1. Art and Diseño

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Characterization of T Cells Immortalized by Taxl of Human T-cell
Leukemia Virus Type 1
By Tsuyoshi Akagi, Hiroaki Ono, and Kunitada Shimotohno
Peripheral blood T cells were immortalized in vitro by introduction of the Taxl gene of human T-cell leukemia virus
type 1 (HTLV-1)with a retroviral vector and were characterized for transformation-associated markers. Long-term observation showed that these Taxl-immortalized T cells eventually exhibitedvery similar features
that were characteristic
of HTLV-1-immortalized T cells, ie, increased expressionof
egr-l, c-fos, IL-ZRa, and Lyn and decreased expression of
Lck and cell-surface CD3 antigen. Among thesechanges, an
increase in tho expression of Lyn and a decrease in the expression of Lck and cell-surface CD3 antigen were observed
H
UMAN T-CELL leukemia virus type 1 (HTLV-l) is
etiologically associated with adult T-cell leukemia
(ATL) and tropical spastic paraparesis (TSP)/HTLV-1-associated myelopathy (HAM).’.’ This virus characteristically
has the ability to immortalize normal T cells invitro.’,’
HTLV-l-immortalized T cells differ inmany ways from
normal T cells. The interleukin-2 (IL-2) requirement for cell
growth is decreased inmany
HTLV-1-immortalized T
cell^.^,^ HTLV-1-immortalized T cells show altered cellsurface phenotypes; eg, expression of CD25 (L-2Ra) and
major histocompatibility complex (MHC) class I1 antigen is
constitutively augmented, whereas expression of the CD31
T-cell receptor (TCR) complex d e c r e a s e ~ . ~T”cells
~ immortalized by HTLV-1 constitutively express many kinds of
immediate early serum responsive genes.”.‘’ Furthermore,
many HTLV-1 -immortalized T cells show changed expression patterns of src-family tyrosine kinases. HTLV-1-immortalized T cells, especially IL-2-independent cells, frequently express a decreased level of Lck, a major tyrosine
kinase in normal T cells.I3 In contrast, many HTLV-1-immortalized T-cell lines show high-level expression of Lyn
that is barely detectable in normal T cells.I4
Tax l , a transcriptional trans-activator of this virus, interacts with at least three distinct cellular transcriptional factors:
cyclic AMP-responsive element-binding proteidactivating
transcription factor (CREB/ATF), nuclear factor KB(NFKB),
and serum responsive factor (SRF).I5 Through the interactions of these transcriptional factors, Taxl transcriptionally
activates not only its own long terminal repeat (LTR), but
also some immediate early serum-responsive genes (c-fos,
c-jun, egr-l, etc), cytokine genes (IL-2,
IL-3,GM-CSF, TGF01, etc), and its receptor gene (IL-2Ra).’*I5
This activation
of cellular genes is thought to leadto T-cell transformation.l2l5We and others have shown that primary human T
cells can be immortalized by the introduction of the Taxl
In our previous study, we characterized the growth
properties of Tal-immortalized T cells at early passage
(several months after the introduction of Taxl) and reported
that these T cells showed a greatly increased proliferative
response to stimulation with anti-CD3 antibody, mainly
through an L-2-independent pathway.16
In this study, we have investigated the Taxl-immortalized
T cells for a long period (up to 3 years) and found that these
T cells eventually showed very similar phenotypes to those
of T cells immortalized by HTLV-1. Many of the characteristic features of HTLV-l -immortalized T cells appear to be
attributable to the function of Tax 1.
Blood, Vol86, No 1 1 (December l), 1995:pp 4243-4249
only in Taxl-immortalized T cells after long-term culture.
The expression level of Taxl protein did not differ significantly between early andlate passage of cells, and
the cellular clonalitywas found to bethe same by the analysis ofthe
retroviral vector integration site and the T-cell receptor pchaingenerearrangement pattern. Thesechanges in the
expression of Lyn, Lck, and cell-surface CD3 antigen probably resultedfrom indirect effectsof Taxl that appeared after
extended culture.
0 1995 by The American Society of Hematology.
