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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1386-1392
IMMUNOBIOLOGY
Abundant Tax protein expression in CD4+ T cells infected with
human T-cell lymphotropic virus type I (HTLV-I) is prevented by
cytotoxic T lymphocytes
Emmanuel Hanon,
Sarah Hall,
Graham P. Taylor,
Mineki Saito,
Ricardo Davis,
Yuetsu Tanaka,
Koichiro Usuku,
Mitsuhiro Osame,
Jonathan N. Weber, and
Charles R. M. Bangham
From the Department of Immunology and Genito-Urinary Medicine and
Communicable Diseases, Imperial College School of Medicine, St Mary's
Campus, London, United Kingdom; Department of Infectious Disease and
Immunology, Okinawa-Asia Research Center of Medical Science, Faculty of
Medicine, University of The Ryukyus, Nishihara, Okinawa, Japan; and
Department of Medical Informatics and Third Department of Internal
Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan.
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Abstract |
The role of the cellular immune response in human T-cell leukemia
virus type I (HTLV-I) infection is not fully understood. A persistently
activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in
the majority of infected individuals. However, it remains unclear
whether this CTL response is protective or causes tissue damage. In
addition, several observations paradoxically suggest that HTLV-I is
transcriptionally silent in most infected cells and, therefore, not
detectable by virus-specific CTLs. With the use of a new flow
cytometric procedure, we show here that a high proportion of naturally
infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10%
and 80%) are capable of expressing Tax, the immunodominant target
antigen recognized by virus-specific CTLs. Furthermore, we provide
direct evidence that autologous CD8+ T cells rapidly kill CD4+
cells naturally infected with HTLV-I and expressing Tax in vitro by a
perforin-dependent mechanism. Consistent with these observations, we
observed a significant negative correlation between the frequency of
Tax11-19-specific CD8+ T cells and the percentage of
CD4+ T cells in peripheral blood of patients infected with HTLV-I.
Those results are in accordance with the view that virus-specific CTLs
participate in a highly efficient immune surveillance mechanism that
persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo.
(Blood. 2000;95:1386-1392)
© 2000 by The American Society of Hematology.
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Introduction |
Human T-cell leukemia virus type I (HTLV-I), which
belongs to the HTLV-BLV subfamily of retroviruses, infects an estimated 10 million people worldwide.1 Unlike human immunodeficiency virus, HTLV-I causes no disease in a majority of infected subjects (asymptomatic carriers). However, approximately 2%-3% develop an
aggressive T-cell malignancy, adult T-cell leukemia/lymphoma, and
another 2%-3% develop a disabling chronic inflammatory disease, involving the central nervous system (HTLV-I-associated
myelopathy/tropical spastic paraparesis; HAM/TSP), the eyes, the lungs,
or the skeletal muscles.2
HTLV-I shares with other retroviruses the three main genomic regions of
gag, pol, and env, but, unlike most leukemia
viruses, it has an additional region called pX that codes for
two transcriptional regulatory proteins, the Tax and Rex
proteins.2 These proteins are the homologues
of the Tat and Rev proteins of human immunodeficiency virus.3 The Rex protein stabilizes viral messenger RNAs
(mRNAs) and regulates their splicing and transport. The Tax protein is of central importance in virus dynamics because, as well as
transactivating viral transcription, it is thought to drive host-cell
proliferation.2 Furthermore, Tax is the dominant target
antigen recognized by HTLV-I-specific cytotoxic T lymphocytes (CTL) in
most responding individuals.4-8 Thus, the Tax protein is at
the center of both efficient HTLV-I replication and the host attack on
the virus.
The risk of HAM/TSP disease is positively correlated with the magnitude
of the proviral load in the blood.9 It is, therefore, important to identify the host factors that determine the magnitude of
the proviral load in vivo. In this regard, the role of the immune
response in HTLV-I infection is still not clear. A high frequency of
circulating Tax-specific CTLs can be found in a majority of
HTLV-I-infected individuals.4-8 However, controversy exists over whether this strong CTL response causes or prevents
HAM/TSP.10,11 Our recent immunogenetic data favor the
possibility that a strong HTLV-I-specific CTL response indeed reduces
proviral load and protects against HAM/TSP.12 However,
direct evidence that Tax-specific CTLs are able to eliminate
HTLV-I-infected cells in vivo has not been obtained. In addition, if
HTLV-I-specific CTLs play a role in the reduction of the proviral load,
a large proportion of infected cells should be capable of expressing at
least the Tax protein, the dominant target antigen recognized by CTLs.
