This work was supported by the Key Projects of Chinese Academy of Sciences (KFZD-SW-205), Strategic Biological Resources Technology Support System of Chinese Academy of Sciences (CZBZX-1 and ZSSB-004), and by Grants from National Science Foundation of China (81401640) and&#160; Shanghai Natural Science Fund (14ZR1434800).

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
<ol>
	<li>Purify primary PBMC from 2 mL of whole blood of CAEBV patients by gradient (e.g., Ficoll-plaque) centrifugation followin...

<ol>
	<li>Cohen, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+infection.">Epstein-Barr virus infection.</a> N Engl J Med. 343 (7), 481-492 (2000).</li><li>Cohen, J. I., Kimura, H., Nakamura, S., Ko, Y. H., Jaffe, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus-associated+lymphoproliferative+disease+in+non-immunocompromised+hosts:+a+status+report+and+summary+of+an+international+meeting,+8-9+September+2008.">Epstein-Barr virus-associated lymphoproliferative disease in non-immunocompromised hosts: a status report and summary of an international meeting, 8-9 September 2008.</a> Ann Oncol. 20 (9), 1472-1482 (2009).</li><li>Oshimi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+and+managing+natural+killer-cell+malignancies.">Progress in understanding and managing natural killer-cell malignancies.</a> Br J Haematol. 139 (4), 532-544 (2007).</li><li>Kawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+allergy+and+Epstein-Barr+virus-associated+T/natural+killer-cell+lymphoproliferative+disease.">Mosquito allergy and Epstein-Barr virus-associated T/natural killer-cell lymphoproliferative disease.</a> Blood. 98 (10), 3173-3174 (2001).</li><li>Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+chronic+active+Epstein-Barr+virus+infection:+is+this+an+infectious+disease,+lymphoproliferative+disorder,+or+immunodeficiency?.">Pathogenesis of chronic active Epstein-Barr virus infection: is this an infectious disease, lymphoproliferative disorder, or immunodeficiency?.</a> Rev Med Virol. 16 (4), 251-261 (2006).</li><li>Kimura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+virologic+characteristics+of+chronic+active+Epstein-Barr+virus+infection.">Clinical and virologic characteristics of chronic active Epstein-Barr virus infection.</a> Blood. 98 (2), 280-286 (2001).</li><li>Ohshima, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposed+categorization+of+pathological+states+of+EBV-associated+T/natural+killer-cell+lymphoproliferative+disorder+(LPD)+in+children+and+young+adults:+overlap+with+chronic+active+EBV+infection+and+infantile+fulminant+EBV+T-LPD.">Proposed categorization of pathological states of EBV-associated T/natural killer-cell lymphoproliferative disorder (LPD) in children and young adults: overlap with chronic active EBV infection and infantile fulminant EBV T-LPD.</a> Pathol Int. 58 (4), 209-217 (2008).</li><li>Okano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+and+problematic+standpoints+of+severe+chronic+active+Epstein-Barr+virus+infection+syndrome.">Overview and problematic standpoints of severe chronic active Epstein-Barr virus infection syndrome.</a> Crit Rev Oncol Hematol. 44 (3), 273-282 (2002).</li><li>Straus, S. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+expansion+of+Vgamma9-JgammaP/Vdelta2-Jdelta3+gammadelta+T+cells+in+nasal+T-cell+lymphoma+and+chronic+active+Epstein-Barr+virus+infection.">Preferential expansion of Vgamma9-JgammaP/Vdelta2-Jdelta3 gammadelta T cells in nasal T-cell lymphoma and chronic active Epstein-Barr virus infection.</a> Am J Pathol. 162 (5), 1629-1638 (2003).</li><li>Imadome, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mouse+xenograft+models+reveal+a+critical+role+of+CD4++T+cells+in+the+proliferation+of+EBV-infected+T+and+NK+cells.">Novel mouse xenograft models reveal a critical role of CD4+ T cells in the proliferation of EBV-infected T and NK cells.</a> PLoS Pathog. 7 (10), e1002326 (2011).</li><li>Shibuya, A., Nagayoshi, K., Nakamura, K., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine+requirement+for+the+generation+of+natural+killer+cells+from+CD34++hematopoietic+progenitor+cells.">Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells.</a> Blood. 85 (12), 3538-3546 (1995).</li><li>Spits, H., Yssel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+Human+T+and+Natural+Killer+Cells.">Cloning of Human T and Natural Killer Cells.</a> Methods. 9 (3), 416-421 (1996).</li><li>Liu, X., Xu, C., Duan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Red+Blood+Cell+Lysis+Method+for+the+Establishment+of+B+Lymphoblastoid+Cell+Lines.">A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines.</a> J Vis Exp. (119), (2017).</li><li>Kawabe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+flow+cytometric+in+situ+hybridization+assay+to+Epstein-Barr+virus-associated+T/natural+killer+cell+lymphoproliferative+diseases.">Application of flow cytometric in situ hybridization assay to Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.</a> Cancer Sci. 103 (8), 1481-1488 (2012).</li></ol>

