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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-08-2477.
IMMUNOBIOLOGY
From the Third Department of Internal Medicine, Center
for Chronic Viral Diseases, and Department of Medical Informatics,
Faculty of Medicine, Kagoshima University, Kagoshima,
Japan
Human T-lymphotropic virus type 1 (HTLV-1)-associated
myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease caused by HTLV-1 infection, in which
HTLV-1-infected CD4+ T cells and HTLV-1-specific
CD8+ T cells may play a role in the disease pathogenesis.
Patients with HAM/TSP have high proviral loads despite vigorous
virus-specific CD8+ T-cell responses; however, it is
unknown whether the T cells are efficient in eliminating the virus in
vivo. To define the dynamics of HTLV-1-specific CD8+
T-cell responses, we investigated longitudinal alterations in HTLV-1
proviral load, amino acid changes in an immunodominant viral epitope,
frequency of HTLV-1-specific T cells, and degeneracy of T-cell
recognition in patients with HAM/TSP. We showed that the frequency and
the degeneracy of the HTLV-1-specific CD8+ T cells
correlated well with proviral load in the longitudinal study. The
proviral load was much higher in a patient with low degeneracy of
HTLV-1-specific T cells compared to that in a patient with comparable
frequency but higher degeneracy of the T cells. Furthermore, in a
larger number of patients divided into 2 groups by the proviral load,
those with high proviral load had lower degeneracy of T-cell
recognition than those with low proviral load. Sequencing analysis
revealed that epitope mutations were remarkably increased in a patient
when the frequency and the degeneracy were at the lowest. These data
suggest that HTLV-1-specific CD8+ T cells with degenerate
specificity are increased during viral replication and control the
viral infection.
(Blood. 2003;101:3074-3081) Virus-specific CD8+ T cells recognize
short peptide fragments bound to the cleft of major histocompatibility
complex (MHC) class I molecules, which are endogenously processed
within virus-infected cells.1,2 The CD8+ T
cells play a pivotal role in controlling viral infection in both the
acute and chronic phase. Human T-lymphotropic virus type 1 (HTLV-1) is
a retrovirus that preferentially and persistently infects
CD4+ lymphocytes in vivo.3 Even though HTLV-1
infection is lifelong, fewer than 1% of the individuals infected with
HTLV-1 develop a neurologic disease termed HTLV-1-associated
myelopathy/tropical spastic paraparesis (HAM/TSP) or a hematologic
disease termed adult T-cell leukemia (ATL); the vast majority of
individuals remain in the asymptomatic carrier state.4-6
HAM/TSP is an inflammatory disease in the spinal cord where
inflammatory cells, predominantly CD8+ T cells, infiltrate
the perivascular area.7 Patients with HAM/TSP show spastic
paraparesis and sphincter dysfunction with mild sensory dysfunction,
which are consistent with the pathologic changes in the spinal
cord.8 It has been demonstrated that the HTLV-1 proviral
load and the frequency of HTLV-1-specific CD8+ T cells are
increased in the peripheral blood in patients with HAM/TSP compared
with HTLV-1 carriers.9-11 In the cerebrospinal fluid in
patients with HAM/TSP, HTLV-1-specific CD8+ T cells
accumulate and HTLV-1-expressing CD4+ T cells are
increased.11,12 Furthermore, HTLV-1-specific
CD8+ T cells and the expression of HTLV-1 gene products
have been demonstrated in the central nervous system.13,14
Therefore, it has been suggested that these cells may play an important
role in the pathogenesis of HAM/TSP.15
HTLV-1-specific T cells show strong killing activity toward
HTLV-1-infected cells in vitro and the frequency is extraordinarily high in the circulation of patients with HAM/TSP,10
reaching over 10% of the CD8+ cell
population.16 The CD8+ T-cell response is
preferentially directed against the HTLV-1 Tax protein. In
HLA-A2+ patients especially, it has been shown that HTLV-1
Tax 11-19 is an immunodominant epitope and is one of the strongest
binding peptides to the HLA-A2 molecule.17 Despite the
vigorous T-cell response to the virus, patients with HAM/TSP show
increased proviral load. Therefore, the question of whether the
increased HTLV-1-specific CD8+ T cells are effective in
killing virus-infected cells in vivo arises. To answer this question, a
longitudinal study was conducted to examine the viral state and
virus-specific CD8+ T-cell responses in patients with
HAM/TSP, in which alterations in proviral load, amino acid changes in
the epitope, HTLV-1-specific T-cell frequency, and degeneracy of
antigen recognition of the T cells were investigated. The high
frequency of the circulating HTLV-1-specific CD8+ T cells
to the immunodominant Tax 11-19 and increased proviral load afford a
unique opportunity to examine detailed virus-host immunity interactions
focusing on a single epitope in bulk peripheral blood mononuclear cells
(PBMCs) in patients with HAM/TSP.