MATERIALS AND METHODS
Cells and cell culture. Primary human T cells derived from the
peripheral blood of a healthy donor were infected with a taxl-expressing retroviral vector, DGL-Taxl, or a control vector, DGL,
followed by selection with G418. Detailed experimental procedures
for establishment of these retroviral vector-infected cells were described previously.’8 These infectants were maintained inAIM-V
medium (GIBCO, Grand Island, NY) supplemented with 10% fetal
calf serum (FCS), 10 ng/mL recombinant IL-2 (Takeda, Osaka, Japan), and 0.05 mmol/L 2-mercaptoethanol. Frozen stocks of these
cells from various culturing periods were used in this study. KN6HT is an HTLV-1-infected human CD4+ helper T-cell clone,” and
FU-KOS-3 is an HTLV-l-uninfected normal human CD4+ helper
T-cell clone derived from the peripheral blood of a healthy donor.’”
These cells were also maintained in the same medium as described
above.
Southern blot analysis. Genomic DNA (10 pp) was digested
with restriction endonucleases, separated by electrophoresis in 0.8%
agarose, and blotted onto a nitrocellulose membrane. The membrane
was hybridized with3’P-labeled
Taxl-specific probe (a 1.3-kb
BamHI-Sal I fragment from pUC/Taxl), as described previously.’*
The membranes were then washed in 0.2X SSC and 0.1% sodium
dodecyl sulfate (SDS) at 65°C for 1 hour and exposed to x-ray films
with intensifying screens at -70°C.
Northern blot analysis. Total cellular RNA (10 pg) was electrophoresed in 1.O% agarose containing formaldehyde and MOPS and
blotted onto a nitrocellulose membrane. The membrane was hybridized with ’*P-labeled probe, as described previously,18washed with
0.2X SSC and 0.1% SDS at 65°C for 1 hour, and exposed to x-ray
film with intensifying screens at -70°C. The DNA fragments used
as probes were as follows: human c-fos cDNA, a 2.1-kb EcoRI
fragment of pSFT-fos cDNA’’; human egr-l cDNA, nucleotides
2530 to 3109 of ETR103”; human IL-2 cDNA, a 0.6-kb Pst I-
Fromthe Virology Division, National Cancer Center Research
Institute, Tsukiji, Chuo-ku, Tokyo, Japan.
Submitted April IO, 1995; accepted August I , 1995.
Supported in part by Grants-in-Aid for Cancer Research and
Grants-in-Aid for a Comprehensive IO-Year Strategy for Cancer
Control from the Ministry of Health and Welfare of Japan.
Address reprint requests to Kunitada Shimotohno, PhD, Virology
Division, National Cancer Center Research Institute, 5-I - I Tsukiji,
Chuo-ku, Tokyo 104, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8611-0033$3.00/0
4243
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AKAGI, ONO, AND SHIMOTOHNO
4244
D m I fragment of p3-16”; human IL-2Ra cDNA, a 1.3-kb Hind111
fragment of pKCR. Tac-2.AZ4; human Lck cDNA. a I .8-kb NCO IHind111 fragment of YT16”; and human Lw cDNA, nucleotides 264
to 1949 of PLY-30.’‘ TheseDNAfragmentswerelabeledwith
a
multiprimeDNAlabelingsystem(Amersham,AmershamPlace,
m,,
UK).
I
F/Nnrescence-activoted cell sorter (FACS)ona/wis. Fluorescein
isothiocyanate (F1TC)-conjugated monoclonalantibodiesagainst
CD2 (LeuS), CD3 (Leu4). CD4 (Leu3a). CD5 (Leul), CD25 (IL2R1). CD28 (KOLT-2). CD4SRA (2H4). and CD4SRO (UCHLI)
to
were used to detect the cell-surface marker. Cells were allowed
react with optimal concentrations of these antibodies and were analyzed with flow cytometer, Cytoron-Absolute (Ortho, Raritan, NJ).
T-cell proliferorinn ussa~. T-cellproliferationwasassayed
by
measuring the incorporation of ’H-thymidine. T cells were cultured
in AIM-V medium containing 10% FCS either in the absence or in
the presence of IL-2 ( I 0 ng/mL) in flat-bottom 96-well plates (4 X
IO4 cellsper well). After 48 hours,each well was pulsed for 18
hourswith I pCi ’H-thymidine. The cellswereharvestedand
the
radioactivity of the 5% trichloro acetic acid-insoluble fraction was
measured in a liquid scintillation counter. Results are presented a s
the means of triplicates f. SD.
