|Year : 2015 | Volume
| Issue : 2 | Page : 78-84
Generation of a genetically engineered aggressive Nk-Cell leukemia cell line with stable IL2 expression
Xiaozhou Hu1, Wing C Chan2, Can Kűcűk1
1 Izmir Biomedicine and Genome Institute (iBG-izmir), Dokuz Eylul University, İzmir, Turkey; Faculty, Department of Pathology, City of Hope Medical Center, Duarte, CA, USA
2 Faculty, Department of Pathology, City of Hope Medical Center, Duarte, CA, USA
|Date of Web Publication||5-Jul-2017|
Assistant Professor of Medical Biology, Izmir Biomedicine and Genome Center (iBG-izmir), Dokuz Eylul University Health Campus, Room 2006.35340 Balçova/Izmir
Source of Support: None, Conflict of Interest: None
Introduction: Aggressive NK-cell leukemia (ANKL) is a highly aggressive disease with extremely poor prognosis. A few malignant NK cell lines reflecting ANKL biology have been generated; however, these NK cell lines require the inclusion of exogeneous IL2 in the culture medium continuously, which increases the cost of cell culture significantly. Methods: IL2 coding sequenced was cloned into MSCV-IRES-YFP (PMIY) vector by directional PCR-cloning. IL2 was ectopically expressed in KHYG1 cell line through retroviral transduction. The transduced cells were cultured in limiting IL2 concentrations during which they were quantified with flow cytometry in regular time intervals to track the transduced population. IL2 transduced KHYG1 cells were then sorted to generate IL2-KHYG1 cells. PRDM1 was ectopically expressed in IL2-KHYG1 cells through retroviral transduction. Trypan blue count was performed to test proliferation of IL2-KYHG1 cells in the absence of IL2. Results: IL2-KHYG1 cells were enriched (from ~7% to ~16% YFP+ cells) during cell culture in limiting (6.25IU) IL2 concentrations. Complete removal of IL2 further enriched the transduced portion to 27% YFP-positivity, which did not increase with additional culturing. IL2 expressing KHYG1 cells were further enriched by sorting transduced IL2-KHYG1 cells with high level IL2 expression. IL2-KHYG1 cells survived and continued to grow without IL2. IL2-KHYG1 cells were transduced successfully using a PRDM1 construct having GFP as a marker under cell culture conditions having no IL2. Conclusion: IL2-KHYG1 cell line may decrease the cost associated with culturing ANKL cell lines, and it may facilitate in vitro investigation of the molecular basis of this malignancy.
Keywords: Aggressive NK-cell leukemia, Genetic engineering, IL2, PRDM1
|How to cite this article:|
Hu X, Chan WC, Kűcűk C. Generation of a genetically engineered aggressive Nk-Cell leukemia cell line with stable IL2 expression. Acta Med Int 2015;2:78-84
| Introduction|| |
Natural killer cell malignancies (NKCLs) are rare and aggressive subtypes of non-Hodgkin lymphomas that are classified into two major categories: Extra- nodal NK/T cell lymphoma of nasal type (ENKTL) and aggressive NK cell leukemia (ANKL). Several malignant NK cell lines were generated from patients having ENKTL., NKCLs are more frequent in East Asian and Central/South American populations compared to other populations of the world. Functional analyses performed with these cell lines complemented with genomics studies.,, on ENKTL tumor cases facilitated identification of several oncogenes or tumor suppressor genes deregulated through genetic and epigenetic mechanisms in ENKTLs including PRDM1(9), HACET-,,, B1MİBCL2L11) or STAT., These studies provided invaluable information regarding the etiology of ENKTL. However, there are very few studies performed to date to characterize aberrant oncogenes and tumor suppressor genes in ANKL to our knowledge. The lack of investigation of ANKL is mainly related to the devastating nature of the disease; the median survival was reported to be only 47 days. Consequently, it is very challenging to generate ANKL cell lines or perform studies on tumor cases due to its extremely aggressive nature.
A few NK cell lines have been generated from ANKL patients,, and these cell lines proved to be highly useful in in vitro studies including, but not limited to the identification of oncogenes or tumor suppressive genes. However, these NK cell lines require the addition of exogenous IL2 for continuous growth in vitro., In the absence of IL2, most NK-cell lines can not survive more than 3-4 days in vitro., Therefore, the cost of malignant NK cell line culture is more expensive than most malignant B or T cell lines which do not require the addition of a growth factor.,, In addition, IL2 is not stable for long periods of time in cell culture which may lead to technical challenges during the cell culture of malignant NK cell lines.
