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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2296-2304
Human T-Cell Leukemia Virus Type II Directly Acts on
CD34+ Hematopoietic Precursors by Increasing Their
Survival Potential. Envelope-Associated HLA Class II Molecules Reverse
This Effect
By
Claudio Casoli,
Maria Carla Re,
Paola Monari,
Giuliano Furlini,
Giovanna Tosi,
Chiara Gradozzi,
Pier Paolo Dall'Aglio, Umberto Bertazzoni, and
Roberto S. Accolla
From the Institute of Medical Pathology, University of Parma, Parma;
the Department of Clinical and Experimental Medicine, the Division of
Microbiology, University of Bologna, Bologna; the Institute of Biology
and Genetics, University of Verona, Verona; the Chair of General
Pathology, Medical School II, University of Pavia at Varese; and
Advanced Biotechnology Center, Genova, Italy.
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ABSTRACT |
The role of human T-cell leukemia virus type II (HTLV-II) in human
lymphoproliferative and hematopoietic abnormalities in which the
retrovirus can be isolated is still elusive. Here we show that the C344
T-cell-derived lymphotropic HTLV-II type IIa Mo strain acts directly
on CD34+ hematopoietic precursors by rescuing them from
apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is
viral strain-specific, as it is not observed with the B-lymphotropic
HTLV-II type IIb Gu strain, it does not require infection of the
hematopoietic precursors, and, interestingly, it is strongly dependent
on the infected cellular host from which the virus was
derived. Indeed, growth adaptation of the Mo strain to the permissive
B-cell line, BJAB, renders the virus no longer capable of mediating the
antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo
strain with antibodies specific for HLA class II, but not class I,
histocompatibility antigens restores the antiapoptotic potential of the
virus. These results constitute the first evidence that HTLV-II
retrovirus can directly influence the homeostasis of human progenitors,
without infecting them, and that this crucial activity is strongly
inhibited by the presence of host-derived envelope-associated HLA class II antigens.
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INTRODUCTION |
THE HUMAN T-CELL leukemia-lymphoma
viruses types I and II (HTLV-I and -II) are closely related oncogenic
retroviruses. Infection is lifelong and transmissible vertically by
breast feeding and horizontally by sexual intercourse, transfusion of
infected blood, and intravenous drug usage. There is strong evidence
associating HTLV-I infection with subsequent development of adult
T-cell leukemia/lymphoma (ATLL), tropical spastic paraparesis (TSP),
and other conditions of inflammatory nature.1 No definitive
relationship between HTLV-II infection and specific human diseases has
been established, although neurodegenerative and lymphoproliferative
disorders can be observed.2 HTLV-II infection has been
shown to be endemic in a large number of native American Indian
populations and high rates of infection have also been shown in
intravenous drug abusers in North America and Europe.2
HTLV infections lead to several immune dysfunctions, including
spontaneous proliferation of T cells, which have been attributed to the
effect of the trans-regulatory protein tax, encoded by the px
viral gene.3,4 In fact, beside the action on the viral long
terminal repeat (LTR) to regulate viral RNA transcription, tax is also capable of trans-activating heterologous eukaryotic promoters of genes involved in T-cell activation and proliferation, including a large array of cytokines.3 Further studies have shown that antibodies to tax are often found in the serum of infected individuals,5,6 and a cell-mediated immune response
directed toward the tax protein is detected in a large proportion of
HTLV-associated myelopathy (HAM) and TSP patients7; in
addition, tax can be found in the cell-free supernatant of cells
expressing the viral product.8 Taken together these
observations suggest that HTLV-I tax can have important biologic
functions outside the infected cell.
Very little is known of the function of HTLV virus-encoded envelope
structures, as well as the actual composition of the viral envelope,
particularly with regard to host cell membrane-derived components. In
other retroviruses, such as human immunodeficiency virus (HIV)-1,
cell-derived molecules have been shown to be present in large amounts,
in a rather selective fashion, and to influence the biologic
characteristics of the virus life cycle.9 HTLV-I and -II
are mostly T lymphotropic showing an in vivo preferential targeting to
CD4 and CD8 T cells, respectively. However, in some patients, it has
been recently shown that both HTLVs can infect non-T-cell populations,
including monocytes and B cells.10-12 In light of these
findings, it would be pertinent to assess the presence and the relative
proportion of envelope-associated cell-derived structures.