This interpretation seems to conflict with the observation that HTLV-I
provirus is transcriptionally silent in a high proportion of T-cell
clones derived from infected patients,13,14 which suggests
that HTLV-I might be latent in most infected peripheral blood
mononuclear cells (PBMCs) in vivo. Consistent with this observation,
with the use of conventional techniques, Tax protein expression cannot be detected in freshly isolated PBMCs, and, indeed, the existence of a
serum factor that represses HTLV-I transcription in vivo has been
postulated.15,16 However, the low frequency of Tax expression in fresh PBMCs could be the consequence of an efficient immune surveillance mechanism mediated by the host cellular immune response.11,12 Therefore, to understand the role of
HTLV-I-specific CTLs in vivo, it is necessary to determine the
proportion of infected PBMCs that are capable of expressing at least
the Tax protein and to characterize qualitatively and quantitatively
their interactions with autologous HTLV-I-specific CD8+ T lymphocytes.
To date, HTLV-I-specific CTLs have been characterized, using as target
cells either leukemic cells4 or transformed cell line
treated with peptides or infected with recombinant
viruses.6,7,17 In this study, we used a sensitive flow
cytometric technique to study intracellular Tax protein expression in
naturally infected PBMCs18 of patients with HAM and
asymptomatic carriers of HTLV-I. Our observations suggest that
Tax-specific CTLs play an important role in reducing the frequency of
Tax-expressing CD4+ T lymphocytes in vivo.
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Materials and methods |
Patients and cells
MT-2 cells were cultured in RPMI 1640 medium (Gibco, Paisley, UK)
supplemented with 10% fetal calf serum (FCS) (Sigma, Dorset, UK), 2 mmol/L glutamine (Gibco), 100 IU/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco).
PBMCs were obtained from 8 patients with a clinical diagnosis of
HAM/TSP, 5 asymptomatic carriers, and 1 normal individual. PBMCs were
isolated on Histopaque®-1077 (Sigma) density gradient and washed
three times with phosphate buffered saline (PBS). Anti-CD8 or CD4
paramagnetic beads (Miltenyi Biotec Ltd, Surrey, UK) were used
according to the manufacturer's instructions to deplete or enrich the
respective PBMC subpopulation. Cells were cultured in RPMI 1640 medium
supplemented with 10% FCS, 2 mmol/L glutamine, 100 IU/mL penicillin,
and 100 µg/mL streptomycin. Alternatively, whole blood was directly
cultivated after being diluted in an equivalent volume of RPMI 1640 medium supplemented with 2 mmol/L glutamine, 100 IU/mL penicillin, and
100 µg/mL streptomycin. PBMCs were then isolated from cultivated
blood as described above. In some experiments, 20 nmol/L of
Concanamycin A (CMA) (Sigma) was added to the culture medium.
Immunofluorescence
Concomitant detection of Tax, CD4, and CD45RO.
After being harvested, cells were fixed in PBS containing 2%
paraformaldehyde (Sigma) for 20 minutes and resuspended in PBS at
4°C until use. Fixed cells were washed with PBS containing 7% of
normal goat serum (Sigma) and incubated with PC5-labeled anti-CD4 and
FITC-labeled anti-CD45RO monoclonal antibodies (MAb) (Beckman Coulter, Bedfordshire, UK) for 15 minutes at room temperature. The cells were then washed and permeabilized with PBS containing 0.1%
Triton X-100 (Sigma) for 10 minutes at room temperature. Permeabilized
cells were washed and resuspended in PBS/7% normal goat serum
containing an anti-Tax MAb (Lt-4)19 or an isotype control
MAb (IgG3) (Southern Biotechnology Associates, Birmingham, AL) for 20 minutes at room temperature. The cells were then washed twice and resuspended in PBS/7% normal goat serum containing
FITC-labeled goat F(ab')2 anti-mouse IgG3
serum (Southern Biotechnology Associates) for 20 minutes at room
temperature. Finally, the cells were washed twice and analyzed by flow
cytometry on a Coulter EPICS® XL (Beckman Coulter).
Concomitant detection of Tax and p24 (HTLV-I gag antigen).