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...

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 leukemia1,2,3. 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 cells4,5,6,7, 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 blood8,9. Patients with CAEBV have a poor prognosis10,11, 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 techniques12,13,14,15,16. 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-213,17. 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.

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

an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection

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.

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 &#215; 106. In this period, cells should be expanded by division into two or thre...

Watch this Scientific Journal Video about An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection at JoVE.com

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

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.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

The overall goal of this procedure is to obtain NK or T cell lines from CAEBV patients which has great significance of research on this fatal disease. This method helps to obtain NK or T cell lines for research on the mechanism of CAEBV, such as studying the raw VBV on cell proliferation, screening inhibitors that interfere NK/T cell proliferation. The main advantage of this technique is that it is a very simple and efficient which requires only a small amount of peripheral blood and a low dose of iron two to establish NK or T cell lines from CAEBV patients.Generally, individuals new to this method will struggle, because cell survival at the first of two weeks of the culture is uncertain and this method will show you how to determine cell proliferation of the early culture. When we first had the idea for this method, we found that human serum iron two can include PBMC for up to two weeks. We showed demonstration of this method is critical, because it shows the shape of the cells and the cause of proliferation.And that extension of cells and numbering the Y-bar cells To begin this procedure, purify primary PBMC from two milliliters of whole blood of CAEBV patients by gradient centrifugation. Alternatively, thaw cryo-preserved PBMC from liquid nitrogen with RPMI-1640 medium. Next, add two microliters of 0.4%trypan blue to 18 microliters of cell suspension to stain the cells for three minutes then transfer the suspension to the cell counter plate and measure the cells concentration and viability with an automated cell counter.Prepare the cell suspension at a density of two to three times 10-to-the-sixth cells per milliliter, supplemented with complete RPMI-1640 culture medium containing 20%human serum, two millimolar L-glutamine and antibiotics. Following that, seed one milliliter of PBMC suspension per well into the 24 well cell culture plates. Next, add 150 units per milliliter rhIL-2 to each well.Place the culture plates in a 5%carbon dioxide incubator at 37-degrees Celsius. On day two, observe the cell morphology under a microscope at 200-times magnification. Cells are in good condition when they are bright and round.In this step, add two microliters of 0.4%trypan blue to 18 microliters of cell suspension and calculate the cells'concentration. Next, observe the cell morphology and monitor the condition of cell growth by numbering the cells before changing medium. It is important to monitor the cell morphology and the condition.We can judge whether the cells survive by the polymorphic cells and the cells'condition at the early stage of a culture. Afterward, remove 500 microliters of the medium from each well of the 24-well plate and add 500 microliters of fresh complete culture medium to it. Then add 150 units per milliliter rhIL-2 to each well.Change the medium twice a week. Plot the cell growth curve. When the viable cell concentration exceeds five times 10-to-the-sixth cells per milliliter, divide cells into new wells at a concentration of one to two times 10-to-the-sixth cells per milliliter in each well.This process takes two to four weeks. When the cells have expanded, collect one times 10-to-the-sixth cells to determine the phenotypes of the cell lines. Centrifuge them at 240 times g for five minutes at room temperature to precipitate the NKNT cells.After that, discard the supernatant, wash the precipitation by adding seven