Imrnunohlotfing lmmunoblottingwasperformedessentially
as
described.”’ Briefly, S X IO5 cells were pelleted and lysed in 100 pL
of 2X SDS-polyacrylamide pel electrophoresis (SDS-PAGE) sample
buffer. After quantitating the protein by BCA protein assay reagent
(Pierce, Rockford, IL), each lysate corresponding to S p g of protein
was fractionated on 10% SDS-PAGE. and the proteins were electrophoreticallytransferred to Immobilon(Millipore,Bedford,MA).
After blocking with 3% bovine serum albumin in TBS (10 mmol/L
Tris-HCI, pH 7.4, 140 mmol/L NaCI) overnight at room temperature.
themembraneswereincubated
with mouseanti-Taxmonoclonal
antibody TAXY7” in the blocking buffer forI hour. The membranes
were then washed extensively with TBS containing0.1% Tween 20,
incubated for I hour at room temperature with horseradish peroxidase-conjugated sheep antimouse IgG antibody, washed, and developed with theAmershamECLchemiluminescencereagentasdirected by the manufacturer.
RESULTS
Immortalization of Taxl-transduced peripheralblood
lymphocytes (PBLs). Continuously growing cells canbe
obtained from PBLs after infection with the Tax I -expressing
retroviral vector, DGL-Tax1 . Two cell lines designated PBL/
DGL-Tax 1 A and PBLIDGL-Tax 1 B were established from
two independent infection experiments. Expression of Tax 1
in these cells was confirmed as described previously.’” PBL/
DGL-Tax 1A and PBLIDGL-Tax1 B have continued to grow
in IL-2-containing medium for nearly 4 and 2 years. respectively. Such long-term proliferating cells were not obtainable
from the PBLs that were infected with a control vector DGL.
AlthoughPBLIDGL could proliferate for several months
after infection in IL-2-containing culture medium, its rate
of proliferation gradually declined thereafter andfinally
ceased to grow. Early passage of PBWDGL-Tax I A did not
differ from PBWDGL in its morphology. but, after culturing
over 2 years, a morphologic change in the phenotype of
PBLIDGL-Tax 1 A could be observed. These cells increased
in size and formed large, irregular aggregates.
Analysis of the clonalie. We maintainedtheDGLTax1 -infected PBLs as a bulk population without cell cloning after infection. We later determined the clonal composition of these cells by analyzing the integration sites of the
5s
PBUDGL-TaxlA
d
E
C
L
*I
I
I
d
d
3
4
E
Pa
m
1
2
21 kb-
5 kb4 kb5
Fig 1. Southern blot analysis of the retroviral vector integration
site. Each DNA was digested with BamHI, which cut once within
the retroviral vector, DGL-Taxl, and was subjected to Southern blot
analysis with Taxl-specific probe. Lane1,
PBL/DGL-TaxlA at3
months PI; lane 2, PBL/DGL-TaxlA at 6 months PI; lane 3, PBL/DGLTaxlA at24 months PI; lane 4, PBL/DGL-TaxlA at 36 months PI; lane
5, PBL/DGL-TaxlB at 22 months PI.
retroviral vector by Southern blottingwith Taxl-specific
probe. PBLIDGL-TaxIA at 3. 6, 24. and 36 months postinfection (PI) and PBUDGL-TaxIB at 22 months PIwere
examined (Fig l ) . In PBL/DGL-TaxIA, one major and one
minorband were detected at 3 months PI (lane I ) . At 6
months PI (lane 2), only the major band was seen. and this
pattern did not change thereafter (lanes 3 and 4). Thus. PBU
DGL-Tax 1 A consisted of a mixed population of at least two
clones at 3 months after infection that became monoclonal
at 6 months and remained so up to at least 36 months. PBL/
DGL-TaxIB was monoclonal at 22 months PI (lane S ) . The
integration pattern of the retroviral vector differed between
PBLIDGL-Tax 1 A and PBLIDGL-Tax I B; therefore. these
two were different clones. The same resultwas obtained
from an analysis for clonality by Southern blottingwith a
probe for the TCR &chain gene (data not shown).