It has not been firmly established whether HTLVs can infect
hematopoietic precursors, and the hematologic abnormalities present in
ATLL patients can simply reflect alterations in environmental factors
such as cytokine,13 some of which, however, are essential for maintaining survival and/or for promoting differentiation of hematopoietic precursors. For example, withdrawal of interleukin-3 (IL-3) from immature hematopoietic cells has been shown to lead to
internucleosomal DNA cleavage and other events characteristic of
apoptosis.14,15 In various viral infections, either an
accelerated apoptosis or a protection from it can be
observed.16-19
For all these reasons, we set out to investigate the biologic effects
of HTLV-II virus on a specific cell lineage, which could be involved in
the hematologic abnormalities associated with HTLV infection. We
focused our study on CD34+ cells either directly isolated
from healthy bone marrow (BM) donors or as a cell line (TF-1). The
effect of two distinct HTLV-II viral strains, Mo and Gu, belonging to
subtype IIa and IIb, respectively, were investigated. Mo and Gu differ
in their proviral genomic sequence by 4.8%; at the level of the
env region, a 95.4% nucleotide sequence identity is observed,
resulting in 2.9% divergence in the predicted amino acid sequence (14 amino acid substitutions, of which five are
nonconservative).20 The two viral subtypes encode different
tax proteins, as shown by an additional 25 amino acids in the carboxy
terminus of the Gu-derived protein, and this may lead to differences in
tax function in vivo.20 Several parameters were analyzed:
(1) CD34+ cell viability in response to HTLV-II treatment,
as assessed by apoptotic cell death analysis after IL-3 deprivation;
(2) CD34+ cell permissivity to infection, as assessed by
polymerase chain reaction (PCR) of viral genes in the treated cells;
(3) correlation between virus-derived, as well as virus-associated cell
host-derived, structures and biologic effects on CD34+
hematopoietic precursors. The results show a critical role for HTLV-II
virions in mediating survival and growth effects on CD34+
hematopoietic precursors. Importantly, membrane components of the
cellular type generating the virus assumed significance in the
virus-mediated effect on hematopoietic progenitors, as host-derived envelope-associated HLA class II, but not class I, molecules appeared to interfere directly with the capacity of the virus to inhibit apoptosis of CD34+ cells.
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MATERIALS AND METHODS |
Cells.
The IL-3-dependent human erythromyeloid cell line, TF-1, was obtained
through the courtesy of Dr B.R. Davis (Medical Research Institute, San
Francisco, CA). TF-1 expresses the CD34 marker in greater than 95% of
the cells.21 It was routinely cultured in RPMI-1640 (GIBCO,
Grand Island, NY) plus 10% fetal calf serum (FCS) (GIBCO) and 1 ng/mL
of human rIL-3 (Genzyme, Boston, MA) in 24-multiwell plates (Falcon,
Oxnard, CA) at the optimal density of 3 to 6 × 105
cells/mL. The C344 cell line, kindly provided by Dr R.C. Gallo (National Cancer Institute, Bethesda, MD) is an HTLV-II-infected T-cell line harboring the HTLV-II Mo provirus.22 The BJAB
cell line is an Epstein-Barr virus (EBV) negative B-cell
line able to support the HTLV-II replication.23 C344 and
BJAB were used as producers for the viral isolates Mo and Gu,
respectively. All cultures were maintained in RPMI 1640 plus FCS and
were kept at 37°C in a 5% CO2 humidified atmosphere.
BM samples.
Samples were obtained from five HIV-1 and HTLV seronegative subjects
after informed consent, according to the Helsinki Declaration of 1975. BM CD34-positive (BM CD34+) cells were purified as
described previously.24 Briefly, after phycoll
separation and washings in Iscove's modified Dulbecco's medium (IMDM)
(GIBCO) plus 10% FCS, light density mononuclear cells were deprived of
adherent cells by two successive 1-hour adherence steps in plastic
petri dishes (Costar, Cambridge, MA). A total of 6 × 106 cells were then reacted with mouse monoclonal antibody
(MoAb) anti-CD2, -CD3, -CD8, -CD11, -CD19, and -CD20 (Becton Dickinson, Mountain View, CA) for 1 hour in ice under agitation. After washing, cells were incubated for 30 minutes on ice with immunomagnetic beads
coated with IgG antimouse (MCP 450 Dynabeads; Dynal, Oslo, Norway) at a
immunomagnetic bead/target cell ratio of 10:1 in a final volume of 400 µL. Lineage-positive cells were then removed by a magnet (MPC1
Dynabeads). The remaining cells were positively selected by My-10
anti-CD34 MoAb (Tecnogenetics, Milan, Italy) and the immunobead
procedure described above, using an immunomagnetic bead-to-cell ratio
of 3:1. CD34+ cells were collected by magnet and
resuspended in 1 mL IMDM plus 10% FCS. After overnight incubation at
37°C, CD34+ cells were gently pipetted 30 to 40 times
to mechanically separate them from immunomagnetic beads, which were
eliminated by a rapid passage on MPC1. The CD34+-enriched
populations were determined to be more than 95% pure at FACScan
analysis by a subsequent staining with a mouse MoAb that recognizes a
separate epitope of CD34 (HPCA-2; Becton Dickinson).
Viral stocks.
Supernatants obtained from HTLV-II-infected C344 and BJAB cell lines
were clarified at low speed centrifugation (1,000g for 10 minutes) and passage through 0.45-µm filters. To obtain concentrated virus preparations, the clarified supernatants were centrifuged at
50,000g and the sedimented particles then purified by
ultracentrifugation on sucrose gradient (25% to 60%). The 1.16 to
1.18 g/mL density fractions were pooled, dialyzed, and pelleted by
centrifugation. Virus particles were resuspended in medium at one tenth
of the original volume. The viral titers are defined by cpm reverse
transcription (RT) activity and levels of p24Ag in
culture fluids. In the supernatants, p24Ag assay showed values ranging
between 200 to 600 pg/mL corresponding to 3 to 8 × 103 cpm/mL of RT activity; the HTLV-II Mo virus preparation
expressed 310 × 103 cpm/mL of RT and contained 48 ng/mL of p24 HTLV-II core antigen. Purified virus from the BJAB cell
line infected with Gu viral strain,20,23 contained 370 ng/mL of p24, whereas those from the BJAB cell line infected with Mo
strain, expressed 40 ng/mL of p24. Heat inactivation of HTLV-II was
performed by incubating the viral preparations at 56°C for 30 minutes.