The cells were processed as described above, but an anti-p24 MAb
(MAB8817: IgG1) (Chemicon International, Temecula, CA) was used in addition to the Lt-4 MAb. The anti-p24 MAb was then detected with a RPE-labeled goat F(ab')2 anti-mouse
IgG1 serum (Southern Biotechnology Associates).
Concomitant detection of Tax and cell mortality.
After being harvested, the cells were incubated for 10 minutes in the
presence of 5 µg/mL of propidium iodide (Sigma), then washed twice
with PBS, fixed in PBS containing 2% paraformaldehyde for 20 minutes,
and resuspended in PBS at 4°C until use. Fixed cells were then
washed with PBS/7% normal goat serum and processed as described above
to detect the Tax protein.
Quantification of tax mRNA
RNA was isolated from PBMCs with the use of a High Pure RNA
Isolation Kit (Boehringer Mannheim, Lewes, UK). Complementary DNA
(cDNA) was then synthesized with the use of 1st Strand cDNA Synthesis
Kit for RT-PCR AMV (Boehringer Mannheim). Polymerase chain reactions
(PCRs) were carried out in 50 µL containing cDNA prepared from 10 ng
of RNA with primers for HTLV-I Tax or human beta-actin. Tax primers
were 5'-TCG CTG CCG ATC ACG ATG CGT TTC C-3' and
5'-AAC ACG TAG ACT GGG TAT CC-3'. Human beta-actin primers were 5'-AAG AGA GGC ATC CTC ACC CT-3' and 5'-TAC ATG
GCT GGG GTG TTG AA-3'. Cycle conditions for amplification of the
tax sequence were one cycle at 95°C for 5 minutes followed by 45 cycles at 95°C for 60 seconds, 58°C for 75 seconds, 72°C
for 90 seconds, and one final cycle at 72°C for 10 minutes. Cycle
conditions for amplification of the beta-actin sequence were one cycle
at 95°C for 5 minutes followed by 30 cycles at 95°C for 60 seconds, 58°C for 75 seconds, 72°C for 90 seconds, and one
final cycle at 72°C for 10 minutes. In preliminary experiments, the
number of cycles was varied between 25 and 50 to determine the
appropriate number of cycles for a semiquantitative PCR (data not shown).
Quantification of proviral load
To measure the proviral load, DNA was extracted from
2 × 106 PBMCs by the proteinase K method. Replicate
serial dilutions of the DNA were amplified with the use of a nested PCR
technique that reliably detected a single copy of HTLV-I Tax proviral
DNA in DNA from 105 cells.20 The proviral DNA
titer was calculated from the Poisson distribution of negative samples
at the cut-off dilution. Interassay variability (0.3 log10)
was determined by repeated testing of a random selection of patient samples.
Detection of Tax11-19-specific CTL and CD4+
cells
In the chronically activated CTL response to HTLV-I, several
peptides derived from the immunodominant Tax protein are restricted by
HLA*A02.17
Tax11-19 is a dominant A*02-restricted
epitope.4 Analysis of PBMCs for the presence of
Tax11-19-specific CTLs was, therefore, performed by the use
of fluorescent-labeled tetramers of
HLA-A*0201 +  2microglobulin + Tax11-19
peptide.12,21 PBMCs were incubated with
Tax11-19 tetramer at 37°C for 30 minutes and anti-CD4 antibody on ice for 30 minutes. The cells were then washed three times
in ice-cold PBS, fixed in 1% paraformaldehyde for 30 minutes at
4°C, and analyzed by flow cytometry on a Coulter EPICS® XL (Beckman Coulter).
| |
Results |
Tax protein expression in PBMCs isolated from infected patients
We devised a sensitive flow cytometric assay to detect intracellular
Tax protein expression. Preliminary experiments were done with the use
of the MT-2 cell line, which is chronically infected with HTLV-I. Those
experiments indicated that 98% of MT-2 cells expressed high levels of
the Tax protein (data not shown). We next checked whether this new
procedure could detect the expression of Tax in PBMCs isolated from
patients infected with HTLV-I. PBMCs were isolated from blood samples
and harvested directly or after 24 hours in vitro culture (in the
absence of interleukin-2 or mitogen). After being harvested, cell
samples were fixed and processed to detect concomitantly Tax and CD4
expression by flow cytometry. Figure 1
shows that a small fraction of CD4+ lymphocytes express detectable
levels of the Tax protein after 24 hours of cultivation. In contrast,
no expression of Tax was observed in freshly isolated PBMCs (Figure 1).
