Download Correlation of human T-cell lymphotropic virus type 1 (HTLV

From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Correlation of human T-cell lymphotropic virus type 1 (HTLV-1) mRNA with
proviral DNA load, virus-specific CD8⫹ T cells, and disease severity in
HTLV-1–associated myelopathy (HAM/TSP)
Yoshihisa Yamano, Masahiro Nagai, Meghan Brennan, Carlos A. Mora, Samantha S Soldan, Utano Tomaru, Norihiro Takenouchi,
Shuji Izumo, Mitsuhiro Osame, and Steven Jacobson
To investigate the role of viral expression
in individuals infected with human T-cell
lymphotropic virus type 1 (HTLV-1), a
real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR)
of HTLV-1 tax messenger RNA (mRNA)
using ABI Prism 7700 Sequence Detection System was developed. Using this
system, the HTLV-1 tax mRNA load was
compared with HTLV-1 proviral DNA load,
HTLV-1 Tax protein expression, HTLV-1
Tax-specific CD8ⴙ T-cell frequency, and
disease severity of HTLV-1–associated
myelopathy/tropical spastic paraparesis
(HAM/TSP). This approach was a sensitive and specific technique for the precise
quantification of HTLV-1 tax mRNA. The
total amount of HTLV-1 tax mRNA and
mRNA expression level in HTLV-1–infected cells (mRNA/DNA ratio) were higher
in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The HTLV-1 tax mRNA
load correlated with the HTLV-1 proviral
DNA load ex vivo, the Tax protein expres-
sion in vitro, and the Tax-specific CD8ⴙ
T-cell frequency ex vivo. The HTLV-1 tax
mRNA load also correlated with disease
severity in HAM/TSP patients. These data
suggest that increased HTLV-1 expression plays an important role in the pathogenesis of HAM/TSP, and the HTLV-1 tax
mRNA level could be a useful predictor of
disease progression in patients with HAM/
TSP. (Blood. 2002;99:88-94)
© 2002 by The American Society of Hematology
Introduction
Human T-cell lymphotropic virus type 1 (HTLV-1) is a human
retrovirus that infects approximately 10 million people worldwide.1
The majority of infected individuals remain healthy lifelong
asymptomatic carriers (ACs), approximately 0.25% to 3% develop
an inflammatory disease of the central nervous system termed
HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/
TSP),2-5 and another 2% to 3% develop an aggressive mature T-cell
malignancy termed adult T-cell leukemia (ATL).6,7 Patients with
HAM/TSP generally harbor a much larger population of HTLV-1–
infected T cells in peripheral blood mononuclear cells (PBMCs)8-10
and a higher immune response against HTLV-1 antigens than
ACs.11-13 These findings indicate that an abundant viral load plays
an important role in the pathogenesis of HAM/TSP, suggesting that
high viral expression may be associated with the high immune
response against the virus.
Even though a high proviral DNA load is characteristic of
patients with HAM/TSP, the expression of HTLV-1 in PBMCs
appears to be very low,14-18 which suggests that HTLV-1 may be
latent in peripheral blood. However, this suggestion is difficult
to reconcile with the observation of high activated immune
responses in patients with HAM/TSP.11-13 Moreover, it has been
reported that a significant proportion of PBMCs infected with
HTLV-1 are capable of expressing HTLV-1 proteins after
short-term cultivation in vitro,17,19 supporting the hypothesis
that HTLV-1 is not inherently latent. Recently, a correlation
between the HTLV-1 proviral DNA load and the frequency of
HTLV-1 Tax-specific CD8⫹ T cells20,21 has been described in
patients with HAM/TSP. Collectively, these data suggest HTLV-1
antigens continuously restimulate the cellular immune system in
vivo. However, no reports have characterized the HTLV-1 tax
messenger RNA (mRNA) expression in the relationship between
the HTLV-1 proviral DNA load and the virus-specific T-cell
immune response in individuals infected with HTLV-1. In this
study, to determine the importance of HTLV-1 tax mRNA
expression in that relationship and in the pathogenesis of
HAM/TSP, a rapid, sensitive, and precise quantification of
HTLV-1 tax mRNA expression was developed by a real-time
reverse transcription-polymerase chain reaction (RT-PCR)
method using the ABI Prism 7700 Sequence Detection System.
Because the level of human immunodeficiency virus type 1
(HIV-1) RNA has been found to be a valid predictor of the
clinical progression of HIV-1–related disease and efficacy of
anti-HIV therapy,22,23 it was also of interest to determine if the
HTLV-1 tax mRNA load as quantified by this novel method
might be a marker of disease activity and of relative prognostic
value in HAM/TSP.
In this study, the HTLV-1 tax mRNA load in PBMCs from
patients with HAM/TSP and ACs was measured and compared
with HTLV-1 proviral DNA load, HTLV-1 Tax protein expression,
HTLV-1 Tax-specific CD8⫹ T-cell frequency, and disease severity
in patients with HAM/TSP. The data presented demonstrate that
increased HTLV-1 expression plays an important role in the
pathogenesis of HAM/TSP, and HTLV-1 tax mRNA level could be
a useful predictor of disease progression in HAM/TSP patients.
From the Viral Immunology Section, Neuroimmunology Branch, National
Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, MD; Center for Chronic Viral Diseases, Faculty of Medicine,
Kagoshima University, Japan; and Third Department of Internal Medicine,
Faculty of Medicine, Kagoshima University, Japan.
Reprints: Steven Jacobson, NIH/NINDS/NIB Bldg 10, Rm 5B-16, Bethesda,
MD 20892; e-mail: [email protected].
Submitted July 24, 2001; accepted September 6, 2001.
© 2002 by The American Society of Hematology
88
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.
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
QUANTITATIVE HTLV-1 mRNA LOAD IN HAM/TSP
Patients, materials, and methods
Subjects and cells
The study population consisted of 16 patients with nontreated HAM/
TSP (HAM-1 to HAM-16), 8 asymptomatic HTLV-1 carriers (AC-1 to
AC-8), and 5 seronegative healthy donors (HDs). Infection of HTLV-1
was confirmed by enzyme-linked immunosorbent assay (Abbot Laboratories, North Chicago, IL) and Western blot analysis (Gene Labs,
Singapore). The diagnosis of HAM/TSP was assessed according to the
World Health Organization guidelines.24 The HAM-2 to -5, -11, -13, and
-16 and AC-4, -6, and -7 individuals were positive for HLA-A*0201. All
subjects were screened for HIV-1/2, hepatitis virus type B/C, HTLV-2,
Epstein-Barr virus, Borrelia burgdorferi, and syphilis antibodies. HAM-8
and HAM-16 were coinfected with hepatitis virus type C. All samples
were taken under informed consent. The clinical features of the patients
included in this study are shown in Table 1. Disease severity was
measured by using an expanded disability status scale (EDSS).25 MT-2
cells and Hut 102 cells are lines of human CD4⫹ T cells infected with
HTLV-1.26,27 Jurkat cells are human T-cell lines not infected with HTLV-1.