Inoculation of the human progenitor TF-1 and BM CD34+
cells with HTLV-II.
Cells to be tested for their susceptibility to HTLV-II were washed
twice in serum-free RPMI-1640 medium and plated at a concentration of 5 ×105 cells/mL in virus-containing medium (0.5 to 1 ng/mL HTLV-IIp24 Ag equivalent, see below). The following virus
preparations were used: (1) purified Mo viral particles generated from
C344 cell line; (2) clarified supernatant from C344 culture containing
Mo virus; (3) purified virions obtained from BJAB cells infected with
HTLV-II Gu strain; (4) clarified supernatant from BJAB cells infected
with HTLV-II Gu strain; (5) clarified supernatant from uninfected BJAB
cell line; (6) purified virions generated from BJAB cells infected with
HTLV-II Mo strain; (7) clarified supernatant from BJAB cells infected
with HTLV-II Mo strain. After a 24-hour incubation at 37°C with the
different inocula, the cells were washed and resuspended in RPMI-1640
medium plus 10% FCS and cultured in absence of IL-3. Due to the low
number of purified BM CD34+ cells obtainable after negative
and positive immunomagnetic bead selection, the HTLV-II action on these
cells was only performed by treatment with HTLV-II Mo strain.
Detection of HTLV-II in Cell Cultures
Antigen detection.
Supernatants from TF-1 cultures were checked for the presence of
HTLV-I/II p24 core antigen by an enzyme immunoassay (Retrovirology Coulter Corp, Hialeah, FL). The concentration of antigen in the samples
was determined by a linear regression analysis using different p24
standard amounts (from 250 to 15.6 pg) performed and interpreted according to the manufacturer's instructions.
PCR analysis.
To verify the presence of a viral infection after HTLV-II inocula, 3 × 105 TF-1 or BM CD34+ cells were
analyzed twice a week for proviral DNA presence by PCR. Briefly, DNA
was extracted from cells using a commercial kit (IsoQuick, Microprobe
Corp, Bothell, WA). PCR amplification of a 159-bp HTLV-II tax
sequence was performed using 50 pmol of each primer SK43-SK44 (Perkin
Elmer Cetus, Norwalk, CT) in 50 µL volume of reaction solution
containing Tris-HCl 10 mmol/L, pH 8.3, KCl 50 mmol/L, MgCl2
2.5 mmol/L, gelatine 1 mg/mL, dNTPs 0.2 mmol/L each, Taq
polymerase 1.25 U. Each amplification was performed by using 1.5 µg
of cellular DNA. The reactions were performed in a System 9600 thermal
cycler (Perkin Elmer Cetus) programmed for 35 cycles of denaturation at
94°C for 10 seconds, annealing at 58°C for 13 seconds, and
extension at 72°C for 10 seconds (first denaturation step: 1 minute; last extension step: 2.30 minutes). For Southern blot analysis,
one third of each amplified sample was subjected to electrophoresis on
2.8% agarose gel (NuSieve:SeaKem 3:1, FMC; Rockland, ME) and then
transferred to a nylon membrane (HyBond N, Amersham, Little Chalfont,
Buckinghamshire, UK). The specific SK45 probe (Perkin Elmer Cetus) was
labeled with fluorescein (3 -oligolabeling system, Amersham) and after
overnight hybridization and filter washes, a detection chemiluminescent
technique (ECL, Amersham) was used. The light signal was detected by
exposure to Hyperfilm-MP (Amersham) at room temperature for about 1 hour. PCR runs included several samples containing all the reagents except DNA or DNA from uninfected cells as negative controls. Positive
control samples consisted of DNA extracted from C344 cells.
Apoptosis assays.
Apoptosis was induced by culturing the CD34+ cells in
absence of their growth factor. Apoptosis was measured by three
different methods. Flow cytometry analysis: cells were fixed in 2 mL
cold 70% ethanol at 4°C for 1 hour, washed in PBS, and treated
with 0.5 mg RNAse (Type I-A, Sigma, St Louis, MO) in 0.4 mL of PBS for
1 hour at 37°C. Propidium iodide (PI; Sigma) was added to a final
concentration of 40 µg/mL, and the samples incubated in the dark at
4°C for 10 minutes. The PI fluorescence of individual nuclei was
measured using a FACStar plus Flow cytometer (Becton Dickinson)
equipped with an Argon ion laser (Spectra Physics 2020) tuned to 514 nm
wavelength, 300 mW output. PI fluorescence was measured on a log
instead of a linear scale to allow for better identification of
apoptotic cells as a subdiploid cell peak. The threshold was triggered
on the same F12 (PI fluorescence) signal in which a clear-cut
distinction between cell debris and apoptotic cells could always be
identified. Quantitative evaluation of apoptotic cells was performed by
the lysis II analysis software (Becton Dickinson) and data were
expressed as the percentage of apoptotic versus nonapoptotic cells,
regardless of the specific cell cycle phase. DNA laddering: the absence
or the presence of fragmentation of chromosomal DNA to the size of
oligonucleosomes was measured according to the method of Facchinetti et
al.25 Briefly, 5 × 106 cells were
resuspended in 500 µL of ice-cold lysing buffer (NaCl 150 mmol/L,
Tris 50 mmol/L [ph 7.6], Triton X-100 1%, EDTA 10 mmol/L) containing
1 µg DNase-free Rnase; after 10 minutes on ice, the sample was low
speed centrifuged and the supernatant recovered. After phenol
extraction and ETOH precipitation, the samples were
resuspended in 25 µL TE. After 5 minutes at 60°C, the fragments
were loaded on a 1.5% agarose gel and run at 7.5 V/cm for 3 hours.