The use of an isotype control MAb (Figure 1) confirmed the specificity
of the detection. Moreover, uninfected PBMCs cultivated in similar
conditions for 24 hours remained negative for Tax expression (data not
shown). Three-color analysis indicated that Tax-expressing CD4+
lymphocytes were also positive for the CD45RO differentiation antigen
(Figure 2). This result is in accordance
with Richardson et al18 who observed a similar phenotype of
HTLV-I-infected cells in vivo. In addition, we assayed PBMCs for the
concomitant expression of Tax and p24 (HTLV-I Gag antigen) after in
vitro cultivation for 24 hours. Because Tax is a powerful
transactivator of viral transcription, Tax-positive cells should
express other viral proteins, including p24. The analysis confirmed
that a large proportion (51%) of Tax-positive PBMCs also expressed the
p24 protein after culture for 24 hours. In contrast, only 1.9% of
Tax-negative PBMCs were positive for the p24 protein.
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| Fig 1.
Concomitant detection of Tax and CD4 antigens in
peripheral blood mononuclear cells isolated from a patient infected
with human T-cell leukemia virus type I.
Cells were cultivated for 24 hours (C, D) or analyzed fresh (A, B). The
Tax protein was detected with the Lt-4 monoclonal antibody (B, D), and
an irrelevant monoclonal antibody was used as a control isotype (A, C).
One representative experiment of 3 is shown.
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| Fig 2.
Concomitant detection of Tax, CD4, and CD45 antigens in
peripheral blood mononuclear cells isolated from a patient infected
with human T-cell leukemia virus type I and cultivated for 24 hours.
One representative experiment of 3 is shown.
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Those results demonstrate that flow cytometry can be used to
specifically detect intracellular Tax expression in PBMCs isolated from
infected patients. With the use of this procedure, it is, therefore,
feasible to quantify the proportion of naturally infected CD4+
lymphocytes that are capable of expressing the Tax protein and to
determine whether this expression is under the control of Tax-specific
CTLs or not.
Time course study of Tax expression in PBMCs isolated
from infected patients
The time course of Tax expression was determined in PBMCs isolated
from both asymptomatic carriers and patients with HAM/TSP. PBMCs were
cultivated for various times, harvested, and then processed to detect
concomitantly Tax and CD4 expression by flow cytometry. Figure
3A and Table 1
show the evolution of the percentage of Tax-positive PBMCs at 0, 6, 12, 24, and 48 hours. Tax expression reaches a maximum at 6-12 hours and
then decreases by 50% during the next 12-36 hours. In contrast to Tax
expression, the percentage of CD4+ lymphocytes remained constant
throughout the time course (data not shown). This constant
percentage indicates that the observed changes in Tax
expression over the time could not be due to a dramatic change in the
proportion of the different PBMC subpopulations. In each patient, the
comparison of Tax positivity with the proviral load indicated that a
large fraction (from 10% to 80%) of infected cells were able to
express the Tax protein at a given time point (Table 1).
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| Fig 3.
Time course study of Tax protein (A) or messenger RNA (B)
expression in peripheral blood mononuclear cells (PBMCs) isolated from
asymptomatic carriers of human T-cell leukemia virus type I (HTLV-I)
(HAP, HAW), from patients with HTLV-I-associated myelopathy/tropical
spastic paraparesis (TBA, TAN), or from an uninfected individual (UA).
PBMCs were cultivated for various times, harvested, and then processed
for the detection of Tax antigen (A: HAP, TBA) or mRNA (B: HAW, TAN,
UA) expression. As a control, beta-actin messenger RNA expression was
also investigated. One representative experiment of 3 is shown.
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Table 1.
Percentage of Tax-expressing cells and proviral load in
peripheral blood mononuclear cells (PBMCs) isolated from 2 asymptomatic carriers (AC) of the virus and 4 patients with
HTLV-I-associated myelopathy/tropical spastic paraparesis
(HAM/TSP)
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To confirm the increase followed by a decrease in Tax-positive cells,
the expression of tax mRNA was investigated by the use of a
semiquantitative reverse PCR. As a control, the expression of
beta-actin mRNA was also investigated. For this experiment, PBMCs were
cultivated for 0, 24, and 48 hours and then treated as described in
"Materials and Methods" for PCR analysis. As shown in Figure 3B,
constitutive expression of the tax mRNA was detected without
cultivation of the PBMCs. The highest level of tax mRNA was observed at
24 hours after cultivation, and then it decreased at 48 hours. In
contrast, the level of beta-actin mRNA remained constant throughout the
time course.