Real-time RT-PCR of complementary DNA
RNA was extracted from PBMCs using RNeasy Mini Kit (Qiagen,
Valencia, CA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using TaqMan Gold RT-PCR Kit
(Applied Biosystems, Foster City, CA). For cDNA synthesis from
extracted mRNA, 3 ␮g mRNA, 10 ␮L 10 ⫻ TaqMan RT buffer, 22 ␮L
89
MgCl2 (25 mM), 20 ␮L dNTPs mixture (at a final concentration of 500
␮M each), 5␮L random hexamers (50 ␮M), 2 ␮L RNase inhibitor (20
U/␮L), and 2.5 ␮L (50 U/␮L) Moloney murine leukemia virus reverse
transcriptase were added to a total volume of 100 ␮L. Samples were
incubated at 25°C for 10 minutes and 48°C for 30 minutes. Reactions
were stopped by heating to 95°C for 5 minutes.
Primers used for the amplification of HTLV-1 tax cDNA were
slightly modified from the sequence previously reported18 to be suitable
for this system. The forward primer RPX-3 (5096-5115; 5⬘-ATCCCGTGGAGACTCCTCAA-3⬘) and the reverse primer RPX-4 ⫹ 3 (73607338; 5⬘-CCAAACACGTAGACTGGGTATCC-3⬘) were located upstream and downstream of the second splice junction site of HTLV-1 pX
(tax/rex) mRNA. The amplified DNA was 147 base pairs (bp). The probe
(5172-5183, 7302-7312; 5⬘-TCCAACACCATG-GCCCACTTCCC-3⬘)
surrounded the second splice junction site of HTLV-1 pX (tax/rex)
mRNA. We used the human housekeeping gene hypoxanthine ribosyl
transferase (HPRT) primers and probe set (Applied Biosystems) for
internal calibration. This internal control appeared to be most stable
within PBMCs from HDs, ACs, HAM/TSP patients, and ATL patients by
using the TaqMan Human Endogenous Control Plate (Applied Biosystems). PCR conditions were as follows: 10 ␮L cDNA solution synthesized from 300 ng RNA was added to 40 ␮L reaction mixture
containing10 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM EDTA, 60 nM
ROX (passive reference dye to normalize receptor signal), 5.5 mM
MgCl2, 0.3 ␮M each primer, 0.2 ␮M TaqMan probe, 200 ␮M each of
dATP, dGTP, and dCTP, 400 ␮M dUTP, 0.5 U AmpErase uracil-Nglycosylase (UNG), and 0.25 U AmpliTaq Gold DNA polymerase
(AmpliTaq Gold; Applied Biosystems). The thermal cycler conditions
Table 1. HTLV-1 tax mRNA load, HTLV-1 proviral DNA load, mRNA/DNA ratio, HTLV-1 Tax-specific CD8ⴙ T-cell frequency, and clinical profiles of HAM/TSP
patients and ACs
Patients
Origin
tax mRNA
load
Proviral DNA
load
Sex
HAM-1
38
F
USA
0.06
1.62
3.70
—
5.5
6
HAM-2
47
F
USA
1.61
11.92
13.51
0.52
6.0
10
HAM-3
63
M
USA
2.32
25.16
9.22
5.70
1.5
18
HAM-4
78
F
USA
5.00
23.20
21.55
3.53
2.0
12
HAM-5
46
F
USA
5.95
12.38
48.06
4.21
3.5
12
HAM-6
47
F
Caribbean
9.76
9.37
104.16
—
6.5
2
HAM-7
57
F
USA
11.79
20.98
56.20
—
6.5
3
HAM-8
48
F
USA
12.32
11.70
105.30
—
6.0
4
HAM-9
56
F
USA
13.62
24.76
55.01
—
6.0
13
HAM-10
64
M
USA
17.78
20.67
86.02
—
6.0
12
HAM-11
59
M
Jamaica
20.08
21.99
91.31
4.71
7.0
10
HAM-12
45
F
USA
25.44
23.09
110.18
—
6.0
3
HAM-13
59
M
USA
71.18
22.89
310.97
17.64
7.0
14
HAM-14
44
M
USA
109.10
47.14
231.44
—
5.5
2
HAM-15
79
M
USA
137.70
69.49
198.16
—
8.0
12
HAM-16
49
M
USA
187.90
60.16
312.33
24.35
8.0
6
39.48
25.41
109.82
8.67
Mean
Median
mRNA/DNA ratio
Tax-specific CD8⫹
T-cell frequency, %
Age, y
EDSS
Duration of illness, y
12.97*
22.44†
88.67‡
AC-1
61
F
USA
0.00
0.00
0.00
—
—
—
AC-2
62
M
USA
0.00
0.07
0.00
—
—
—
AC-3
49
F
USA
0.01
0.02
50.00
—
—
—
AC-4
62
F
USA
0.11
4.35
2.53
0.06
—
—
AC-5
37
M
USA
0.51
2.92
17.47
—
—
—
AC-6
66
F
USA
2.87
7.97
36.01
0.45
—
—
AC-7
23
M
USA
2.94
3.61
81.44
1.64
—
—
AC-8
44
F
Japan
5.58
3.10
180.00
—
—
—
Mean
1.50
2.76
45.93
0.72
Median
0.31*
3.01†
26.74‡
The mRNA/DNA ratio was calculated by the following formula: mRNA/DNA ratio ⫽ (HTLV-1 tax mRNA load/HTLV-1 proviral DNA load) ⫻ 100.
The Tax-specific CD8⫹ T-cell frequency is expressed as a percentages of HTLV-1 Tax11-19/HLA-A*0201 tetramer-specific CD8⫹ T cells in the total amount of CD8⫹ T cells.
— indicates subjects who are not HLA-A*0201.
HTLV-1 tax mRNA load, HTLV-1 proviral DNA load, and mRNA/DNA ratio were higher in HAM/TSP patients than in ACs with statistical significance by the Mann-Whitney U
test: *P ⫽ .0014; †P ⫽ .0003; ‡P ⫽ .0433. The EDSS score significantly correlated with both HTLV-1 tax mRNA load and mRNA/DNA ratio (P ⫽ .0109 and .0089, respectively),
but not with HTLV-1 proviral DNA load (P ⫽ .5236) by Spearman rank correlation analysis.
