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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1374-1383
Characterization of a Novel Human Natural Killer-Cell Line (NK-YS)
Established From Natural Killer Cell Lymphoma/Leukemia Associated
With Epstein-Barr Virus Infection
By
Junjiro Tsuchiyama,
Tadashi Yoshino,
Masaharu Mori,
Eisaku Kondoh,
Takeshi Oka,
Tadaatsu Akagi,
Akio Hiraki,
Hiroyuki Nakayama,
Akira Shibuya,
Yuxiang Ma,
Teruyuki Kawabata,
Shigeru Okada, and
Mine Harada
From the Second Department of Internal Medicine, the Second
Department of Pathology, the Department of Immunology, and the First
Department of Pathology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, Japan and the Faculty of Health and Welfare
Science, Okayama Prefectural University, Soja, Japan.
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ABSTRACT |
A novel cell line was established from a patient with a
leukemic-state nasal angiocentric natural killer (NK) cell lymphoma with systemic skin infiltration. The morphology of the leukemic cells
was large-granular-lymphocyte (LGL), and their immunophenotype was
CD2+, CD3 , CD5+,
CD7+, CD16, CD56+, and
CD57. The presence of Epstein-Barr viral (EBV) genome
was shown in specimens from the patient's nose, skin, and
peripheral blood by in situ hybridization using an EBV-encoded
small RNA-1 probe or by Southern blotting using a terminal-repeat probe
of the EBV genome. Leukemic cells were cocultured with a mouse stromal
cell line (SPY3-2) in the presence of 100 U/mL recombinant human
interleukin-2 and a novel stromal cell-independent cell line, NK-YS,
was established. The NK-YS cells showed LGL morphology and expressed
surface CD2, CD5, CD7, CD25, CD56, and CD95. The NK-YS cells retained
cytotoxicity against K562 and Jurkat cells. A Southern blotting using a
terminal-repeat probe of EBV showed that NK-YS and fresh leukemic cells
had a clonal EBV genome, whereas the T-cell receptor  and  chain
genes of NK-YS were not rearranged. In an immunocytochemical analysis, the NK-YS cells showed a type-II latent infection of EBV. The NK-YS cells preserved the original characteristics of NK cell lymphoma/leukemia and will be a useful tool for the study of
biological characteristics of EBV-associated nasal angiocentric
NK cell lymphoma/leukemia.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
LETHAL MIDLINE granulomatosis and
lymphomatoid granulomatosis have each been thought of as a type of
granulomatous angiitis similar to Wegener's
granulomatosis.1-3 Histological and immunohistochemical studies performed for lethal midline granulomatosis showed that these
lymphoid cells infiltrating in an angiodestructive manner showed
atypical morphologies and a phenotype of mature T cells.4,5 The disease was thus regarded as a kind of peripheral T-cell lymphoma termed as angiocentric immunoproliferative lesions.6
However, many cases of nasal angiocentric immunoproliferative lesions
have been described in Asian countries, and they are reported to be
immunophenotyped as CD3CD56+, not rearranged
for T-cell receptor genes, and to be carrying the Epstein-Barr viral
(EBV) genome. Accordingly, this lymphoma is now considered a clonal
proliferative disorder of natural killer (NK) cells associated with EBV
infection.7-15 In a recent workshop held in Hong Kong on
nasal and related extranodal angiocentric T/NK cell lymphomas, both
Western and Asian types of angiocentric lymphomas were confirmed to be
a distinct clinicopathologic entity, ie, nasal and extranodal
angiocentric T/NK cell lymphoma.16 The diagnostic criteria
of nasal angiocentric T/NK cell lymphoma include (1) angiocentric
infiltration with or without necrosis, (2) extranodal infiltration into
skin, soft tissue, testis, upper respiratory tract, or gastrointestinal
tract, (3) EBV association, (4) an immunophenotype of CD56+
CD3 (NK cell type) or
CD56CD3+ (T cell type), and (5) no
rearrangement of T-cell receptor genes (NK cell type).16 It
is well known that nasal angiocentric T/NK cell lymphoma associated
with EBV infection is refractory to conventional chemotherapy, and its
clinical course is highly aggressive.17
We recently treated a patient with typical nasal angiocentric NK cell
lymphoma, who developed skin infiltration and leukemia with
proliferation of large granular lymphocytes (LGLs). We cocultured leukemic cells from the patient with a mouse stromal cell line (SPY3-2)18 in the presence of recombinant human interleukin 2 (rhIL-2). After a 3-month cultivation, the leukemic cells grew independent of the stromal cells, and a novel cell line, NK-YS, was
established. This is the first cell line of NK leukemic cells originating from a typical case of EBV-associated nasal angiocentric NK
cell lymphoma/leukemia. In the present study, we characterized the
biological features of this unique cell line at the cellular and
molecular levels.
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MATERIALS AND METHODS |
Case report.
A 19-year-old Japanese female had been complaining of persistent nasal
obstruction with serous rhinorrhea and low-grade fever for 2 months. A
left nasal obstructive tumor was found and diagnosed by biopsy as a
nasal angiocentric lymphoma (Fig 1A).
