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IMMUNOBIOLOGY
From the Viral Immunology Section, Neuroimmunology
Branch, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, MD.
Peripheral blood mononuclear cells (PBMCs) from patients with
human T-cell lymphotropic virus type I (HTLV-I)-associated
myelopathy/tropical spastic paraparesis (HAM/TSP) proliferate
spontaneously in vitro. This spontaneous lymphoproliferation (SP)
is one of the immunologic hallmarks of HAM/TSP and is considered to be
an important factor related to the pathogenesis of HAM/TSP. However,
the cell populations involved in this phenomenon have not yet been
definitively identified. To address this issue, the study directly
evaluated proliferating cell subsets in SP with a flow cytometric
method using bromodeoxyuridine and Ki-67. Although both
CD4+ and CD8+ T cells proliferated
spontaneously, the percentage of proliferating CD8+ T cells
was 2 to 5 times higher than that of CD4+ T cells. In
addition, more than 40% of HTLV-I Tax11-19-specific CD8+
T cells as detected by an HLA-A*0201/Tax11-19 tetramer proliferated in
culture. In spite of this expansion of HTLV-I-specific
CD8+ T cells, HTLV-I proviral load did not decrease. This
finding will help elucidate the dynamics of in vivo virus-host
immunologic interactions that permit the coexistence of high
HTLV-I-specific CD8+ cytotoxic T-lymphocyte responses and
high HTLV-I proviral load in HAM/TSP.
(Blood. 2001;98:1506-1511) Human T-cell lymphotropic virus type I (HTLV-I) is
an exogenous human retrovirus that has been demonstrated to be the
etiologic agent in adult T-cell leukemia and a progressive neurologic
disease called HTLV-I-associated myelopathy/tropical spastic
paraparesis (HAM/TSP).1,2 HAM/TSP is caused by
preferential damage of the thoracic spinal cord and is characterized
clinically by muscle weakness, hyperreflexia, spasticity in the lower
extremities, and urinary disturbance. Although development of
this disease is not completely understood, virus-host immunologic
interactions have been suggested to play a role in the pathogenesis of
this disorder.
Although several immunologic parameters have been shown to be elevated
in patients with HAM/TSP, including high frequencies of circulating
HTLV-I-specific CD8+ cytotoxic T lymphocytes (CTL), high
antibody titers against HTLV-I antigens are found in both peripheral
blood and cerebrospinal fluid, as well as an increased expression of
cytokine and chemokines.3-7 The immunologic hallmark of
HTLV-I-infected individuals is the capacity of peripheral blood
mononuclear cells (PBMCs) to spontaneously proliferate in
vitro,8-11 that is, extraordinarily high uptake of
3H-thymidine (typically >100 000 cpm after 4 days in
culture) in the absence of exogenous antigens or stimulants. Moreover,
the magnitude of this spontaneous lymphoproliferation (SP) is more pronounced in HAM/TSP patients than in asymptomatic HTLV-I
carriers8-11 and has been used as an evaluation of
treatment for this disorder.12,13 However, it is still
unknown if this spontaneous proliferation is solely a consequence of
activation of CD4+ HTLV-I-infected T-cells or may in part
be due to expansion of CD8+ virus-specific lymphocytes.
HTLV-I has a preferential tropism for CD4+ T cells in
vivo,14 and a high amount of HTLV-I proviral load has been
reported in patients with HAM/TSP.15 The tax
region of HTLV-I encodes the Tax protein, a strong transactivator of
both viral and host genes, including interleukin 2 (IL-2) and IL-2
receptor (IL2r).16,17 Although HTLV-I Tax protein could
not be detected in fresh PBMCs ex vivo, Tax protein was found after
several hours of culture in vitro.18 These
observations suggested that the high SP particularly demonstrated from
PBMCs of patients with HAM/TSP may be due to HTLV-I-infected
CD4+ T cells proliferating by an IL2-IL2r autocrine loop
in vitro.19 Additionally, SP of specific cell subsets has
been examined directly from PBMCs of patients with HAM/TSP. With the
use of purified cell separation techniques, SP was observed in T cells
but not in B cells or monocytes.11 Moreover,
CD4+ cells were found to proliferate spontaneously in vitro
and to express HTLV-I antigens, whereas purified CD8+ cells
did not.20 However, purified CD8+ cells were
still capable of proliferating when cultured with irradiated autologous
HTLV-I-infected CD4+ cells in the presence of exogenous
IL-2.20 In addition, expansion of CD8+ cells
in SP in HAM/TSP has also been reported on the basis of a proportional
change of cell surface markers.21 These studies suggest
that both CD4+ and CD8+ cells proliferate in
SP. However, the methods used did not allow identification or
quantification of specific phenotypic cell subsets in SP.