Patients
Peptides
Intracellular cytokine detection by flow cytometry The assay was conducted by a modified protocol as previously described.16 Briefly, Hmy2.C1R cells transfected with HLA-A*0201 (Hmy-A2) were prepulsed with 1 µM Tax 11-19 or APL for 1 hour and washed. Cryopreserved PBMCs were quickly thawed and washed. Then 5 × 105 PBMCs were cocultivated with the same number of peptide-prepulsed Hmy-A2 cells for 6 hours. Brefeldin A (Sigma, Tokyo, Japan) was added to the cells at a final concentration of 10 µg/mL at the beginning of the culture to minimize the expression of HTLV-1 protein in the infected cells, which may lead to activation of HTLV-1-specific T cells.22 After culture, cells were harvested, washed, and stained with antihuman CD8 antibody conjugated with PC5 (Beckman-Coulter, Tokyo, Japan) at 4°C for 20 minutes. Cells were washed and fixed with 4% paraformaldehyde for 5 minutes, then washed again. The cells, resuspended in 50 µL permeabilization buffer containing 0.1% saponin (Sigma), were stained with antihuman interferon (IFN-) antibody conjugated with
fluorescein isothiocyanate (FITC; Pharmingen, San Diego, CA) for 20 minutes. Epics-XL flow cytometer and SYSTEM II software were used for
fluorescent signal detection and data analysis (Beckman-Coulter).
Lymphocytes were readily distinguished from Hmy-A2 cells by size and
were gated on forward and side scatter image. Ten thousand
CD8+ cells were further gated and the proportion of
IFN-+ cells in the CD8+ cell population was
analyzed. The frequency of peptide-specific CD8+ T cells
was obtained by subtracting the percentage of IFN-+
cells without peptide from that with a peptide. The degeneracy of
T-cell recognition is evaluated according to the degree by which the T
cell recognizes altered peptides from a cognate peptide at the T-cell
clone level. Therefore, the total degeneracy of virus-specific T-cell
population in the PBMCs could be evaluated by relative T-cell
recognition of an APL against the cognate peptide. The relative T-cell
recognition of an APL against Tax 11-19 was given by the following
formula: (frequency of APL-specific T cells)/(frequency of Tax
11-19-specific T cells) × 100. The functional T-cell avidity to the
antigen was estimated as the peptide concentration required for
half-maximum IFN- production.
Quantitative PCR for HTLV-1 proviral load The method used has been previously described.9 Briefly, gDNA was extracted from PBMCs by using Qiagen DNA extraction kit (Qiagen, Tokyo, Japan). Approximately 100 µg DNA was subjected to a real-time PCR using a TaqMan probe. The fluorescent signal was detected by an ABI PRISM 7700 sequence detector (Applied Biosystems, Chiba, Japan). The copy number of the target gene in the sample was estimated by the standard curves. HTLV-1 copy number per 104 PBMCs was calculated according to the following formula: (copy number of HTLV-I tax gene in the sample)/((copy number of -globin gene in the
sample)/2) × 10.4 The assay was conducted in triplicate.
Sequencing analysis of HTLV-1 tax gene The method was previously described.18 Briefly, 100 ng DNA was amplified by 35 cycles of PCR using primers PXO1+: 5'-TCGAAACAGCCCTGCAGATA-3' at position 7257-7276 and PX22 :
5'-TGGTGGGCAAACAGTCTTCG-3' at position 7928-7947. One microliter of the
first PCR products was further used for 20 cycles of nested PCR using
internal primers PXI1+: 5'-ATACAAAGTTAACCATGCTT-3' at position
7274-7293 and PXI1: 5'-GGGTTCCATGTATCCATTTC-3' at position 7644-7663. Amplified DNA products were purified using QIA quick purification kit
(Qiagen). The purified tax gene was subcloned into pCR-Blunt
II-TOPO cloning vector (Invitrogen, Burlingame, CA). The vector
was linearized by EcoRI digestion, which does not cut the
tax gene, and purified by the QIA quick purification kit.