Cell-surfcrce phenotyprs. Cell-surface expression of
CD3. CD4, CD25 (IL-2Ra). and CD28 antigens in PBL/
DGL-TaxIA and PBUDGL-Tax I B was analyzed byflow
cytometry. PBL/DGL at 6 months PI was also examined as
a control. As shown in Fig 2. PBLIDGL. PBLIDGL-TaxIA
(except for 36 months PI), and PBUDGL-Tax 1 B all had the
phenotype of helper T cells, CD3’ and CD4’. In contrast.
PBLIDGL-TaxIA at 36 months PI showed a markedly decreased level of CD3 expression and a slight reduction in
CD4 expression. Expression of CD25 (IL-2Ra) was higher
in all the DGL-Tax 1 infectants than in PBWDGL, especially
in long-term cultured DGL-Tax I infectants (PBLIDGLTaxlA at 24 months PIand 36 months PI and PBL/DGLTax I B at 22 months PI). Percentages of CD28’ cells were
70% to 80% in PRLlDGL at 6 months PI and in PBL/DGLTax I A at 6 and 24 months PI and were 90% t o 95% in PBL/
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Tax1 IMMORTALIZED T CELLS
4245
CD3
CD28
C D4
96
PBUDGL
6m P.I.
64
32
32
-
6
64
128
192
256
64192 128
256
256
64
128
192
256
64192 128
256
PBU
DGL-Tax1 A
6m P.I.
24m P.I.
64
32
36m P.I.
32
PBU
D G L - T a lB
64
"1
64
128
192
258
64
128
64
192
g61
64
64
l
Fig 2. Analysis of cell-surface phenotypes. Cells cultured in IL-2-containing medium were subjected to FACS analysis. Cytofluorometric
profiles of cells stained with anti-CD3 antibody, anti-CD4 antibody, anti-CD25 antibody, or anti-CD28 antibody are shown. The names of the
cells examined are indicated on the left. The vertical axis indicates relative cell numbers. The horizontal axis indicates relative fluorescence
intensities. We confirmed that the fluorescence intensity of the staining with negative control antibody, FITC-conjugated normal mouse IgG,
was less than 64 in each case.
DGL-Tax1 A at 36 months PI and PBL/DGL-Tax 1 B at 22
months PT. As for other cell-surface markers, we found that
all of these Tax I -immortalized T cells are CD2' , CD5 ' , and
CD45RO' (data not shown).
IL-2 dependrwcy on cell proliferution. Growth properties of PBL/DGL-Tax IA and PBWDGL-TaxlBwithor
without IL-2 were analyzed. KN6-HT,an HTLV- I -immortalized human CD4' helper T-cell clone, and a PBL/DGL
culture at 6 months PT were also examined as controls. As
shown in Fig 3, all of these cells could proliferate in the
presence of 1L-2. In the absence of IL-2, significant levels
of proliferation were observed in PBUDGL-Tax I A of late
passages (24 and 36 months PI) and in PBL/DGL-Tax1B at
22 months PI, but these levels were much lower than that
of KN6-HT. However, theseTax1-immortalized
T cells
could not be maintained for more than 1 month after completedepletion of TL-2, whereas KN6-HT could (data not
shown). Therefore, Tax1-immortalized T cells retained their
IL-2 dependency even at 36 months PI.
Analysis of cellular gene expression. We further characterized the Tax 1 -immortalized T cells by using Northern blot
analysis to examine the expression of several cellular genes
(Fig 4A). Initially, we focused on genes of e g r - l , c-fos, IL,
are known to betrans-activated by
2, and I L - ~ R c Ywhich
Tax I . Compared with PBL/DGL, all of the DGL-Tax1 infectants expressed egr-l and c:fo.r at a much higher level.