In this study, we describe a flow cytometric method for directly
identifying proliferating cell subsets in SP. Bromodeoxyuridine (BrdU)
is a thymidine analog that is incorporated into DNA during the S phase
of the cell cycle.22 Ki-67 is a nuclear cell
proliferation-associated antigen expressed in proliferating cells
during the late G1 to M phase of the cell cycle.23 BrdU
incorporation and Ki-67 expression can be detected and quantified by
using fluorescence-conjugated monoclonal antibodies (mAbs). We also
examined the proliferation of HTLV-I-Tax-specific CD8+ T
cells during SP by using a combination of BrdU or Ki-67 and an
HLA-A*0201/Tax11-19 tetramer. Using these methods, we demonstrated predominant expansion of CD8+ T cells, including
HTLV-I-Tax-specific CD8+ T cells, during SP. This report
is the first direct evidence of the proliferation of specific cell
subtypes in SP in HAM/TSP. In addition, we investigated the amount of
HTLV-I provirus and HTLV-I Tax protein expression during SP. The
predominant involvement of CD8+ cells in SP may underlie
the pathogenesis and progression of HAM/TSP.
Subjects
Proliferation assays
Because 3H-thymidine assays do not allow distinction of phenotypes of proliferating cells, proliferation was also measured by using a BrdU Flow Kit (Pharmingen, San Diego, CA). This procedure uses a fluorescence-conjugated anti-BrdU mAb to label BrdU that has been incorporated into newly synthesized DNA within dividing cells. Concomitant staining with mAbs against surface markers permits identification of phenotypic subsets of proliferating cell populations by using flow cytometry. BrdU (10 µM) was added to the cell culture for 16 hours before staining and fixation. Cells were washed with phosphate-buffered saline containing 1% FCS and 0.1% NaN3 and incubated with anti-human CD4-phycoerythrin (PE; Caltag Laboratories, Burlingame, CA) and antihuman CD8-RPE/Cy-5 (Dako Laboratories, Denmark) or antihuman CD8-PE (Caltag Laboratories) and antihuman CD4-Tri-Color (Caltag Laboratories) for 20 minutes at 4°C. Cells were then fixed with a paraformaldehyde solution for 30 minutes at 4°C, permeabilized with saponin for 10 minutes at 4°C, and re-fixed with paraformaldehyde for 5 minutes at 4°C. Following fixation, cells were treated with 300 µg/mL DNase for 1 hour at 37°C, then incubated with anti-BrdU-fluorescein isothiocyanate (FITC) or a matched isotype control mAb for 20 minutes at room temperature, as per kit instructions. Flow cytometric analyses were performed by using a FACS Calibur (Becton Dickinson, Mountain View, CA). Proliferation of T-cell subsets identified with the BrdU assay was further confirmed by measuring the expression of a nuclear-proliferating cell antigen, Ki-67. Cultured cells were washed and labeled with antihuman CD4-PE (Caltag Laboratories) and antihuman CD8-RPE/Cy-5 (Dako) for 30 minutes at 4°C. They were then fixed and permeabilized with a paraformaldehyde and saponin solution from a Cytoperm/Cytofix kit (Pharmingen) for 20 minutes at 4°C. Fixed cells were labeled with either antihuman Ki-67-FITC mAb or a matched isotype control antibody for 30 minutes at 4°C and analyzed by flow cytometry. Identification of Tax-specific CD8+ cells The HTLV-I Tax 11-19 peptide is a dominant epitope that is recognized by HLA-A2-restricted CD8+ CTLs in patients with HAM/TSP.7,24 Analysis of Tax-11-19-specific CD8+ cells was performed by using a PE-conjugated HLA-A*0201 tetramer (provided by NIAID MHC Tetramer Core Facility, Atlanta, GA; and NIH AIDS Research and Reference Reagent Program). The tetramer was loaded with HTLV-I Tax 11-19 peptide (LLFGYPVYV), which was synthesized and 95% purified by high-performance liquid chromatography (New England Peptide, Fitchburg, MA). Tetramer loaded with HIV Gag77-85 peptide (SLYNTVATL) was used as a negative control. Cultured cells were washed and incubated with HLA-A2 tetramer, antihuman CD4-FITC mAb (Caltag Laboratories), and antihuman CD8-RPE/Cy5 mAb (Dako) for 30 minutes at 4°C, then analyzed by flow cytometry. Tetramer-labeled cells were also fixed and permeabilized as described earlier and stained for BrdU incorporation or Ki-67 expression.Identification of HTLV-I Tax-expressing cells PBMCs (5 × 105) were placed on a culture well (round bottom 96-well plate) in 200 µL RPMI-1640 