The tax gene was sequenced with PXI1+ or PXI1 primer using
dye terminator DNA sequencing kit (Applied Biosystems) in an automatic
sequencer (377 DNA Sequencer, Applied Biosystems). Approximately 50 clones were sequenced in each sample and mutations in the
tax gene encoding Tax 11-19 peptide were confirmed by
sequencing in the reverse direction.
Frequency of HTLV-1-specific CD8+ T cells correlated with the proviral load To optimize a concentration of peptide, which induced cytokine production in antigen-specific T cells, we conducted a peptide titration assay. IFN- production in bulk PBMCs in response to Tax
11-19 peptide appeared at 0.001 nM and reached the maximum over 1 nM,
whereas weak responses to some analog peptides were observed at 1000 nM
(data not shown). Therefore, we used peptides at 1 µM in the
subsequent study. Figure 1 shows a
representative IFN- production from CD8+ cells in PBMCs
from patient no. 31.
A high frequency of Tax 11-19-specific CD8+ T cells, with
a lesser extent to APLs, and a low frequency to antigen-presenting cells (APCs) without peptide (NP, background) were detected. The background was 0.42% ± 0.42% (mean ± SD) in 53 HLA-A2
HAM/TSP patients. Peptide-specific CD8+ T-cell frequencies
were estimated by subtraction of percentage of IFN- As shown in Figure 2A-B, in patient no. 31, the alterations in proviral load and frequency of HTLV-1 Tax 11-19-specific CD8+ T cells during 8 years ranged from 506 to 674 copies/104 PBMCs and from 6.7% to 19.5%, respectively. The CD8+ T cells were effectively increased in parallel with the slight increase in the proviral load. The alterations in patient no. 38 were from 1595 to 2225 copies/104 PBMCs and from 2.2% to 3.1%, respectively; the increase in virus-specific T-cell frequency was small. In patient no. 48, the alterations were from 93 to 605 copies/104 PBMCs and from 0.8% to 5.5%, respectively. The degree of the increase in virus-specific T-cell frequency to the increase of proviral load was different in each patient; however, HTLV-1 Tax 11-19-specific CD8+ T-cell frequency correlated well with the proviral load in each patient, suggesting that the HTLV-1 Tax 11-19-specific CD8+ T cells proliferate to control viral replication. Degeneracy of T-cell recognition in HTLV-1-specific CD8+ T cells correlated with the proviral load To test whether T-cell recognition of the antigen may be altered during the clinical course, especially when the proviral load was increased, a combination of intracellular cytokine detection and APL was used. With this method, the degeneracy of T-cell recognition in the HTLV-1-specific CD8+ T-cell population can be evaluated. For example, in Figure 1, the Tax 11-19-specific CD8+ T cells were relatively tolerated with the alanine substitution at position 6, and, to a lesser extent at position 5, whereas rarely at positions 4 and 8. The relative T-cell responses to the APL against those to the Tax 11-19 peptide were calculated according to the formula mentioned in "Patients, materials, and methods." For example, the calculated value was 14.9% for G4A, 37.9% for Y5A, 66.2% for P6A, and 7.7% for Y8A from the data in Figure 1. The relative T-cell response to the APL was plotted according to the time course in the 3 patients (Figure 2C) and mean fluorescence intensity (MFI) of IFN--FITC in the peptide-specific CD8+ T cells was
graphed (Figure 2D). Although the relative T-cell response in patient
no. 31 varied according to each APL, P6A was highly tolerated over
40%, and Y5A recognition was approximately 30%, but the recognitions
for G4A and Y8A were low. In patient no. 38, the relative APL
recognition was low compared to that in patient no. 31, in whom the
responses to all the APLs were less than 17%. In patient no.