Giemsa-stained cytospin preparations: cytospin of CD34+
cells were analyzed by light microscopy and the percentage of cells
with the morphologic features of apoptosis were calculated based on a
sample of 200 cells.
Proliferation assay.
The capacity of HTLV-II-treated CD34+ cells to overcome
apoptotic death after IL-3 withdrawal was further assayed by
3H-thymidine incorporation during time to assess potential
viral-dependent mitogenic effect. Briefly, cells were incubated with 1 µCi/mL of 3H-thymidine (6 Ci/mmol) for 6 hours starting
at different times after virus treatment. Cells were then harvested,
washed, and 3H-thymidine DNA content measured by liquid
scintillation counter.
Sequence analysis of the env gene of proviral DNA from T-
and B-HTLV-II-infected cells.
The genomic DNA of isolates Mo and Gu was prepared from infected cells
as described previously.23 Sequencing of the env gp46 region of these isolates was performed by cloning the PCR product
of the env gp46 region using the pCRTM11 plasmid
(Invitrogen Corp, San Diego, CA) under conditions recommended by the
manufacturer. The two primers used were nucleotides (nt) 5100-5130 upstream and nt 6170-6200 downstream. The reaction mixture contained,
in a 100-µL volume, 200 µmol/L dNTPs, 20 pmol of each primer, 2.5 U
of Taq polymerase, 1 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), and 20 to 40 ng of genomic DNA as template. The PCR
reaction was performed for 60 cycles (denaturation at 92°C for 60 seconds, annealing at 62°C for 60 seconds, extension at 72°C
for 60 seconds). DNA sequencing with M13 reverse and forward primers
was performed using the dideoxy chain termination method (T7 sequencing
kit; Pharmacia, Uppsala, Sweden). At least three clones of each isolate
were sequenced to ensure the reliability of the method.
Immunoblot analyses of HTLV-II virions.
Cell-free supernatants containing HTLV-II virions generated from C344
established cell line or in infected BJAB cells were ultracentrifuged
at 150,000g for 1 hour and the pellets were resuspended in
TNE buffer (Tris 10 mmol/L [pH 8.0], NaCl 100 mmol/L,
EDTA 1 mmol/L) at 1/100 of initial volume. To conserve envelope
glycoprotein integrity, the virion concentrates were purified on
Sepharose CL-4B (Pharmacia) chromatography column and the fractions
containing virus were pooled and centrifuged in microfuge for 90 minutes. The pellets were disrupted in lysis buffer containing 0.5%
Triton X-100 and analyzed by immunoblot in a 10% polyacrylamide gel in nonreducing conditions. Blots of HTLV-II extracts from T and B cells
were probed with: (1) MoAb to anti-HTLV-II gp46 (Cellular Products,
Buffalo, NY) to determine structural changes of viral envelope protein;
(2) MoAbs specific for common determinants of HLA class I, MoAb
B9.12.1,26 for common determinants of HLA class II
molecules, MoAb Tu35,27 or for the class II DR subset, MoAb
D1.1228 to identify specific cellular histocompatibility
antigens associated with virions. Immunoreactivity was visualized by
ECL detection system.
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RESULTS |
HTLV-II prolongs CD34+ hematopoietic precursor cell
survival and suppresses apoptosis in the absence of IL-3.
In a first set of experiments, TF-1 cells were deprived of IL-3 and
after 24 hours of culture inoculated with either one of the two HTLV-II
viral strains, Mo and Gu. Untreated TF-1 cells, maintained in the
absence or presence of IL-3, were used as controls. To establish the
relative extent of programmed cell death induced in the various
experimental conditions, quantitative fluorescence staining of nuclear
DNA was performed. Results of a typical experiment are reported in
Table 1. As expected, IL-3-deprived TF-1
cells showed a dramatic increase in the percentage of apoptotic cells, already appreciable at day 3 of culture when compared with TF-1 cells
grown in the presence of the cytokine. Interestingly, IL-3-deprived TF-1 cells inoculated with the Mo virus originated from the C344 T-cell
line, either as clarified supernatant or as purified viral preparation,
showed a drastically reduced percentage of apoptotic cells, closely
approaching the values obtained in untreated TF-1 cells grown in the
presence of IL-3. On the contrary, IL-3-deprived TF-1 cells inoculated
with Gu virus originated from the B-cell line, BJAB, did not show
significant protection from apoptosis. To investigate whether the
findings obtained in the TF-1 cell line could be reproduced in normal
hematopoietic precursors, experiments were performed on BM
CD34+ cells obtained from five healthy subjects. The low
number of CD34+ cells obtainable after the various
purification steps prevented an extensive kinetics analysis of the
normal BM-derived cells with both virus strains. Only three time points
(0-, 3-, and 7-day point) after inoculation of C344-derived HTLV-II Mo
strain were considered. As shown in Table
2, the susceptibility of BM-derived CD34+ cells to
apoptosis was reduced to near basal levels, similar to the conditions
obtained in TF-1 cells grown in the presence of IL-3 and in
IL-3-deprived TF-1 cells inoculated with the same virus. From these
data, we conclude that the physiologic tendency to apoptosis of BM
CD34+ cells cultured in the absence of IL-3 can be overcome
by HTLV-II Mo virus strain.
