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

Summary

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

Abstract

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

Introduction

Epstein-Barr virus (EBV) is ubiquitous and infects not only B cells, but also T and natural killer (NK) cells, which causes a number of EBV-associated NK/T lymphoproliferative diseases (LPD) and lymphoma/leukemia, such as EBV-associated hemophagocytic lymphohistiocytosis, hydroa vacciniforme-like lymphoma, extranodal NK/T-cell lymphoma, nasal type and aggressive NK cell leukemia^1,2,3. Among these is severe chronic active EBV (SCAEBV) disease, which is incident mainly in East Asia, and which is now considered to be a LPD caused by clonal expansion of EBV-infected T or NK cells....

Protocol

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

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

1. Isolation of PBMC from CAEBV Patients

1. Purify primary PBMC from 2 mL of whole blood of CAEBV patients by gradient (e.g., Ficoll-plaque) centrifugation following the manufacturer's instructions. Alternatively, thaw cryopreserved PBMC from liquid nitrogen with RPMI 1640 medium.
2. Add 2 µL of t....

Results

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

Discussion

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

Materials

List of materials used in this article

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