Flow cytometric and PCR analyses indicate that the level of Tax
expression in cultivated PBMCs increases rapidly to reach a maximum
during the first 12 hours and then decreases during the next 36 hours.
The observed increase in Tax expression was not the consequence of a
nonspecific mitogenic activation of infected cells by FCS. Indeed, a
higher increase in Tax expression (Figure 4) was observed in PBMCs cultivated in
whole blood (autologous human serum) than in PBMCs cultivated in the
presence of FCS. A similar increase in Tax expression was observed both
in heparin- and EDTA-treated blood (Figure 4). This experiment also
excluded the possibility that the observed increase in Tax expression
is due to the removal of a serum factor that represses the expression of viral protein in vivo.15,16
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| Fig 4.
Concomitant detection of Tax and CD4 antigens in
peripheral blood mononuclear cells (PBMCs) isolated from a patient
infected with human T-cell leukemia virus type I (TAR).
PBMCs were isolated from blood either before or after cultivation for
12 hours. Either heparin or EDTA was used as anticoagulant. The Tax
protein was detected with the Lt-4 monoclonal antibody (D, E, F), and
an irrelevant monoclonal antibody was used as a control isotype (A, B,
C). One representative experiment of 3 is shown.
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Presence of CD8+ lymphocytes decreases the frequency
of Tax expression
A freshly activated anti-Tax CTL activity has been identified in the
blood of the majority of patients infected with HTLV-I.4-7 The activity of these Tax-specific CTLs could be responsible for the
decrease in the percentage of Tax-expressing cells observed between 12 and 48 hours of cultivation (Figure 3 and Table 1). To test this
hypothesis, PBMCs were selectively depleted of CD8+ lymphocytes and
cultivated for 24 hours. As a control, PBMCs were also depleted of
CD56+ lymphocytes. Figure 5A shows that the
depletion of CD8+ lymphocytes is associated with an increase in the
percentage of Tax expression in CD4+ lymphocytes. In contrast, the
depletion of CD56+ lymphocytes has no significant effect on the
frequency of Tax expression. A similar effect of CD8+ lymphocyte
depletion on Tax expression has been observed in three asymptomatic
carriers of the virus and in 3 patients with HAM/TSP. Furthermore,
within a single patient (HT) the results were quantitatively
reproducible: the same results have been obtained in 3 independent
experiments, using blood samples of the same patient taken at different
times (data not shown). We also determined the effect of an artificial enrichment of autologous CD8+ lymphocytes. The results of these experiments are presented in Figure 5B and shows that, for each patient, Tax expression was reduced in a dose-dependent manner when the
frequency of CD8+ lymphocytes was increased. These results demonstrate
that the presence of CD8+ lymphocytes is associated with a reduction in
the frequency of Tax expression in CD4+ lymphocytes.
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| Fig 5.
Effect of CD8+ lymphocytes on the frequency of Tax
expression in CD4+ lymphocytes.
(A) Peripheral blood mononuclear cells (PBMCs) were isolated from an
asymptomatic carrier (HT) and cultivated for 24 hours either directly
or after depletion of CD8+ or CD56+ lymphocytes, or both. The cells
were then harvested and processed to perform the concomitant detection
of Tax and CD4 antigens. One representative experiment of 3 is shown.
(B) PBMCs were isolated from 3 asymptomatic carriers and from 3 patients with HTLV-I-associated myelopathy/tropical spastic paraparesis
depleted of CD8+ cells and then cultivated for 24 hours after
re-addition of a different number of autologous CD8+ cells. Tax
positivity in CD4+ lymphocytes was then plotted as a function of the
percentage of CD8+ lymphocytes in each culture. HTLV-I = human T-cell
leukemia virus type I.
