An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

Summary

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Abstract

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

Introduction

Epstein-Barr virus (EBV) is ubiquitous and infects not only B cells, but also T and natural killer (NK) cells, which causes a number of EBV-associated NK/T lymphoproliferative diseases (LPD) and lymphoma/leukemia, such as EBV-associated hemophagocytic lymphohistiocytosis, hydroa vacciniforme-like lymphoma, extranodal NK/T-cell lymphoma, nasal type and aggressive NK cell leukemia^1,2,3. Among these is severe chronic active EBV (SCAEBV) disease, which is incident mainly in East Asia, and which is now considered to be a LPD caused by clonal expansion of EBV-infected T or NK cells^4,5,6,7, but without the apparent immunodeficiency present in infectious mononucleosis (IM)-like symptoms including fever, hepatosplenomegaly, lymphadenopathy, and liver dysfunction persistently or recurrently, as well as high EBV-DNA load in the peripheral blood^8,9. Patients with CAEBV have a poor prognosis^10,11, and its pathogenesis and the role of the EBV is unclear. Therefore, cell lines derived from EBV-associated NK/T lymphoproliferative diseases and lymphomas are very helpful as cell models for clarifying the mechanism of EBV induced NK or T cell proliferation and its relationship with high incidence of leukemia or lymphoma.

To date, several cell lines have been established with different techniques^{12,13,14,15,16}. A human NK cell line, NK-YS, was established from NK cell lymphoma/leukemia, by co-culturing with a mouse stromal cell line as feeder, and in the presence of rhIL-2 at a concentration of 20U per mL¹⁵. KAI3 was another NK cell line established from patients with a severe mosquito allergy or SCAEBV with autologous lymphoblastoid cell line (LCL), B cells transformed by EBV, as feeder cells and addition of rhIL-2 at 100U/mL¹⁶. SNK6 and SNT8 were derived from tumor tissues of nasal NK/T cell lymphoma patients by adding high-dose of rhIL-2 (700U/mL)¹². With similar technique, the SNK-1 cell was from CAEBV patients, cultured from PBMC by removing T cells and adding 700U/mL of rhIL-2^13,17. SNT13 and SNT15 were established by removing CD4+ and CD8+ cells¹⁸. So far, other T cell and NK cell lines from EBV-NK/T LPD patients were all developed with this method¹⁹.

The disadvantages of the existing methods mentioned above include the employment of feeder cells, the requirement of a high-dose of IL-2, the utilization of as much as 10 mL whole blood, or the purification of NK/T cells, which is very challenging in the clinic due to the necessity of verifying which types of cell that EBV latently infects before beginning to culture. As CAEBV mainly occurs in children in Asia, 10 mL blood is not easy to acquire in all regions. In this study, we developed a new simple method with a high success rate to establish NK and/or T cell lines by culturing PBMC from CAEBV patients using a low-dose of rhIL2 and a volume of 2 mL of whole blood without feeder cells. The results of this method have proven its high efficiency and time saving.

Protocol

This study was approved by the Ethics Committee of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the protocol follows the institutional guidelines for human welfare.

NOTE: See Figure 1 for a schematic of the workflow.

1. Isolation of PBMC from CAEBV Patients

1. Purify primary PBMC from 2 mL of whole blood of CAEBV patients by gradient (e.g., Ficoll-plaque) centrifugation following the manufacturer's instructions. Alternatively, thaw cryopreserved PBMC from liquid nitrogen with RPMI 1640 medium.
2. Add 2 µL of trypan blue (0.4%) to 18 µL cell suspension, wait for 3 min to stain cells, transfer the suspension to the cell counter plate, and measure the cells' concentration and viability with an automated cell counter.
3. Prepare the cell suspension at a density of 2-3×10⁶ cells/mL supplemented with complete RPMI 1640 culture medium containing 20% human serum (heat inactivated at 56 °C), 2 mM L-glutamine, and antibiotics (100 µg/mL streptomycin, 100 U/mL penicillin). Seed 1 mL of PBMC suspension per well into 24-well cell culture plates.
4. Add 150 U rhIL-2 per well.
5. Place the culture plates in a 5% CO₂ incubator at 37 °C.
6. On day 2, observe the cell morphology under a microscope (200X magnification); cells are in good condition when they are bright and round.

2. Expansion of Cells and Numbering the Viable Cells

1. Observe the cell morphology under a microscope (200X magnification), and monitor the condition of cell growth by numbering the cells before changing medium: add 2 µL of trypan blue (0.4%) to 18 µL cell suspension, and calculate the cells' concentration (see 1.2, Figure 2).
2. Discard 500 µL of the medium in a 24-well plate, and add 500 µL fresh complete culture medium. Add 150U rhIL-2 per well.
3. Change the medium twice a week as in step 2.2.
4. Draw the cell growth curve (Figure 3).
5. When the viable cells' concentration exceeds 5×10⁶/mL, divide cells into new wells at a concentration of 1-2×10⁶ cells/mL in each well. This process takes 2-4 weeks.