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Table 2.
Decrease in the Percentage of Apoptosis in BM
CD34+ Cells Seeded in Liquid Cultures in the Absence of
IL-3 After HTLV-II Contact
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These results show for the first time that HTLV-II virus can directly
modify the cell growth of both a CD34+ cell line and, more
importantly, of normal CD34+ hematopoietic precursors. They
also point to a difference in the biologic properties of the two
HTLV-II viral strains used.
HTLV-II virus adapted to a distinct susceptible cellular host no
longer retains the capacity to protect hematopoietic precursors from
IL-3 withdraw-dependent apoptosis.
The capacity of the Mo virus to protect CD34+ cells from
apoptosis as opposed to the incapacity to do so of the Gu virus may be
the consequence of several causes, either single or combined, including
viral structural differences, cell type tropism, phenotype adaptation
to distinct cellular environments, and capacity to infect the target.
To examine these questions in more detail, the following experiments
were performed. Uninfected BJAB cells, maintained in exponential phase
of growth, were cocultured for 48 hours with the same share of C344
Mo-infected cells in transwell-col units to avoid direct cell-to-cell
contact. After removal of C344 cells and washing, BJAB cells were
expanded and controlled in purity by flow cytometric analysis. Transfer
of infection from T- to B-cell line was obtained, as HTLV-II Mo
produced by BJAB cells was evident after 20 days of culture in the
absence of C344 T cells, as assessed by PCR analysis (data not shown)
and detection of p24Ag in supernatants (80, 145, >220 pg/mL at 7, 14, and 20 day point). After various serial passages, purified virus or
clarified supernatants from BJAB cells infected with HTLV-II Mo virus
strain were used to inoculate IL-3-deprived TF-1 cells. The results of a typical experiment are shown in Table 1. It can be clearly seen that
both forms of HTLV-II Mo virus derived from BJAB cells, in contrast to
those derived from C344 T-cell line, were unable to protect from
apoptosis. Indeed, apoptosis was accelerated because at day 6, only
17% and 28% of TF-1 viable cells, depending on the virus preparations
used, remained in the culture. The above results were confirmed both by
DNA analysis, which showed the characteristic features of DNA
degradation observed in apoptotic dead and Giemsa-stained cytospins
showing apoptotic nuclei (data not shown).
These data indicate that suppression of apoptosis in IL-3-deprived
TF-1 cells is not only related to the HTLV-II viral strain used, but
also to virus adaptation to the permissive cellular host.
Susceptibility of TF-1 and BM-derived CD34+ cells to
HTLV-II infection.
The results reported above show that, in appropriate conditions,
hematopoietic progenitors are protected from apoptosis when confronted
with HTLV-II virus. To determine whether block of apoptosis requires
active infection of the cells, culture of TF-1 cells treated with
C344-derived HTLV-II Mo strain or BJAB-derived HTLV-II Gu strain were
analyzed for the presence of p24 Ag in the supernatant twice a week
beginning at day 5 and up to day 25 and by PCR for the presence of
integrated proviral sequences in the cellular DNA. In experimental
conditions allowing the infection of umbilical cord blood mononuclear
cells with the above virus preparations, none of the samples tested
showed a detectable amount of p24Ag (data not shown), indicating the
lack of an active viral replication in these cells. Moreover, TF-1
cells analyzed at day 3 and 7 after the inoculum of the C344-derived Mo
strain did not show the presence of HTLV-II tax-specific
sequences in their genomes (Fig 1, lanes 5 and 3, respectively). Superimposable results were obtained with the
normal BM CD34+ cells treated with the same viral
preparation (data not shown). HTLV-II virus of the Mo strain adapted to
BJAB cells, under the form of either purified particles or cleared
supernatants, was also tested for the ability to infect TF-1.
Interestingly, unlike its equivalent derived from the C344 T-cell line,
the Mo strain adapted to BJAB cells, was infectious for hematopoietic
progenitors. As shown in Fig 1, by day 3 (lane 4) and particularly by
day 7 (lane 2) after virus inoculum, proviral DNA sequences were found in TF-1 cells. Furthermore, heat-inactivated HTLV-II Mo strain derived
from the C344 T-cell line was still capable of protecting from
apoptosis TF-1 cells (Table 1) and normal BM CD34+ cells
(Table 2) deprived of IL-3.
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| Fig 1.
Assessment of HTLV-II proviral DNA in TF-1 cell line by
PCR amplification of a tax gene sequence. TF-1 cells treated
with HTLV-II Mo strain derived from C344 T cells (lanes 3 and 5) or BJAB cells (lanes 2 and 4) were cultured for 3 days (lanes 4 and 5) or
7 days (lanes 2 and 3). DNA was extracted, amplified, and electrophoresed. Detection of the amplified product was performed by
using a tax specific probe. Lane 1, negative control (untreated TF-1 cells); lane 6, positive control represented by PCR-amplified DNA
from C344 T cells; lane 7, molecular weight markers.
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These results show that infection is not required for generating
HTLV-II-mediated protection from apoptosis of hematopoietic precursors
and, by consequence, they rule out also the possibility that newly
produced soluble tax protein is involved in this event. Taken together,
these results show that integration of HTLV-II provirus is not required
for blocking or suppressing apoptosis.