Immunohistochemical staining and in situ hybridization of the
paraffin-embedded specimens disclosed that the lymphoma cells were
positive for polyclonal CD3, CD45RO, and EBV-encoded small RNA-1
(EBER-1). The patient was referred to us for treatment of the disease.
Her own and her family's histories were not contributory. The physical
examination showed no significant findings except swelling in the left
root of the nose, and laboratory tests showed no remarkable
abnormalities. A magnetic resonance imaging analysis of the head and
neck showed the tumor located in the nasal cavity without the
involvement of cervical lymph nodes, tonsils, or the central nervous
system. Chest and abdominal computed tomography scans disclosed no
involvement in these areas. Bone marrow involvement was not detected in
the aspirated specimen. The patient was initially treated with
conventional chemotherapy, the CHOP (cyclophosphamide, adriamycin,
vincristine, and prednisolone) regimen, but no response was achieved.
Local radiotherapy at a total dose of 50 Gy was then used, and a
considerable regression of the nasal tumor was observed. However, at
the end of the radiation therapy the patient developed multiple
erythematous or purpuric skin lesions with ulceration. A biopsied
specimen of the skin lesion (Fig 1B) showed that lymphoma cells invaded both the dermis and epidermis. These infiltrating lymphoma cells on
frozen sections were shown to be immunohistochemically positive for
CD56, but negative for CD3, CD4, CD8, CD16, negative CD19, CD20, and
CD57. Southern blotting analysis of DNA extracted from the skin lesion
showed no rearrangement of the immunoglobulin heavy chain gene or
T-cell receptor and chain genes. In situ hybridization
disclosed the presence of EBER-1 in the nuclei of the lymphoma cells.
These results indicated that this lymphoma was compatible with the
diagnostic criteria of angiocentric NK cell lymphoma. The skin lesions
showed spontaneous regression and relapse in an alternating fashion
until the final stage, ie, the development of liver infiltration and
leukemic change. Seven days after the seventh course of chemotherapy,
the patient's white blood cell count was increased to 1 × 104/mm3; 99% of the cells were abnormal LGLs
having coarse azurophilic granules (Fig 1C). These abnormal LGLs
expressed CD2, CD5, CD7, and CD56 antigens (Table 1)
and EBER-1 (data not shown), but were
negative for CD3. A monoclonal band of EBV terminal repeat was
documented. These characteristics were compatible with those of the
skin lesions stated previously.
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| Fig 1.
Pathology of lymphoma in the (A) nose, (B) skin, and (C)
peripheral blood of the patient. (A) Original nasal angiocentric lymphoma shows vascular infiltration with prominent necrosis
(hematoxylin-eosin [HE] staining, original magnification × 40).
(B) Lymphoma cells infiltrate into the epidermis and dermis (HE
staining, original magnification × 40). (C) Leukemic cells in the
terminal stage show the morphology of LGLs with coarse azurophilic
granules and abundant cytoplasm (May-Grünwald Giemsa staining,
original magnification × 500).
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Establishment of NK cell lymphoma/leukemia cell line.
Mononuclear cells were isolated by the Ficoll-Hypaque method from the
patient's peripheral blood after the appearance of circulating malignant cells, and were suspended in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Sanko Junyaku Co, Tokyo, Japan). This cell preparation was composed of more than 99% CD56-positive cells. The
mononuclear cells (1 × 106) were inoculated into a
25-cm2 culture flask (Falcon 3013; Becton Dickinson,
Oxnard, CA) with or without a confluent layer of the mouse spleen
stromal cell line SPY3-2 as a feeder layer in 5 mL of IMDM supplemented
with 10% FBS containing 100 units of rhIL-2 (Shionogi and Co, Osaka, Japan).18 This flask was maintained in a humidified
atmosphere at 37°C with 5% CO2 in air. Half of the
medium was collected twice a week and centrifuged at 1,000 rpm for 3 minutes in a plastic tube and the pellet was resuspended in 2.5 mL of
the fresh medium. This suspension was returned to the initial culture
flask. After 2 weeks of culture, the feeder layer was found to be
destroyed or killed by the cultured leukemic cells when observed under
a phase-contrast microscope; the leukemic cell suspension was then transferred to a newly prepared culture flask with a confluent monolayer of SPY3-2. After we confirmed the proliferation of NK leukemia cells in the culture fed by SPY3-2 at 12 weeks, we cloned them
by limiting dilution and established a cell line, NK-YS, which could
proliferate in the liquid culture system independently of the stromal
cell line used for a feeder layer.
Morphological evaluation.
Cytocentrifuge smears of peripheral blood mononuclear cells from the
patient and cells from NK-YS cells were prepared and stained with
May-Grünwald Giemsa for observation under a light microscope. For
electron microscopic observation, cell pellets from NK-YS were fixed in
1.5% glutaraldehyde for 2 hours, followed by postfixation with 1%
osmium tetraoxide for 2 hours. The preparations were then dehydrated in
graded ethanol, and embedded in SPUR. Ultra-thin sections
were made for double-staining with uranyl acetate and lead citrate. The
stained sections were observed under a JEM 1010 electron microscope
(NIHON DENSHI, Tokyo, Japan) at 70 kV.