48, the T-cell response was relatively high to all the APLs except for
Y8A, which reached up to 90% for P6A. This indicated that T-cell
recognition of HTLV-1 Tax 11-19 peptide is strict in patient no. 38, but not in patient no. 48. These data suggest that not only the
virus-specific T-cell frequency but also the extent of degeneracy in
T-cell responses differ among the patients.
In the longitudinal study (Figure 2C), it is striking that the
recognition for some APLs correlated with the frequency of the
virus-specific T cells and the proviral load. This was particularly dramatic in patient no. 31, who showed a high frequency of HTLV-1 Tax
11-19-specific CD8+ T cells. For example, the T-cell
recognitions of P6A and Y5A were well associated with the proviral load
and the virus-specific T cell frequency (Figure 2A-C), whereas the
recognition of G4A and Y8A was not changed. In patient no. 38, the
relative T-cell responses to P6A and Y5A correlated with the proviral
load; however, the G4A recognition correlated with neither the proviral
load nor the T-cell frequency (Figure 2A-C). In patient no. 48, the curves of CD8+ T-cell recognition of P6A and G4A correlated
with the curves of the proviral load and the frequency, but were not
clear in the response to Y5A and Y8A (Figure 2A-C). Although
alterations in MFI of IFN- To compare the effect of HTLV-1-specific T cells on the proviral load
among these 3 patients, we calculated the average of each factor during
the time course (Table 1). Patient no. 38 had the highest proviral load with the lowest frequency and degeneracy of the virus-specific T cells. Patient no. 48 had the same frequency of
the HTLV-1-specific T cells as patient no. 38, but increased degeneracy of 52.3% for the APLs and the lowest proviral load. Patient
no. 31 had extremely high virus-specific T-cell frequency of 14.0%,
but the proviral load was higher than that in patient no. 48. This
patient showed a moderate extent of degeneracy in T-cell recognition
among all the patients included.
To investigate whether or not the degeneracy of T-cell recognition is
associated with the proviral load as observed in patients no. 38 and
no. 48, we measured degeneracy and proviral load in a series of 22 patients with HAM/TSP who had HLA-A*02 haplotype. The proviral load of
these patients ranged from 2230 to 130 copies/104 PBMCs,
with the median of 481 copies/104 PBMCs. We divided the
patients into 2 groups according to the following: patients with
proviral load more than the median were included in high proviral load
group (n = 11), whereas patients with proviral load less than the
median were included in low proviral load group (n = 11). As shown in
Figure 3, the patients with low proviral
load showed an increased degeneracy of HTLV-1 Tax 11-19-specific T
cells as compared to the patients with high proviral load. This was
observed when Y5A peptide was used, but not when the other peptides
were used. These data suggest that virus-specific T cells with
increased degeneracy have an advantage to control viral infection.
To test whether the increased degeneracy of T-cell recognition resulted from increased avidity of Tax 11-19-specific CD8+ T cells to the APCs, we conducted antigen titration assays using wild-type Tax 11-19 peptide at various concentrations ranging from 0.001 to 1000 nM. As shown in Figure 4A, the antigen
concentration required to reach the half-maximum number of
IFN-
HTLV-1-specific T-cell frequency was higher in patients with HAM/TSP than in asymptomatic HTLV-1 carriers It would be of interest to know if degeneracy of T-cell recognition in HTLV-1-specific T cells differed between patients with HAM/TSP and asymptomatic HTLV-1 carriers. Therefore, we measured frequencies of HTLV-1 Tax 11-19-specific CD8+ T cells in CD8+ T-cell population both in 47 patients with HAM/TSP and 32 asymptomatic HTLV-1 carriers, who had HLA-A*02 haplotype. The frequency was 1.90% ± 2.68% (mean ± SD) and 0.25% ± 0.21%, respectively. The frequency of HTLV-1 Tax 11-19-specific CD8+ T cells was significantly higher in HAM/TSP patients than in the carriers (P < .0001; Figure 5). Unfortunately, it was difficult to investigate degeneracy of HTLV-1 Tax 11-19-specific T cells in the carriers because the frequency of the T cells in the carriers was too small to evaluate T-cell degeneracy.
