Name: an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection
Uploaded: 2018-03-30T13:00:00
Description: 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

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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronic+mononucleosis+syndrome.">The chronic mononucleosis syndrome.</a> J Infect Dis. 157 (3), 405-412 (1988).</li><li>Jones, J. F., 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=T-cell+lymphomas+containing+Epstein-Barr+viral+DNA+in+patients+with+chronic+Epstein-Barr+virus+infections.">T-cell lymphomas containing Epstein-Barr viral DNA in patients with chronic Epstein-Barr virus infections.</a> N Engl J Med. 318 (12), 733-741 (1988).</li><li>Kikuta, 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=Epstein-Barr+virus+genome-positive+T+lymphocytes+in+a+boy+with+chronic+active+EBV+infection+associated+with+Kawasaki-like+disease.">Epstein-Barr virus genome-positive T lymphocytes in a boy with chronic active EBV infection associated with Kawasaki-like disease.</a> Nature. 333 (6172), 455-457 (1988).</li><li>Nagata, 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=Characterization+of+novel+natural+killer+(NK)-cell+and+gammadelta+T-cell+lines+established+from+primary+lesions+of+nasal+T/NK-cell+lymphomas+associated+with+the+Epstein-Barr+virus.">Characterization of novel natural killer (NK)-cell and gammadelta T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus.</a> Blood. 97 (3), 708-713 (2001).</li><li>Nagata, 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=Presence+of+natural+killer-cell+clones+with+variable+proliferative+capacity+in+chronic+active+Epstein-Barr+virus+infection.">Presence of natural killer-cell clones with variable proliferative capacity in chronic active Epstein-Barr virus infection.</a> Pathol Int. 51 (10), 778-785 (2001).</li><li>Tabata, N., 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=Hydroa+vacciniforme-like+lymphomatoid+papulosis+in+a+Japanese+child:+a+new+subset.">Hydroa vacciniforme-like lymphomatoid papulosis in a Japanese child: a new subset.</a> J Am Acad Dermatol. 32 (2 Pt 2), 378-381 (1995).</li><li>Tsuchiyama, J., 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=Characterization+of+a+novel+human+natural+killer-cell+line+(NK-YS)+established+from+natural+killer+cell+lymphoma/leukemia+associated+with+Epstein-Barr+virus+infection.">Characterization of a novel human natural killer-cell line (NK-YS) established from natural killer cell lymphoma/leukemia associated with Epstein-Barr virus infection.</a> Blood. 92 (4), 1374-1383 (1998).</li><li>Tsuge, I., 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=Characterization+of+Epstein-Barr+virus+(EBV)-infected+natural+killer+(NK)+cell+proliferation+in+patients+with+severe+mosquito+allergy;+establishment+of+an+IL-2-dependent+NK-like+cell+line.">Characterization of Epstein-Barr virus (EBV)-infected natural killer (NK) cell proliferation in patients with severe mosquito allergy; establishment of an IL-2-dependent NK-like cell line.</a> Clin Exp Immunol. 115 (3), 385-392 (1999).</li><li>Zhang, Y., 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=Common+cytological+and+cytogenetic+features+of+Epstein-Barr+virus+(EBV)-positive+natural+killer+(NK)+cells+and+cell+lines+derived+from+patients+with+nasal+T/NK-cell+lymphomas,+chronic+active+EBV+infection+and+hydroa+vacciniforme-like+eruptions.">Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions.</a> Br J Haematol. 121 (5), 805-814 (2003).</li><li>Oyoshi, M. 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

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.