Flow cytometric analysis.
Leukemic cells from the patient and NK-YS cells were analyzed by
single-color immunofluorescence with a flow cytometer (FACS Calibour;
Becton Dickinson, Mountain View, CA) for the expression of surface
markers. The fluorescein isothiocyanate (FITC)- or phycoerythrin
(PE)-conjugated antibodies used were as follows: Leu5b (CD2), Leu4
(CD3), Leu3a (CD4), Leu1 (CD5), Leu9 (CD7), Leu2a (CD8), CALLA (CD10),
LFA1 (CD11a), Leu15 (CD11b), LeuM5 (CD11c), LeuM7 (CD13), LeuM3
(CD14), LeuM1 (CD15), Leu11 (CD16), Leu12 (CD19), Leu16 (CD20), CR2
(CD21), IL2R (CD25), LeuM9 (CD33), HPCA1 (CD34), HLe1 (CD45), Leu19
(CD56), Leu7 (CD57), TCR /, and SmIg ( +  ) from
Becton-Dickinson; OKT6 (CD1) from Ortho Diagnostic Systems (Raritan,
NJ); B1 (CD21) from Coulter Immunology (Hialeah, FL); LFA3 (CD58), Fas
(CD95) from Immunotech (Marseilles, France); TCR / from T Cell
Sciences (Cambridge, MA). Throughout the flow cytometric analysis,
FITC- or PE-conjugated mouse IgG was used as the negative control.
Cytotoxic assay.
The conventional 51Cr release assay was used to evaluate
the NK cell activity of NK-YS cells.19 Briefly, NK-YS cells
were cultured with IMDM supplemented with 10% FBS and 100 U/mL of
rhIL-2 for 3 days, and then they were mixed and incubated for 4 hours with 51Cr-labeled target cells such as K562 cells or Jurkat
cells at various effector/target ratios. The specific release of
51Cr from the target cells into the supernatant was
measured with a gamma counter. All experiments were performed in
triplicate, and the percentage lysis was determined with the following
equation: (experimental mean cpm  spontaneous release mean
cpm)/(total release mean cpm spontaneous release mean cpm) × 100 = % specific lysis.
Inteferon- (IFN-) and tumor necrosis
factor- (TNF-) production.
One million NK-YS cells or control Jurkat cells were cultured with IMDM
supplemented with 10% FBS and 100 U/mL of rhIL-2 for 48 hours, and at
the end of the culture period, the culture media were harvested by
centrifugation at 500g for 5 minutes at 4°C after the
culture. The concentrations of IFN- and TNF- in these culture
media were determined by an enzyme-linked immunosorbent assay (ELISA; EASIA kit; MEDGENIX, Fleurus, Belgium).
Chromosomal analysis.
NK-YS cells in the logarithmic phase of cell growth were arrested with
0.01 mg of colcemid for 30 minutes before hypotonic treatment with
0.075 mol/L KCl. The cells were fixed with a mixture of methanol and
acetic acid at a 3:1 ratio. Cell suspensions in the fixatives were
dropped onto a glass slide and flame dried. A conventional G-banding
method was used for karyotyping.20
Southern blotting analysis of T-cell receptor genes.
The rearrangement of the T-cell receptor and chain genes was
evaluated according to the standard method.12,14,21
Briefly, 5 µg DNA from NK-YS cells was extracted according to the
standard methods, digested with EcoRI, BamHI, or
HindIII, electrophoresed through a 0.6% agarose gel and
transferred onto a nitrocellulose filter. DNA extracted by human
placenta was used as the negative control. The filter was hybridized
with a 32P-labeled C1 or J probe and washed under
appropriate stringency conditions, and bands were visualized by
autoradiography.
Detection of EBV genome.
DNA samples from NK-YS cells, an EBV-transformed B lymphoblastoid cell
line (LAD),22,23 as a positive control and Jurkat cells as
a negative control were extracted according to the standard method,
digested with EcoRI and PstI restriction enzymes,
electrophoresed, and blotted to nitrocellulose filters. These filters
were hybridized with a 32P-labeled cDNA probe of the EBV
terminal repeat and washed under appropriate stringency conditions, and
the bands were then visualized by autoradiography.24
Immunocytochemical study for EBV products.
To investigate and determine the latent infection pattern of EBV, we
stained NK-YS cells with anti-EBV nuclear antigen-2 (EBNA-2; Novocostra
LAB, Newcastle-upon-Tyne, UK), anti-latent membrane protein-1 (LMP-1;
Novocostra), and anti-ZEBRA (immediate-early BZLF1 gene product)
antibody (Dakopatts, Glostrup, Denmark) using an
avidin-biotin-peroxidase complex (ABC) method.18
Cytocentrifuge smears of NK-YS and LAD cells (positive control) were
stained. Briefly, NK-YS cells were cytocentrifuged at 1,500 rpm, and
the slide preparations were air dried and fixed with acetone for 5 minutes at 4°C. These slides were pretreated with normal serum and
incubated with the primary antibody for 2 hours. After sufficient washing with phosphate-buffered saline (PBS), the slides were incubated
with a biotinylated secondary antibody for 30 minutes. After a further
washing with PBS, they were incubated with ABC for 60 minutes. After
further washing with PBS, they were stained for peroxidase activity
with 0.2 mg/mL of 3,3 diaminobenzidine tetrahydrochloride and 0.0075%
of hydrogen peroxide in PBS.18 We then observed the stained
slides under a light microscope.