supplemented with L-glutamine, penicillin, streptomycin, and 5% human AB serum. Harvested cells were washed with phosphate-buffered saline containing 1% FCS and 0.1% NaN3 and incubated with antihuman CD4-PE mAb (Caltag Laboratories) and antihuman CD8-RPE/Cy5 mAb (Dako) for 20 minutes at 4°C. Cells were then fixed and permeabilized with 4% formaldehyde and 0.1% saponin (CytoFix/Cytoperm Kits; Pharmingen) for 20 minutes at 4°C. After washing with 0.1% saponin buffer (Perm/Wash solution; Pharmingen), the cells were incubated with anti-HTLV-I Tax mAb (the reagent was 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, FITC-conjugated goat F(ab')2 antimouse immunoglobulin G2a mAb (Southern Biotechnology Associates, Birmingham, AL) was used as a second antibody for labeling anti-HTLV-I Tax mAb. Flow cytometric analyses were performed by using a FACS Calibur (Becton Dickinson).Quantitative polymerase chain reaction HTLV-I proviral load was measured by using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA) as previously described.15 DNA was extracted from 1 × 106 cells by using Puregene DNA Isolation Kit (Gentra, Minneapolis, MN) and was adjusted to 10 ng/µL. Polymerase chain reaction (PCR) conditions were as follows: 10 µL DNA solution was added to 40 µL reaction mixture containing 10 mM Tris-HCl; 50 mM KCl; 10 mM EDTA; 60 nM ROX (passive reference dye); 5.5 mM MgCl2; 0.2 µM of each primer; 0.1 µM TaqMan probe; 200 µM each of dATP, dGTP, and dCTP; 400 µM dUTP; 0.5 U uracil-N-glycosylase; and 1.25 U Taq polymerase (AmpliTaq Gold; Applied Biosystems).The primer set for HTLV-I pX region was
5'-ACAAAGTTAACCATGCTTATTATCAGC-3' positioned at 7276-7302 and
5'-ACACGTAGACTGGGTATCCGAA-3' positioned at 7355-7334. The primer set
for The amount of HTLV-I proviral DNA was calculated by the following
formula: copy number of HTLV-I (pX) per 100 cells = [(copy number of
pX) / (copy number of
Cultured PBMCs from patients with HAM/TSP contain 2 cell populations distinguished by size (large cells and small cells).25 These populations can be identified with a flow cytometer by plotting forward scatter and side scatter of the cells. The phenotypic composition of each cell population was analyzed by using mAbs against CD3 (T lymphocytes), CD16 (natural killer cells), CD14 (monocytes), and CD19 (B cells). Before culture, the smaller cell population consisted of lymphocytes and the larger cell population consisted of monocytes. After SP of HAM/TSP PBMCs, the large cell population contained lymphocytes, shown by an increased percentage of CD3+ cells (data not shown).25 In contrast, the percentage of CD3+ cells in the large cell population did not change in an unstimulated culture of healthy PBMCs. This finding supports a morphologic change on blasting and suggests that the lymphocytes in the large cell population may be preferentially activated.25 PBMCs from patients with HAM/TSP were cultured for 6 days and assayed for proliferation and lymphocyte phenotypic composition. Proliferation observed using the 3H-thymidine assay was consistent with earlier descriptions of SP in HAM/TSP.8-11 During SP of HAM/TSP PBMCs, 3H-thymidine counts increased over time in culture, reaching maximal levels of 30 000 to 130 000 cpm on day 4 or day 5 (data not shown). Unstimulated PBMCs from healthy donors incorporated less then 1000 cpm at the same time points, whereas PHA-stimulated PBMCs from the same donors typically peaked around 80 000 to 100 000 on day 3 or day 4 (data not shown). On days 0, 2, 4, and 6 of the proliferation, PBMCs were harvested
and stained with CD4-FITC and CD8-RPE/Cy5 and analyzed by flow
cytometry. CD4+ and CD8+ subsets were
quantified and used to calculate a ratio of CD4+ cells to
CD8+ cells. Data from 5 patients with HAM/TSP are
summarized in Figure 1. As shown, the
ratio of CD4+ cells to CD8+ cells decreased for
each patient over 6 days. Surprisingly, this change in ratio appeared
to be driven by the increasing CD8+ population more than by
a decrease in CD4+ percentages. Occasional drops in
CD8+ and/or CD4+ cells by day 6 are likely a
consequence of maintaining cell cultures for prolonged periods without
supplementing the media and are consistent with decreases in
proliferation observed in 3H-thymidine incorporation assays
after day 4, as described earlier.
