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NEOPLASIA
Characterization of novel natural killer (NK)–cell and ␥␦ T-cell lines
established from primary lesions of nasal T/NK-cell lymphomas
associated with the Epstein-Barr virus
Hiroshi Nagata, Akiyoshi Konno, Nobuhiro Kimura, Yu Zhang, Michiko Kimura, Ayako Demachi,
Teruaki Sekine, Kohtaro Yamamoto, and Norio Shimizu
Studies on nasal T/natural killer (NK)–cell
lymphoma have been hampered by its tendency to cause necrosis. Thus, the establishment of cell lines of this neoplasm would
seem to be valuable. This study attempted
to establish cell lines from primary lesions
of this tumor, and successfully obtained 2
novel Epstein-Barr virus (EBV)–positive cell
lines, SNK-6 and SNT-8, by means of highdose recombinant interleukin 2. Flow cytometry showed that SNK-6 had an NK-cell
phenotype, CD3ⴚCD4ⴚCD8ⴚCD19ⴚCD56ⴙ Tcell receptor (TCR) ␣/␤ⴚ TCR ␥/␦ⴚ, whereas
SNT-8 was CD3ⴙCD4ⴚCD8ⴚCD19ⴚCD56ⴙ
TCR ␣/␤ⴚ TCR ␥/␦ⴙ. These were consistent with immunophenotypes of their
original tumors, and the cell lines had
monoclonal EBV clones identical to ones
in their original tumors. Thus, the cell
lines developed from cells forming the
primary lesions. Genotypic analysis
showed that SNK-6 had unrearranged TCR
and immunoglobulin heavy-chain genes,
supporting the conclusion that SNK-6 was
of NK-cell lineage. On the other hand,
SNT-8 had rearranged TCR ␤-, ␥-, and
␦-chain genes, and together with its phenotype, SNT-8 proved to be a ␥␦ T-cell
line. This is the first report of the establishment of cell lines from primary lesions of
nasal T/NK cell lymphomas, and the results demonstrated that there are at least
2 lineages, NK- and ␥␦ T-cell, in this
neoplasm. Moreover, it has been suggested that nasal T/NK cell lymphomas of
these lineages may belong to the same
clinicopathologic entity because both
types of cases shared common clinical
and histopathologic features. (Blood.
2001;97:708-713)
© 2001 by The American Society of Hematology
Introduction
Nasal T/natural killer (NK)–cell lymphoma is a distinct clinicopathologic entity characterized by progressive necrotic lesions in the
nasal cavity, nasopharynx, and palate.1 It has been called angiocentric lymphoma, lethal midline granuloma, or polymorphic reticulosis.2 It is a relatively rare disease associated with a poor prognosis
and more often seen in Asia than in the United States and
Europe.3-14 Nasal T/NK-cell lymphomas can exhibit a broad
histologic spectrum and usually show invasion of the vascular
walls, which is associated with prominent ischemic necrosis of
both tumor cells and normal tissue.