RT-PCR analysis for mRNA expression of EBNA-2 and LMP-1.
Total RNA was extracted from LAD cells, NK-YS cells, and Jurkat cells
with an RNA Zol kit (Biotex, Houston, TX). First strand cDNA was
synthesized from 4 µg of the total RNA with oligo-dT primers
(Promega, Madison, WI) and reverse transcriptase (Superscript II;
GIBCO-BRL, Gaithersburg, MD) in a total of 20 µL of the reaction buffer. Two microliters of the reaction buffer containing the cDNA
sample was amplified by using polymerase chain reaction (PCR) Core Kit
(Boehringer Mannheim, Indianapolis, IN) and specific oligonucleotide
primers for EBNA-2; 5 -AGG CTG CCC ACC CTG AGG AT-3, 5-GCC ACC TGG
CAG CCC TAA AG-3, and LMP-1; 5-CGG AAG AGG TGG AAA ACA AA-3, 5-GTG
GGG GTC GTC ATC ATC TC-3, according to the previous
reports.25 This primer pair of LMP-1 can detect the
deletion in LMP-1 gene product.25 The PCR parameters for EBNA-2 and LMP-1 were identical. The PCR cycle was repeated 30 times,
and a fraction of each sample was electrophoresed on a 2% agarose gel,
stained with ethidium bromide, and visualized under ultraviolet
illumination.
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RESULTS |
Establishment of the NK-YS cell line.
In the primary culture of leukemic cells with a stromal cell line,
SPY3-2, the leukemic cells proliferated slowly during the first 6 weeks
of culture. Thereafter, the proliferation rate accelerated, and the
total number of leukemic cells reached a level more than 1 × 108 cells at 12 weeks of the primary culture. Some of these
cells were cryopreserved for subsequent studies. In the coculture, some of the leukemic cells adhered to stromal cells and migrated under the
stromal cell layer. Within 2 weeks after the start of coculture, the
adherent stromal cell layer was destroyed; first, the stromal cells in
contact with each other shrank and detached, then the cell membranes of
the stromal cells broke and the debris of the stromal cells resided in
the adherent layer, and finally no stromal cells were seen. This
leukemic cell-mediated cytotoxicity against a stromal cell layer was
never observed in cocultures with other myeloid or B lymphoid leukemic
cell lines (data not shown). Thus, this cytotoxicity was considered to
be derived from the natural killing activity of the NK leukemic cells.
At 12 weeks of the primary culture, the leukemic cells showed stable
proliferation, growing independently of the stromal cells. We then
performed a cloning procedure using a limiting dilution method and
established the stromal cell-independent cell line NK-YS. The doubling
time of this cell line was approximately 48 hours (data not shown). The
cytotoxicity of the NK-YS cells against the stromal cells was retained
after the cloning procedure.
Morphology.
NK-YS cells showed LGL morphology when evaluated by light microscope.
They have large nuclei with coarse chromatin and conspicuous nucleoli,
and they have abundant basophilic cytoplasm with many azurophilic
granules (Fig 2A). These azurophilic
granules were negative for peroxidase staining (data not shown).
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| Fig 2.
Morphology of the novel human NK cell line of NK-YS (A)
under light microscopy (May-Grünwald Giemsa staining, original
magnification × 500) and (B) under electron microscopy (original
magnification × 3,800). The NK-YS cells show LGL morphology
identical to that of leukemic cells in the peripheral blood of the
patient from whom the cell line was derived.
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When NK-YS cells were evaluated by electron microscope, they showed
dense granules 500 µm in diameter corresponding to the azurophilic
granules, and the granules were also negative for peroxidase staining
under electron microscopic observation (data not shown). The cells
showed mitochondria and polysomes in the cytoplasm. They were
characterized by convoluted nuclei with prominent polarity against
dense granules and pseudopodia (Fig 2B).
Immunophenotyping.
The immunophenotyping results are summarized in Table 1 and Fig
3. The NK-YS cells expressed CD2, CD5, CD7,
CD25, CD56, CD58, and CD95 (Fas) antigens but did not show detectable
levels of surface CD3, CD4, CD8, CD16, or CD21 antigens. Leukemic cells from the peripheral blood expressed CD2, CD5, CD7, and CD56. Thus, the
NK-YS cells reflected the phenotype of the original leukemic cells.
CD25 (IL-2 receptor ) seemed to be induced during the culture
process. CD95 (Fas) expression was observed in 96% of the NK-YS cells.
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| Fig 3.
Surface antigen expression of NK-YS cells, analyzed by
flow cytometry after the cells were stained with FITC- or PE-labeled antibodies.
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Cytotoxic activity.
The cytotoxic activity of NK-YS cells against K562 and Jurkat cells is
shown in Fig 4. The cytotoxicity increased
linearly along with the logarithm of effector/target ratios. The degree of the cytotoxicity against K562 cells was not different from that
against Jurkat cells.