HTLV-II virus-dependent protection from IL-3 withdraw-induced
apoptosis in TF-1 cells is related to host-derived HLA class II
molecules associated with the virus.
As shown above, protection from apoptosis does not require infection of
the cell. Thus, the results obtained raise the possibility that HTLV-II
Mo virus adapted to BJAB cells may have undergone structural
modifications, particularly in its envelope components that first
contact the cellular membrane, which make it no more capable to exert
its biologic function. To assess this possibility, HTLV-II Mo provirus
isolates from C344 T cells and from BJAB cells were sequenced and
compared in the env region. In addition, the two virus isolates
were examined for structural changes or modification of gp46 envelope
glycoprotein by immunoblot analysis. Neither differences between the
proviral env gene sequences nor important posttranscriptional
structural changes such as different glycosylation patterns in the
corresponding gp46 were found (data not shown).
We then investigated whether host-derived cell surface structures, and
particularly HLA-encoded glycoproteins, were differentially represented
in the envelope of the two virus isolates. For these studies MoAbs
directed against monomorphic determinants of either HLA class I or
class II, were used in immunoblot analysis. Preliminary experiments had
shown that both HLA class I and class II antigens were expressed on the
cell surface of the C344 and BJAB cell lines, the expression of both
markers being stronger in the B-cell line, particularly for HLA class
II molecules (data not shown). Figure 2
shows the results of these experiments. HTLV-II Mo strain isolates from
both T- and B-cell lines presented in their envelope substantial amounts of HLA class I antigens represented by a major band of about 60 kD in nonreducing conditions. Of note and in agreement with the surface
expression in the cellular hosts, HLA class I antigens were more
abundant on the B-cell-derived Mo strain. Similarly, the MoAb D1.12
specific for monomorphic determinants of HLA class II DR molecules,
recognized a viral envelope-associated protein of about 65 kD in the
BJAB-derived isolate, consistent with the size of an HLA-DR antigen in
nonreducing conditions. This antibody was not able to show sufficient
amounts of a similar antigen in the C344-derived viral lysate. However,
probing with the MoAb Tu35, recognizing all HLA class II antigens,
including DR, DP, and DQ, resulted in a visible band of the same
molecular weight as the one detected by the D1.12 antibody in both
BJAB- and C344-derived viruses, suggesting that the lack of detection
of DR antigens in the envelope of T cell-derived Mo strain could be due
to qualitative more than quantitative differences.
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| Fig 2.
Immunoblot analysis of HTLV-II virus lysates after
reaction with MoAbs specific for HLA class-I (B9.12.1) or class-II
(D1.12 and Tu35). Pellets of purified HTLV-II virions generated from C344 cell line (T) or from BJAB cells (B) were incubated in lysis buffer containing 0.2% sodium dodecyl sulfate (SDS) and equivalent concentrations of extracts, as shown by protein staining, analyzed by
immunoblot in a 10% polyacrylamide gel containing 0.1% SDS. Immunoreactivity was visualized by chemiluminescence detection system.
The size (in kilodaltons [k], indicated on the right) were determined
by comparison with protein markers.
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It was then of interest to analyze whether the presence of HLA
glycoproteins on the viral envelope of the two HTLV-II Mo isolates could be associated with the distinct apoptotic behavior observed in
the TF-1 cell line inoculated with the two isolates.
Table 3 summarizes the results of a typical
experiment of susceptibility to apoptosis, as described in Table 1, in
which the C344-derived and the BJAB-derived Mo strains were incubated
with saturating concentrations of the various anti-HLA MoAbs, before
using for inoculating TF-1 cells. The percentage of apoptotic cells was then calculated after 1, 4, and 8 days of culture. It can be seen that
preincubation of the BJAB-derived Mo strain with anti-HLA class II, but
not with anti-HLA class I, antibodies resulted in strong protection
from apoptosis of TF-1 cells, similar to the protection observed when
the C344-derived Mo strain was used. In this latter case, no
significant further protection or opposite effects, such as block of
protection from apoptosis, were observed.
View this table:
[in this window]
[in a new window]
|
Table 3.
HTLV-II-Mediated Protection From Apoptosis of TF1 Cells
After Treatment of the Virus With Anti-HLA Class I (B9.12.1) or
Anti-HLA Class II (Tu35, D1.12) Antibodies
|
|
From these data, we conclude that host-derived HLA-class II, but not
class I, molecules present in the envelope of HTLV-II Mo strain
originated from the B-cell line BJAB are associated with the block of
protection from apoptosis observed in TF-1 hematopoietic progenitor
cells.
Treatment with C344-derived Mo strain or with BJAB-adapted Mo strain
treated with anti-class II antibody is mitogenic for CD34+
TF-1 cells.
To assess whether CD34+ cell protection from apoptosis by
HTLV-II virus was accompanied by a mitogenic effect induced by the virus, the DNA content of TF-1 cells undergoing different treatments was measured by 3H-thymidine incorporation. As shown in
Fig 3, withdrawal of IL-3 resulted in a
sharp decrease in cell proliferation analyzed over a 16-day period.
