Name: An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection (Video) | JoVE Journal
Uploaded: 2018-03-30T13:00:00.000+00:00
Duration: 9 min 43 s
Description: Learn an efficient method to establish NK and T cell lines from CAEBV patients using minimal peripheral blood and low-dose IL-2. This technique enables research on chronic active Epstein-Barr virus mechanisms and immune cell proliferation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 following the manufacturer&#39;s instructions. Alternatively, thaw cryopreserved PBMC from liquid nitrogen with RPMI 1640 medium.</li>
	<li>Add 2 &#181;L of trypan blue (0.4%) to 18 &#181;L cell suspension, wait for 3 min to stain cells, transfer the suspension to the cell counter plate, and measure the cells&#39; concentration and viability with an automated cell counter.</li>
	<li>Prepare the cell suspension at a density of 2-3&#215;106 cells/mL supplemented with complete RPMI 1640 culture medium containing 20% human serum (heat inactivated at 56 &#176;C), 2 mM L-glutamine, and antibiotics (100 &#181;g/mL streptomycin, 100 U/mL penicillin). Seed 1 mL of PBMC suspension per well into 24-well cell culture plates.</li>
	<li>Add 150 U rhIL-2 per well.</li>
	<li>Place the culture plates in a 5% CO2 incubator at 37 &#176;C.</li>
	<li>On day 2, observe the cell morphology under a microscope (200X magnification); cells are in good condition when they are bright and round.</li>
</ol>
2. Expansion of Cells and Numbering the Viable Cells
<ol>
	<li>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 &#181;L of trypan blue (0.4%) to 18 &#181;L cell suspension, and calculate the cells&#39; concentration (see 1.2, Figure 2).</li>
	<li>Discard 500 &#181;L of the medium in a 24-well plate, and add 500 &#181;L fresh complete culture medium. Add 150U rhIL-2 per well.</li>
	<li>Change the medium twice a week as in step 2.2.</li>
	<li>Draw the cell growth curve (Figure 3).</li>
	<li>When the viable cells&#39; concentration exceeds 5&#215;106/mL, divide cells into new wells at a concentration of 1-2&#215;106 cells/mL in each well. This process takes 2-4 weeks.</li>
</ol>
3. Cell Phenotyping by Flow Cytometry
<ol>
	<li>When cells have expanded, collect 1&#215;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).</li>
	<li>Discard the supernatant, wash the precipitation by adding 7 mL PBS. Centrifuge for 5 min at 240 x g at room temperature. Repeat once.</li>
	<li>Add 200 &#181;L human serum to each tube, mix well and incubate for 10 min at room temperature.</li>
	<li>Centrifuge cells at 240 x g for 5 min at room temperature. Discard the supernatant and re-suspend the cells at 1&#215;106/mL with cold PBSA (PBS+0.2%BSA). Divide cells into 5 tubes, each tube containing 2&#215;105 cells.</li>
	<li>Centrifuge cells at 240 x g for 5 min at 4 &#176;C. Discard the supernatant and re-suspend the cells with PBSA buffer or PBSA containing 10 &#181;L PE (or PE-Cy7) and 10 &#181;L 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.</li>
	<li>Incubate the cells with antibodies for 20-30 min on ice in the dark.</li>
	<li>Wash the cells twice with cold PBSA, re-suspend the cells with 300 &#181;L cold PBSA.</li>
	<li>Analyze the cell phenotype with a flow cytometer.
	<ol>
		<li>Set up the Flow Cytometer in &#34;Create worksheet&#34; condition. Set up the experimental template with a dot plot that displays forward scatter (FSC) versus side scatter (SSC).</li>
		<li>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.</li>
		<li>Perform compensation using the isotype control and a single positive control in each 2-color analysis group.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Expansion and Cryopreservation of NK/T Cells
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C per min to -80 &#176;C and then transfer directly into liquid nitrogen.</li>
</ol>