The most characteristic feature of the disease is a consistent and
strong association with the Epstein-Barr virus (EBV); indeed,
almost all cases are positive for EBV irrespective of ethnic
difference,15-22 yet the precise role of the virus in the etiology of the
disease is poorly understood. The cellular origin of the nasal
T/NK-cell lymphoma has been controversial. It was initially
thought to originate from the T-cell lineage based on immunophenotype.23-27 Later studies using combined immunophenotypic and
genotypic analysis have suggested that most nasal T/NK-cell
lymphomas are of NK-cell lineage, being CD56⫹, negative for
surface CD3 (Leu4), and unassociated with rearrangements of the
T-cell receptor (TCR) genes.11,28-30 On the other hand, some studies
have reported the existence of T-cell lymphomas that express TCR
proteins, that is ␣/␤ and ␥/␦ chains, associated with rearranged
TCR genes; these were presumably ␣␤ T-cell lymphomas10,30 and
␥␦ T-cell lymphomas.18,31 In addition, there have been reports of
atypical cases in which cell surface antigens were expressed
discordantly; these were CD3(Leu4)⫹CD56⫹ cases without TCR
gene rearrangements,32 and cases with TCR proteins expressed
discordantly with their rearrangement status of the TCR genes.10,29,30
Moreover, it has recently been suggested that nasal T/NK-cell
lymphomas of NK- and ␥␦ T-cell lineages are derived from
activated cytotoxic cells, have common clinical and histopathologic features, and belong to the same clinicopathologic entity.31
Therefore, precise phenotypes of this neoplasm including the T-cell
subset in cases of T-cell lymphomas and their relationships to
clinicopathologic features need to be studied. However, it is often
difficult to obtain a sufficient amount of tissues from such a
necrotic lesion. To solve this problem, the propagation of tumor
cells in vitro seems to be useful. Recently, a novel NK-cell line,
NK-YS, was established from peripheral blood of a patient with a
leukemic-state nasal NK-cell lymphoma.33 However, because nasal
T/NK-cell lymphomas infrequently develop leukemia, a more
applicable method for establishing a cell line is needed.
From the Department of Otorhinolaryngology, School of Medicine, Chiba
University, Chiba; First Department of Internal Medicine, School of Medicine,
Fukuoka University, Fukuoka; and Department of Virology, Division of Virology
and Immunology, Medical Research Institute, Tokyo Medical and Dental
University, Tokyo, Japan.
Reprints: Norio Shimizu, Department of Virology, Division of Virology and
Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan; e-mail: nshivir
@mri.tmd.ac.jp.
Submitted May 30, 2000; accepted September 1, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by Grants-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports and Culture of Japan (no. 11670207 and
09253103) and Grants-in-Aid for Education and Research Promotion Program
1999 from Tokyo Medical and Dental University.
708
© 2001 by The American Society of Hematology
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
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BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
NK- AND ␥␦ T-CELL LINES OF NASAL LYMPHOMAS
709
Flow cytometric analysis
The SNK-6 and SNT-8 cells were analyzed by 2-color immunofluorescence
with a flow cytometer (EPICS XL, Beckman Coulter, Hialeah, FL) for the
expression of surface markers. The following antibodies conjugated with
fluorescein isothiocyanate (FITC) or phycoerythrin (PE) (Becton Dickinson) were used: anti-CD3, -CD4, -CD8, -CD16, -CD19, -CD21, -CD25,
-CD56, -CD57, -HLA-DR, -TCR ␣/␤, and -TCR ␥/␦.
Southern blot analysis for TCR and immunoglobulin genes
Figure 1. Endoscopic findings. The patients’ right nasal cavities show granulomatous tumors and white necrosis. Left panel, patient 1; right panel, patient 2.
Therefore, in the present study, we aimed to establish cell lines
from primary lesions of nasal T/NK-cell lymphomas. We established 2 novel EBV-positive cell lines of NK- and ␥␦ T-cell
lineages, SNK-6 and SNT-8. In the present report, we describe
immunologic and molecular biologic features of these cell lines.
The SNK-6 and SNT-8 cells were analyzed for rearrangements of the TCR
␤, ␥, and ␦ chains and the immunoglobulin heavy-chain genes by Southern
blotting. DNA (3 ␮g) extracted from each of the cell lines was digested with
a restriction endonuclease BamHI, EcoRI, or HindIII (TAKARA, Kyoto,
Japan), electrophoresed through a 0.7% agarose gel and transferred onto a
nylon membrane. Human placenta DNA was used as a germline control.
The membrane was subjected to a fluorescein-labeled C␤1, J␥1, J␦1, or J␦3
probe for the TCR genes, or a JH probe for the immunoglobulin heavy-chain
gene.35-39 The J␦3 probe was a 1.5-kb XbaI genomic fragment of the J␦3
region. In addition, DNA was digested with KpnI for the J␥1 probe, and
processed in the same manner. Hybridizations were visualized by a
Fluorescein Gene Images System (Amersham, Buckinghamshire, UK)
according to the manufacturer’s instructions.
Patients, materials, and methods
Assessment of clonality of EBV
Patients and nasal tumors
DNA extracted from the cell lines as well as the frozen tumor tissues were
digested with BamHI, separated on a 0.7% agarose gel, and transferred to a
nylon membrane. A 1.9-kb XhoI subfragment of BamHI-Dhet derived from
EBV termini was used as a probe for terminal repeats of the EBV DNA40
according to the methods for detecting signals described above. Raji and
B95-8 cells were used as controls for cell expansion with monoclonal EBV
infection and polyclonal EBV replication, respectively.
Tumor tissues obtained from 2 Japanese patients with nasal T/NK-cell
lymphomas were used in this study. Patient 1 was a 62-year-old man and
patient 2 was a 48-year-old woman. Both patients had tumors on their right
inferior turbinates through the nasal vestibula. The tumors were composed
of granulomatous parts and white necrotic parts (Figure 1). Biopsies were
performed at the granulomatous parts, but small necrotic tissues were
included. Freshly biopsied tissues were cut into several pieces, and portions
of these tissues were used for establishing cell lines. The remaining tissues
were fixed in 10% buffered formalin for paraffin sections or frozen in liquid
nitrogen for immunophenotyping and molecular analysis.
Immunohistochemistry and in situ hybridization
Immunophenotyping of cells for B-, T-, and NK-cell differentiation
antigens was performed on frozen sections as well as on paraffin sections.
The antibodies used for the paraffin sections were anti-CD3⑀ polyclonal
antiserum (Dako, Glostrup, Denmark), L26 (anti-CD20, Dako), UCHL-1
(anti-CD45RO, Dako), and anti-CD56 (Novocastra, Newcastle, UK) monoclonal antibodies. The antibodies used for the frozen sections were
anti-CD2 (Leu5b) and anti-CD3 (Leu4) monoclonal antibodies (Becton
Dickinson, San Jose, CA). A standard peroxidase antiperoxidase complex
method followed by diaminobenzidine reaction was used to detect positive
signals. Paraffin sections were also used for in situ hybridization with a
digoxigenin-labeled oligonucleotide probe complementary to the EBVencoded RNA (EBER-1): the sequence is 5⬘-AGACACCGTCCTCACCACCCGGGACTTG TA-3⬘. Hybridization was detected by the procedure
described by Chang and colleagues.34
Cell culture and morphologic evaluation
The tumor tissues were minced, and strained through a 70-␮m cell strainer
(Becton Dickinson). The cells obtained through the strainer were then
suspended in RPMI 1640 medium supplemented with antibiotics, 10%
heat-inactivated human serum, and 700 U/mL of recombinant human
interleukin 2 (IL-2). The cells were cultured in a humidified atmosphere at
37°C with 5% CO2 in air. One half of the medium was exchanged with fresh
medium twice a week. The cultured cells from patients 1 and 2 have been
maintained in the presence of IL-2 for over 20 months and 17 months,
respectively, and designated as SNK-6 and SNT-8. Cytocentrifuge smears
of the cells were prepared and stained with May-Giemsa for morphologic evaluation.
Results
Primary lesions
The tumors in both patients shared common histologic features.
The normal structure of the nasal mucosa disappeared and was
replaced by tumors, and nonsuppurative necrosis was readily
noted. The tumors were composed of pleomorphic, medium- to
large-sized atypical lymphoid cells. Infiltration of neoplastic cells
to small vessels was observed (Figure 2). In situ hybridization
showed that both tumors were positive for EBV. Immunophenotypes of both tumors were CD3⑀⫹ CD20⫺CD56⫹, whereas the
tumors showed a difference in positivity for the surface CD3
(Leu4) antigen. The tumor in patient 1 was CD3 (Leu4)⫺, and the
tumor in patient 2 was CD3 (Leu4)⫹. These results indicated that
the tumor in patient 1 had a phenotype of NK cells. The results are
summarized in Table 1.