Incubation with C344-derived Mo strain resulted in maintenance of cell
proliferation, although the proliferative capacity evaluated in the
whole cell population was slower than the one observed in the presence
of IL-3. On the contrary, treatment of TF-1 cells with BJAB-derived Gu
strain or Mo strain adapted to BJAB both resulted in a rapid decrease
of 3H-thymidine incorporation. Incubation of TF-1 cells
BJAB-derived Mo strain treated with anti-class II antibodies restored,
instead, the capacity of the cells to proliferate in absence of IL-3,
with a kinetics superimposable to the one observed for TF-1 cells
treated with C344-derived Mo strain.
View larger version (16K):
[in this window]
[in a new window]
| Fig 3.
Measurement of TF-1 cell proliferation after incubation
with HTLV-II virus. TF-1 cells were deprived of IL-3 for 24 hours and
then seeded at a concentration of 2.5 × 105 cells/well in
a volume of 300 µL of culture medium. At this time, cells were either
supplemented with IL-3 (subsequently added every 4 days) or treated
with the various stimuli listed in the bottom panel. Treatments with
HTLV-II strains were for 24 hours. Cell proliferation was measured by
3H-thymidine incorporation (cpm in the ordinate) over time
(days in the abscissa).
|
|
From these results, we conclude that the antiapoptotic effect on TF-1
cells by C344-derived Mo strain and by the same strain adapted to a
B-cell environment and treated with anti-class II antibodies is
accompanied by a mitogenic effect.
| |
DISCUSSION |
HTLV-II is known for its capacity to deregulate T-lymphocyte growth,
most likely through constitutive activation of cellular genes mediated
by the viral trans-regulatory protein tax. The present study was
undertaken to investigate whether other cellular systems, and
particularly hematopoietic precursors, could be sensitive to the
HTLV-II action, as hematologic abnormalities other than T-cell
proliferation are associated with the virus infection. Our results show
unambiguously and for the first time that hematopoietic precursors,
represented both by the TF-1 CD34+ positive cell line and,
more importantly, by normal BM-derived CD34+ cells, can
directly be affected by the HTLV-II virus. In fact, it was found that
the HTLV-II Mo viral strain, belonging to the subtype IIa, grown in a
T-cell host was able to block the apoptosis induced after IL-3
withdrawal in hematopoietic precursors represented by the TF-1 cell
line and by normal BM-derived CD34+ cells. The contact with
the virus was necessary and sufficient to mediate the biologic effect
as suggested by the fact that heat-inactivated virus was still able to
block apoptosis and that no integration and viral replication was
demonstrated in TF-1 cells undergoing protection from apoptosis.
Protection from apoptosis was accompanied by a direct mitogenic effect
of the virus because TF-1 cells continued to proliferate after viral
treatment in the absence of IL-3, although to a lesser extent with
respect to cells treated with IL-3 alone. The relatively slower
replication time of TF-1 cells treated with the virus should be framed,
however, with the likely possibility of the more heterogeneous response
of the cultured cells to the virus stimulus as compared with the IL-3
growth factor stimulus. Taken together, these results provide evidence
that HTLV-II virions are critically involved in mediating survival and
growth effects on CD34+ hematopoietic precursors and
suggest that the hematologic abnormalities found during HTLV-II
infection can depend, at least in part, on a direct virus-cell
interaction.
The mitogenic activity triggered by HTLV-II contact with the cellular
target is reminiscent of a similar effect of HTLV-I virus on resting T
cells.29,30 Several studies have strongly suggested that
HTLV-I tax protein is implicated in the virus-dependent mitogenic
activity.31,32 In the present study, the HTLV-II mitogenic
effect was exerted in the absence of infection, simply by contact with
the virus; more importantly, the effect persisted even in the presence
of heat-inactivated viral particles, indicating that tax did not play a
significant role in the protection from apoptosis.
All of these observations suggested, instead, that structural
components of the HTLV-II viral envelope could be involved in the
triggering of the mitogenic effect on contact with CD34+
cells. We first focused our attention on viral encoded structures such
as gp46 envelope glycoprotein for several reasons: (1) the HTLV-I gp
46, closely related in sequence to HTLV-II counterparts, has been
reported to be involved in the triggering of human peripheral T-lymphocyte proliferation when these cells are confronted with the
virus32,33; (2) our results showed that the capacity to
deregulate the programmed cell death of the hematopoietic cellular
targets was not shared by a distinct HTLV-II isolate, the Gu strain,
grown in the B-cell host BJAB; (3) more importantly, we found that the
T-cell-derived HTLV-II Mo strain, adapted to infect and replicate the
B-cell BJAB environment, was no more capable of protecting
hematopoietic precursors from apoptosis. It is known that modification
in HIV-1 gp120 acquired by passages through different cell types can
produce few amino acid substitutions in specific protein domains that can affect binding, cell fusion, infectivity, and tropism of the virus.34 In contrast to HIV, whose frequency of mutations
is very high, HTLV-II viral strains are very much
conserved.2 Nevertheless, we decided to investigate whether
the adaptation in two distinct cellular environments of the same
HTLV-II virus, the Mo strain, could generate mutations in the HTLV-II
gp46 encoding gene offering a hint to explain the distinct biologic
behavior of BJAB-derived on hematopoietic precursors. Comparative
sequence analysis of gp46 encoding env genes from HTLV-II Mo
strain derived from T cells or from B cells showed, instead, no
differences. We therefore concluded that a major component of the viral
envelope such as gp46 was not responsible for the biologic effect
observed.
