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J Virol, January 1998, p. 841-846, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human T-Lymphocyte Transformation with Human T-Cell
Lymphotropic Virus Type 2
Sara L.
Tarsis,1
Ming-Tsung
Yu,2
Elizabeth S.
Parks,1
Deborah
Persaud,3
José L.
Muñoz,4 and
Wade P.
Parks1,*
Department of Pediatrics, New York University
Medical Center,1
Prenatal Diagnosis
Laboratory of New York City/MHRA,2 New York,
New York 10016;
Department of Pediatrics, Johns Hopkins Medical School,
Baltimore, Maryland 212053; and
Department of
Pediatrics, New York Medical College, Valhalla, New York
105954
Received 19 February 1997/Accepted 28 September 1997
 |
ABSTRACT |
Human T-cell lymphotrophic virus type 2 (HTLV-2), a common
infection of intravenous drug users and subpopulations of Native Americans, is uncommon in the general population. In contrast with the
closely related HTLV-1, which is associated with both leukemia and
neurologic disorders, HTLV-2 lacks a strong etiologic association with
disease. HTLV-2 does shares many properties with HTLV-1, including in
vitro lymphocyte transformation capability. To better assess the
ability of HTLV-2 to transform lymphocytes, a limiting dilution assay
was used to generate clonal, transformed lymphocyte lines. As with
HTLV-1, the transformation efficiency of HTLV-2 producer cells was
proportionately related to the number of lethally irradiated input
cells and was comparable to HTLV-1-mediated transformation efficiency.
HTLV-2-infected cells were reproducibly isolated and had markedly
increased growth potential compared to uninfected cells; HTLV-2
transformants required the continued presence of exogenous interleukin
2 for growth for several months and were maintained for over 2 years in
culture. All HTLV-2-transformed populations were CD2 and/or CD3
positive and B1 negative and were either CD4+ or
CD8+ populations or a mixture of CD4+ and
CD8+ lymphocytes. Clonality of the HTLV-2 transformants was
confirmed by Southern blot analysis of T-cell receptor 
chain
rearrangement. Southern blot analysis revealed a range of integrated
full-length genomes from one to multiple. In situ hybridization
analysis of HTLV-2 integration revealed no obvious chromosomal
integration pattern.
| |
TEXT |
The first identified human
retroviruses, the human T-cell lymphotropic virus types 1 and 2 (HTLV-1
and -2), have the ability to transform lymphocytes in vitro
(10, 11, 51, 62). T lymphocytes infected with HTLV
demonstrate enhanced growth potential, marked by seemingly unlimited
entry into the cell cycle. The two HTLV serotypes are approximately
65% homologous on the nucleotide level (56, 59) and
demonstrate a correspondingly high serologic cross-reactivity
(37). Nevertheless, they have distinct seroepidemiologic and
clinical profiles. HTLV-1 is associated etiologically with adult T-cell
leukemia (16, 26, 63) as well as with a peripheral neuropathy known as tropical spastic paraparesis or HTLV-associated myelopathy (1, 20, 31, 48). HTLV-1 is endemic to southern Japan, the Caribbean basin, central Africa, northeastern South America,
and regions of the southeastern United States (3-6, 8, 25, 38,
43, 54). HTLV-2 is occasionally found in Native American Indians
(12-15, 28, 34, 39, 41) as well as in a significant
proportion of intravenous drug users (IVDUs) (2, 33, 35,
53). Recently, Hall et al. defined two subtypes of HTLV-2,
HTLV-2a and HTLV-2b, isolated from peripheral blood lymphocytes (PBLs)
of IVDUs (23). HTLV-2 has not been associated with any
disease to date, though there have been isolated reports of
HTLV-2-associated neuropathy mostly from south Florida and the
Caribbean (24, 27, 30, 42, 58).
Persaud et al. (50) reported a limiting dilution
infectivity assay for HTLV-1 which allowed early detection of infected cultures and interleukin 2 (IL-2)-driven expansion of clonal
populations. The present study extends this method to HTLV-2
and characterizes the resultant transformants. Clonal HTLV-2
populations were routinely and reproducibly generated with the HTLV-2a
laboratory strain LAMP/MO. Furthermore, Southern blot analysis was used
to characterize both the number and size of proviral integrations in
the transformants derived from the in vitro system. These results
are compared with those previously noted for HTLV-1. Additionally,
chromosomal in situ hybridization was used to localize both HTLV-1 and
HTLV-2 proviral integrations.
Infection-transformation efficiency and assay reproducibility.