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AKAGI, ONO, AND SHIMOTOHNO
4246
-
id
12
’-
4
-
T
IL-2 ( )
Fig 3. Analysis of IL-2 dependency on cell proliferation. Cell proliferationinthe absence I-) or the
presence (+) of IL-2 was measured by 3H-thymidine
incorporation assay. The names of the cells examined are indicated below. Columns and bars show
means and standard deviations, respectively, for
triplicate samples.
That level was almost the same or even higher than that of
an HTLV-l-infected T-cell clone, KN6-HT. None of the
cells expressed IL-2 mRNA.The expressionlevel of ZL2Ra was slightly higher in PBL/DGL-TaxlA from an early
passage (6 months PI) and much higher in later passages of
PBL/DGL-Tax I A (24 and 36 months PI) andin PBLIDGLTaxlB at 22 months PI. The expression level of these genes
in those long-term cultured cells was almost the same as
that
of KN6-HT.
We then analyzed the expression of src-family tyrosine
kinases, Lek and Lyn (Fig 4A), because altered production
of these kinases in HTLV-I -infected T cells is r e p ~ r t e d . ’ ” ’ ~
Lek, which is knowntobethemajortyrosinekinase
in
in PBLIDGL and most of
normal T cells,”~26 was expressed
the DGL-Tax 1 infectants, except for PBL/DGL-Tax1 A at
36 months PI. Expression of Lck was markedly decreased
in KN6-HT and PBWDGL-Tax1 A at 36 months PT. We
could hardly detect Lck mRNA in PBUDGL-TaxlA at 36
of the gene was evident
months PI, whereas slight expression
in KN6-HT. On the qther hand,increased expression of Lyn,
which is known to be expressed ata very low level in normal
T ~ e l l s , ’ was
~ ~ *observed
~
in KN6-HT and in the long-term
cultured DGL-Tax 1 infectants, PBLDGL-Tax1 A at 24 and
36 months PI, and PBL/DGL-TaxlB at 22 months PI. We
confirmed that the expression levels of Lck and Lyn in a
normal helper T-cell clone, FU-KOS-3,’” were quite similar
to those of PBUDGL and markedly different from those of
PBL/DGL-Tax I A at 36 months PI (Fig 4B).
Analysis of Tux1 expression. Therewas a clear difference in theexpression of genes for Lek and Lyn between the
early and latepassages of DGL-Tax1 infectants, as indicated;
therefore, weexaminedthepossiblerole
of Taxl in the
altered expression of these genes. There were no significant
differences in the amount of Tax1 protein in these DGLTaxl infectants (Fig 5). Thus, the difference in the cellular
gene expression could not be explainedby the level of Taxl
protein.
DISCUSSION
Primary human T cells derived from PBLs wereimmortalized by transduction of Tux1 using a retroviral vector.
Al-
though we maintained the Tax1 -transduced PBLs as bulk
populations in IL-2-containing medium, the immortalized
cells weremonoclonal CD4’ T cells at 6 months PI. We
estimated the infection efficiency to be around 0.1% (data
not shown); therefore, clonal selection of the infected cells
should have occurred. Such clonal selection is reported for
the course of T-cell immortalization by HTLV-1 .27.28 Two
Taxl-immortalized T-cell clones were established from two
independent infection experiments.
These
two
clones
showeddifferentintegration
patterns of retroviralvector.
Therefore, it is less likelythat
the immortalization was
caused by the insertional activation of some cellular oncogene by the
retroviral
vector
at
the integration
site.
Grassmannet all7 also reportimmortalizationofprimary
human T cells by Taxl. They used Herpesvirus Suirniri vector, which retained most of the large viral genome, including
the recently identified genes for cyclin-like protein and Gprotein-like protein.*””’ In contrast, the retroviral vector
used in this work encodes only Taxl and neoI8 and seems
to be preferable for evaluating the effect of Tax 1.