Morphology and phenotypes of the cell lines
The morphologies of the SNK-6 and SNT-8 cells were similar to
each other, except for their sizes. The SNK-6 cells were composed
Figure 2. Histopathology. Primary lesions show invasion of tumor cells to small
blood vessels. (A) Patient 1. (B) Patient 2. (Hematoxylin-eosin, final
magnification ⫻ 140).
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710
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
NAGATA et al
Table 1. Summary of phenotypes of primary tumors
Case
CD2
(Leu5b)
CD3
(Leu4)
CD3⑀
CD20
CD45RO
CD56
EBER-1
Developed
cell line
1
⫹
⫺
⫹
⫺
⫹
⫹
⫹
SNK-6
2
⫹
⫹
⫹
⫺
⫹
⫹
⫹
SNT-8
of medium to large cells, whereas the SNK-8 cells included small
cells as well as medium to large ones (Figure 3). Large cells in both
cell lines had large nuclei and abundant basophilic cytoplasm.
Nuclei as well as cytoplasm became smaller and pycnotic as the
cell sizes became smaller. Both cells had a number of cytoplasmic
vacuoles, but scarcely any azurophilic granules.
Flow cytometry showed that the SNK-6 cells were CD3⫺
CD4⫺CD8⫺CD16⫺CD19⫺CD21⫺CD25⫹CD56⫹CD57⫺HLA-DR⫹,
and TCR ␣/␤⫺ TCR ␥/␦⫺ (Figure 4A). Thus, SNK-6 was shown to have
an NK-cell phenotype. On the other hand, the SNK-8 cells were
CD3⫹CD4⫺CD8⫺CD16⫺CD19⫺CD21⫺CD25⫹CD56⫹CD57⫺HLADR⫹, and TCR ␣/␤⫺ TCR ␥/␦⫹ (Figure 4B).
Southern blot analysis for TCR and immunoglobulin genes
Neither of the cell lines showed a rearrangement of the immunoglobulin heavy-chain gene. In terms of the TCR genes, the SNK-6
cells showed no rearrangements of the TCR ␤-, ␥-, and ␦-chain
genes, supporting that SNK-6 is of NK-cell lineage (Figure 5). On
the other hand, the SNT-8 cells showed rearrangements of the ␤-,
␥-, and ␦-chain genes. The rearrangements of the ␥-chain gene
were initially not detected by the J␥1 probe hybridized with
BamHI, EcoRI, or HindIII digest (Figure 5B). Then, we evaluated
hybridization of the probe with the KpnI digest, because it has been
reported that the V␥9-J␥P rearrangement frequently observed in
rearranging ␥ loci can be detected with the KpnI digest, but the
BamHI rearranged band has virtually the same size as the germline
bands and the EcoRI and HindIII bands are in germline configuration in this rearrangement.41,42 As expected, a 9-kb germline band
derived from a KpnI fragment including the J␥P gene changed to a
12-kb band probably by assignment of the V␥9 gene to the J␥P
gene (Figure 5B). With respect to the ␦-chain gene, deletion was
initially found in the J␦1 region in the SNT-8 cells (Figure 5C),
suggesting that rearrangements of the ␣-chain gene or deletion of
the ␦-chain gene occurred in the cells.43 Subsequently, we sought
rearrangements of the ␦-chain gene by hybridizing the blots with
the J␦3 probe, because the SNT-8 cells showed expression of the
TCR ␥/␦ receptor by flow cytometry. As a result, the J␦3 probe
successfully detected rearranged bands, indicating that the deletion
of the J␦1 gene was a consequence of the usage of the J␦3 gene in
rearrangements of the ␦ locus. (Figure 5D).
lines corresponded to the bands in their respective original tumors
(Figure 6).