milliliters of PBS. Centrifuge it for five minutes at 240 time g at room temperature. Repeat the procedure once more, then add 200 microliters of human serum to each tube, mix well and incubate for 10 minutes at room temperature Centrifuge the cells at 240 times g for five minutes at room temperature.Discard the supernatant and re-suspend the cells at one times 10-to-the-sixth cells per milliliter with cold PBSA. Subsequently, divide the cells into five tubes each containing two times 10-to-the-fifth cells. After that, centrifuge the cells at 240 times g for five minutes at four-degrees Celsius Discard the supernatant and re-suspend the cells with PBSA buffer or PBSA containing 10 microliters of PE and 10 microliters of FITC labeled antibodies to stain cell receptors of T or NK cells on ice.Cells suspended with PBSA buffer are used as a negative control. Next, incubate the cells with antibodies for 20 to 30 minutes on ice in the dark. Wash the cells twice with cold PBSA and re-suspend the cells with 300 microliters of cold PBSA before the analysis with the flow cytometer.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 rhIL-2 at 300 units per milliliter to the plates, then change half of the medium every three days until the cell clusters are clearly visible under the microscope. After that, transfer the cells from the 24-well plates to T-25 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 to 15 milliliters. Following that, add rhIL-2 with a concentration of 150 units per milliliter to it. Change the medium 24 hours before freezing after two to three weeks growing, when the cell mass can be observed with the naked eye.Measure the cell concentration with the cell counter. Then, centrifuge the cell suspension for five minutes at 240 times g and re-suspend the cell pellet at a density of five to 10 times 10-to-the-sixth cells per milliliter with the frozen stock solution which contains 90%fetal bovine serum and 10%DMSO. Freeze it at a rate of one-degree Celsius per minute to minus 80-degrees Celsius and then transfer directly into liquid nitrogen.After three days culture during the establishment of cell lines, the polymorphic cells begin to appear. After seven days, cells grow quickly as both the number and viability of cells increased at a high rate. Small clusters of cells are clearly visible after 10 to 14 days growing, when the cell concentration exceed three to six times 10-to-the-sixth.In this period, cells should be expanded by division into two or three wells of the culture plate. About a month later, once the cell number reaches three to five times 10-to-the-seventh, the count is high enough for preservation. Another important issue is to determine the phenotypes after the cells have been cultured successfully.Our results indicate that T and NK cells can be cultured by the method described above and cell lines can be established with this technique as the cells grew more than three months in good condition. This method, this technique, can be done even more. It's easy to perform properly.While attempting this procedure, it's important to remember to keep the variability of PBMC. For this procedure, either method, like MTT-assay kit 8, can be performed in order to answer additional questions like the toxicity of NK or T cell lines which may be used for adoptive immunotherapy. After each development, this techniques paves the way for researchers in the field of CAEBV to explore mechanism of EBV associated NK/T that involve operative diseases in the cell model system.After watching this video, you should have a good understanding of how to establish an expand the NK and the T cell lines following this protocol. Don't forget that working with a pathogen can be extremely hazardous and precautions should always be taken where performing this procedure.

an-efficient-simple-method-to-establish-nk-t-cell-lines-from-patients

Research

JoVE Journal

Immunology and Infection

9.0K vistas.  Chinese Academy of Sciences. The overall goal of this procedure is to obtain NK or T cell lines from CAEBV patients which has great significance of research on this fatal disease. This method helps to obtain NK or T cell lines for research on the mechanism of CAEBV, such as studying the raw VBV on cell proliferation, screening inhibitors that interfere NK/T cell proliferation. The main advantage of this technique is that it is a very simple and efficient which requires only a small amount of peripheral blood and a low do...