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
90
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
YAMANO et al
were 2 minutes at 50°C to activate AmpErase UNG enzyme, 10 minutes
at 95°C to inactivate UNG and activate AmpliTaq Gold DNA polymerase, and then 45 cycles of 15 seconds at 95°C (denaturation) followed by
1 minute at 60°C (annealing and extension).
Standard curves for the value of HTLV-1 tax mRNA and HPRT mRNA were
generated using cDNAfrom MT-2 cells. MT-2 cDNAwas serially diluted 10-fold
with diethyl pyrocarbonate (DEPC) H2O down to a 10⫺4 dilution, and sample
cDNA from 300 ng RNA per well was applied and analyzed by this system. The
amplification of standard cDNA and sample cDNA was carried out in a 96-well
reaction plate. All standards and samples were assayed in triplicate. Each plate
always contained the same standard. When a serial dilution of 100 to 10⫺4 of the
MT-2 cell cDNA was used as the template for the real-time PCR, a specific signal
of each increased in accordance with the increase of PCR cycles, but not in the
negative control. The threshold cycle (Ct) values were used to plot a standard
curve in which Ct decreased in linear proportion to the log of the template copy
number. The correlation values of standard curves were always more than 99%.
The relative HTLV-1 tax mRNA load was calculated by the following
formula: HTLV-1 tax mRNA load ⫽ (value of tax)/(value of HPRT)
⫻ 10 000.
Real-time PCR of DNA
The HTLV-1 proviral DNA load was measured using this same TaqMan
system as previously described.10,21 DNA was extracted from 1 ⫻ 106 cells
using Puregene DNA Isolation Kit (Gentra, Minneapolis, MN) and 100 ng
of the sample DNA solution per well was analyzed by this system. The
HTLV-1 proviral DNA load was calculated by the following formula: copy
number of HTLV-1 (pX) per 100 cells ⫽ (copy number of pX)/(copy
number of ␤-actin/2) ⫻ 100.
HTLV-1 Tax protein expression in PBMCs
cytometry. The frequencies of HTLV-1 Tax-specific CD8⫹ T cells were
expressed as a percentage of total CD8⫹ T cells.
Statistical analysis
The Mann-Whitney U test was used to compare the data between patients
with HAM/TSP and ACs. Simple regression analysis was used to test the
correlation between HTLV-1 proviral DNA load, HTLV-1 tax mRNA load,
mRNA/DNA ratio, and HTLV-1 Tax-specific CD8⫹ T-cell frequency. To
test the correlation of EDSS with HTLV-1 proviral DNA load, HTLV-1 tax
mRNA load and mRNA/DNA ratio, the Spearman rank correlation
was used.
Results
Accuracy of real-time RT-PCR
To determine the interassay coefficient of variance of this real-time
HTLV-1 tax RT-PCR system, cDNA from the HTLV-1–infected
MT-2 cell was diluted serially with cDNA from the HTLV-1–
noninfected Jurkat cell, and HTLV-1 tax mRNA load of each
dilution was measured 5 times (Figure 1A). In this study, the
HTLV-1 tax mRNA load was calculated as the value of HTLV-1 tax
mRNA divided by the value of HPRT as described in “Patients,
materials, and methods.” To estimate the accuracy of this system,
we calculated the intercoefficient of variance (CV%) in each
amount. The mean interassay CV% was 19.3%, which is consistent
with the CV% measuring HTLV-1 proviral DNA using real-time
PCR methods.10,29
To test the intra-assay coefficient of variance of this real-time
The PBMCs (5 ⫻ 105) were placed on a culture well (round bottom 96-well
plate) in 200 ␮L RPMI 1640 supplemented with L-glutamine, penicillin,
streptomycin, and 5% human AB serum. Harvested cells were washed with
phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS) and
0.1% NaN3 and incubated with antihuman CD4-PE (Caltag Laboratories,
Burlingame, CA) and antihuman CD8-Tricolor monoclonal antibodies
(mAbs; Caltag Laboratories) for 20 minutes at 4°C. Following washing
with PBS containing 1% FCS and 0.1% NaN3, cells were fixed and
permeabilized with 4% formaldehyde and 0.1% saponin (CytoFix/
Cytoperm kits, Pharmingen, SanDiego, CA) for 20 minutes at 4°C. After
washing with 0.1% saponin buffer (Perm/Wash solution, Pharmingen), cells
were incubated with anti-HTLV-1 Tax mAb (obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID, NIH:
HTLV-1 Tax hybridoma 168A51-42 from Dr Beatrice Langton) for 30
minutes at 4°C. Followed by washing unbound antibody with 0.1% saponin
buffer, fluorescein isothiocyanate (FITC)–conjugated goat F(ab⬘)2 antimouse IgG2a was used as second antibody for labeling anti-HTLV-1 Tax
mAb. After a 20-minute incubation, cells underwent a final wash. Flow
cytometric analyses were performed using a FACS Calibur (Becton
Dickinson, Mountain View, CA).
Identification of HTLV-1 Tax-specific CD8ⴙ T cells
The HTLV-1 Tax 11-19 peptide is a dominant epitope recognized by HLA-A2–
restricted CD8⫹ T cells in HAM/TSP patients.13,28 HTLV-1 Tax11-19/HLA
A*0201-specific CD8⫹ T cells were analyzed using a phycoerythrin (PE)–
conjugated HLA-A*0201 tetramer (provided by NIAID MHC Tetramer Core
Facility, Atlanta, GA, and NIH AIDS Research and Reference Reagent Program)
for HLA A*0201 subjects. The tetramer was loaded with HTLV-1 Tax 11-19
peptide (LLFGYPVYV; single-letter amino acid codes), which was synthesized
and 95% purified by high-performance liquid chromatography (HPLC; New
England Peptide, Fitchburg, MA). Tetramer loaded with HIV Gag 77-85 peptide
(SLYNTVATL) was used as a negative control. PBMCs from HLA-A*0201
subjects were washed and incubated with PE-conjugated HLA-A*0201 tetramer,
antihuman CD4-FITC (Caltag Laboratories), and antihuman CD8-Tricolor
mAbs (Caltag Laboratories) for 30 minutes at 4°C, and then analyzed by flow
Figure 1. Validation of real-time quantitative RT-PCR assay. (A) To determine
interassay CV% for this real-time RT-PCR assay, cDNA from the HTLV-1–infected
MT-2 cell line was serially diluted with cDNA from the uninfected Jurkat cell line and
HTLV-1 tax mRNA load was measured 5 times per dilution. (B) The intra-assay CV%
was determined by measuring mRNA load from the PBMCs of 3 HAM/TSP patients in
5 separate experiments. The CV% represents (SD of HTLV-I tax mRNA load/average
of HTLV-I tax mRNA load) ⫻ 100.