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| Fig 4.
Assay of the natural killing activity of NK-YS against
K562 and Jurkat cells. The specific lysis (%) was plotted against the effector/target ratio. The effector/target ratio was altered from 1:1
to 40:1. The NK-YS cells show natural killing activity against the K562
and the Jurkat cells.
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Production of IFN- and TNF-.
High levels of IFN- were produced by NK-YS cells when cultured in
the standard culture medium containing rhIL-2. The TNF- concentration in the culture medium was undetectable with the ELISA
used in this study (Table 2).
Chromosomal analysis.
The fresh leukemic cells showed a complex karyotype of 46, XX,
der(4)t(1;4)(q12;p16) in 7 of the 10 metaphases analyzed (data not
shown). Additional chromosomal abnormalities such as 9 and +mar1,
add(3)(q21) and add(15)(p17), or add(5)(p11), 7, and
+mar2 were also observed. The G-banding analysis showed that the
NK-YS cells had a karyotype of 46, XX, add (3) (q26. 2),
der(4)t(1;4)(q12;p16) in 10 of the 10 metaphases analyzed (Fig
5). The NK-YS cells preserved the
common chromosomal abnormalities of
der(4)t(1;4)(q12;p16) observed in the original
leukemic cells.
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| Fig 5.
Karyotype of the NK-YS cells. The karyotype is 46, XX,
add (3) (q26.2), der(4)t(1;4)(q12;p16) according to the ISCN
system.
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Southern blotting analysis of T-cell receptor genes.
The T-cell receptor and chain genes of the NK-YS cells showed a
germline configuration. (Fig 6). This
observation was identical to those of biopsied samples from the skin
lesion as noted in the case report (data not shown). Because the sample of fresh leukemic cells was limited, the rearrangement of the T-cell
receptor and chain genes in those cells was not analyzed.
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| Fig 6.
Southern blotting analysis of the NK-YS genome for the
rearrangement of the T-cell receptor (A) and (B) chain genes.
DNA extracted from human placenta was used as a negative control (NC). (A) BamHI- or EcoRI-digested DNA was electrophorased in
the left or right lane, respectively. (B) EcoRI-,
BamHI-, or HindIII-digested DNA was electrophorased in
the left, center, or right lane, respectively. The NK-YS cells show the
germ-line configuration of T-cell receptor and chain genes.
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Detection of EBV terminal repeat.
The Southern blotting analysis showed that freshly isolated leukemic
cells and the NK-YS cells had an identical monoclonal band of the EBV
terminal repeat (Fig 7), indicating that
the fresh leukemic cells and NK-YS cells are derived from the same
clone and that the EBV genome in the NK-YS cells was infected in vivo rather than during the culture process in vitro.
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| Fig 7.
Southern blotting analysis of genomic DNA extracted from
NK-YS cells and fresh leukemic cells after restriction enzyme digestion using EcoRI and PstI with the EBV terminal repeat.
EBV-transformed B lymphoblastoid cell line, LAD cells, and Jurkat cells
were used for positive and negative controls, respectively. The NK-YS
cells showed a monoclonal band of EBV terminal repeat identical to that of the fresh leukemic cells, but not identical to that of the LAD
cells.
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Expression of EBV latent proteins and replication-associated
proteins.
The immunocytochemical analysis showed that the NK-YS cells expressed a
low level of LMP-1 but not EBNA-2 or ZEBRA protein in contrast to the
LAD cells, which expressed EBNA-2, LMP-1, and ZEBRA proteins (Fig
8). These results indicated that the NK-YS cells had a type-II latent infection of EBV, whereas the LAD cells had
a type-III latent infection.26 The lack of ZEBRA protein production indicated that EBV did not replicate in the NK-YS
cells.27-29
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| Fig 8.
Expression of EB viral latent proteins by NK-YS cells and
by LAD cells used as a positive control. The LAD cells expressed EBNA-2, LMP-1, and ZEBRA proteins. NK-YS cells expressed a low level of
LMP-1, but no EBNA-2 or ZEBRA protein (original
magnification × 200).
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Expression of EBNA-2 and LMP-1 mRNA.
By RT-PCR, it was shown that the NK-YS cells expressed low levels of
LMP-1 mRNA but no EBNA-2 mRNA, whereas the LAD cells expressed high
levels of EBNA-2 and LMP-1 mRNA (Fig 9).
This result is compatible with the immunocytochemistry. The LMP-1
reverse transcription PCR products of the NK-YS cells were about 30 bp shorter than those of the LAD cells. This result suggests that EBV in
the NK-YS may have a deletion in the LMP-1 gene.25
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| Fig 9.
Reverse transcription-PCR analysis of EBNA-2 and LMP-1
mRNA expression in Jurkat cells (lane 1 and 4), NK-YS cells (lane 2 and
5), and LAD cells (lane 3 and 6). Ten microliters of reaction solution
was applied to each lane of gel. The LAD cells expressed both
EBNA-2 (195 bp) and LMP-1 (160 bp) mRNA. NK-YS cells expressed a
low level of LMP-1 (130 bp) mRNA with about 30 bp deletion, but no
EBNA-2 mRNA. ø X174 HaeIII digest was electrophorased in the MW
lane.