<ol><li>Cohen, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epstein-Barr+virus+infection%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">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/pubmed?term=((Epstein-Barr+virus-associated+lymphoproliferative+disease+in+non-immunocompromised+hosts%3A+a+status+report+and+summary+of+an+international+meeting%2C+8-9+September+2008%5BTitle%5D)+AND+%22Ann+Oncol%22%5BJournal%5D)">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/pubmed?term=((Progress+in+understanding+and+managing+natural+killer-cell+malignancies%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">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/pubmed?term=((Mosquito+allergy+and+Epstein-Barr+virus-associated+T%2Fnatural+killer-cell+lymphoproliferative+disease%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Pathogenesis+of+chronic+active+Epstein-Barr+virus+infection%3A+is+this+an+infectious+disease%2C+lymphoproliferative+disorder%2C+or+immunodeficiency%3F%5BTitle%5D)+AND+%22Rev+Med+Virol%22%5BJournal%5D)">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/pubmed?term=((Clinical+and+virologic+characteristics+of+chronic+active+Epstein-Barr+virus+infection%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Proposed+categorization+of+pathological+states+of+EBV-associated+T%2Fnatural+killer-cell+lymphoproliferative+disorder+%28LPD%29+in+children+and+young+adults%3A+overlap+with+chronic+active+EBV+infection+and+infantile+fulminant+EBV+T-LPD%5BTitle%5D)+AND+%22Pathol+Int%22%5BJournal%5D)">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/pubmed?term=((Overview+and+problematic+standpoints+of+severe+chronic+active+Epstein-Barr+virus+infection+syndrome%5BTitle%5D)+AND+%22Crit+Rev+Oncol+Hematol%22%5BJournal%5D)">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/pubmed?term=((The+chronic+mononucleosis+syndrome%5BTitle%5D)+AND+%22J+Infect+Dis%22%5BJournal%5D)">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/pubmed?term=((T-cell+lymphomas+containing+Epstein-Barr+viral+DNA+in+patients+with+chronic+Epstein-Barr+virus+infections%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">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/pubmed?term=((Epstein-Barr+virus+genome-positive+T+lymphocytes+in+a+boy+with+chronic+active+EBV+infection+associated+with+Kawasaki-like+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+novel+natural+killer+%28NK%29-cell+and+gammadelta+T-cell+lines+established+from+primary+lesions+of+nasal+T%2FNK-cell+lymphomas+associated+with+the+Epstein-Barr+virus%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Presence+of+natural+killer-cell+clones+with+variable+proliferative+capacity+in+chronic+active+Epstein-Barr+virus+infection%5BTitle%5D)+AND+%22Pathol+Int%22%5BJournal%5D)">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/pubmed?term=((Hydroa+vacciniforme-like+lymphomatoid+papulosis+in+a+Japanese+child%3A+a+new+subset%5BTitle%5D)+AND+%22J+Am+Acad+Dermatol%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+a+novel+human+natural+killer-cell+line+%28NK-YS%29+established+from+natural+killer+cell+lymphoma%2Fleukemia+associated+with+Epstein-Barr+virus+infection%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+Epstein-Barr+virus+%28EBV%29-infected+natural+killer+%28NK%29+cell+proliferation+in+patients+with+severe+mosquito+allergy%3B+establishment+of+an+IL-2-dependent+NK-like+cell+line%5BTitle%5D)+AND+%22Clin+Exp+Immunol%22%5BJournal%5D)">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/pubmed?term=((Common+cytological+and+cytogenetic+features+of+Epstein-Barr+virus+%28EBV%29-positive+natural+killer+%28NK%29+cells+and+cell+lines+derived+from+patients+with+nasal+T%2FNK-cell+lymphomas%2C+chronic+active+EBV+infection+and+hydroa+vacciniforme-like+eruptions%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">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/pubmed?term=((Preferential+expansion+of+Vgamma9-JgammaP%2FVdelta2-Jdelta3+gammadelta+T+cells+in+nasal+T-cell+lymphoma+and+chronic+active+Epstein-Barr+virus+infection%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Novel+mouse+xenograft+models+reveal+a+critical+role+of+CD4%2B+T+cells+in+the+proliferation+of+EBV-infected+T+and+NK+cells%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">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/pubmed?term=((Lymphokine+requirement+for+the+generation+of+natural+killer+cells+from+CD34%2B+hematopoietic+progenitor+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Cloning+of+Human+T+and+Natural+Killer+Cells%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">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/pubmed?term=((A+Simple+Red+Blood+Cell+Lysis+Method+for+the+Establishment+of+B+Lymphoblastoid+Cell+Lines%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Application+of+flow+cytometric+in+situ+hybridization+assay+to+Epstein-Barr+virus-associated+T%2Fnatural+killer+cell+lymphoproliferative+diseases%5BTitle%5D)+AND+%22Cancer+Sci%22%5BJournal%5D)">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>

D.Z., X.Z., and X.C. are co-inventors on pending patent applications covering potential uses of this method for establishing NK/T cell lines and the cell lines established in this study. D.Z., X.Z., and X.C. declare no competing financial interests in this work. The remaining authors declare they have no competing financial interests.

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

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 × 106. In this period, cells should be expanded by division into two or three wel...

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 ...

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

Research

JoVE Journal

Immunology and Infection

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

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.

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

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 naîve T cells.

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

Source: Xu, C., et al. An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection. J. Vis. Exp. (2018).This video demonstrates the phenotyping of EBV-infected T and NK cell lines using flow cytometry, highlighting the preparation, antibody staining, and analysis steps to identify lineage-specific surface markers. It shows how marker profiles confirm distinct T-cell and NK-cell populations.

Phenotyping EBV-Infected Cell Lines Using Flow Cytometry

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Functional Characterization of Endogenously Expressed Human RYR1 Variants

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

This protocol permits the isolation of Epstein-Barr virus particles from the human P3HR1 cell line upon inducing the viral lytic cycle with phorbol 12-myristate 13-acetate. DNA is subsequently extracted from the viral preparation and subjected to real-time PCR to quantify the viral particle concentration.

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line

Learn an efficient method to establish NK and T cell lines from CAEBV patients using minimal peripheral blood and low-dose IL-2. This technique enables research on chronic active Epstein-Barr virus mechanisms and immune cell proliferation.

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

Explore More Videos