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
QUANTITATIVE HTLV-1 mRNA LOAD IN HAM/TSP
91
HTLV-1 tax RT-PCR system, PBMCs from 3 patients with
HAM/TSP were separated into 5 samples per individual. RNA was
extracted from each sample, cDNA was synthesized, and HTLV-1
tax mRNA load for each sample was determined (Figure 1B). The
CV% was calculated for each patient. The mean value of intraassay CV% was 19.2%, which is consistent with the CV%
measuring HIV-1 RNA.30 These data demonstrate that this realtime HTLV-1 tax RT-PCR assay is accurate and reliable for
quantitation of HTLV-1 tax mRNA.
Sensitivity and specificity of real-time RT-PCR
To determine the sensitivity of this real-time RT-PCR assay, the
HTLV-1 tax mRNA load was determined from MT-2 cells diluted
serially with PBMCs from a healthy donor not infected with
HTLV-1. As shown in Figure 2A, the HTLV-1 tax mRNA signal
could be detected in a dose-dependent manner with a sensitivity
limit as low as one MT-2 cell in 106 PBMCs.
To test the specificity of this assay, cDNA from HTLV-1–
noninfected Jurkat cells and PBMCs from HTLV-1–noninfected
HDs (n ⫽ 5) were analyzed. No signal was observed from any of
these HTLV-1⫺ cells (Figure 2B). MT-2 cDNA without RT also
showed no signal. Two HTLV-1–infected cell lines were tested for
expression of HTLV-1 tax mRNA by this system (Figure 2B). The
HTLV-1 tax mRNA was detected in these 2 cell lines, with Hut 102
cells expressing 20 times more HTLV-1 tax mRNA than MT-2
cells. This difference is consistent with previous reports demonstrating that Hut 102 cells have 10 to 17 HTLV-1 proviral copies per cell
compared to MT-2 cells, which have 2 to 8 HTLV-1 proviral copies
per cell.29,31-33
HTLV-1 tax mRNA load in HAM/TSP patients and ACs
Using this novel method, HTLV-1 tax mRNA load was analyzed in
PBMCs from HAM/TSP patients and ACs. As demonstrated in
Table 1 and Figure 3, the HTLV-1 tax mRNA load ranged from 0.06
to 187.90 in HAM/TSP patients and 0.00 to 5.58 in ACs. The
HTLV-1 tax mRNA load in HAM/TSP patients was significantly
Figure 3. HTLV-I tax mRNA load in HAM/TSP patients and ACs. HTLV-1 tax mRNA
load was assessed in PBMCs from 16 HAM/TSP patients (HAM) and 8 asymptomatic
HTLV-I carriers (AC). HTLV-1 tax mRNA load was significantly higher (P ⫽ .0014) in
HAM/TSP patients compared to ACs.
higher than that in ACs (P ⫽ .0014, Mann Whitney U test) as
reported previously.34 The absolute amount of HTLV-1 tax mRNA
before adjusting by the HPRT value in HTLV-1–infected individuals was 103 to 106 times less than that in MT-2 cells, which is
consistent with previous reports.18,34
Comparison of HTLV-1 tax mRNA load with HTLV-1 proviral
DNA load ex vivo
To determine if there was a correlation between the HTLV-1 proviral
DNA load and the HTLV-1 tax mRNA load in HTLV-1–infected
individuals, a real-time quantitative HTLV-1 DNA PCR assay21 was
used to measure the HTLV-1 proviral DNA load in PBMCs from
HAM/TSP patients and ACs. As previously reported,10,21 the HTLV-1
proviral DNA load, which represented a population of infected cells in
PBMCs, was greater in HAM/TSP patients than ACs (P ⫽ .0003, Mann
Whitney U test, Table 1). Furthermore, the HTLV-1 proviral DNA load
was correlated significantly with the HTLV-1 tax mRNA load in
HAM/TSP patients (P ⬍ .0001, r2 ⫽ 0.812, simple regression analysis,
Figure 4).
To adjust the HTLV-1 tax mRNA expression level in HTLV-1–
infected PBMCs, we calculated the mRNA/DNA ratio by dividing
the HTLV-1 tax mRNA load by HTLV-1 proviral DNA load. The
mean value of the mRNA/DNA ratio was 109.82 for HAM/TSP
patients and 45.93 for ACs. The mRNA/DNA ratio of HAM/TSP
patients was statistically higher than that of AC (P ⫽ .0433, Mann
Whitney U test, Table 1).
Comparison of HTLV-1 tax mRNA load with HTLV-1 Tax protein
expression in vitro
Because increased levels of mRNA may be associated with
increased protein production, it was important to determine if there
was a correlation between the HTLV-1 tax mRNA load and the
amount of HTLV-1 Tax protein expression. PBMCs from 4
Figure 2. Sensitivity and specificity of real-time RT-PCR assay. (A) To determine the
sensitivity of this real-time RT-PCR assay, HTLV-1 tax mRNA load was determined from
MT-2 cells serially diluted with PBMCs from an HTLV-1⫺ healthy donor. HTLV-I tax mRNA
could be detected from as little as a 10⫺6-fold dilution of MT-2 cells. (B) The specificity of this
assay was determined by measuring HTLV-1 tax mRNA expression of PBMCs from 5
HTLV-1–noninfected HDs, HTLV-1–uninfected Jurkat cells, and 2 HTLV-1–infected cell
lines (MT-2 and Hut 102). HTLV-I tax mRNA was detected in the 2 infected cell lines, but not
in uninfected cells.
Figure 4. Correlation between HTLV-1 proviral DNA load and HTLV-1 tax mRNA
load in HAM/TSP patients. A statistically significant correlation (P ⬍ .0001,
r2 ⫽ 0.812) between the HTLV-1 proviral DNA load and HTLV-1 tax mRNA load was
observed in PBMCs from HAM/TSP patients.