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DISCUSSION |
We described the establishment of a novel cell line, NK-YS, derived
from a patient with NK cell lymphoma/leukemia. The clinical manifestations and course of this patient were typical for
EBV-associated nasal angiocentric NK cell lymphoma, as reported
previously.16 Although the patient responded transiently to
radiotherapy, the disease was resistant to conventional chemotherapy
and progressed to disseminated skin infiltration and peripheral blood
in the terminal stage. By Southern blotting analysis using an EBV
terminal repeat probe, the NK-YS cells possessed the clonality
identical to that of the leukemic cells in the patient's peripheral
blood, but it was not confirmed that leukemic cells proliferating in the peripheral blood originated from the nasal lymphoma, because the
biopsied specimen from the nasal lesion was not available for
investigation. However, the presence of EBER-1 in the nasal and skin
lesions of the lymphoma and in the leukemic cells strongly suggests
that all shared the common origin of lymphoma/leukemia cells.
Several cell lines of NK leukemia have been reported.30-33
Among them, the presence of EBV genome was shown in a cell line (YT)
established in Japan. However, in that case, the patient's diagnosis
was lymphoblastic lymphoma with thymoma. The morphological, phenotypic,
and cytogenetic characteristics of the original lymphoma cells of the
YT cell line and their association with EBV infection were not fully
described.31,34 Other cell lines were established from
aggressive NK leukemia in Western countries. Among them, the NK92 cell
line was reported not to carry the EBV genome,32 but the
association with EBV infection was not determined in other cell
lines.30,33 NK-YS is the first cell line of an NK lineage originating from a clinically documented EBV-associated nasal angiocentric NK cell lymphoma and retaining all of the cytological characteristics of NK cell lymphoma reported by Jaffe.16
The vigorous cytotoxicity of NK-YS cells against the stromal cell line
used for the feeder layer and against leukemic cell lines suggests that
lymphoma cells may be actively involved in the prominent necrosis of
the nasal lesion, which is the histological hallmark of nasal
angiocentric NK cell lymphoma. Thus, NK-YS cells fulfill the
requirements for a representative cell line of EBV-associated nasal
angiocentric T/NK cell lymphoma.
It was recently reported that the apoptosis of normal NK cells and
cells of NK92 cell line can be induced in vitro when they are
stimulated with both IL-2 and IL-12; this apoptosis is mediated by high
levels of IFN- and TNF- produced by these NK cells.35 However, stimulation with IL-2 alone did not induce the apoptosis of
the NK cells. When NK-YS cells were cultured with 100 U/mL of IL-2,
they produced high levels of IFN- but not TNF-, as mentioned
previously. This IFN- production seems to be much higher than that
of normal NK cells and the NK92 cell line stimulated with IL-2 and
IL-12.35 This result indicates that NK-YS cells are highly
resistant to apoptosis inducible by IFN-. This resistance to
apoptotic stimulation by IFN- seems to be one of the important features of NK-YS cells.
NK cell malignancy associated with a cytogenetic abnormality in the
long arm of chromosome 1 has been reported. Fernandez et
al30 reported a case of NK-LGL leukemia with the
chromosomal duplication of (1) (q21; q31), and Robertson33
et al described another case showing add (1) (q42). Similarly, the
chromosomal abnormality in the NK-YS cells and original leukemic cells
involved der(4)t(1;4)(q12;p16) in the chromosomal analysis, although
the pattern and location of these abnormalities in the long arm of chromosome 1 were not consistent. It is not yet known whether a
specific oncogene or antioncogene for NK cell malignancy is involved in
the long arm of chromosome 1.
African Burkitt's lymphoma is the first lymphoid malignancy in which
the EBV genome was shown in relation to the development of the
disease.36 However, the role of EBV products in this classic EBV-associated lymphoid malignancy has not been fully documented. Recent studies clarified the roles of EBNA-2 and LMP-1 in
the development of the immortality of EBV-infected B
cells.37,38 The role of EBV products in relation to the
immortality of NK cells has not been investigated to our knowledge. The
infection status of EBV in NK-YS cells indicated type-II
latency26 as shown in Fig 8, which is consistent with
previous observations of nasal angiocentric T/NK cell
lymphomas,7,16 and the deleted LMP-1 of the NK-YS cells may
play an important role in the transformation of NK-cells.25
This cell line should be of value in pursuing genes involved in NK cell
lymphomagenesis and in understanding the role of the EBV gene products
in nasal NK lymphoma biology.
| |
FOOTNOTES |
Address reprint requests to Junjiro Tsuchiyama, MD,
Department of Internal Medicine, Okayama Municipal Hospital, 6-10 Amase, Okayama 700, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr T. Takenouchi, Chiba Prefectural Cancer Center, for
providing the DNA probe for EBV terminal repeat. We thank Mrs T. Takabatake for her excellent technical support and Mrs Y. Watanabe for
her assistance in the laboratory. We also thank the laboratory staff of
Matsuo Hospital for providing technical support. We thank Mrs R. Oonishi and Mr Oohashi, SRL laboratory, for their assistance in the
chromosomal analysis of cell line.
| |
REFERENCES |
1.