3. Cell Phenotyping by Flow Cytometry

1. When cells have expanded, collect 1×10⁶ cells to determine the phenotypes of cell lines. Centrifuge cells at 240 x g for 5 min at room temperature (NK/T cells will precipitate at the bottom of the tube).
2. Discard the supernatant, wash the precipitation by adding 7 mL PBS. Centrifuge for 5 min at 240 x g at room temperature. Repeat once.
3. Add 200 µL human serum to each tube, mix well and incubate for 10 min at room temperature.
4. Centrifuge cells at 240 x g for 5 min at room temperature. Discard the supernatant and re-suspend the cells at 1×10⁶/mL with cold PBSA (PBS+0.2%BSA). Divide cells into 5 tubes, each tube containing 2×10⁵ cells.
5. Centrifuge cells at 240 x g for 5 min at 4 °C. Discard the supernatant and re-suspend the cells with PBSA buffer or PBSA containing 10 µL PE (or PE-Cy7) and 10 µL FITC labeled antibodies to stain cell receptors of T or NK cells on ice as is indicated by Figure 4. Cells suspended with PBSA buffer are used as a negative control.
6. Incubate the cells with antibodies for 20-30 min on ice in the dark.
7. Wash the cells twice with cold PBSA, re-suspend the cells with 300 µL cold PBSA.
8. Analyze the cell phenotype with a flow cytometer.
   1. Set up the Flow Cytometer in "Create worksheet" condition. Set up the experimental template with a dot plot that displays forward scatter (FSC) versus side scatter (SSC).
   2. Load the isotype control tube to optimize the FSC and SSC voltages, and optimize the FSC threshold value to eliminate debris without interfering with the cell population of interest. Delete all parameters except FSC, SSC, FITC, PE and PE-Cy7.
   3. Perform compensation using the isotype control and a single positive control in each 2-color analysis group.
   4. Load samples and create HLA-DR VS CD19, CD4 VS CD8, CD56 VS CD16 and CD3 VS CD16 dot plots showing different population of cells.
      NOTE: PBMC were used to perform compensation using the negative/isotype control and the single positive control. 2-color immunofluorescence with flow cytometer was used routinely to analyze the expression of surface markers. The following antibodies are included: anti-HLA-DR, anti-CD4, anti-CD16 conjugated with fluorescein isothiocyanate (FITC), anti-CD8, anti-CD56, CD3 conjugated with phycoerythrin (PE), and anti-CD19 conjugated with PE-Cy7.

4. Expansion and Cryopreservation of NK/T Cells

1. Change the medium when the cell phenotype analysis is completed. Carefully remove half of the supernatant and avoid touching the cells at the bottom of the plate. Add the same volume of fresh culture medium containing 300 U/mL rhIL-2 to the cell plates.
2. Change half of the medium every 3 days (as 2.2) until the cell clusters are clearly visible under the microscope (Figure 2). Typically, this process takes 2-3 weeks.
3. Transfer the cells from 24-well plates to T25 culture flasks after mixing different wells of the same lineage. Double the volume of culture medium as it turns yellow until the volume of the medium expands to 10-15 mL. Add rhIL-2 with the concentration of 150 U/mL.
4. Change medium 24 h before freezing after 2-3 weeks growing, when the cell mass can be observed with the naked eye. Measure cell concentration with a cell counter.
5. Centrifuge the cell suspension for 5 min at 240 x g, and re-suspend cell pellet at a density of 5-10 x 10⁶ cells/mL with the frozen stock solution which contains 90% fetal bovine serum and 10% dimethylsulfoxide (DMSO). Freeze at a rate of 1 °C per min to -80 °C and then transfer directly into liquid nitrogen.

Results

After 3 days culture during the establishment of cell lines, the polymorphic cells begin to appear (Figure 2). After 7 days, cells grow quickly, as both the number and viability of cells are increased at a high rate (Figure 3). Small clusters of cells are clearly visible after 10-14 days growing, when the cell concentration can exceed 3-6 × 10⁶. In this period, cells should be expanded by division into two or three wel...

Discussion

In this protocol, a novel technique for establishing NK/T cell lines from whole blood of CAEBV patients has been developed. Compared with the existing methods, the major advantage of this method is its simplicity and its requirement of only a small volume of blood, while it exhibits a high success rate and good conditions of cell viability. Furthermore, NK/T cell lines can be established by culturing PBMC without determining the cell types the EBV latently infected in advance, as the determination would consume more bloo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hu HLA-DR FITC	BD	560944	antibody for FACS
Hu CD4 FITC	BD	555346	antibody for FACS
Hu/NHP CD8 PE	BD	557086	antibody for FACS
Hu CD3 PE	BD	555340	antibody for FACS
Hu CD16 FITC 3G8	BD	555406	antibody for FACS
Hu/NHP CD56 PE MY31	BD	556647	antibody for FACS
hu/CD19 PE-Cy7	BD	560728	antibody for FACS
human IL-2	Roche	11147528001
human serum	MRC	CCC118-125
CO2 incubator	SANYO
Centrifuge	Techcomp	CT6T	Centrifugation
microscope	OLYMPUS CKX53	CKX53
Automated Cell Counter	Countstar	IC 1000	For cell counting
24 well cell culture cluster	Corning Incorporated	3524	Polystyrene plates
25cm2 cell culture flask	Nest	707001	Polystyrene
FBS	Gibco	10270	Component of cell medium
RPMI Medium 1640	life	22400089	For cell medium
L-Glutamine	Amresco	374	Component of cell medium
100 x streptomycin penicillin solution BioRoYeeBRY-2309	BioRoYee	BRY-2309	Component of cell medium
Ficoll paque plus	GE Healthcare	17-1440-03	For in vitro isolation of lymphocyte
DMSO	Sigma	D2650	For freezing cells
flow cytometer	BD	BD FACSAria II
soft ware	BD	BD FACSDiva soft ware	FACS analysis