Here, we report establishment and characterization of an IL2 independent aggressive NK leukemia cell line, IL2-KHYG1, through stable expression of ectopic IL2 such that it can grow continuously in the absence of IL2. IL2-KHYG1 cell line generated here has the potential to significantly decrease the cost associated with in vitro culture and, hence, functional studies on ANKL.
| Material & Methods|| |
Cell Line Material
The characteristics of KHYG1 cell line were reported earlier. Briefly, KHYG1 cells originated from a patient with aggressive NK cell leukemia having a p53 point mutation. KHYG1 cells were cultured in RPMI-1640 (Gibco-Invitrogen, Carlsbad, CA) supplemented with 10% FBS; penicillin G (100 units/mL) and streptomycin (100 μg/mL); and 60-70 IU IL-2 (R&D Bioscience) at 37 °C in 5% CO2.
Generation of The Retroviral Constructs
IL2- pDNR-LIB (Thermoscientific, Rockford, IL) was used as the IL2 source sequence in the cloning of the retroviral expression plasmid. The NCBI accession number used for IL2 is BC070338.1. IL2 coding sequence was PCR- cloned using the high-fidelity PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies, Santa Clara, CA) upstream of IRES using EcoRI and XhoI sites into PMIY [Figure 1]a. Sequence validation of the insert was performed with diagnostic restriction mapping and Sanger sequencing. We used the same retroviral PRDM1 construct described and reported earlier.
|Figure 1: Transduction of KHYG1 cells with IL2 and cell culture in limiting doses of IL2. (a) The IL2-PMIY retroviral construct generated for ectopic IL2 expression. (b) The FACS profile showing the % of YFP+ cells of the empty vector or IL2 transduced KHYG1 cells after culturing with progressively decreasing doses of IL2 for 7 days. (c) Quantification of the % of YFP+ cells in vector or IL2 transduced KHYG1 cells 7 days after culturing cells with progressively decreasing doses of IL2. EV: Empty vector|
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Reconstitution of Il2 or Prdm1 in Khyg1 Cells
Retroviruses were generated by cotransfection of 4 μg of empty plasmid vector, vector containing IL2 or PRDMlα construct with 4 μg of the packaging plasmid pCL-Ampho using Turbofect (Fermentas) into ~70% confluent 293T cells seeded 24 h earlier. 24 h after transfection, the medium was replaced with 3 ml fresh medium, and 48 h after transfection, the supernatants were collected, spun down at 2,095 x g for 5 min to remove the cellular debris, and filtered with a 0.22-μm filter. A total of 500,000 cells were mixed with 1 ml retroviral supernatant in 12-well plates, with 10 μg/mL Polybrene (Chemicon-Millipore) included. Then, cells were spinoculated at 524 x g for 90 min at 4 °C. After centrifugation, cells were incubated in a humidified, 5% CO2 incubator at 37 °C for 7 h. 7h after transduction, transduced cells were spun down at 300g for 5 min and the retroviral supernatant was replaced with the regular NK cell growth medium. Retroviral transduction efficiency was evaluated two or three days after transduction with flow cytometry.
Culture of Il2 Transduced Khyg1 Cells with Progressively Decreasing Concentrations of Il2
In the first experiment, 1 × 106 vector or IL2 transduced KHYG1 cells were seeded in 1ml into 24 well plates and cultured with 50, 25, 12.5, 6.25 IU of IL2. In all the subsequent experiments, 0.5 × 106 IL2 transduced KHGY1 cells were seeded in 1ml into 12 well plates and cultured with 50, 25, 12.5 or 6.25 IU of IL2. In every 3 days, cells were spun down at 300g for 5 min and the medium was replaced with fresh IL2 having corresponding IL2 concentrations to keep the dose of IL2 stable.
Transduced KHYG1 cells were washed once and re-suspended in 1x PBS + 0.1% BSA at 0.5 x 106 cells/ml for FACS analysis. FACS Calibur or BD LSR II flow cytometers (BD Biosciences) were used for the analysis. Forward and side scatter gated cells were used for the calculations. Untransduced cells were analyzed in parallel to set the thresholds.