Liebow AA,
Carrington CB,
Friedman RJ:
Lymphomatoid granulomatosis.
Hum Pathol
3:457,
1972[Medline]
[Order article via Infotrieve]
2.
Kassel SH,
Echevarria RA,
Guzzo FP:
Midline malignant reticulosis (so called lethal midline granuloma).
Cancer
23:920,
1969[Medline]
[Order article via Infotrieve]
3.
DeRemee RA,
Weiland LH,
McDonald TJ:
Polymorphic reticulosis, lymphomatoid granulomatosis: Two diseases or one?
Mayo Clin Proc
53:634,
1978[Medline]
[Order article via Infotrieve]
4. Ishii Y, Yamanaka N, Ogawa K, Yoshida Y, Takami T, Matsuura A,
Isago H, Kataura A, Kikuchi K: Nasal T-cell lymphoma as a type of
so-called "lethal midline granuloma." Cancer 50:2336, 1982
5.
Jaffe ES:
Pathologic and clinical spectrum of post-thymic T-cell malignancies.
Cancer Invest
2:413,
1984[Medline]
[Order article via Infotrieve]
6.
Lipford EH,
Margolick JB,
Longo DL,
Fauci AS,
Jaffe ES:
Angiocentric immunoproliferative lesions: A clinicopathologic spectrum of post-thymic T-cell proliferations.
Blood
72:1674,
1988[Abstract/Free Full Text]
7.
Harabuchi Y,
Yamanaka N,
Katakura A,
Imai S,
Kinoshita T,
Mizuno F,
Osato T:
Epstein-Barr virus in nasal T-cell lymphomas in patients with lethal midline granuloma.
Lancet
335:128,
1990[Medline]
[Order article via Infotrieve]
8.
Arber DA,
Weiss LM,
Albujar PF,
Chen YY,
Jaffe ES:
Nasal lymphomas in Peru, high incidence of T-cell immunophenotype and Epstein-Barr virus infection.
Am J Surg Pathol
17:392,
1993[Medline]
[Order article via Infotrieve]
9.
Su I-J,
Hsieh HC,
Lin KH,
Uen WC,
Kao CL,
Chen CJ,
Cheng AL,
Kadin ME,
Chen JY:
Aggressive peripheral T-cell lymphomas containing Epstein-Barr viral DNA: A clinicopathologic and molecular analysis.
Blood
77:799,
1991[Abstract/Free Full Text]
10.
Ho FCS,
Choy D,
Loke SL,
Kung ITM,
Fu KH,
Liang R,
Todd D,
Khoo RKK:
Polymorphic reticulosis and conventional lymphomas of the nose and upper aerodigestive tract: A clinicopathologic study of 70 cases, and immunophenotypic studies of 16 cases.
Hum Pathol
21:1041,
1990[Medline]
[Order article via Infotrieve]
11.
Ho FCS,
Srivastava G,
Loke SL,
Fu KH,
Leung BPY,
Liang R,
Choy D:
Presence of Epstein-Barr virus DNA in nasal lymphomas of B and T cell type.
Hematol Oncol
8:271,
1990[Medline]
[Order article via Infotrieve]
12.
Ferry JA,
Sklar J,
Zukerberg LR,
Harris NL:
Nasal lymphoma: A clinicopathologic study with immunophenotypic and genotypic analysis.
Am J Surg Pathol
15:268,
1991[Medline]
[Order article via Infotrieve]
13.
Kanavaros P,
Lescs ML,
Biere J,
Divine M,
Gelateau F,
Joab I,
Bosq J,
Farcet JP,
Reyes F,
Gaulard P:
Nasal T-cell lymphoma: A clinicopathologic entity associated with peculiar phenotype and with Epstein-Barr virus.
Blood
81:2688,
1993[Abstract/Free Full Text]
14.
Kaneko T,
Fukuda J,
Yoshihara T,
Zheng H,
Mori S,
Mizoguchi H,
Oshimi K:
1995. Nasal natural killer (NK) cell lymphoma: Report of a case with activated NK cells containing Epstein-Barr virus and expressing CD21 antigen, and comparative studies of their phenotype and cytotoxicity with normal NK cells.
Br J Hematol
91:355,
1995[Medline]
[Order article via Infotrieve]
15.
Jaffe ES:
Nasal and nasal type T/NK cell lymphoma: A unique form of lymphoma associated with the Epstein-Barr virus.
Histopathology
27:581,
1995[Medline]
[Order article via Infotrieve]
16.
Jaffe ES,
Chan JKC,
Su IJ,
Frizzera G,
Shigeo M,
Feller AC,
Ho FCS:
Report of the workshop on nasal and related extranodal angiocentric T/natural killer cell lymphomas.
Am J Surg Pathol
20:103,
1996[Medline]
[Order article via Infotrieve]
17.
Liang R,
Todd D,
Chan TK,
Chiu E,
Lie A,
Kwong YL,
Choy D,
Ho FCS:
Treatment outcome and prognostic factor for primary nasal lymphoma.
J Clin Oncol
13:666,
1995[Abstract/Free Full Text]
18.