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Effect of CD8+ lymphocytes on the frequency of Tax
expression in autologous or heterologous CD4+ lymphocytes
To characterize the mechanism by which CD8+ T lymphocytes reduce Tax
expression in PBMCs, we next determined whether CD8+ lymphocytes were
able to reduce Tax expression in heterologous CD4+ lymphocytes. For
this purpose, CD4+ and CD8+ lymphocytes from 2 different patients (TAZ,
TAT), respectively HLA-A*2402/2601, B*5101/52 011, Bw4,
Cw*1202/1402 and HLA-A*0201/6801, B*1503/3501, Cw*0202/1601, were isolated. CD4+ lymphocytes from each patient were cultivated for 24 hours in the absence or in the presence of
autologous or heterologous CD8+ lymphocytes. As shown in Figure 6, the reduction of Tax expression in CD4+
lymphocytes only occurred significantly in the presence of autologous
CD8+ lymphocytes. In contrast, heterologous CD8+ lymphocytes only
slightly decreased the frequency of Tax expression. This observation is
compatible with a dominant major histocompatibility complex class I
restricted mechanism of cellular cytotoxicity.22
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| Fig 6.
Effect of CD8+ lymphocytes on the frequency of Tax
expression in autologous or heterologous CD4+ lymphocytes.
Various combinations of purified CD4+ and CD8+ lymphocytes of two
patients with HTLV-I-associated myelopathy/tropical spastic paraparesis
(TAZ and TAT) were cultivated for 24 hours and then processed for the
concomitant detection of Tax and CD4 antigens. One representative
experiment of 2 is shown. HTLV-I = human T-cell leukemia virus type
I.
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CD8+ lymphocytes kill Tax-expressing cells by a perforin-dependent
mechanism
Cytotoxic CD8+ lymphocytes can mediate their antiviral effect
through perforin-dependent lysis of infected cells.23 To
further determine the mechanism by which CD8+ lymphocytes reduce Tax
expression, PBMCs from 2 patients were cultivated for 24 hours with or
without 20 nmol/L of CMA, an inhibitor of the perforin-dependent
cytotoxic pathway.23 As shown in Figure
7, the presence of CMA increased the
frequency of Tax expression to that caused by the depletion of CD8+
lymphocytes. In contrast to Tax expression, the percentage of CD4+
lymphocytes remained constant in cultures treated in the presence or in
the absence of CMA (data not shown).
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| Fig 7.
Effect of Concanamycin A (CMA) on the frequency of Tax
expression in CD4+ lymphocytes.
Peripheral blood mononuclear cells (PBMCs) were isolated from an
asymptomatic carrier (HT) and from a patient with HTLV-I-associated
myelopathy/tropical spastic paraparesis (TAZ), cultivated with or
without CMA for 24 hours, and then processed for the concomitant
detection of Tax and CD4 antigens. The effect of CMA was compared with
the effect of CD8+ lymphocyte depletion. One representative experiment
of 3 is shown. HTLV-I = human T-cell leukemia virus type I.
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The reduction of Tax expression by a perforin-dependent mechanism has
two implications. First, the level of mortality in Tax-positive cells
should be higher than that found in Tax-negative cells. Second, the
presence of CMA should specifically reduce this higher level of
mortality in Tax-positive cells. To test these predictions, PBMCs
isolated from 2 infected patients were cultivated in the presence or in
the absence of 20 nmol/L of CMA for 24 hours, harvested, and incubated
for 10 minutes with propidium iodide, which labels dead cells. Samples
were then fixed and processed to detect concomitantly Tax and propidium
iodide by flow cytometry. Table 2 shows
that, in the absence of CMA, the level of mortality in Tax-expressing cells was indeed more than tenfold higher than the Tax-negative population. As predicted, in the presence of CMA, the level of mortality dropped in Tax-expressing cells, whereas it slightly increased in the Tax-negative population.
To confirm that CD8+ lymphocytes were responsible for the increase in
mortality in the Tax-positive population, CD4+ lymphocytes were
purified and cultivated for 24 hours in the absence or in the presence
of increasing numbers of CD8+ lymphocytes. Cells were then harvested,
incubated for 10 minutes with propidium iodide, fixed, and processed to
detect concomitantly Tax and propidium iodide by flow cytometry. The
analysis showed that the level of mortality in Tax-expressing CD4+
cells increased from 7.8% in the absence of CD8+ cells to 10.3%,
17.3%, and 20.4% when 5%, 12.5%, and 25% of CD8+ cells were added
to the culture, respectively. Those results provide strong evidence
that CD8+ lymphocytes selectively kill Tax-expressing CD4+ lymphocytes
in vitro.