Discussion
The present study established 2 novel cell lines of T/NK-cell
lineage from the primary lesions of nasal T/NK-cell lymphomas by
means of a simple technique using high-dose IL-2. Established cell
lines had monoclonal EBV clones identical to those in their
respective original lesions. Moreover, the flow cytometric phenotypes of the cell lines corresponded with the immunohistochemical
phenotypes of the primary lesions. Therefore, the cell lines were
demonstrated to have developed from cells that formed the nasal
T/NK-cell lymphomas in the patients. As far as we know, this is the
first report describing the establishment of cell lines from primary
lesions of the nasal T/NK-cell lymphoma.
Clonality of EBV
Southern blot analysis for the number of EBV terminal repeats
showed a monoclonal band in each of the SNK-6 cells, the SNT-8
cells, and their original tumors. In addition, the bands in the cell
Figure 3. Morphology. Established cell lines show rich cytoplasmic vacuoles and no
azurophilic granules. (A) SNK-6. (B) SNT-8. (Wright-Giemsa, final magnification ⫻ 440)
Figure 4. Expression of cell surface markers for subset of T cells and NK cells
by established cell lines. (A) SNK-6. (B) SNT-8.
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BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Figure 5. Southern blot analysis. Southern blot analysis shows no rearrangements
of TCR genes in SNK-6 and rearrangements of the ␤-, ␥-, and ␦-chain genes in
SNT-8. Panels show hybridization with the following probes: (A) the C␤1 probe, (B)
the J␥1 probe, (C) the J␦1 probe, (D) the J␦3 probe. Human placenta DNA was used
as a germline control. The letters above the pictures are BamHI (B), EcoRI (E),
Hind III (H), and KpnI (K) digest. Note that the rearrangements of the ␥-chain gene in
SNT-8 cannot be detected by the BamHI, EcoRI, or HindIII digest. The lack of a 5-kb
Hind III band hybridizing with the J␥1 probe in SNT-8 is due to a polymorphism for a
Hind III cleavage site.41
The establishment of tumor cell lines seems to be valuable for
the study of the nasal T/NK-cell lymphoma because the tendency
of the disease to cause necrosis has hampered detailed analyses of
the tumor. Recently, a novel NK-cell line NK-YS was established
from the peripheral blood of a patient with a leukemic-state nasal
NK-cell lymphoma.33 However, an association of leukemia with
this disease is not common. Thus, we believe that a technique to
establish a cell line from a primary lesion is more applicable to the
disease. We have tried to date to establish cell lines from 5 primary
lesions obtained from 5 independent patients and have succeeded
in obtaining the 2 cell lines and in maintaining 2 other cells for over
1 month. Therefore, we believe that the technique described here
will be generally useful for establishing cell lines from more cases
of nasal T/NK-cell lymphomas.
The establishment of tumor cell lines appears to have advantages over biopsies for phenotypic and genotypic studies. In
phenotypic studies, the expression of cell surface antigens can be
analyzed more accurately by flow cytometry for suspended cells
than by immunohistochemistry on tissue sections because most
monoclonal antibodies for cell surface antigens were originally
developed for flow cytometry, and the results of immunohistochemistry are occasionally difficult to interpret. Moreover, a cytoplasmic
molecule such as CD3⑀ in NK cells, which is not expressed on the
cell surface,30,44,45 may lead to false-positive results by immunohistochemistry, and can be assessed accurately by flow cytometry.
With respect to genotypic studies, propagation of a clonal population of tumor cells appears to generate a clear result, whereas
analysis of a biopsied tissue is associated with potentially equivocal findings because of the presence of nontumorous cells.46
NK- AND ␥␦ T-CELL LINES OF NASAL LYMPHOMAS
711
Furthermore, the availability of a large amount of DNA allows us to
repeat analysis and to try various DNA probes to confirm the
reproducibility of our results.