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
92
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
YAMANO et al
HAM/TSP patients (HAM-1, -11, -14, -15) were cultured for 48
hours in culture medium without mitogenic stimuli. PBMC samples
were taken after 0, 12, 24, and 48 hours, respectively, and the
HTLV-1 tax mRNA load and HTLV-1 Tax protein expression level
as measured by FACS analysis using monoclonal anti-Tax antibodies were determined. As shown in Figure 5, a close correlation was
found between the HTLV-1 tax mRNA load and the percentage of
cells expressing HTLV-1 Tax protein. The detection of HTLV-1 tax
mRNA and the expression of HTLV-1 Tax protein were coincident
and peaked at approximately 12 to 24 hours of in vitro culture
(Figure 5).
Comparison of HTLV-1 tax mRNA load with HTLV-1 Tax-specific
CD8ⴙ T-cell frequency ex vivo
The high levels of HTLV-1 proviral DNA load detected in
PBMCs from HAM/TSP patients have been shown previously to
correlate with high frequencies of virus-specific CD8⫹ T
cells.20,21 It was therefore of interest to determine if HTLV-1 tax
mRNA load also correlated with the frequency of HTLV-1
Tax-specific CD8⫹ T cells. The frequency of HTLV-1 Tax
11-19/HLA A*0201-specific CD8⫹ T cells was analyzed using a
PE-conjugated HLA-A*0201 tetramer for HLA-A*0201 subjects (HAM-2-5, -11, -13, -16 and AC-4, -6, -7). The percentages
of HTLV-1 Tax 11-19/HLA A*0201-specific CD8⫹ T cells in the
total amount of CD8⫹ T cells are summarized in Table 1. As
reported previously,20,21 the HTLV-1 proviral DNA load significantly correlated with the frequency of HTLV-1 Tax-specific
CD8⫹ T cells in HAM/TSP patients (P ⫽ .0191, r2 ⫽ 0.699,
simple regression analysis, Table 1). Furthermore, the HTLV-1
tax mRNA load was also significantly correlated with the
frequency of HTLV-1 Tax-specific CD8⫹ T cells in HAM/TSP
patients (P ⫽ .0013, r2 ⫽ 0.893, simple regression analysis;
Table 1 and Figure 6). HTLV-1 Tax 11-19/HLA A*0201-specific
CD8⫹ T cells have also been reported in PBMCs of ACs35 and
although in this study the sample size of HLA A*0201⫹ ACs was
too small to compare statistically, increased mRNA load in this
group was associated with a higher frequency of HTLV-1
Tax-specific CD8⫹ T cells (Table 1).
Figure 6. Correlation of HTLV-1 Tax-specific CD8ⴙ T-cell frequency with HTLV-1 tax
mRNA load. The frequency of HTLV-1 Tax 11-19/HLA-A*0201-specific CD8⫹ T cells
(expressed as a percentage of total CD8⫹ T cells) was analyzed using a PE-conjugated
HLA-A*0201 tetramer for HLA-A*0201 subjects. A significant correlation (P ⫽ .0013,
r2 ⫽ 0.893) was demonstrated between HTLV-1 tax mRNA load and the frequency of
HTLV-1 Tax-specific CD8⫹ T cells in HAM/TSP patients.
Comparison of disease severity with HTLV-1 proviral DNA load,
HTLV-1 tax mRNA load, and mRNA/DNA ratio in
HAM/TSP patients
Clinical features of HAM/TSP patients included in this study are shown
in Table 1. Because HAM/TSP is a chronic progressive disease, patients
with a long duration of illness are usually severely affected.36 However,
there were 3 patients (HAM-3, -4, -5) who showed relatively mild
progression and had a long duration of illness as reflected by relatively
low scores on the EDSS (Table 1). The EDSS index is a neurologic
measure of disability that is weighted toward ambulation.25 Of interest is
the observation that the HTLV-1 tax mRNA loads of all 3 of these
patients were low, even though 2 of the 3 patients (HAM-3, -4) harbored
average levels of HTLV-1 proviral DNA load. Furthermore, patients
with high HTLV-1 tax mRNA load and a long duration of illness
(HAM-11, -13, -15, -16) had more severe disease with EDSS scores
above 7 (patients essentially restricted to a wheelchair).
To examine the possibility that quantitative real-time PCR for
HTLV-1 DNA and RNA determinations might have prognostic value in
HAM/TSP disease progression, the EDSS values of these patients were
statistically compared with HTLV-1 proviral DNA load, HTLV-1 tax
mRNA load, and mRNA/DNA ratio by Spearman rank correlation
analysis (Table 1). The EDSS score significantly correlated with both
HTLV-1 tax mRNA load and mRNA/DNA ratio (P ⫽ .0109 and .0089,
respectively), but not with HTLV-1 proviral DNA load (P ⫽ .5236).
Thus, HTLV-1 tax mRNA load and mRNA/DNA ratio may have
relative prognostic value for the assessment of disease progression in
HAM/TSP.
Discussion
Figure 5. Correlation between HTLV-1 tax mRNA load and HTLV-1 Tax protein
expression in vitro. PBMCs from 4 HAM/TSP patients were cultured for 48 hours.
Cultured PBMC samples were harvested after 0, 12, 24, and 48 hours to estimate
HTLV-1 tax mRNA load and the percentage of HTLV-1 Tax protein expressing cells.
HTLV-1 Tax protein expression closely followed HTLV-1 tax mRNA expression in the
PBMCs from these individuals.
Using a newly established real-time RT-PCR assay to quantitate
HTLV-1 tax mRNA load, this study demonstrated a significant difference in the HTLV-1 tax mRNA load between patients with HAM/TSP
and ACs. Furthermore, HTLV-1 tax mRNA load significantly correlated
with the HTLV-1 proviral DNA load, the HTLV-1 Tax protein expression, and the frequency of HTLV-1 Tax 11-19/HLA A*0201-specific
CD8⫹ T cells. Collectively, these results support previous studies
demonstrating a relationship between the HTLV-1 proviral DNA load
and the frequency of HTLV-1 Tax-specific CD8⫹ T cells in HAM/TSP
patients,20 the existence of chronically activated HTLV-1 Tax-specific
CD8⫹ T cells,21 and the existence of high anti-HTLV-1 IgM antibodies
suggesting continuous immune stimulation in vivo.37 These results
indicate that HAM/TSP patients have more HTLV-1 proviral DNA load
than ACs, which may be associated with higher mRNA expression
resulting in heightened virus-specific immune responses. This profile
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
QUANTITATIVE HTLV-1 mRNA LOAD IN HAM/TSP
does not characterize all retroviruses. For example, in individuals
infected with HIV-1, the plasma viral RNA load, which is known to be
significantly correlated with HIV-1 RNA load in PBMCs,38,39 is
inversely proportional to the frequency of HIV-1–specific CD8⫹ T
cells,40,41 which suggests an effective immunologic elimination of
HIV-1 antigens. However, in HTLV-1 infection, as the virus antigen
expression increases, HTLV-1–specific CD8⫹ T cells are generated,
which appear to be unable to eliminate infected cells. Therefore, these
results support the hypothesis that expansion of HTLV-1 can occur in the
presence of an activated immune system that is trying to control the
infection. This novel assay is also useful to define the role of HTLV-1
viral expression in patients with ATL. In preliminary data, HTLV-1 tax
mRNA load was generally low in patients with ATL even in individuals
with a high proviral DNA load (data not shown).