T cells immortalized by HTLV-l are known to have several characteristic features that are distinct from normal T
cells. In thisstudy, we showed that TaxI-immortalized T
cells eventually exhibited features that were very similar to
those characteristic features of T cellsimmortalized by
HTLV- l , ie, increased expression of egr-I, c7fos, IL-2Ra,
and Lyn and decreased expression of Lek and cell-surface
CD3 antigen. In addition, we previouslyreportedthatthe
Tax 1 -immortalized T cells resembled HTLV- 1 -immortalized T cells in the aberrant expression of G D 2 g a n g l i o ~ i d e . ~ ~
Investigation of one of theTaxl-immortalizedT-cell
clones at various times in culture showed that, among these
alterations associated with transformation, an increase in the
expression of Lyn and a decrease in the expression of Lck
and cell-surface CD3 antigen were observed only in longterm cultured cells. This finding was in marked contrast to
theincreased expression of egr-l, c-fos, and IL-2Ra observed both in early and latepassages. However, augmented
expression of IL-2Ra was detectedinlate-passagedcells.
Because the Tax1 -transduced T cells became monoclonal as
early as 6 months P1 and retained their clonality as shown
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Taxl IMMORTALIZED T CELLS
4247
B
n
egf- l
-
3
v)
0
Y
3
LL
3.1kb
..r). 2.2kb
4
l.Okb
4
3.5kb
..- -
IL-2Ra
3.8kb
LYn
4
1.5kb
Lck
*2.2kb
LYn
-3.8kb
1""r
4
~
rRNA
~
4 18s
rRNA
1
28s
18s
2
3
4
5
6
1
2
3
Fig 4. Northern blot analysis of cellular gene expression. Total RNA was isolated from cells cultured in IL-2-containing medium and
subjected t o Northern blot analysis. The names of the probesused are indicated on the left.
The sizes of thebands are indicated on the right.
Ethidium bromide staining of rRNAs is shown at the bottom. (A)
Lane 1, KN6-HT, an HTLV-l-immortalized human CD4' helper T-cell clone;
lane 2, PBL/DGL at 6 months PI; lane 3, PBL/DGL-TaxlA at 6 months PI; lane 4, PBL/DGL-TaxlA at 24 months PI; lane 5, PBL/DGL-TaxlA at
36 months PI; lane 6, PBL/DGL-TaxlB at 22 months PI. (B) Lane 1, FU-KOS-3, a normal humanCD4' helper T-cell clone; lane 2, PBL/DGL at 6
months PI; lane 3, PBL/DGL-TaxlA at 36 months PI.
by analysis of the retroviral vector integration site and the
TCR rearrangement pattern, it is unlikely that a preexisting
minor clone with a different phenotype from the major population in the early passage became dominant after long-term
culture. Moreover, there was no significant difference in the
amount of Taxl protein between the early and late passages
of Taxl-transduced T cells. Considering these results, the
changes in the expression of Lyn. Lck, and cell-surface CD3
antigen may have been indirect effects of Tax 1 that appeared
only after the long culturing period. That the induced expression of Tax1 in JURKAT cells stably transfected with f a r 1
gene does not change the expression levels of Lck and the
&chain of CD3 also supports the indirect involvement of
Tax1 in the decreased expression of these genes.'I There
are many reports describing the progressive changes in the
properties of T cells infected with HTLV- 1 ."',33-35 Based on
long-term observation, Yssel et alJSreported that, after infection of a T-cell clone with HTLV-I, two distinct phases,
phase I and 11, are observed. Phase I cells are seen early
after infection, proliferate inan IL-2-dependent way, and
are not different from the uninfected parental clone in morphology or cell-surface expression of the CD3iTCR complex. Phase I1 cells, which emerge later, proliferate in the
absence of IL-2 in large dense aggregates, have a giant celllike appearance, do not express the CD31TCR complex on
their surface, and therefore cannot respondto the signals
through the CD3iTCR complex. Although the expression of
src-family tyrosine kinases in phase I1 HTLV-I -infected T
cells was not investigated in their study. Bolen et al" described that decreased IL-2 dependency in HTLV-I-infected T cells correlates with decreased amounts of Lck and
significant increases in the expression of Lyn. Based on those
results, altered expression of src-family tyrosine kinases is
expected to occur in phase 11 HTLV-I-infected T cells.