The decreasing CD4+/CD8+ ratio is simple
numerical evidence, suggesting that CD8+ cells are involved
in in vitro proliferation. However, because the
3H-thymidine method of measuring proliferation is not
capable of identifying phenotypic subsets of cells, we used 2 nonradioisotopic reagents, BrdU and Ki-67, coupled with flow cytometry.
Proliferation of each T-cell subset was determined by the percentage of
CD4+ or CD8+ cells also labeled with anti-BrdU
or anti-Ki-67. Representative data from the BrdU incorporation assay
are shown in Figure 2. The histogram
cells are based on their expression of CD4 or CD8 and BrdU. Both
CD4+ and CD8+ cells gated by the small cell
population did not show proliferation. However, proliferation is
clearly seen in both CD4+ and CD8+ populations
gated by the large cell population on days 3 and 4. The percentage of
proliferating CD4+ (or CD8+) cells in total
CD4+ (or CD8+) cells is graphed in Figure
3A (see legend about calculation). The
CD8+ subset proliferated to a greater degree than the
CD4+ subset. Some CD4
CD8+ cells specific for HTLV-I antigens are prominent in
the peripheral blood of patients with HAM/TSP and may be important in
progression of the disease.5,26 Proliferation of HTLV-I antigen-specific CD8+ cells might, therefore, be expected
to contribute to CD8+ cell expansion. To address this
issue, CD8+ cells specific for the HTLV-I Tax 11-19 peptide
were identified in 3 HLA-A2 HAM/TSP patients, using an HLA-A*0201/Tax
11-19 peptide tetramer. Tetramer-positive cells were measured in both
the small cell and large cell populations. Results from one
representative patient with HAM/TSP (HAM-4) are shown in Figure
4A. On day 0 and day 1, most Tax-specific
CD8+ cells were found in the gate small cell population,
and some Tax-specific CD8+ cells were BrdU negative in the
gated large cell population. Over 4 days in culture, the Tax-specific
CD8+ cells increased in the large cell population from
22.0% to 40.2% of total CD8+ cells in the large cell
population. Interestingly, Tax-specific CD8+ cells in the
small cell population decreased from day 1 to day 4 in the same patient
(data not shown), suggesting that these cells became activated in vitro
and underwent morphologic changes that shifted them into the population
of larger cells. To identify proliferation of these Tax-specific
CD8+ cells, PBMCs were concomitantly labeled with Tax
tetramer and either BrdU or Ki-67. After gating on the
tetramer-positive CD8+ population, histograms were
generated to demonstrate the increase in the percentage of cells
positive for BrdU (Figure 4B showed representative data of HAM-4).
After 4 days in culture, 40.2% (HAM-2), 70.0% (HAM-3), and 74.6%
(HAM-4) of Tax-specific CD8+ cells were labeled with BrdU
in 3 patients with HAM/TSP, respectively. Similar Tax-specific
CD8+ cell proliferation was measured by Ki-67 expression
(data not shown). The expansion of Tax-specific CD8+ cells
in SP supports the view that HTLV-I-specific CD8+ T
lymphocytes may play a critical role in the inflammatory process of
HAM/TSP. This view is further supported by evidence of the elevated
production of proinflammatory cytokines, such as interferon
The observed expansion of Tax-specific CD8+ cells in SP
might also be expected to reduce the amount of HTLV-I present. In
support of this hypothesis, an increased frequency of CD8+
cells has been associated with reduced Tax expression in vivo. Similarly, the frequency of Tax-specific CTL was negatively correlated with the percentage of CD4+ cells in fresh peripheral blood
leukocytes from patients with HAM/TSP.18 To investigate a
possible relationship between HTLV-I load and Tax-specific
CD8+ cell proliferation more directly, proviral load was
determined in patients with HAM/TSP during SP by using quantitative
real-time PCR. Results are summarized in Figure
5. HTLV-I proviral load did not
significantly change during culture. Although the CD4/CD8 ratio at day
4 in Figure 1 decreased compared with that at day 0, HTLV-I proviral
load did not change (Figure 5). If HTLV-I were to infect only
CD4+ cells, then the HTLV-I proviral load might have
decreased at day 4 as the percentage of CD4+ also appeared
to be decreasing over time (Figure 1). However, because the HTLV-I
proviral load was relatively stable during this 4-day culture period
when the percentage of CD4+ cells was decreased, it
suggests that other cells (possibly CD8+) may be HTLV-I
infected. This finding is consistent with the recent report that
CD8+ T cells may also be infected with
HTLV-I.27 Therefore, expansion of HTLV-I-infected
CD8+ T cells may contribute to the proviral load in SP.