Quantitative measurements of HTLV-1 tax mRNA load in PBMCs
from HAM/TSP patients and ACs continue to highlight differences
between these 2 groups. This study demonstrated that adjusting the
HTLV-1 tax mRNA load with HTLV-1 proviral DNA load (mRNA/
DNA ratio) was higher in HAM/TSP patients than in ACs. These results
indicate that in HAM/TSP patients a higher proportion of HTLV-1–
infected cells express HTLV-1 tax mRNA at higher levels than in ACs.
These observations support and extend previous reports describing a
number of host genetic and virologic differences between HAM/TSP
patients and ACs including differences in human leukocyte antigen
haplotypes,35,42 differences in the amount of soluble suppressive factors16,43 and CD8⫹ T-cell responses,19-21,44 and differences in HTLV-1
tax genomic sequences.45 Many of these observations distinguishing
HAM/TSP patients from carriers may also be related to higher proviral
DNA load, higher mRNA load (as demonstrated in this study), and
higher immune response against HTLV-1.10-13 The mRNA/DNA ratios
as determined by quantitative DNA and RNA PCR assays presented
here differed from a previous report34 that showed that the mRNA/DNA
ratio was similar in both HAM/TSP patients and ACs although the total
HTLV-1 tax mRNA load was higher in HAM/TSP patients than in ACs
(also demonstrated in the present study). This may be due to differences
in the technology and accuracy of the assays used. Therefore, it is
important to extend the observations on differences in the mRNA/DNA
ratio between HAM/TSP patients and ACs to larger groups of individuals infected with HTLV-1.
Because HAM/TSP is a chronic progressive disorder,36 clinical end
points are cumbersome when evaluating disease activity. Thus, a clinical
and laboratory goal is to define surrogate markers of HTLV-1 infection
that can be used to monitor HAM/TSP disease activity. Surrogate
markers of HAM/TSP disease activity have been reported. Neopterin
levels in cerebrospinal fluid (CSF), a marker of cell-mediated immune
activation,46,47 have been used as an immunologic marker for monitoring disease activity and treatment efficacy.48 Monitoring of ex vivo
spontaneous lymphoproliferation and the frequency of HTLV-1 Taxspecific CD8⫹ T cells, which have reported to be associated with the
pathogenesis of HAM/TSP,12,20,21 have also been used as potential
surrogate immunologic markers of disease progression.49 In this present
report, HTLV-1 tax mRNA load was correlated with HTLV-1 Tax-
93
specific CD8⫹ T-cell frequencies in HAM/TSP patients, suggesting that
the quantitative analysis of HTLV-1 tax mRNA might also be used to
monitor HAM/TSP disease activity. In this study, the frequency of
CD8⫹ T cells specific only to the HTLV-1 Tax region was analyzed;
however, other HTLV-1 regions have also been shown to be important
as targets for CD8⫹ T-cell immune responses.50,51
In the assessment of any new HTLV-1–associated immunologic
assay, it is of interest to determine if it can be used as a prognostic
indicator of disease progression in HAM/TSP. For example, it has been
reported that the anti-HTLV-1 antibody titers and the neopterin levels in
CSF are high in patients with acute progressive HAM/TSP and low in
those with slowly progressive disease.36 The HTLV-1 tax mRNA load
measured by this novel real-time RT-PCR method was demonstrated to
generate a quantitative prognostic index of clinical progression in a
limited cohort of HAM/TSP patients. The EDSS value is a commonly
used neurologic measure of disability25 and significantly correlated with
both HTLV-1 tax mRNA load and mRNA/DNA ratio (P ⫽ .0109 and
.0089, respectively), but interestingly not with HTLV-1 proviral DNA
load (P ⫽ .5236). This tendency has been also observed in HIV-1
infection.22,23 These findings suggest that the amounts of expressed
antigen and induced immune response may be more sensitive than
proviral DNA load as a prognostic indicator in virus-associated diseases.
As with any chronic, progressive neurologic disease, it is difficult to
predict clinical progression. However, the HTLV-1 tax mRNA load was
demonstrated to reflect viral activity and could be an important marker
to be used to predict long-term outcome in HAM/TSP patients. We are
currently evaluating virologic and immunologic markers described here
over time in HAM/TSP patients with or without treatment. HTLV-1
proviral DNA load, HTLV-1 tax mRNA load, and HTLV-1 Tax-specific
CD8⫹ T-cell frequency are generally stable over time in untreated
patients (data not shown). Further longitudinal and controlled studies
enrolling a large number of patients must be conducted.
In summary, a real-time quantitative RT-PCR of HTLV-1 tax mRNA
has been developed. This approach proved to be a sensitive and specific
technique for precise quantification of HTLV-1 tax mRNA. The novel
assay demonstrated a significant correlation between HTLV-1 DNA,
RNA, protein, and HTLV-1–specific T-cell immune response. Additionally, HTLV-1 expression levels in virus-infected cells (mRNA/DNA
ratio) were higher in HAM/TSP patients than in ACs, suggesting the
existence of genetic or virologic differences. Furthermore, comparison
of molecular results with clinical features suggested that HTLV-1 tax
mRNA load and mRNA/DNA ratio may be valid predictors of disease
progression. These data support the hypothesis that higher HTLV-1
expression plays an important role in the pathogenesis of HAM/TSP.
Acknowledgments
We thank Nazli Azimi, National Cancer Institute, for her kind help
and James M. Dambrosia, Biostatistics Branch, National Institute
of Neurological Disorders and Stroke, for his support in statistical
analysis.
References
1. de The G, Bomford R. An HTLV-I vaccine: why,
how, for whom? AIDS Res Hum Retroviruses.
1993;9:381-386.
2. Osame M, Usuku K, Izumo S, et al. HTLV-I associated myelopathy, a new clinical entity [letter].
Lancet. 1986;1:1031-1032.