Variant epitopes were increased in a patient with low frequency and limited degeneracy in the virus-specific T cells To determine if degeneracy or frequency of the HTLV-1-specific CD8+ T cells may have an effect on the viral epitope variations, the HTLV-1 tax gene containing the sequence coding Tax 11-19 peptide was sequenced at 3 time points in the 3 patients (Table 2).
For this experiment 2 DNA samples with low virus-specific T-cell frequencies and one sample with the highest frequency during the time course were chosen. In patient no. 31, the amino acid mutation rate in the Tax 11-19 ranged from 2.0% to 6.3%. In patient no. 48, the mutation rate was also not widely changed from 2.1% to 6.3%. However, in patient no. 38 who had relatively high proviral load and low frequency of the HTLV-1-specific T cells (Figure 2; Table 1), the variation rate reached 20.4% in the sample drawn on June 24, 1999. In this time point, the frequency and the degeneracy of the virus-specific T cells were the lowest in the time course (Figure 2B-C). The numbers of amino acid substitutions of Tax11-19 in the overall 445 clones sequenced were 2, 1, 3, 9, 4, 1, 3, 2, and 1 in the order of the amino acid position, respectively (Table 2). This indicates that the substitutions were frequently observed at positions 4 and 5. The substitution of glycine by arginine at position 4 was most frequently observed with 9 of 26 variants (34.6%). When we predicted the HLA-A2-binding affinity of the variants through the Internet, only 6 of 26 variants would make a lower HLA-A2-binding affinity than Tax 11-19 (Table 2). There was no accumulation of variant epitope during the time course as seen in HIV infection.23-25
In this longitudinal study, we have shown that: (1) the HTLV-I Tax 11-19-specific CD8+ T-cell frequency correlated with the proviral load during the time course of the disease; (2) the T cells showed an increased degeneracy of T-cell recognition when the T-cell frequency and the proviral load were increased; (3) in 2 patients with comparable virus-specific T-cell frequency, the proviral load was much higher in the patient who had low degeneracy of the T cells as compared to the other patient who had high degeneracy and the patients with high proviral load had lower degeneracy than those with low proviral load; (4) the degeneracy of T-cell recognition was independent of the T-cell avidity; (5) the frequency of HTLV-1 Tax 11-19-specific T cells is significantly higher in HAM/TSP patients than in the asymptomatic HTLV-I carriers; (6) the epitope variants were remarkably increased in a patient when the frequency and the degeneracy of the T cells were the lowest in the time course; (7) the amino acid changes in the epitope predominantly occurred in the central positions of the epitope; and (8) there is no accumulation of any variant epitope during the time course of the disease. When the proviral load was increased, HTLV-1-specific CD8+
T cells were increased and the T-cell population that recognized APLs
substituted at positions 5 and 6 was increased in patients no. 31 and
no. 38 (Figure 2A-C). Then, according to the decrease in proviral load,
both the frequency and the degeneracy of virus-specific T cells were
decreased. This suggests that the fine specificity of HTLV-1-specific
CD8+ T cells is altered according to the viral load in vivo
and that the degenerate specificity may give virus-specific T cells an advantage to eliminate the virus. When we compared several factors including viral load, frequency and degeneracy of virus-specific T
cells among the patients, patients no. 38 and no. 48 had comparable virus-specific T-cell frequencies (Table 1), and MFI curves of IFN- The increased frequency and degeneracy of HTLV-1-specific
CD8+ T cells in response to the increased proviral load may
have another biologic role. If virus-specific CD8+ T cells
have limited degeneracy, they could eliminate wild-type viruses.
However, some viruses with a mutation at the epitope would escape from
the T cells, thereby increasing the proportion of the mutant viruses.
The rate of epitope variants in patient no. 38 on June 24, 1999 was as high as 20.4%, whereas the rates were 0% or 5.9% at other
time points (Table 2). However, the degree of IFN- The HTLV-1-specific T-cell frequency correlated well with the proviral load in each individual. Recently, it has clearly been shown that HTLV-1 mRNA load correlates with proviral DNA load and virus-specific CD8+ T-cell frequency by a quantitative method.33 If the increased proviral load during the clinical course in our patients suggests an elevated level of HTLV-1 mRNA load, then increased Tax protein expression would effectively stimulate the Tax 11-19-specific T cells in vivo (Figure 2A-B). In HTLV-1 infection, it has been suggested that HTLV-1 predominantly replicates as a provirus via mitotic proliferation of the infected cells in vivo.3 High virus-specific T-cell response observed in patients with HAM/TSP suggests that the immune system is continuously exposed to the viral antigens in vivo.10 Recently, it has been shown that circulating CD4+ T cells infected with HTLV-1 rarely express viral antigens; however, they readily express the viral proteins in a 6-hour ex vivo culture and are subsequently killed by the CD8+ CTLs in vitro.34 Patient no. 31 showed a marked increase of HTLV-1-specific T-cell frequency (2.9 times elevation from 6.7% to 19.5%) corresponding to the increase of proviral load (1.3 times elevation from 506 to 674 copies/104 PBMCs) during the course (Figure 2A-B), suggesting that the T cells proliferated by a stimulation from HTLV-1. The proviral load was then reduced, implying that the T cells killed the virus-infected cells in vivo. However, the proviral load finally reached a final level of 506 copies/104 PBMCs despite continuous high virus-specific T-cell frequency on March 10, 1999. This suggests that silent HTLV-1-infected cells, which do not express the viral antigens, may survive in the body of the infected individual. A marked increase in proviral load is a virologic hallmark in patients with HAM/TSP as compared to asymptomatic HTLV-1 carriers.9 However, it is still controversial as to whether HTLV-1-specific T cells are increased in patients with HAM/TSP as compared to the carriers. It has been reported that circulating HTLV-1-specific T cells are significantly increased in patients with HAM/TSP as compared to asymptomatic HTLV-1 carriers with the use of CTL assay, limiting dilution assay, and intracellular cytokine assay.10,11,16 However, it has also been reported that HTLV-1-specific T cells are increased both in HAM/TSP patients and asymptomatic HTLV-1 carriers.35 It was not clear what causes this discrepancy. One possibility may be that the numbers of subjects included were small in both studies. In this study with a larger number of subjects, HTLV-1 Tax 11-19-specific T-cell frequency was significantly increased in the patients with HAM/TSP as compared to the carriers. It has been shown that HTLV-1 Tax 11-19-specific T cells accumulate and are activated in cerebrospinal fluid in patients with HAM/TSP as compared to the periphery and that CD8+ T cells accumulate in the spinal cords of patients with HAM/TSP.7,11,13 Collectively, these data suggest that HTLV-1-specific CD8+ T cells may play an important roll in the pathogenesis of HAM/TSP.15 Crystal structure analysis of the HLA-A2/Tax 11-19/ TCR complex in a
Tax 11-19-specific T-cell clone clearly shows that the amino acid at
position 5 fits into the pocket formed by the TCR V The naturally occurring variant epitope altered from glycine to
arginine at position 4 of Tax 11-19 epitope, designated as G4R
(LLFRYPVYV), is of interest, because the variant was predominantly investigated among the 3 patients (9 of 26 variants; Table 2). This
mutation has previously been demonstrated in HLA-A*02+
HAM/TSP patients and asymptomatic HTLV-1 carriers with different ethnic
origins and in HLA-A*02+ ATL patients.18,36 In
the present study, the variant did not become dominant among the
patients during the time course of the disease. The predicted
HLA-A2-binding affinity of G4R is comparable to that of cognate Tax
11-19 peptide (Table 2). If the variant peptide is endogenously
processed and expressed efficiently with HLA on the infected cells, it
is expected that G4R-specific CD8+ T cells might emerge and
kill the cells infected with the mutant virus. Therefore, we conducted
an experiment to investigate whether G4R-specific CD8+ T
cells may emerge during the time course. However, CD8+ T
cells in PBMCs did not recognize the G4R peptide during the course in
the 3 patients (data not shown), suggesting that the nonpredominance of
G4R did not result from an appearance of G4R-specific CD8+
T cells. It has been demonstrated that Tax protein acts as a trans-activator on its long terminal repeat and activates a
number of cellular genes such as interleukin 2 through nuclear
transcription factor NF- In conclusion, HTLV-1 persistently infects humans despite a vigorous virus-specific T-cell response, in part because the majority of the virus-infected cells rarely express viral antigens in vivo. During viral replication, HTLV-1-specific CD8+ T cells show increases in both frequency and degeneracy. Thereafter, the interaction between the T cells and the virus may transiently reach an equilibrium state. This high immune response to the virus may play a crucial role in the pathogenesis of HAM/TSP. The elucidation of the underlying mechanisms by which HTLV-1-infected cells rarely express the viral antigens in vivo will be beneficial in designing patient therapies.
We thank Ms Y. Nishino and T. Muramoto for the excellent technical assistance, and Samantha S. Soldan and Arlene R. Ng for the critical reading of the manuscript.
Submitted August 19, 2002; accepted December 2, 2002.
Prepublished online as Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood-2002-08-2477.
Supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research (OPSR) in Japan.
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.
Reprints: Ryuji Kubota, Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan; e-mail: kubotar{at}m2.kufm.kagoshima-u.ac.jp.
1. Zinkernagel RM, Doherty PC. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature. 1974;248:701-702[CrossRef][Medline] [Order article via Infotrieve]. 2. Townsend AR, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986;44:959-968[CrossRef][Medline] [Order article via Infotrieve].
3.
Richardson JH, Edwards AJ, Cruickshank JK, Rudge P, Dalgleish AG.
In vivo cellular tropism of human T-cell leukemia virus type 1.
J Virol.
1990;64:5682-5687
4.
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 5. 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[CrossRef][Medline] [Order article via Infotrieve]. 6. Osame M, Usuku K, Izumo S, et al. HTLV-I associated myelopathy, a new clinical entity. Lancet. 1986;1:1031-1032[Medline] [Order article via Infotrieve]. 7. Umehara F, Izumo S, Nakagawa M, et al. Immunocytochemical analysis of the cellular infiltrate in the spinal cord lesions in HTLV-I-associated myelopathy. J Neuropathol Exp Neurol. 1993;52:424-430[Medline] [Order article via Infotrieve]. 8. Osame M, Matsumoto M, Usuku K, et al. Chronic progressive myelopathy associated with elevated antibodies to human T-lymphotropic virus type I and adult T-cell leukemialike cells. Ann Neurol. 1987;21:117-122[CrossRef][Medline] [Order article via Infotrieve]. 9. 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[Medline] [Order article via Infotrieve]. 10. 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[CrossRef][Medline] [Order article via Infotrieve].
11.
Greten TF, Slansky JE, Kubota R, et al.
Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19-specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.
Proc Natl Acad Sci U S A.
1998;95:7568-7573 12. Moritoyo T, Izumo S, Moritoyo H, et al. Detection of human T-lymphotropic virus type I p40tax protein in cerebrospinal fluid cells from patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis. J Neurovirol. 1999;5:241-248[Medline] [Order article via Infotrieve].
13.
Levin MC, Lehky TJ, Flerlage AN, et al.
Immunologic analysis of a spinal cord-biopsy specimen from a patient with human T-cell lymphotropic virus type I-associated neurologic disease.
N Engl J Med.
1997;336:839-845 14. Moritoyo T, Reinhart TA, Moritoyo H, et al. Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes. Ann Neurol. 1996;40:84-90[CrossRef][Medline] [Order article via Infotrieve]. 15. Jacobson S. Cellular immune responses to HTLV-I: immunopathogenic role in HTLV-I-associated neurologic disease. J Acquir Immune Defic Syndr Hum Retrovirol. 1996;13(suppl 1):S100-106.
16.
Kubota R, Kawanishi T, Matsubara H, Manns A, Jacobson S.
Demonstration of human T lymphotropic virus type I (HTLV-I) tax-specific CD8+ lymphocytes directly in peripheral blood of HTLV-I-associated myelopathy/tropical spastic paraparesis patients by intracellular cytokine detection.
J Immunol.
1998;161:482-488 17. 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;151:3874-3883[Abstract].
18.
Furukawa Y, Kubota R, Tara M, Izumo S, Osame M.
Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia.
Blood.
2001;97:987-993 19. Utz U, Koenig S, Coligan JE, Biddison WE. Presentation of three different viral peptides, HTLV-1 Tax, HCMV gB, and influenza virus M1, is determined by common structural features of the HLA-A2.1 molecule. J Immunol. 1992;149:214-221[Abstract]. 20. Garboczi DN, Ghosh P, Utz U, Fan QR, Biddison WE, Wiley DC. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature. 1996;384:134-141[CrossRef][Medline] [Order article via Infotrieve]. 21. Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol. 1994;152:163-175[Abstract]. 22. Nuchtern JG, Bonifacino JS, Biddison WE, Klausner RD. Brefeldin A implicates egress from endoplasmic reticulum in class I restricted antigen presentation. Nature. 1989;339:223-226[CrossRef][Medline] [Order article via Infotrieve]. 23. Phillips RE, Rowland-Jones S, Nixon DF, et al. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature. 1991;354:453-459[CrossRef][Medline] [Order article via Infotrieve]. 24. Borrow P, Lewicki H, Wei X, et al. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat Med. 1997;3:205-211[CrossRef][Medline] [Order article via Infotrieve]. 25. Evans DT, O'Connor DH, Jing P, et al. Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef. Nat Med. 1999;5:1270-1276[CrossRef][Medline] [Order article via Infotrieve].
26.
Kalams SA, Johnson RP, Trocha AK, et al.
Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.
J Exp Med.
1994;179:1261-1271
27.
Marten NW, Stohlman SA, Smith-Begolka W, et al.
Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system.
J Immunol.
1999;162:3905-3914
28.
Silins SL, Cross SM, Elliott SL, et al.
Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.
J Exp Med.
1996;184:1815-1824
29.
McHeyzer-Williams MG, Davis MM.
Antigen-specific development of primary and memory T cells in vivo.
Science.
1995;268:106-111 30. Bachmann MF, Speiser DE, Ohashi PS. Functional maturation of an antiviral cytotoxic T-cell response. J Virol. 1997;71:5764-5768[Abstract]. 31. Bachmann MF, Barner M, Viola A, Kopf M. Distinct kinetics of cytokine production and cytolysis in effector and memory T cells after viral infection. Eur J Immunol. 1999;29:291-299[CrossRef][Medline] [Order article via Infotrieve].
32.
Horwitz MS, Yanagi Y, Oldstone MB.
T-cell receptors from virus-specific cytotoxic T lymphocytes recognizing a single immunodominant nine-amino-acid viral epitope show marked diversity.
J Virol.
1994;68:352-357
33.
Yamano Y, Nagai M, Brennan M, et al.
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).
Blood.
2002;99:88-94
34.
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 35. Parker CE, Daenke S, Nightingale S, Bangham CR. Activated, HTLV-1-specific cytotoxic Tlymphocytes are found in healthy seropositives as well as in patients with tropical spastic paraparesis. Virology. 1992;188:628-636[CrossRef][Medline] [Order article via Infotrieve]. 36. Niewiesk S, Daenke S, Parker CE, et al. Naturally occurring variants of human T-cell leukemia virus type I Tax protein impair its recognition by cytotoxic T lymphocytes and the transactivation function of Tax. J Virol. 1995;69:2649-2653[Abstract].
37.
Siekevitz M, Feinberg MB, Holbrook N, Wong-Staal F, Greene WC.
Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus, type I.
Proc Natl Acad Sci U S A.
1987;84:5389-5393
© 2003 by The American Society of Hematology.
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  R. Fukumoto, M. Dundr, C. Nicot, A. Adams, V. W. Valeri, L. E. Samelson, and G. Franchini Inhibition of T-Cell Receptor Signal Transduction and Viral Expression by the Linker for Activation of T Cells-Interacting p12I Protein of Human T-Cell Leukemia/Lymphoma Virus Type 1 J. Virol., September 1, 2007; 81(17): 9088 - 9099. [Abstract] [Full Text] [PDF]
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 R. Kubota, K. Hanada, Y. Furukawa, K. Arimura, M. Osame, T. Gojobori, and S. Izumo Genetic Stability of Human T Lymphotropic Virus Type I despite Antiviral Pressures by CTLs J. Immunol., May 1, 2007; 178(9): 5966 - 5972. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) measured by a quantitative real-time PCR
(copy/104 PBMCs). (B) Frequency of HTLV-1 Tax
11-19-specific CD8+ T cells in CD8+ cell
population (
), which were measured as shown in Figure 
and V
B.