The HTLV-2 transformation assay was performed as described by Persaud
for HTLV-1 (50). Briefly, LAMP/MO cells (gift of Robert Gallo, National Cancer Institute, Bethesda, Md.) were exposed on ice to
11,700 rads (390 rads/min for 30 min) from a gamma source. Various
numbers of irradiated cells (10, 100, and 1,000) were cocultivated with
104 activated peripheral blood mononuclear cells (PBMCs) in
round-bottom 96-well plates in the presence of IL-2 (10 U/ml)
(Boehringer-Mannheim Corp., Indianapolis, Ind.). HTLV-2-transformed
cell populations were identified as cells that continued to proliferate
beyond 6 weeks, the point at which most of the activated PBMCs not
exposed to the HTLV-2-producing cells no longer proliferate in medium containing IL-2, and also by the continued production of HTLV p24
antigen (determined by enzyme-linked immunosorbent assay; (Coulter
Immunology, Hialeah, Fl) >1,000 pg/ml) in the culture supernatant.
Control wells containing only activated PBMCs or only irradiated
LAMP/MO (104 cells/well) were maintained in parallel. After
6 to 9 weeks of culture, cocultures which continued to grow were
expanded in growth medium supplemented with 10 U of IL-2/ml.
HTLV-2-transformed clones were defined as cells exposed to HTLV-2 that
continued to proliferate in the presence of IL-2 and that continuously
produced p24 antigen (>1,000 pg/ml) by 12 weeks after the initial
coculture. Control wells, which contained 104
phytohemagglutinin-activated PBMCs in the absence of HTLV-2-infected cells, proliferated for approximately 4 weeks and were never
p24+; wells which contained only HTLV-2-irradiated cells
never exhibited growth and remained positive for p24 at low levels for
approximately 3 to 6 weeks after the assay set-up.
Transformation efficiency was defined as the percentage of the total
cultures exposed to irradiated LAMP/MO HTLV-2 producer cells that met
the criteria for transformation. The lymphocyte transformation
efficiency of HTLV-2 is demonstrated in Table
1, which summarizes the results of three
different experiments. Different donor PBMCs were used for each
experiment. With 1,000 HTLV-2-irradiated input cells, the overall
transformation efficiency was 71%, with a range of 40 to 81%. With
100 and 10 input cells, the overall transformation efficiencies dropped
to 58 and 10%, respectively, demonstrating that transformation
efficiency is related to the number of input infected cells.
By linear regression analysis, the log input number of
LAMP/MO cells correlated with the efficiency of transformation with an r value of 0.88 and a P value of <0.0001.
There was no evidence of a significant difference in donor lymphocytes
relative to lymphocyte transformation. All continuous lymphocyte growth
was HTLV related: there were no cultures which were positive for growth
and negative for p24.
Time course of HTLV-2-mediated T-cell transformation.
In one
representative experiment (Table 1, experiment C), at 9 weeks after
coculture with 104 PBMCs (the earliest time point tested,
to allow residual p24 to reach undetectable levels), 67% of the wells
with 100 LAMP/MO cells/well were p24+, indicating
infection. By week 10, 70% of the wells with 103
LAMP/MO cells/well were p24 positive. By week 12, all cultures which demonstrated growth had detectable p24, and no additional positive cultures were noted. Supernatant from cells in control wells
containing only irradiated LAMP/MO cells were p24 negative at the
earliest time point tested. Most of the wells that continued to
proliferate could be readily expanded and continued to produce p24.
These cultures have been maintained in culture for over 2 years with
the addition of IL-2. Abrupt removal of IL-2 results in cessation of
cell growth, but it is possible to generate IL-2 independence of
HTLV-2-transformed cells (data not shown). We conclude that these
HTLV-2 lymphocyte lines are IL-2 dependent in the initial phases of
transformation.
Cell surface phenotypes of HTLV-2 transformants.
The cell
surface phenotypes of 41 cultures from experiment C (Table 1) were
determined at the earliest time possible after coculture (approximately
12 weeks). As shown in Table 2, all HTLV-2-transformed cultures were positive for the T-lymphocyte markers
CD2 and/or CD3 and negative for the B1 cell surface antigen. Forty-four
percent of the HTLV-2 transformants generated consisted of pure
(>95%) populations of CD4+ or CD8+ T
lymphocytes, and the remainder consisted of mixtures of the two
subsets. A greater percentage of the transformants (39 versus 5%) were
CD4+ than CD8+ in this system. In
contrast, analyses of lymphocytes in vivo indicate that HTLV-1
is detected primarily in the CD4+ subset (52)
and HTLV-2 is found in the CD8+ population
(29). The findings reported here indicate that in vitro, the CD4+ cell is at least as susceptible to
infection and transformation with HTLV-2 as the CD8+ T
cell. One possible explanation for the difference between the in vitro
and in vivo observations is that HTLV-2-infected CD4+ T
lymphocytes may be eliminated in vivo (45) or
suppressed by the host immune system.