3. Gessain A, Barin F, Vernant JC, et al. Antibodies
to human T-lymphotropic virus type-I in patients
with tropical spastic paraparesis. Lancet. 1985;2:
407-410.
4. Kaplan JE, Osame M, Kubota H, et al. The risk of
development of HTLV-I–associated myelopathy/
tropical spastic paraparesis among persons infected with HTLV-I. J Acquir Immune Defic Syndr.
1990;3:1096-1101.
5. Trujillo JM, Concha M, Munoz A, et al. Seroprevalence and cofactors of HTLV-I infection in Tumaco, Colombia. AIDS Res Hum Retroviruses.
1992;8:651-657.
6. Uchiyama T, Yodoi J, Sagawa K, Takatsuki K,
Uchino H. Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood. 1977;50:
481-492.
7. Uchiyama T. Human T cell leukemia virus type I
(HTLV-I) and human diseases. Annu Rev Immunol. 1997;15:15-37.
8. Gessain A, Saal F, Gout O, et al. High human Tcell lymphotropic virus type I proviral DNA load
with polyclonal integration in peripheral blood
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
94
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
YAMANO et al
mononuclear cells of French West Indian, Guyanese, and African patients with tropical spastic
paraparesis. Blood. 1990;75:428-433.
9. Kira J, Koyanagi Y, Yamada T, et al. Increased
HTLV-I proviral DNA in HTLV-I–associated myelopathy: a quantitative polymerase chain reaction study [published erratum appears in Ann
Neurol 1991;29:363]. Ann Neurol. 1991;29:194201.
10. Nagai M, Usuku K, Matsumoto W, et al. Analysis
of HTLV-I proviral load in 202 HAM/TSP patients
and 243 asymptomatic HTLV-I carriers: high proviral load strongly predisposes to HAM/TSP.
J Neurovirol. 1998;4:586-593.
11. Jacobson S, Zaninovic V, Mora C, et al. Immunological findings in neurological diseases associated with antibodies to HTLV-I: activated lymphocytes in tropical spastic paraparesis. Ann Neurol.
1988;23:S196–200.
12. Jacobson S, Gupta A, Mattson D, Mingioli E, McFarlin DE. Immunological studies in tropical spastic paraparesis. Ann Neurol. 1990;27:149-156.
13. Parker CE, Daenke S, Nightingale S, Bangham
CR. Activated, HTLV-1–specific cytotoxic T-lymphocytes are found in healthy seropositives as
well as in patients with tropical spastic paraparesis. Virology. 1992;188:628-636.
14. Shimoyama M, Minato K, Tobinai K, et al. Atypical
adult T-cell leukemia-lymphoma: diverse clinical
manifestations of adult T-cell leukemia-lymphoma. Jpn J Clin Oncol. 1983;13:165-187.
15. Sugamura K, Fujii M, Kannagi M, et al. Cell surface phenotypes and expression of viral antigens
of various human cell lines carrying human T-cell
leukemia virus. Int J Cancer. 1984;34:221-228.
16. Tochikura T, Iwahashi M, Matsumoto T, et al. Effect of human serum anti-HTLV antibodies on viral antigen induction in vitro cultured peripheral
lymphocytes from adult T-cell leukemia patients
and healthy virus carriers. Int J Cancer. 1985;36:
1-7.
17. Gessain A, Saal F, Giron M, et al. Cell surface
phenotype and human T lymphotropic virus type
1 antigen expression in 12 T cell lines derived
from peripheral blood and cerebrospinal fluid of
West Indian, Guyanese and African patients with
tropical spastic paraparesis. J Gen Virol. 1990;
71:333-341.
18. Kinoshita T, Shimoyama M, Tobinai K, et al. Detection of mRNA for the tax1/rex1 gene of human
T-cell leukemia virus type I in fresh peripheral
blood mononuclear cells of adult T-cell leukemia
patients and viral carriers by using the polymerase chain reaction. Proc Natl Acad Sci U S A.
1989;86:5620-5624.
19. Hanon E, Hall S, Taylor GP, et al. 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. Blood.
2000;95:1386-1392.
20. Kubota R, Kawanishi T, Matsubara H, Manns A,
Jacobson S. HTLV-I specific IFN-gamma⫹ CD8⫹
lymphocytes correlate with the proviral load in
peripheral blood of infected individuals. J Neuroimmunol. 2000;102:208-215.
21. Nagai M, Kubota R, Greten T, et al. Increased
activated human T cell lymphotropic virus type I
(HTLV-I) Tax11–19-specific memory and effector
CD8⫹ cells in patients with HTLV-I-associated
myelopathy/tropical spastic paraparesis: correlation with HTLV-I provirus load. J Infect Dis. 2001;
183:197-205.
22. O’Brien W, Hartigan P, Martin D, et al. Changes in
plasma HIV-1 RNA and CD4⫹ lymphocyte counts
and the risk of progression to AIDS. Veterans Af-
fairs Cooperative Study Group on AIDS. N Engl
J Med. 1996;334:426-431.
23. Mellors JW, Munoz A, Giorgi JV, et al. Plasma
viral load and CD4⫹ lymphocytes as prognostic
markers of HIV-1 infection. Ann Intern Med. 1997;
126:946-954.
24. Osame M. Reveiw of WHO Kagoshima meeting
and diagnostic guidelines for HAM/TSP. In: Blattner W, ed. Human Retrovirology HTLV. New York,
NY: Raven Press; 1990:191-197.
38.
39.
25. Kurtzke J. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale
(EDSS). Neurology. 1983;33:1444-1452.
26. Miyoshi I, Kubonishi I, Yoshimoto S, et al. Type C
virus particles in a cord T-cell line derived by cocultivating normal human cord leukocytes and
human leukaemic T cells. Nature. 1981;294:770771.
27. Poiesz BJ, Ruscetti FW, Gazdar AF, et al. Detection and isolation of type C retrovirus particles
from fresh and cultured lymphocytes of a patient
with cutaneous T-cell lymphoma. Proc Natl Acad
Sci U S A. 1980;77:7415-7419.
28. Koenig S, Woods RM, Brewah YA, et al. Characterization of MHC class I restricted cytotoxic T cell
responses to tax in HTLV-1 infected patients with
neurologic disease. J Immunol. 1993;151:38743883.
29. Kamihira S, Dateki N, Sugahara K, et al. Realtime polymerase chain reaction for quantification of HTLV-1 proviral load: application for analyzing aberrant integration of the proviral DNA
in adult T-cell leukemia. Int J Hematol. 2000;72:
79-84.