In the present study, the results of phenotypic and genotypic
analysis completely agreed with each other in the SNK-6 cells. The
SNK-6 cells had an NK-cell phenotype CD3(Leu4)⫺CD4⫺
CD8⫺CD19⫺CD56⫹TCR ␣/␤⫺ TCR ␥/␦⫺ and unrearranged TCR
genes. On the other hand, the SNT-8 cells had a ␥␦ T-cell
phenotype CD3(Leu4)⫹CD4⫺CD8⫺CD19⫺TCR ␣/␤⫺ TCR ␥/␦⫹
and showed rearrangements of the ␥- and the ␦-chain genes of the
TCR, yet it was also CD56⫹ and associated with a rearranged
␤-chain gene. Because it was reported that peripheral ␥␦ T-cell
lymphomas frequently expressed CD5631,47-49 and showed rearrangements of the ␤-chain gene,47,49,50 SNT-8 was considered to be a
tumor cell line of ␥␦ T-cell lineage. Hence, the nasal T/NK-cell
lymphoma in case 2 proved to be a ␥␦ T-cell lymphoma. On the
basis of previous reports, NK-cell is the predominant phenotype in
nasal T/NK-cell lymphomas, and T cell is a minor phenotype.11,18,28,30,32 Therefore, SNT-8 should be useful for the study on
the nasal T/NK-cell lymphoma of the ␥␦ T-cell subset.
The genotypic studies of the SNT-8 cells gave us 2 lessons in
evaluating rearrangements of the TCR genes in T-cell lymphomas.
First, for analysis of the TCR ␥-chain gene by using the J␥1 probe,
KpnI digest should be analyzed, when BamHI, EcoRI, or HindIII
digest does not show a rearrangement.41,42 Second, when no signal is detected by the J␦1 probe, another probe for the ␦-chain
gene should be applied to determine whether it is deleted or
rearranged.39,43 However, to determine the subset of T-cell lymphomas, expression of the ␣/␤ or ␥/␦ TCR should be confirmed,
because rearrangements of the ␥-chain gene are commonly found
in T-cell lymphomas irrespective of the type of TCR that is
expressed51 and rearrangements of the ␤-chain gene are also found
in ␥␦ T-cell lymphomas as described above. In addition, consistency between the genotype and the phenotype seems necessary.
Reviewing previously reported cases with nasal T-cell lymphomas
with these considerations, we found only a small number of cases
that can be clearly categorized as ␣␤ or ␥␦ T-cell lymphoma. Two
of the 4 cases with nasal T-cell lymphomas reported by Chiang and
coworkers29,30 (case 2 in reference 29 and case 9 in reference 30), a
case in a series of 10 cases of T-cell lymphomas by Harabuchi and
colleagues10 (case 17), and a CD3⫹ TCR ␣/␤⫹ sinonasal case by
Kanavaros and associates12 appeared to be ␣␤ T-cell lymphomas.
Interestingly, in contrast to most ␥␦ T-cell lymphomas, 3 of these 4
cases were negative for CD56 and the other one was only weakly
Figure 6. Clonality of EBV. Southern blot analysis for clonality of EBV as evidence
for monoclonal expansions of EBV-positive cells in primary tumor in patient 1 (lane 1),
SNK-6 cells (lane 2), primary tumor in patient 2 (lane 3), and SNT-8 cells (lane 4).
Controls are B95-8 cells (B) and Raji cells (R) for cells with monoclonal EBV
expansion and polyclonal EBV replication, respectively.