PBLDGL-Tax1 A showed very similar phenotypic changes
during extended time in cultures, as described here. More-
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AKAGI,ONO,
4248
PBU
DGL-Tax1A
l
1
2
3
4
5
6
Fig 5. lmmunoblot analysis of Taxl protein. Whole cell lysate was
prepared from cells cultured in IL-2-containing medium. Each lysate
correspondingto 5 p g of protein was subjected to immunoblot analysis with mouse anti-Tax monoclonal antibody TAXY7. The band for
p40TnX'
is indicated by an arrow. Lane 1, KN6-HT, an HTLV-l-immortalized human CD4' helper T-cell clone; lane 2, PBLlDGL at 6 months
PI; lane 3, PBL/DGL-TaxlA at 6 months PI; lane 4, PBL/DGL-TaxlA at
24 months PI; lane 5, PBL/DGL-TaxlA at 36 months PI; lane 6, PEL/
DGL-TaxlB at 22 months PI.
over, as reported previously," whereas Taxl-transduced T
cells at early passage (corresponding to PBL/DGL-TaxIA
at 6 months PI in this study) showed hyper-responsiveness
to stimulation through the CD31TCR complex, PBL/DGLTaxlA at 36 months PI did not respond (data not shown).
In this way, PBUDGL-Tax1A at 36 months PI resembled
phase I1 HTLV- I -infected T cells in many aspects. In PBL/
DGL-TaxIB cultured for 22 months, expression of Lyn increased notably and Lck expression decreased slightly, but
cell-surface expression of CD3 antigen did not decrease significantly. This phenotype is very similar to that of PBL/
DGL-Tax I A at 24 months PI, and we expect that PBL/DGLTaxlB will exhibit the same phenotypic changes as PBL/
DGL-TaxIA after more prolonged culture. Considering the
close similarities between PBL/DGL-Tax1 A at 36 months PI
and many HTLV-I-immortalized T cell^,'^^"^" Tax1 itself
seems to play a crucial role in the expression of many characteristic features of HTLV-I -immortalized T cells. The molecular mechanisms underlying the progressive changes observed in TaxI-immortalized T cells are not clear. Tax1 is
reported to repress the expression of the DNA pol p gene
that is involved in DNA repair and thereby induces DNA
damage.'".37 Accumulation of DNA damage during long culturing periods may lead to these phenotypic changes. But,
considering the particular phenotype that appeared in many
HTLV-I -immortalized T cells as well as in Tax 1 -immortalized T cells, involvement of epigenetic change of cellular
gene expression, such as progressive changes with cellular
aging, rather than the accumulation of random mutations
seems more likely. In this context, it is noteworthy that
several transcriptional factors that are known to be regulated
by Taxl, such as AP-I, ATFEREB, and SRF, have been
reported to alter its activities with cellular aging.3x-4'There
is a possibility that, with cellular aging, the function of Tax1
as a transcriptional trans-activator for many cellular genes
AND SHIMOTOHNO
is modulated by these changes in cellular transcriptional factors.
Despite the similarities to phase I1 HTLV-l -infected T
cells, long-term cultured TaxI-immortalized T cells remained IL-2 dependent evenat 36 months PI. Grassmann
et all' also reported the IL-2 dependency of the T cells
immortalized by a Tax 1 -expressing Herpesvirus Scrimin' vector. These results imply that, in addition to toxl. other viral
genes, such as env gene and a recentlyidentifiedp12'
gene"')..'' may be required to establish a fully IL-2-independent growth state.
In this report, we clearly showed the importance of Tax1
in many aspects of phenotypic transformation by HTLV-I.
Further investigation of our Tax I -immortalized T cells will
help to unravel the precise
molecular mechanisms leading
to the transformation of T cells after HTLV-I infection.
ACKNOWLEDGMENT
We thank Dr Y. Yamanashi (University of Tokyo, Tokyo, Japan),
Dr T. Hirano (University of Hiroshima, Hiroshima, Japan), and Dr
Y. Koga (University of Kyushu. Kyushu, Japan) for providing probe
DNAs. We also thank Dr Y. Tanaka (University of Kitasato. Tokyo,
Japan) and Dr M. Yasukawa (University of Ehime, Ehirne. Japan)
fur supplying anribody and the T-cell clone, respectively.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 86: 4243-4249
Characterization of T cells immortalized by Tax1 of human T-cell
leukemia virus type 1
T Akagi, H Ono and K Shimotohno
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