Tsuchiyama J,
Mori M,
Okada S:
Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro.
Blood
85:3107,
1995[Abstract/Free Full Text]
19.
Shibuya A,
Nagayoshi K,
Nakamura K,
Nakauchi H:
Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells.
Blood
85:3538,
1995[Abstract/Free Full Text]
20.
Konishi H,
Sakurai M,
Nakao H,
Maseki N,
Kaneko Y,
Yagiri Y,
Notohara K,
Frizzere G:
Chromosome abnormalities in malignant lymphoma in patients from Kurashiki: Immunological and phenotypic correlations.
Cancer Res
50:2698,
1990[Abstract/Free Full Text]
21.
Shimodaira S,
Ishida F,
Kobayashi H,
Mahbub B,
Kawa-Ha K,
Kitano K:
The detection of clonal proliferation in granular lymphocyte-proliferative disorders of natural killer cell lineage.
Br J Hematol
90:578,
1995[Medline]
[Order article via Infotrieve]
22.
Cao L,
Yoshino T,
Nishiuchi R,
Yamadori I,
Akagi T:
Homotypic cell aggregation via conformational change of CD44 molecule induced by anti-CD44 monoclonal antibodies.
Immunobiol
193:1,
1995[Medline]
[Order article via Infotrieve]
23.
Nishiuchi R,
Yoshino T,
Matsuo Y,
Sakuma I,
Cao L,
Seino Y,
Takahashi K,
Akagi T:
The Fas antigen is detected on immature B cells and the representative cell lines show Fas-mediated apoptosis.
Br J Hematol
92:302,
1996[Medline]
[Order article via Infotrieve]
24.
Raab-Traub N,
Flynn K:
The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation.
Cell
47:883,
1986[Medline]
[Order article via Infotrieve]
25.
Kingma DW,
Weiss WB,
Jaffe ES,
Kumar S,
Frekko K,
Raffeld M:
Epstein-Barr virus latent membrane protein-1 oncogene deletions: Correlations with malignancy in Epstein-Barr virus-associated lymphoproliferative disorders and malignant lymphomas.
Blood
88:242,
1996[Abstract/Free Full Text]
26.
Rowe M,
Lear AL,
Croom-Carter D,
Davies AH,
Rickinson AB:
Three pathways of Epstein-Barr virus gene activation from EBNA 1-positive latency in B lymphocytes.
J Virol
66:122,
1992[Abstract/Free Full Text]
27.
Schepers A,
Pich D,
Mankertz J,
Hammerschmidt W:
cis-Acting elements in the lytic origin of DNA replication of Epstein-Barr virus.
J Virol
67:4237,
1993[Abstract/Free Full Text]
28.
Gruffat H,
Renner O,
Pich D,
Hammerschmidt W:
Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus.
J Virol
69:1878,
1995[Abstract]
29.
Schepers A,
Pich D,
Mankertz J,
Hammerschmidt W:
Activation of oriLyt origin of DNA replication of Epstein-Barr virus by BZLF1.
Virology
220:367,
1996[Medline]
[Order article via Infotrieve]
30.
Fernandez LA,
Pope B,
Lee C,
Zayed E:
Aggressive natural killer cell leukemia in an adult with establishment of an NK cell line.
Blood
67:925,
1986[Abstract/Free Full Text]
31.
Yodoi J,
Teshigawara K,
Nikaido T,
Fukui K,
Noma T,
Honjo T,
Takigawa M,
Sasaki M,
Minato N,
Tsudo M,
Uchiyama T,
Maeda M:
TCGF (IL-2)-receptor inducing factors: Regulation of IL-2 receptors on a natural killer-like cell line (YT cells).
J Immunol
134:1623,
1985[Abstract]
32.
Gong JH,
Maki G,
Klingemann HG:
Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells.
Leukemia
8:652,
1994[Medline]
[Order article via Infotrieve]
33.
Robertson MJ,
Cochran KJ,
Cameron C,
Le JM,
Tantravahi R,
Ritz J:
Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia.
Exp Hematol
24:406,
1996[Medline]
[Order article via Infotrieve]
34.
Yoneda N,
Tatsumi E,
Kawano S,
Teshigawara K,
Oka T,
Fukuda M,
Yamaguchi N:
Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT.
Leukemia
6:136,
1992[Medline]
[Order article via Infotrieve]
35.
Ross ME,
Caligiuri MA:
Cytokine-induced apoptosis of human natural killer cells identifies a novel mechanism to regulate the innate immune response.
Blood
89:910,
1997[Abstract/Free Full Text]
36.
Epstein MA,
Achong BG,
Barr YM:
Virus particles in cultured lymphoblasts from Burkitt's lymphoma.
Lancet
1:702,
1964[Medline]
[Order article via Infotrieve]
37.
Cohen JI,
Wang F,
Mannick J,
Kief E:
Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation.
Proc Natl Acad Sci USA
86:9558,
1989[Abstract/Free Full Text]
38.
Kaye KM,
Izumi KM,
Kief E:
Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation.
Proc Natl Acad Sci USA
90:9150,
1993[Abstract/Free Full Text]
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Abstract
Introduction
Abstract