Frequency of Tax11-19-specific CD8+ T cells
is negatively correlated with the percentage of CD4+ T cells
in peripheral blood
Persistent elimination of Tax-expressing CD4+ T cells by the
abundant Tax-specific CTLs might have a significant impact on the CD4+
T cell population in vivo, especially in patients with HAM/TSP who have
a high frequency of infected CD4+ T cells. To test this hypothesis, we
determined the frequency of Tax11-19-specific CD8+
lymphocytes and the percentage of CD4+ cells in fresh PBMCs isolated
from 19 patients with HAM/TSP who were
HLA-A*02 positive.
Tax11-19-specific CD8+ lymphocytes were detected by using
fluorescent-labeled tetramers of
HLA-A*0201 + 2microglobulin + Tax11-19
peptide.12,21 The analysis showed a significant
negative correlation (2-tailed P = .015; Spearman rank
correlation) between the frequency of Tax11-19-specific CTLs and the percentage of CD4+ cells. This result is consistent with
the view that Tax-specific CTLs participate in an immune surveillance
mechanism that persistently destroys and removes Tax-expressing
infected CD4+ T cells in vivo.
| |
Discussion |
The role of the cellular immune response in HTLV-I infection is not
fully understood. A persistently activated CTL response to HTLV-I is
found in the majority of infected individuals.4-8 However,
several observations paradoxically suggest that HTLV-I is
transcriptionally silent in most infected cells13,14 and, therefore, not detectable by virus-specific CTLs.
The characterization of the immune response against HTLV-I has been
complicated by the difficulty of identifying cells that are naturally
infected in vivo, the CD4+ lymphocytes.18 In this study, we
report a new flow cytometric approach that allows the detection of the
immunodominant T-cell antigen in HTLV-I infection, the Tax protein
(Figure 1). The induction of viral protein expression in some PBMCs
after long-term cultivation in vitro has been known for a long
time.15,16 However, it has been difficult to demonstrate Tax protein expression in freshly isolated PBMCs, although tax mRNA has
been detected in a proportion of individuals infected with
HTLV-I.24-26 We report here that a large proportion of
HTLV-I-infected PBMCs (10%-80%) isolated from infected patients
become positive for Tax protein only after 6 hours of cultivation in
vitro (Figure 3 and Table 1). Most naturally infected cells in vivo
are, therefore, capable of expressing the Tax protein and are
consequently detectable by Tax-specific CTLs. In addition, the use of
flow cytometry allowed the concomitant detection of Tax and cell
surface markers. The majority of cells expressing Tax were
characterized as CD4+ CD45RO+ lymphocytes (Figure 2), in accordance
with Richardson et al18 who observed a similar phenotype in
HTLV-I-infected cells in vivo.
This technique also allowed us to quantify the possible interactions
between Tax-specific CD8+ CTLs and their target cells. With the use of
PBMCs isolated from both asymptomatic carriers and patients with
HAM/TSP, we observed a negative correlation between the frequency of
CD8+ T lymphocytes and the frequency of Tax expression (Figure 5).
Several observations indicate that this effect is the consequence of a
selective killing of Tax-expressing cells by CD8+ T lymphocytes. First,
CMA, an inhibitor of perforin-dependent cytotoxicity,23
allowed the frequency of Tax expression to increase to a similar extent
as CD8+ lymphocyte depletion (Figure 7). Second, CMA reduced the level
of mortality observed in Tax-positive cells but had the opposite effect
in Tax-negative cells (Table 2). Third, the mortality rate in
Tax-positive cells increased in a dose-dependent manner with the
frequency of CD8+ T lymphocytes in culture. Finally, CD8+ T lymphocytes
were capable of reducing Tax expression in autologous but not
heterologous CD4+ T cells, consistent with a major histocompatibility
complex-restricted mechanism of cytotoxicity (Figure 6). These results
provide evidence that CD8+ lymphocytes rapidly kill autologous
Tax-expressing CD4 T cells by a perforin-dependent mechanism.
The results presented in this study raise the possibility that
HTLV-I-specific CTLs participate in an immune surveillance mechanism
that destroys and removes Tax-expressing CD4+ lymphocytes in vivo. If
this mechanism occurs, the persistent elimination of
Tax-expressing CD4+ T cells by the abundant Tax-specific CTLs might
have a significant impact on the CD4+ T cell population in vivo.