T-cell receptor rearrangement of HTLV-2 transformants.
The
cell surface analyses indicated that several of the HTLV-2
transformants were homogenous T-lymphocyte populations. Southern blot
analysis for T-cell receptor beta chain (TCR) gene rearrangement was
performed to confirm lineage and to establish clonality (44, 60). A representative blot is shown in Fig.
1. All 15 cultures analyzed were
confirmed to be T cells by virtue of rearrangement. Ten were determined
to be clonal populations, and 5 were oligoclonal. Notably, the TCR
gene pattern demonstrated by LAMP/MO (Fig. 1, lane MO) was distinct
from that of the other samples analyzed, indicating that the cultures
generated in the infectivity assay were in fact new transformants and
not the result of outgrowth of insufficiently irradiated LAMP/MO.
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FIG. 1.
TCR rearrangement of HTLV-2 transformants. DNAs from
selected HTLV-2 lines were digested with HindIII,
subjected to Southern blotting as previously described (50),
and hybridized with a 0.42-kb probe to the constant regions, CT1 and
CT2, of the chain of the T-cell receptor (Oncor, Gaithersburg,
Md.). CT DNA was labeled with 32P by using a
random-priming kit (GIBCO/BRL) and was purified on a Sephacryl G-50
column (Pharmacia, Piscataway, N.J.). The germline control (lane PBL)
consisted of DNA from normal PBLs and gave the predicted bands of 3.7 and 7.7 kb. LAMP/MO (lane MO) demonstrated a clonal pattern
distinct from the HTLV-2 transformants. Lanes 3, 4, and 5 are clonal
populations by this analysis, and lanes 1 and 2 are oligoclonal.
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Proviral copy number in HTLV-2-transformed cell lines.
LAMP/MO and the 15 HTLV-2 transformants studied above were next
analyzed for viral copy number by Southern blot analysis. Digestion of
DNA from the HTLV-2 transformants with the enzyme HindIII, which does not cut in the proviral sequence,
was utilized to determine the number of integrated proviruses. Each
band generated by HindIII digest on Southern blot
analysis thus represents a distinct proviral integration site. A
representative blot is shown in Fig. 2.
LAMP/MO contained a single major band and several (at least three)
other, much fainter appearing bands. Of the 10 cultures determined to
be clonal by TCR rearrangement, 5 had single integration sites, 3 had a single strong band and several faint bands, and 2 had multiple
bands. The five samples which were oligoclonal by TCR rearrangement
all had multiple integration sites. No difference in growth properties
in cells with single or multiple integration sites was noted (with
regard to IL-2 requirements and generation time).
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FIG. 2.
Detection of HTLV-2 proviral integration sites in
transformed populations of T lymphocytes. Southern blot analyses of DNA
from HTLV-2 transformants determined to be clonal by TCR
rearrangement analysis were digested with HindIII and
hybridized with HTLV-2 probe pMO-4, which consisted of inserts of the
entire HTLV-2 genome (57) (kindly provided by George Shaw,
University of Alabama, Birmingham, Ala.). Lane MO, LAMP/MO; lanes 1 to 9, HTLV-2 transformants; lane 10, negative control (PBLs from
HTLV-negative donor).
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Defective proviral forms associated with HTLV-2.
Clonal
populations of HTLV-1-infected lymphocytes have been shown to
contain both full-length and subgenomic, or defective, proviruses. The
presence of three proviral forms (full-length, 8.8 kb; defective, 6.5 and 3.5 kb) in LAMP/MO has been described previously
(9). However, the transmissibility of these forms has not
been investigated qualitatively in vitro. The HTLV-2 transformants derived from this transformation assay provided the opportunity to
determine whether defective proviral forms are associated with T
lymphocytes transformed in vitro with HTLV-2. For these analyses, a
combination of the enzymes AseI and EcoRV, which
each cut once in the 5' (bp 1032) and 3' (bp 8035) long terminal
repeat, respectively, were used. A full-length provirus digested with
the combination EcoRV/AseI would be visualized at
7.0 kb; a defective with internal deletions would be detected as a
smaller band.
LAMP/MO and a total of 18 HTLV-2 transformants were analyzed for
proviral structure. As demonstrated in Fig.
3, all transformants
contained a
full-length proviral copy and no transformant contained
only a
subgenomic fragment. LAMP/MO (Fig.