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BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
NAGATA et al
positive for CD56. On the other hand, one ␥␦ T-cell case was
reported by Kanavaros and coworkers,18 and 2 more cases were
recently reported by the same group.31 Independently, 7 cases of
possible ␥␦ T-cell lymphomas were reported by Harabuchi and
colleages10; these cases were positive for the ␦-chain protein and
negative for the ␤-chain protein, whereas 2 of them were CD3⫺,
and none of them showed simultaneous rearrangements of the ␥and ␦-chain genes. In addition, our case 2 was a ␥␦ T-cell
lymphoma. Therefore, it may be that the ␥␦ T-cell subset is
relatively common in the nasal T-cell lymphoma. The establishment of more cell lines appears to contribute to clarifying the
features of the subset of the nasal T-cell lymphoma.
In the present study, the NK- and the ␥␦ T-cell lymphomas
formed similar tumors characterized by granulomatous appearance
and necrosis in the nasal mucosa. Moreover, both tumors consisted
of pleomorphic medium- to large-sized cells, which showed
angioinvasion and were positive for EBV and CD56⫹. Recently,
Arnulf and coworkers studied 11 cases of nonhepatosplenic
peripheral ␥␦ T-cell lymphomas31; 3 of these cases presented in the
nasal cavity and the other cases were in other extranodal sites,
including the larynx, lung, gastrointestinal tract, or skin. Interestingly, all of the 3 nasal cases were EBV positive, and 2 of them
were CD56⫹, whereas only a few cases in the other sites were EBV
positive, and none of them was CD56⫹. They concluded that nasal
T/NK-cell lymphomas, whatever their NK- or ␥␦ T-cell origin,
have common clinical and histopathologic features, are associated
with EBV, and are derived from activated cytotoxic cells, and thus
most likely belong to the same clinicopathologic entity. The present
results appear to support their conclusions.
Although the etiology of the nasal T/NK-cell lymphoma is
largely unknown, it has been recognized that the disease occurs in a
consistent and strong association with EBV.15-22 Likewise, SNK 6
and SNT-8 harbored monoclonal EBV clones. However, the
mechanisms by which T/NK cell lineages are affected by the virus
are poorly understood. In terms of the infecting route, CD21 is the
only known receptor for the virus to date.52,53 Kaneko and
colleagues showed that CD21 was expressed on normal NK cells
activated with IL-2 as well as on isolated tumor cells from a nasal
NK-cell lymphoma.54 However, 2 EBV-positive NK-cell lines,
SNK-6 in the present study and NK-YS,33 did not express the CD21
antigen. Likewise, 5 EBV-positive T-cell lines, SNT-8 in the
present study and 4 cell lines reported previously,55 were negative
for the CD21 antigen. With respect to the tumorigenicity of the
virus, the latent membrane protein LMP-1 is known to possess
transforming activity for rodent as well as human cells,56,57 and
may also play a role in the nasal T/NK-cell lymphoma. Cytogenetic
studies have shown frequent abnormalities in chromosome 6 in
nasal T/NK-cell lymphomas,58,59 and there has also been a report
that abnormalities of chromosome 6 were additionally recognized
in cells isolated from patients with chronic active EBV infection
and that the abnormalities might be caused by the integration of the
virus to chromosome 6.60 Therefore, to clarify a role of the EBV in
the etiology of the nasal T/NK-cell lymphoma, virologic, molecular biologic, and cytogenetic studies seem to be necessary, warranting the use of the established cell lines for future studies and
necessitating further efforts to develop more cell lines from nasal
T/NK-cell lymphomas.
Acknowledgment
The authors thank Mrs Aya Kasuya for her technical assistance.
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2001 97: 708-713
doi:10.1182/blood.V97.3.708
Characterization of novel natural killer (NK)−cell and γδ T-cell lines
established from primary lesions of nasal T/NK-cell lymphomas
associated with the Epstein-Barr virus
Hiroshi Nagata, Akiyoshi Konno, Nobuhiro Kimura, Yu Zhang, Michiko Kimura, Ayako Demachi,
Teruaki Sekine, Kohtaro Yamamoto and Norio Shimizu
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