30. Sun R, Ku J, Jayakar H, et al. Ultrasensitive reverse transcription-PCR assay for quantitation of
human immunodeficiency virus type 1 RNA in
plasma. J Clin Microbiol. 1998;36:2964-2969.
31. Kobayashi NK, Sabe H, Shigesada H, Noma K,
Honjo T, Hatanaka M. Genomic structure of HTLV
(human T-cell leukemia virus): detection of defective genome and its amplification in MT-2 cells.
EMBO J. 1984;3:1339-1343.
32. Cimarelli A, Duclos CA, Gessain A, et al. Quantification of HTLV-II proviral copies by competitive
polymerase chain reaction in peripheral blood
mononuclear cells of Italian injecting drug users,
central Africans, and Amerindians. J Acquir Immune Defic Syndr Hum Retrovirol. 1995;10:198204.
33. Albrecht B, Collins ND, Newbound GC, Ratner L,
Lairmore MD. Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative
competitive polymerase chain reaction. J Virol
Methods. 1998;75:123-140.
34. Furukawa Y, Osame M, Kubota R, Tara M, Yoshida M. Human T-cell leukemia virus type-1
(HTLV-1) Tax is expressed at the same level in
infected cells of HTLV-1–associated myelopathy or tropical spastic paraparesis patients as in
asymptomatic carriers but at a lower level in
adult T-cell leukemia cells. Blood. 1995;1:18651870.
35. Jeffery KJ, Usuku K, Hall SE, et al. HLA alleles
determine human T-lymphotropic virus-I (HTLV-I)
proviral load and the risk of HTLV-I-associated
myelopathy. Proc Natl Acad Sci U S A. 1999;96:
3848-3853.
36. Nakagawa M, Izumo S, Ijichi S, et al. HTLV-I-associated myelopathy: analysis of 213 patients
based on clinical features and laboratory findings.
J Neurovirol. 1995;1:50-61.
37. Nagasato K, Nakamura T, Ohishi K, et al. Active
production of anti-human T-lymphotropic virus
type I (HTLV-I) IgM antibody in HTLV-I-associ-
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
ated myelopathy. J Neuroimmunol. 1991;32:
105-109.
Sei S, Kleiner DE, Kopp JB, et al. Quantitative
analysis of viral burden in tissues from adults and
children with symptomatic human immunodeficiency virus type 1 infection assessed by polymerase chain reaction. J Infect Dis. 1994;170:
325-333.
Meylan PR, Burgisser P, Weyrich SC, Spertini F.
Viral load and immunophenotype of cells obtained from lymph nodes by fine needle aspiration as compared with peripheral blood cells in
HIV-infected patients. J Acquir Immune Defic
Syndr Hum Retrovirol. 1996;13:39-47.
Greenough T, Brettler D, Somasundaran M, Panicali D, Sullivan J. Human immunodeficiency virus
type 1-specific cytotoxic T lymphocytes (CTL),
virus load, and CD4 T cell loss: evidence supporting a protective role for CTL in vivo. J Infect Dis.
1997;176:118-125.
Ogg G, Jin X, Bonhoeffer S, et al. Quantitation of
HIV-1-specific cytotoxic T lymphocytes and
plasma load of viral RNA. Science. 1998;279:
2103-2106.
Usuku K, Sonoda S, Osame M, et al. HLA haplotype-linked high immune responsiveness against
HTLV-I in HTLV- I–associated myelopathy: comparison with adult T-cell leukemia/lymphoma. Ann
Neurol. 1988;23:S143–150.
Minato S, Itoyama Y, Goto I, Yamamoto N. Expression of HTLV-I antigen in cultured peripheral
blood mononuclear cells from patients with
HTLV-I associated myelopathy. J Neurol Sci.
1988;87:233-244.
Elovaara I, Koenig S, Brewah AY, et al. High human T cell lymphotropic virus type 1 (HTLV-1)specific precursor cytotoxic T lymphocyte frequencies in patients with HTLV-1–associated
neurological disease. J Exp Med. 1993;177:15671573.
Furukawa Y, Yamashita M, Usuku K, et al. Phylogenetic subgroups of human T cell lymphotropic
virus (HTLV) type I in the tax gene and their association with different risks for HTLV-I–associated
myelopathy/tropical spastic paraparesis. J Infect
Dis. 2000;182:1343-1349.
Nomoto M, Utatsu Y, Soejima Y, Osame M. Neopterin in cerebrospinal fluid: a useful marker
for diagnosis of HTLV-I–associated myelopathy/tropical spastic paraparesis. Neurology.
1991;41:457.
Ali A, Rudge P, Dalgleish AG. Neopterin concentrations in serum and cerebrospinal fluid in
HTLV-I infected individuals. J Neurol. 1992;239:
270-272.
Nakagawa M, Nakahara K, Maruyama Y, et al.
Therapeutic trials in 200 patients with HTLV-I–
associated myelopathy/ tropical spastic paraparesis. J Neurovirol. 1996;2:345-355.
Lehky TJ, Levin MC, Kubota R, et al. Reduction
in HTLV-I proviral load and spontaneous lymphoproliferation in HTLV-I–associated myelopathy/tropical spastic paraparesis patients treated
with humanized anti-Tac. Ann Neurol. 1998;44:
942-947.
Jacobson S, Shida H, McFarlin DE, Fauci AS,
Koenig S. Circulating CD8⫹ cytotoxic T lymphocytes specific for HTLV-I pX in patients with
HTLV-I associated neurological disease. Nature.
1990;348:245-248.
Pique C, Ureta-Vidal A, Gessain A, et al. Evidence for the chronic in vivo production of human T cell leukemia virus type I Rof and Tof
proteins from cytotoxic T lymphocytes directed
against viral peptides. J Exp Med. 2000;191:
567-572.
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
2002 99: 88-94
doi:10.1182/blood.V99.1.88
Correlation of human T-cell lymphotropic virus type 1 (HTLV-1) mRNA with
proviral DNA load, virus-specific CD8 + T cells, and disease severity in
HTLV-1−associated myelopathy (HAM/TSP)
Yoshihisa Yamano, Masahiro Nagai, Meghan Brennan, Carlos A. Mora, Samantha S Soldan, Utano
Tomaru, Norihiro Takenouchi, Shuji Izumo, Mitsuhiro Osame and Steven Jacobson
Updated information and services can be found at:
http://www.bloodjournal.org/content/99/1/88.full.html
Articles on similar topics can be found in the following Blood collections
Clinical Trials and Observations (4525 articles)
Immunobiology (5474 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.