3, lane 2) contains,
in addition
to the full-length provirus (visualized here as a
band of 7.0 kb) one
major defective form of approximately 6.0
kb. One sample (Fig.
3, lane
5) contained, in addition to the
full-length form, a band slightly
larger than the full-length
form. Two other HTLV-2 transformants
harbored a smaller HTLV-2
proviral form in addition to the full-length
provirus (Fig.
3,
lane 3, and data not shown). With these three
exceptions, all
of the other 15 samples examined contained exclusively
a full-length
provirus.
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FIG. 3.
Detection of HTLV-2 proviral integration forms in clonal
populations of T lymphocytes. Southern Blot analyses of HTLV-2
transformants digested with a combination of
AseI/EcoRV as described in the text and
hybridized with probe pMO-4. Lane 1, negative control; lane 2, LAMP/MO; lanes 3 to 10, HTLV-2 transformants. Low-molecular-weight
bands (<1 kb) seen in lanes 2, 4, 5, 7, and 10 most likely represent
cellular-probe degradation products.
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Production of subgenomic, or defective, provirus by HTLV-1 has been
demonstrated in clinical samples (
47). Similarly, analyses
of HTLV-1-transformed T lymphocytes demonstrated that most
transformants
contained defective proviral forms in addition to the
full-length
provirus (data not shown). In contrast to the data for
HTLV-1
transformants, our studies present little evidence for
the existence
of defective HTLV-2 provirus. The findings reported
herein suggest
either that defectives are not efficiently
generated in HTLV-2
replication, and therefore play little role in
HTLV-2-mediated
viral transformation, or that HTLV-2 defectives that
are generated
in vitro are "lethal-dominants" and cannot support
replication.
Chromosomal localization of HTLV integration.
Several studies
have addressed the site specificity of HTLV-1 proviral chromosomal
integration (40, 55, 65). No obvious clustering of proviral
integration has been observed, indicating that HTLV-1 probably
integrates randomly into chromosomal DNA. The localization of HTLV-2
proviral integration has not been previously addressed. In this series
of experiments, chromosomal in situ hybridization of several
HTLV-1 and HTLV-2 transformants was used to determine the proviral
chromosomal localization. If there were a preferential site(s) of
proviral integration, the transformants derived from this assay could
facilitate its localization because multiple transformants from a
single donor can be analyzed. Figure 4
shows an example of in situ hybridization of metaphase spreads of
two HTLV-2-transformed cell lines with probe pMO4: proviral integration
at 2p24 was noted for cell line 7MO3-27, and proviral integration at
1q21-22 was noted for cell line 7MO2-7. A minimum of 20 cells were
analyzed for each cell line. Signals detected more than three times at
the same chromosome locations were scored as specific proviral
integration sites. Eight HTLV-2 transformants were examined, and a
total of 17 integration locations were detected; 10 HTLV-1
transformants were also examined, and a total of 25 integration
locations were noted (data not shown). No specific chromosomal
integration site or pattern was identified with transformants from
either virus.
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FIG. 4.
In situ hybridization of HTLV-2-transformed cell
lines. Metaphase preparations of two HTLV-2 transformants (7MO2-7
and 7MO3-27) were hybridized with probe pMO-4. Cell cultures were
harvested for metaphase preparation with colcemid by standard
cytogenetic laboratory procedures. Probes were labelled by nick
translation with either biotin-dUTP or digoxigenin-dUTP
(Boehringer-Mannheim Corp.) and detected by nonfluorescent methods
which use peroxidase-tagged avidin (Vector Lab Inc., Burlington,
Calif.) or peroxidase-tagged anti-digoxigenin (Boehringer-Mannheim),
followed by diaminobenzidine and silver amplification (Amersham Corp.,
Arlington Heights, Ill.). This method was originally described by Burns
et al. (7), and hybridization and signal detection were
performed as described by Lee et al. (36) with
modifications (61). The probe concentration used was 10 µg/ml, and hybridization was performed overnight by standard in situ
hybridization protocols (Oncor). For each probe and transformant cell
line, several amplification times were tested: the best signal
localization was achieved with the minimal amplification time (ranging
from 20 to 40 min) sufficient to observe specific signal while
minimizing the background. Cells were chosen for further analysis when
the silver signals were confined to a single small dot on each
chromatid. Images were recorded and analyzed with Cytovision software
(Applied Imaging, Pittsburgh, Pa.). These results are representative of
the results obtained with the other HTLV-2-transformed cell lines
analyzed. Similar results were also achieved when
HTLV-1-transformed cell lines were hybridized with the HTLV-1
probe pMT2, a full-length (8.25 kb) HTLV-1 probe as previously
described (57).