We then turned our attention on cell host-derived membrane structures,
which could be acquired by the HTLV-II virus during the budding from
infected cells. In other human retroviruses such as HIV-1, a series of
cell surface antigens have been detected as integral components on
cell-free virions or have been found to be associated with virus
preparations as contaminants that are difficult to
remove.35 Among the various cell membrane components that
could be present in the viral envelope, we set out to investigate HLA
class I and class II antigens because they have been detected in
substantial amounts in other retroviral envelopes such as HIV-1, in
which they can be involved in several aspects of the virus life cycle
and pathogenicity. MoAbs to HLA class I molecules inhibit virus
propagation in in vitro cultures by neutralizing the virus through
interaction with HLA class I molecules associated with the virion
envelope.9,35 In addition, a second antiviral mechanism of
anti-HLA class I antibodies, which affects a postbinding stage of the
virus life cycle and which probably acts through a modulation of the
target T-cell activation, has been recently reported.36 HIV-1 envelope-associated HLA class II antigens can serve as
superantigen-presenting structures to T cells, which are then activated
and proliferate. Moreover, HLA-DR-bearing HIV could, in the continuous
presence of certain superantigens, induce T-cell
apoptosis.37 Little is known about the selective presence
and corresponding functional implication of cell-derived components in
the HTLV envelope, although preliminary studies indicate that HLA
components can be found in the HTLV-I envelope.38 The
results presented in this study clearly show the presence of both HLA
class I and class II antigens on the HTLV-II virus envelope. A
relationship existed between the amount of HLA molecules expressed in
the infected host cell and corresponding detectability in the virus
envelope. Indeed, the BJAB-derived HTLV-II Mo strain presented a more
elevated concentration of both HLA class I and HLA class II antigens
with respect to the T-cell-derived strain. Of particular interest was
the fact that HLA class II, but not class I, molecules were
functionally involved in the mechanism of HTLV-II-mediated protection
from apoptosis. In fact, preincubation of the BJAB-derived virus with antibodies against HLA class II, but not class I, molecules reverted the functional phenotype of the virus to the one of the T-cell-derived virus, and protection from apoptosis of, and consequent mitogenic effect on, hematopoietic precursors was reestablished. The fact that
C344 cells were chronically infected by the HTLV-II Mo strain prevented
the analysis of the biologic effect of the BJAB-derived Gu strain after
possible adaptation to this T-cell environment. It remains to be
investigated whether Gu adaptation to a different T-cell environment
could result in a virus with antiapoptotic potential on
CD34+ cells.
The mechanism by which HLA class II, but not class I, molecules
interfere with the mitogenic effect of HTLV-II is a matter of
speculation. The concentration of class II molecules on the virus
envelope may play a role, as class II molecules are barely appreciable
in the envelope of the mitogenic HTLV-II Mo strain originated from the
C344 T-cell line. However, the results presented in this study
indicated also a differential presence of the class II subsets in the
HTLV-II envelope of the T-cell-derived virus with the major subset,
the HLA-DR, undetectable. This finding is at variance with the
situation observed in the HIV viral envelope, in which, instead, a
presence of DR antigen in the absence of DP and DQ antigens has been
reported.9 Therefore, the possibility that DR, more than DP
and DQ, is involved in the loss of mitogenic potential of HTLV-II Mo
strain derived from BJAB cells cannot be ruled out. HLA class II
antigens are crucial molecules for the correct functioning of the
immune system. Expressed on appropriate antigen-presenting cells, they
act as peptide antigen-presenting structures for the recognition by the
clonotypically distributed T-cell receptor, delivering the specific
first signal which triggers, in T-helper cells, the cascade of events
leading to both cellular and humoral immune responses.39 In
certain circumstances and particularly when stimulation via HLA class
II molecules is not accompanied by accessory signals, T cells fail to
respond, may become anergic, or even receive an apoptotic
signal.40 Moreover, recent studies suggest that HLA class
II itself can promote and transduce an apoptotic signal in the cells
that express it.41,42 It is therefore tempting to speculate
that the abrogation of the mitogenic effect of BJAB-derived HTLV-II Mo
virus on hematopoietic precursors is not a purely passive phenomenon,
but may be due to a direct interaction of viral envelope HLA class II
molecules with CD34+ cell surface structures. If this
interpretation is correct, the natural receptor of HLA class II
molecules, the CD4 molecule, should not be involved, as TF-1 cells do
not express detectable amounts of CD4 (data not shown). Thus,
additional, still elusive, cell surface molecules of hematopoietic
cells may serve as HLA class II functional ligands.
| |
FOOTNOTES |
Submitted May 27, 1997;
accepted November 16, 1997.
Supported in part by Istituto Superiore Sanità (ISS) IX National
Project AIDS No. 9403-16 and No. 9493-02 (to R.S.A.), by Associazione
Italiana per la Ricerca sul Cancro (AIRC) programme 1996, and by ISS
First National Project Tuberculosis (to R.S.A.). G.T. is
the recipient of an I.S.S. AIDS fellowship.
Address reprint requests to Claudio Casoli, PhD, Istituto
di Patologia Medica, Facoltà di Medicina Università di
Parma, v. Gramsci 14, I-43100 Parma, Italy.
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 |
R.S.A. thanks Prof Lorenzo Moretta, IST, Genova for the invaluable
scientific and economic support to his research.
| |
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Abstract
Introduction
Abstract