It was unexpected that HTLV-I proviral load was stable in spite of
Tax-specific CD8+ cell expansion. Therefore, we
investigated HTLV-I Tax protein expression by using an intracellular
protein detection technique. We chose to study the HTLV-I Tax protein
because it is a major target of the HTLV-I-specific CTLs that
pivotally control viral infection. As shown in Figure
6, HTLV-I Tax protein-expressing cells
could not be detected in uncultured PBMCs, but were detectable during
culture, and reached a maximum at 12 hours. After 72 hours, HTLV-I
Tax-expressing cells were no longer detected. This result is consistent
with published data.18 It has been also demonstrated that
CD4+ cells naturally infected with HTLV-I were killed in
vitro by CD8+ lymphocytes by a perforin-dependent cytotoxic
pathway, and CD8+ CTLs controlled HTLV-I antigen-expressing
cells.18 However, the stable proviral load in culture
while Tax-specific CD8+ cells were still expanding suggests
the involvement of an additional mechanism to escape from
CD8+ CTLs. One possible mechanism involves the expansion of
HTLV-I-infected (CD4+ and CD8+) cells by
mitotic proliferation (infected cells do not express HTLV-I antigens)
rather than infectious transmission (infected cells express HTLV-I
antigens and produce HTLV-I virions). This first type of propagation
might result in an increase in integrated HTLV-I without increased
HTLV-I antigen expression. Without expressing HTLV-I Tax, these
infected cells could not be targeted by Tax-specific CD8+
CTLs. As Tax-expressing cells were killed by Tax-specific
CD8+ CTLs, mitotic proliferation (silent provirus) would
selectively predominate. Following the reduction of Tax expression,
Tax-specific CD8+ cell proliferation might be supported by
cytokines, such as IL-15, which is an important factor for maintaining
CD8+ cells.28 Other mechanisms associated with
SP have been previously reported that include interaction between
HTLV-I-infected T cells and noninfected T cells mediated by CD2/LFA-3
and LFA-1/ICAM.29 Further study of this phenomenon may
help us to understand the mechanism that permits coexistence of high
HTLV-I-specific CD8+ proliferative and CTL responses
and high HTLV-I proviral load in HAM/TSP in vivo.30,31 In
a recent HIV study, it has been reported that even after effective
combination therapy that reduced plasma HIV RNA to undetectable levels,
HIV can persist in a latent form in resting CD4+ T
cells.32
Spontaneous proliferation of CD4+ and CD8+ cells may be an important phenomenon not only in vitro but also in situ in HAM/TSP. Ijichi et al33 have proposed that an autoaggressive process against bystander tissue plays a crucial role in the pathogenesis of this disease. In this hypothesis, lymphocytes that include both HTLV-I-infected CD4+ cells and HTLV-I antigen-specific CD8+ cells enter spinal cord lesions and proliferate in situ in a manner similar to in vitro SP. The in situ immune response propagated against viral antigens may act as an autoaggressive effector for the development of central nervous system tissue damages in HAM/TSP. In support of this hypothesis, an immunohistochemical phenotypic analysis of infiltrated mononuclear cells in a spinal cord lesion in a patient with HAM/TSP has showed the presence of both CD8+ cells and CD4+ cells.34,35 This report provides direct evidence that CD8+ cells in vitro predominantly expand in SP of PBMCs from HAM/TSP. Although there is no direct in vivo correlate for spontaneous lymphoproliferating PBMCs, the observations in this study may be used as a surrogate marker for virus-host immunologic interactions that may occur in vivo. Thus, these findings support the ex vivo observations demonstrating expansion of CD8+ cells, including HTLV-I Tax-specific CD8+ lymphocytes that have been suggested to play a critical role in the pathogenesis of HAM/TSP. The elucidation of the role of these and other viral antigen-specific CD8+ cells in HAM/TSP may reflect similar mechanisms in other diseases with viral etiologies.
Submitted November 27, 2000; accepted April 27, 2001.
J.A.S. and M.N. contributed equally to this study.
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: Steven Jacobson, Viral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 5B-16, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: jacobsons{at}ninds.nih.gov.
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© 2001 by The American Society of Hematology.
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Abstract
Introduction
-plate counter (Wallal, Gaithersburg, MD).
and CD8
and
tumor necrosis factor 
, by HTLV-I-specific CD8+ T
lymphocytes in HAM/TSP.