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The chromosomal localization studies of the transformants derived from
this in vitro transformation system confirm and extend
the conclusions
of previous studies for HTLV-1. Analysis of multiple
transformants,
including several from a single donor, have failed
to reveal a
conserved integration locus. However, the possibility
of HTLV
integration adjacent to conserved, genome-wide repetitive
sequences has
not been eliminated. Recent work indicating HTLV
proviral integration
in the GC-rich fraction of the genome (
65)
supports the
concept that HTLV-1 integration may be governed by
yet undefined
rules. Further studies are necessary to elucidate
completely the
relationship between HTLV integration and T-lymphocyte
transformation.
The data presented in this report show that clonal populations of
lymphocytes transformed with HTLV-2 can be reproducibly
generated
by using a modified limiting dilution cocultivation
assay. Clonal
transformants are typically CD3
+, B1
,
CD4
+, or CD8
+ T lymphocytes demonstrating
IL-2-dependent long-term growth and
continuous p24 production. The time
course of transformation and
transformation efficiency, as well as the
phenotypic profile of
the resultant HTLV-2 clones, are comparable
to the results obtained
when HTLV-1 is used to transform PBMCs
(
50). One notable difference
is the frequent detection of
defective proviral formation with
HTLV-1 transformants and the
rarity of defective proviruses noted
with HTLV-2 transformants.
The present results do not illuminate one of the more intriguing
biologic questions about HTLV-1 and HTLV-2, namely,
the viral
properties that account for the markedly different ecologic
niches.
In recent years, the HTLV seroprevalence among IVDUs has been
attributed predominantly to HTLV-2, indicating a possible
quantitative
or qualitative difference in infectivity between the two
viruses.
Since in vitro transformation with HTLV-1 and
HTLV-2 are apparently
equivalent, perhaps other factors are
important determinants of
transmission and explain the seeming
differences in the niches
occupied by these closely related
retroviruses. Particularly puzzling
from a seroepidemiologic
perspective is the relative absence of
HTLV-2 in the general
population and, in spite of a low level
of HTLV-1 in the general
population, the relative absence of HTLV-1
in IVDUs (
2,
33,
35,
53). It is clear that HTLV-1 is
etiologically associated
with clinical disease; it seems possible
that HTLV-2 may be as
virulent as HTLV-1 but the high mortality
of infected patients from
other causes (
49) may obscure HTLV-2-specific
disease.
Alternatively, HTLV-2 may produce only subclinical
infections
in the vast majority of HTLV-2-infected patients and
differ qualitatively
from HTLV-1 in disease potential.
The present studies are the first qualitative analyses of the
HTLV-2 proviral structure in transformed T lymphocytes. Clonal
T-cell populations were shown to contain either single or multiple
integration sites. HTLV-1 proviral integration has been
characterized
by numerous studies: adult T-cell leukemia cells contain
oligo-
or monoclonal viral integration (
64), while in cells
from patients
with tropical spastic paraparesis/HTLV-associated
myelopathy,
there is a notably polyclonal pattern (
21,
22).
Though the
demonstration of immortalization with a single proviral
integration
in both HTLV-1 and HTLV-2 suggests that one
integration site,
or virus by itself, is sufficient for transformation,
additional
cellular events are most likely involved as well in
HTLV-mediated
T-cell transformation. Transformation
associated with multiple
integrants could be due to successive
integration cycles until
one favorable integration event occurs;
alternatively, the transformed
phenotype may represent the cumulative
effect of multiple integrations.
The in vitro data presented above
provide support for the former
hypothesis.
HTLV transformation confers enhanced growth potential on the infected
lymphocyte. This transforming capacity has been exploited
in the
laboratory as a method of immortalizing lymphocytes for
further study,
in a manner similar to Epstein-Barr virus transformation
of B
lymphocytes. PBLs from patients with adenosine deaminase
deficiency
(
32) and paroxysmal nocturnal hemoglobinura (
46)
have been immortalized with HTLV-1, and a series of papers have
examined hormone responsiveness in Pygmy and normal
HTLV-2-transformed
T-cell lines (
17-19). The
transforming ability of HTLV-2, the relative
frequency of
monoclonal integrants, and the absence of disease
association render
HTLV-2 a suitable candidate for the immortalization
of T
lymphocytes from patients for multiple purposes.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, New York University School of Medicine, 550 First Ave., Rm. TH-501A, New York, NY 10016. Phone: (212) 263-6425. Fax: (212) 263-8172. E-mail: PARKSW01{at}mcrcr.med.nyu.edu.
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J Virol, January 1998, p. 841-846, Vol. 72, No. 1
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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