Indeed, we observed, in 19 patients infected with HTLV-I, a significant
negative correlation between the percentages of CD4+ lymphocytes and
Tax11-19-specific CTLs in vivo. Moreover, Jeffery et
al11,12 have recently shown that the common major histocompatibility complex class I type, HLA-A*02, is
associated with both protection from HAM/TSP disease and a significant
reduction in provirus load in asymptomatic carriers of HTLV-I. This
observation is also consistent with the view that HTLV-I-specific CTLs
eliminate HTLV-I-infected cells in vivo. Furthermore, the existence of
this immune surveillance mechanism might explain why a short in vitro incubation (6 hours) of PBMCs is associated with a significant increase
in the percentage of Tax-positive cells (Figure 3 and Table 1). The
observed increase in Tax expression was neither due to the removal of a
serum factor repressing the expression of viral protein nor to a
mitogenic activation by FCS (Figure 4). On the contrary, Tax expression
was even higher when CD8+ lymphocytes were depleted and was reduced in
a dose-dependent manner when the frequency of CD8+ lymphocytes was
increased (Figure 5). We suggest, therefore, that the observed increase
in Tax expression during short-term culture results from the longer
average lifespan of infected CD4+ T cells in vitro. In vivo, the cells
are efficiently mixed, and an infected CD4+ cell is likely to be killed
shortly after it starts to express Tax. In vitro, the cells are
relatively sedentary, so an infected CD4+ T cell can express Tax on
average longer before it is destroyed by CTLs. Tax-specific CTLs could, therefore, play an important role in reducing the frequency of Tax-expressing CD4+ T cells in vivo. This effect could be responsible for the low frequency of Tax expression in fresh PBMCs. However, we
cannot exclude the possibility that another mechanism suppresses HTLV-I
gene expression in peripheral blood.
It is still not known whether HTLV-I-specific CTLs are only protective
or also cause bystander tissue damage. However, a dual role of
virus-specific CTLs has been demonstrated in lymphocytic choriomeningitis virus27 and
influenza28 virus infections. Influenza-specific CTLs have
been shown to confer protection against low-dose viral challenge but
exacerbate viral pathology and cause mortality at high viral
dose.28 In HTLV-I infection, a high proviral load might
similarly increase the probability that virus-specific CTLs cause
bystander tissue damage in the central nervous system. Consistent with
this suggestion, an accumulation of both Tax-expressing CD4+ and CD8+
lymphocytes is found in active central nervous system lesions of
patients with HAM/TSP.29-31 In addition, Kubota et
al32 demonstrated that a high frequency of PBMCs isolated
from patients with HAM/TSP and cultivated in vitro produce large
quantities of pro-inflammatory cytokines, including IFN- and
TNF- , which are neurotoxic. Therefore, in addition to their ability
to eliminate Tax-expressing cells, HTLV-I-specific CTLs might also
produce pro-inflammatory cytokines in situ and consequently contribute to tissue damage of the central nervous system. Further experiments must be done to characterize more precisely the role of both
HTLV-I-specific CTLs and infected cells in HAM/TSP pathogenesis.
In conclusion, the results presented in this study provide evidence
that virus-specific CTLs may participate in an immune surveillance
mechanism in vivo that persistently destroys and removes
HTLV-I-infected cells in peripheral blood. The efficiency of this
immune surveillance mechanism may be of prime importance in the
reduction of HTLV-I replication in vivo.
| |
Acknowledgments |
We thank the staff members and blood donors of the Kagoshima Red Cross
Blood Center and of St. Mary's Hospital. The authors would like to
thank Rebecca Asquith for helpful comments on the manuscript. E. Hanon
is a senior research assistant of the Fonds National Belge de la
Recherche Scientifique (F.N.R.S.).
| |
Footnotes |
Submitted August 25, 1999; accepted October 18, 1999.
Supported by the Program for Promotion of Fundamental Studies in Health
Sciences of the Organization for Pharmaceutical Safety and Research
(OPSR) (Japan), the Wellcome Trust (UK), the Royal Society (UK), and
the Fords National Belge de la Recherche Scientifique (Belgium).
Reprints: Charles Bangham, Department of Immunology, Imperial
College School of Medicine, St Mary's Campus, Norfolk Place, W21PG,
London, United Kingdom; e-mail: c.bangham{at}ic.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction