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IMMUNOBIOLOGY
From the Departments of Immunology and Genito-Urinary
Medicine and Communicable Diseases, Imperial College School of
Medicine, St Mary's Campus, London, United Kingdom; the First
Department of Internal Medicine, Ehime University School of Medicine,
Shigenobu, Ehime, Japan; and the Department of Infectious Disease and
Immunology, Okinawa-Asia Research Center of Medical Science, Faculty of
Medicine, University of The Ryukyus, Nishihara, Okinawa, Japan.
The transactivator protein of human
T-lymphotropic virus I (HTLV-I), Tax, has been associated
with the up-regulation of several host cell genes, including
interleukin 2 (IL-2), the IL-2 receptor- Human T-lymphotropic virus type I (HTLV-I), which
belongs to the HTLV-bovine leukemia virus (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; ACs). However, approximately 2% to 3% develop an aggressive
T-cell malignancy, adult T-cell leukemia/lymphoma (ATL) and another 2%
to 3% develop a disabling chronic inflammatory disease, involving the
central nervous system (CNS) (HTLV-I-associated myelopathy/tropical
spastic paraparesis; HAM/TSP), the eyes, lungs, or skeletal
muscles.2
HTLV-I shares with other replication-competent retroviruses the 3 main
genomic regions of gag, pol, and env,
but unlike most leukemia viruses it has an additional region called
pX that codes for 2 transcriptional regulatory proteins, the
Tax and Rex proteins.2 The Rex protein stabilizes viral
messenger RNAs 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 up-regulate
several cellular genes including interleukin (IL)-2 receptor
(CD25) and IL-2.3-8
Several observations suggest that direct or indirect Tax-induced
up-regulation of cellular genes involved in lymphocyte proliferation or
inflammation plays a role in the pathogenesis of HTLV-I-associated diseases.2,3,9,12 It has been suggested that an autocrine loop involving IL-2 and the IL-2 receptor is an important mechanism by
which HTLV-I-infected cells proliferate in vivo.6-8 The
same mechanism could also play a part in the development of ATL.
Overproduction of interferon The effects of HTLV-I infection on cytokine production have so far been
studied either in mixed cell populations or in in vitro- derived
T-cell clones, because there has been no sensitive assay to identify
HTLV-I protein expression in individual cells. We wished to identify
the cytokines produced by T cells naturally infected with HTLV-I, with
minimal in vitro manipulation. We therefore used our recently described
sensitive flow cytometric assay to detect intracellular Tax protein
expression.13
Patients and cells
Cell surface staining
Detection of intracellular Tax and cytokines After cell surface labeling, cells were washed and permeabilized with PBS/7% normal goat serum containing 0.2% saponin (Sigma) (PBS-SAPO) for 10 minutes at room temperature. Permeabilized cells were washed and resuspended in PBS-SAPO containing an anti-HTLV-I Tax Mab (Lt-4; IgG3)15 or an isotype control mAb (Southern Biotechnology) for 20 minutes at room temperature. The cells were then washed twice and resuspended for 20 minutes at room temperature in PBS-SAPO containing FITC-labeled goat F(ab')2 antimouse IgG3 serum (Southern Biotechnology) and anti-tumor necrosis factor (anti-TNF) (Pharmingen), anti-IFN- (Pharmingen), anti-IL-2 (Beckman Coulter),
or anti-IL-4 (Pharmingen) mAb. Finally, the cells were washed twice and
analyzed by flow cytometry on a Coulter EPICS XL (Beckman Coulter).
Effect of HTLV-I Tax expression on infected PBMCs Short-term cultivation of PBMCs obtained from HTLV-I-infected patients is associated with increased Tax protein expression in infected PBMCs.13 We recently described a sensitive flow cytometric assay to detect intracellular Tax protein expression.13 In this study, we used this flow cytometric assay to characterize the biologic effect of Tax expression on naturally infected PBMCs. For this purpose, PBMCs were isolated from 3 ACs of the virus and 3 HAM/TSP patients and harvested directly or after 6 or 12 hours of cultivation in vitro. After being harvested, cell samples were fixed and processed to detect concomitantly Tax and the expression of 3 lymphocyte surface markers of activation (CD25, CD69) or proliferation (CD71). Figure 1 shows the results of one representative experiment of 6. The results show that Tax protein was not detected in freshly isolated PBMCs. In contrast, after 6 hours of cultivation, a significant proportion (about 55%) of PBMCs expressed the Tax protein. Interestingly, more than 50% of Tax-expressing cells were negative for the CD25, CD69, or CD71 markers at 6 hours. In contrast, after 12 hours of cultivation, the majority (> 75%) of Tax-expressing cells became positive for CD25, CD69, and CD71. These results indicate that a significant proportion of HTLV-I-infected PBMCs do not express activation/proliferation markers in peripheral blood. However, once they express the Tax protein for more than 6 to 12 hours, they become activated, which is consistent with previous studies showing the transactivating effect of Tax.3-7
IFN- and TNF, are thought to be involved in the
pathogenesis of HAM/TSP.9-12 We therefore investigated
whether activation of HTLV-I-infected PBMCs is associated with the
production of these proinflammatory cytokines. We also detected the
production of IL-2 and IL-4 in Tax-expressing lymphocytes, because of
the potential importance of IL-2 in HTLV-I infection6-8 and the role of IL-4 in controlling the balance of Th1/Th2 cells in the
immune response. PBMCs were isolated from 3 ACs of the virus and 3 HAM/TSP patients and cultivated for 6, 12, and 24 hours in the presence
of monensin to induce the accumulation of cytokines within activated
cells.16 In addition, CMA, which is an inhibitor of the
perforin-dependent cytotoxic pathway,17 was added to the
culture medium to prevent the destruction of Tax-expressing PBMCs by
HTLV-I-specific cytotoxic T lymphocytes (CTLs).13,18 The results of this time-course study
are shown in Table 1 and Figure
2. After 6 hours of
cultivation, Tax-expressing PBMCs were negative for IFN-, TNF, IL-2,
or IL-4. After 12 hours, IFN- but not IL-2, IL-4, or TNF was
detected in a proportion of Tax-expressing PBMCs. At this time point,
more than 10% of Tax-expressing PBMCs were capable of producing
IFN-. After 24 hours of cultivation, IL-2, IL-4, or TNF production
remained negative, whereas the frequency of IFN--producing PBMCs
continued to increase within Tax+ cells (Figure
2). Similar conclusions were made using PBMCs obtained from 3 ACs
of the virus and 3 patients with HTLV-I-associated neurological
disease (Table 1). Furthermore, the concomitant detection of IFN-
and another HTLV-I antigen, the p24 Gag protein, gave comparable
results. More than 10% of HTLV-I p24+ PBMCs were positive
for IFN- (data not shown).
The failure to detect IL-2, IL-4, or TNF could be due to poor
sensitivity of the intracellular cytokine detection assay used. To
investigate this possibility, PBMCs from the same 6 HTLV-I-infected individuals were activated using a polyclonal activator (a combination of PMA and A23187). After 6 hours of cultivation, cells were harvested and processed to detect IFN-
The MHC class II antigen presentation pathway is not involved in
the induction of IFN-
by SK/SD-specific CD4+ T cells.20 PBMCs
isolated from one AC and one patient with HAM/TSP were cultivated
overnight in the presence or absence of this anti-MHC class II mAb. The
cells were then harvested and processed for Tax and intracellular
cytokine detection. The results (Figure
4B) show that the presence of this
anti-MHC class II mAb did not inhibit the production of IFN- by
Tax-expressing cells. In contrast, a powerful inhibition of IFN-
production by SK/SD-specific CD4+ T cells was observed in
the presence of the anti-MHC class II mAb (Figure 4A), which confirmed
the ability of this mAb to inhibit the MHC class II presentation
pathway. These observations indicate that the MHC class II antigen
presentation pathway was not involved in the induction of IFN-
production by Tax-expressing PBMCs. The spontaneous IFN- production
associated with Tax expression in CD4+ T cells is therefore
more likely to be due to the direct or indirect transactivating effect
of Tax on certain host cell genes.3-7 In addition, IFN-
might be produced by HTLV-I-specific CD8+ T cells after
stimulation via the class I pathway of antigen presentation.
A proportion of Tax-expressing PBMCs are effector-memory T lymphocytes Previous reports have shown that the majority of HTLV-I-infected PBMCs carry markers of the memory phenotype of T lymphocytes.13,21 In accordance with these reports, we show here using PBMCs isolated from 3 ACs and 3 HAM/TSP patients that 79% to 87% of Tax-expressing PBMCs were indeed CD45RA CD45RO+ (Figure
5 and Table
3). In this context,
Sallusto and colleagues14 have recently shown that the
memory phenotype of T lymphocytes can be divided into 2 functionally
different populations, which they name central-memory and
effector-memory T lymphocytes, on the basis of CCR7 and CD45RA
expression.22 Furthermore, Sallusto and
coworkers14 demonstrated that cells of the central-memory phenotype recirculate within the central lymphoid tissues, whereas those with the effector-memory phenotype migrate in peripheral tissues.
Therefore, to characterize the recirculation pattern of
HTLV-I-infected PBMCs, we investigated the expression of CCR7 and
CD45RA in Tax-expressing PBMCs freshly isolated from 3 ACs and 3 HAM/TSP patients. Table 4 shows that
Tax-expressing PBMCs contain both central-memory T cells and
effector-memory T cells. We then investigated the expression of CD62L,
an adhesion molecule involved in the recirculation of T lymphocytes
through the central lymphoid tissues.14 Consistent with
the results presented above, we observed that a significant proportion
of Tax-expressing cells were CD62Llow (data not shown).
Together, these results indicate that a fraction of HTLV-I-infected
PBMCs expresses the effector-memory phenotype of T lymphocytes. This
implies that these effector-memory HTLV-I-infected PBMCs are able to
leave the circulation and migrate in the parenchyma of
peripheral tissues.
Tax is thought to up-regulate several cellular genes including
IL-2R (CD25) and IL-2.3-8
This abnormal gene expression could play a role in the pathogenesis of
the inflammatory and malignant diseases associated with the virus. In
this study, we used intracellular staining of HTLV-I Tax protein
to investigate the cytokine and surface marker phenotype of cells
that express Tax. Using this technique, we confirmed that
CD4+ lymphocytes naturally infected with HTLV-I
spontaneously up-regulate several cellular genes including markers of
activation (CD25, CD69) and proliferation (CD71) (Figure 1). We also
demonstrate that HTLV-I Tax expression is associated with the
production of IFN- Two hypotheses could explain the observed cellular activation of
Tax-expressing cells. First, Tax expression might directly or
indirectly induce the expression of host genes involved in cellular
activation. Several reports support this hypothesis and show the
transactivating capability of HTLV-I Tax on cellular genes.3-8 However, there is a second possibility. If
HTLV-I-infected cells carry a T-cell receptor (TCR) that is
specific for HTLV-I epitopes,18 HTLV-I protein expression
in vitro could lead to cellular activation of HTLV-I-infected
cells through HTLV-I peptide/MHC class II complex presentation.
However, Figure 4B shows that the presence of anti-MHC class II mAb did
not inhibit the production of IFN- Despite up-regulating CD25, CD69, CD71, and IFN- Cellular activation and proinflammatory cytokine production of
HTLV-I-infected cells could contribute to the pathogenesis of HAM/TSP
disease. It is well established that HTLV-I infection is associated
with a high proviral load in peripheral blood.23 Our
results suggest that infected cells are potentially harmful and could
induce nonspecific inflammatory responses in peripheral tissues. This
conclusion is based on the following observations on freshly isolated
HTLV-I infected cells: (1) There is abundant Tax expression in vitro
and a persistent activation of anti-Tax CTLs13; we
concluded that such Tax expression probably also occurs in vivo. (2)
Tax expression is associated with cellular activation, as shown by the
up-regulation of surface markers of activation and proliferation
(Figure 1). (3) Tax expression is also associated with the expression
of IFN-
In conclusion, we characterized here the phenotype of PBMCs that are
naturally infected with HTLV-I. Tax expression in those cells was
associated with cellular activation and with production of IFN-
We thank the staff and patients of St Mary's Hospital Medical Trust.
Submitted January 22, 2001; accepted March 28, 2001.
Supported by the Wellcome Trust (United Kingdom) and the Fonds National Belge de la Recherche Scientifique (FNRS, Belgium). E.H. is a Senior Research Assistant of the FNRS. The HTLV European Research Network supported by the European Community Biomed Programme provided a forum for critical appraisal of this work.
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: Charles Bangham, Department of Immunology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, W2 1PG, London, United Kingdom; e-mail: c.bangham{at}ic.ac.uk.
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F. Toulza, A. Heaps, Y. Tanaka, G. P. Taylor, and C. R. M. Bangham High frequency of CD4+FoxP3+ cells in HTLV-1 infection: inverse correlation with HTLV-1-specific CTL response Blood, May 15, 2008; 111(10): 5047 - 5053. [Abstract] [Full Text] [PDF]
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 K. S. Jones, C. Petrow-Sadowski, D. C. Bertolette, Y. Huang, and F. W. Ruscetti Heparan Sulfate Proteoglycans Mediate Attachment and Entry of Human T-Cell Leukemia Virus Type 1 Virions into CD4+ T Cells J. Virol., October 15, 2005; 79(20): 12692 - 12702. [Abstract] [Full Text] [PDF]
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 K. S. Jones, S. Akel, C. Petrow-Sadowski, Y. Huang, D. C. Bertolette, and F. W. Ruscetti Induction of Human T Cell Leukemia Virus Type I Receptors on Quiescent Naive T Lymphocytes by TGF-{beta} J. Immunol., April 1, 2005; 174(7): 4262 - 4270. [Abstract] [Full Text] [PDF]
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 Y. Yamano, C. J. Cohen, N. Takenouchi, K. Yao, U. Tomaru, H.-C. Li, Y. Reiter, and S. Jacobson Increased Expression of Human T Lymphocyte Virus Type I (HTLV-I) Tax11-19 Peptide-Human Histocompatibility Leukocyte Antigen A*201 Complexes on CD4+ CD25+ T Cells Detected by Peptide-specific, Major Histocompatibility Complex-restricted Antibodies in Patients with HTLV-I-associated Neurologic Disease J. E |
but not interleukin-2
by human T-lymphotropic virus type I-infected peripheral blood
mononuclear cells
Top
Abstract
Introduction
(IL-2R
indicates control; 
,
SK/SD. (B) Effect of the anti-MHC class II mAb on the production of
IFN-