Trypan Blue Exclusion Assay
Cell suspension was mixed with equal volume of 0.4% isotonic trypan blue solution (Sigma Aldrich, St Louis, MO). Total cell number was determined after 2 min in Fuchs-Rosenthal hemocytometer under light microscope.
| Results|| |
IL2 Transduced KHYG1 Cells were Positively Selected in Culture with Limiting IL2 Concentrations
We asked whether IL2 transduced KHYG1 cells can be enriched by decreasing the IL2 concentration in culture medium. To address this question, we first cultured vector only or IL2-transduced KHYG1 cells with progressively decreasing doses (50IU, 25IU, 12.5IU, 6.25IU) of IL2 for 7 days. We did not observe any dose dependent change (50.5% vs.51.1% for vector and 6.8% vs. 6.4% for IL2 transduced cells when 50IU or 6.25IU IL2 cultured cells compared, respectively) in empty vector or IL2 transduced KHYG1 cells [Figure 1]b, [Figure 1]c. We modified the experiment by decreasing the density of seeded KHYG1 cells from 1 × 106 cells/ml to 0.5 × 106 cells/ml before further culturing of IL2 transduced KHYG1 cells with limiting doses of IL2. After 7 days, we observed a marginal increase (1.4%) in the % of YFP+ cells cultured with 6.25 IU IL2 compared to 50IU IL2 concentration.There was not a significant change in the % of YFP+ cells (-0.6% and -0.03%) when cells cultured with 25 and 12.5 IU were compared to the cells cultured with 50IU IL2 [Figure 2]a. We continued to culture the cells for 7 additional days with the same concentrations of IL2 and observed an increase in the % of YFP+ cells when cells cultured in 25IU, 12.5IU and 6.25IU IL2 concentrations compared to cells cultured in 50IU IL2 concentrations (1.8%, 5.8% and 5.5%, respectively) [Figure 2]b. The same cells were continued to be cultured with the corresponding IL2 concentrations for 14 additional days, and we observed a significant increase in the % of YFP+ cells in all concentrations compared to the % of YFP+ cells determined 20 days ago (2, 5.5,6.3 and 7.3% in 50, 25, 12.5, 6.25 IU IL2 treated cells, respectively). The positive selection of YFP+ cells was inversely proportional to the IL2 concentrations used for culture (7.8, 10.7, 12.1%, 14.5% in 50, 25, 12.5, 6.25IU IL2 treated cells) [Figure 2]c. Positive selection of IL2 transduced KHYG1 cells were slower than we expected so we decided to withdraw all IL2 and continue culturing IL2 transduced KHYG1 cells.
|Figure 2: Positive selection of IL2-KHYG1 cells with limiting doses of IL2 concentrations. The FACS profile showing the % of YFP+ cells of the IL2 transduced KHYG1 cells after culturing cells with progressively decreasing doses of IL2 for 7 (A, left) ,14 (B, left) and 28 days (C, left). Comparison of the % of YFP+ cells in empty vector or IL2 transduced KHYG1 cells 7 (A, right), 14 (B, right) or 28 (C, right) days after culturing cells with progressively decreasing doses of IL2. Data are mean ± SD of two independent experiments|
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IL2 Transduced KHYG1 Cells were Further Enriched After Complete Removal of IL2
During the time period in which IL2 transduced KHYG1 cells were cultured in limiting IL2 concentrations, we had kept a seperate T25 flask of these cells with normal IL2 concentrations which did not have any positive selection as expected. We continued to culture these cells without IL2. The % of YFP+ cells after 7 days of no IL2 was only 6.1 % [Figure 3]a. We continued to culture IL2 transduced KHYG1 cells for 7 additional days and we observed a robust increase (6.1% to 33.4%) in the % of YFP+ cells but a considerable portion of cells having no IL2 transduction continued to grow along with the IL2 transduced cells [Figure 3]-A. We cultured these cells 14 more days; however, the % of YFP+ cells was still 27% ([Figure 3]a, right). It is possible that the IL2 secreted by the transduced KHYG1 cells generated sufficient local IL2 concentrations which kept the untransduced cells alive and allowed them to grow. Therefore, we decided to sort YFP+ cells to generate a pure NK cell population having the capacity to express and secrete IL2. At 83 days after complete withdrawal of IL2, 27% of the cells were YFP+ [Figure 3]b, [Figure 3]c and we sorted IL2-KHYG1 cells with high YFP expression at this time point.
