stem Cell Research Center, Tehran University of Medical Sciences and cooperation of Molecular Medicine Network of Iran.

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