|Figure 3: Positive selection of IL2-KHYG1 cells in the absence of IL2. (a) FACS profile of IL2-KHYG1 cells was determined at the indicated time points after culturing cells with medium having no IL2. (b) FACS profile shows the % of YFP+ cells 83 days after the withdrawal of IL2.This is the time point YFP+ cells were sorted post-transduction of KHYG1 with IL2. (c) Comparison of the % of YFP+ cells at different time points after withdrawal of IL2. Data are mean ± SD of two independent experiments|
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Successful Transduction of Il2-Khyg1 Cells with Prdm1
Next, we wanted to test whether we can ectopically express other genes in sorted IL2-KHYG1 cells cultured without IL2. As YFP was used as the flourophore during the establishment of the IL2-KHYG1 cell line, we transduced the YFP sorted IL2-KHYG1 cells with a retroviral construct having GFP as the marker of transduction. After transduction, most of the cells (92.7% and 94.5%, in empty vector or PRDM1 transduced cells, respectively) were still YFP+ as expected [Figure 4]a. Retroviral transduction of IL2-KHYG1 cells with the empty vector or a tumor suppressor gene, PRDM1, revealed that IL2-KHYG1 cells can be transduced successfully [Figure 4]b. We obtained 34% and 16.5% GFP+/YFP+ cells in empty vector and PRDM1 transduced IL2-KHYG1 cells, respectively, suggesting that these cells can be used to test a variety of experimental endpoints.
|Figure 4: Retroviral transduction of IL2-KHYG1 cells with PRDM1. (a) FACS profile showing the % of YFP+ cells after retroviral transduction of IL2-KHYG1 cells with empty vector (PMIG) or PRDM1. (b) FACS profile showing the % of YFP+, GFP+ or YFP+/GFP+ cells. The percentage of GFP and/or YFP+ cells were determined 62h after transduction with empty vector or PRDM1. EV: Empty vector (PMIG)|
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Il2-Khyg1 Cells Grow at a Comparable Rate to Khyg1 Cells in the Absence of Exogenous Il2
Next, we evaluated whether the speed of growth of the IL2-KHYG1 cell line in the absence of IL2 is comparable to the original KHYG1 cell line cultured in the presence of exogenous IL2. To address this question, we performed trypan blue staining to determine the viable cell numbers. Quantification of the cell numbers in 2 day intervals showed that IL2-KHYG1 cells grow at a similar rate in the absence of IL2 to the IL2-dependent KHYG1 cells cultured in the presence IL2 [Figure 5]a. We then cultured KHYG1 cells in the absence of IL2, and observed halting of cell proliferation and induction of apoptosis by 3 days of IL2 withdrawal as expected [Figure 5]b.
|Figure 5: Enginereed KHYG1 cells grows at a comparable rate to the IL2 dependent KHYG1 cell line in the absence of IL2. (a) The growth capacity of IL2-KHYG1 cells were evaluated in the absence of IL2. Cellular growth was evaluated with trypan blue staining. Cells unstained with trypan blue were considered alive and quantified. ± signs in parenthesis show whether IL2 is included during cell culture or not. Data are means ± SD of three biological replicates|
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| Discussion|| |
There are almost no studies performed investigating the etiology of ANKL despite the aggressive clinical course of this malignancy. NK cell lines representing ANKL pathogenesis are highly useful tools to study the basis of the disease. However, the available ANKL cell lines KHYG1 and IMC1 are IL2 dependent. Continuous IL2 administration is required for in vitro studies which increase the cost of cell culture, and this situation may pose challenges in identification of aberrant genes as knock-down or ectopic expression experiments require enormous amount of time. Additionally, recombinant human (rhIL2) is not stable for a long time and it needs to be replenished continuously. Instability of IL2 in vitro may result in practical problems during long-term in vitro experiments that may lead to cell death of cultured cells without any experimental manipulation. IL2-KHYG1 cells will presumably not have these challenges owing to stable and continuous expression.
ANKL is a very aggressive malignancy with very limiting information regarding the causes of it. Therefore, there is urgent need for development of novel tools to investigate the molecular etiology of ANKL in order to identify and functionally characterize the aberrant oncogenes or tumor suppressor genes associated with ANKL pathogenesis. These genes, in turn, can serve as potential therapeutic targets.
To sum up, we established an IL2 independent version of the KHYG1 cell line by genetically engineering it through stable ectopic expression of IL2. IL2-KHYG1 cells may facilitate in vitro functional studies by eliminating the requirement of IL2 to keep the cell line grow.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]