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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3094-3100
Rapid Determination of Epstein-Barr Virus-Specific CD8+
T-Cell Frequencies by Flow Cytometry
By
Kiyotaka Kuzushima,
Yo Hoshino,
Ken Fujii,
Naoaki Yokoyama,
Masatoshi Fujita,
Tohru Kiyono,
Hiroshi Kimura,
Tsuneo Morishima,
Yasuo Morishima, and
Tatsuya Tsurumi
From the Laboratory of Viral Oncology, Aichi Cancer Center Research
Institute, Nagoya, Japan; the Department of Hematology and
Chemotherapy, Aichi Cancer Center Hospital, Nagoya, Japan; and the
Department of Pediatrics, Nagoya University School of Medicine, Nagoya,
Japan.
 |
ABSTRACT |
We have developed an efficient and rapid method for detection of
Epstein-Barr virus (EBV)-specific CD8+ T-cell frequencies
both in freshly isolated peripheral blood mononuclear cells (PBMCs) and
in vitro established cytotoxic T lymphocyte (CTL) lines. Responder
cells are thereby stimulated with an autologous lymphoblastoid cell
line for 5 hours and intracellular accumulation of interferon  (IFN) is detected by multiparameter flow cytometric analysis.
EBV-specific CD8+ T-cell frequencies ranged between
0.63% and 1.29% in PBMCs of 5 healthy long-term EBV carriers. Using
EBV-specific T-cell lines, it was shown that flow cytometric analysis
is more sensitive than limiting dilution analysis for CTL precursors
and enzyme-linked immunospot assay detecting IFN-producing T cells.
The class I restriction of IFN production was confirmed using an
anti-class I monoclonal antibody (MoAb). Information on other cytokine
production of EBV-specific CTLs could be obtained using combinations of
anti-cytokine MoAbs. The sensitive and rapid nature of the flow
cytometric assay for EBV-specific CD8+ T-cell frequency
has significant advantages for evaluation of EBV-specific
CD8+ T-cell responses in PBMCs of patients with
EBV-related diseases.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
EPSTEIN-BARR VIRUS (EBV) is well known to
be associated with several malignant diseases. These include a
proportion of Hodgkin's lymphomas, nasopharyngeal carcinomas,
Burkitt's lymphomas, and immunoblastic lymphomas seen in
immunocompromised hosts.1
Primary infection with EBV is usually asymptomatic, although some may
suffer from acute infectious mononucleosis.1 After primary
infection, a strong HLA class I-restricted, virus-specific CD8+ cytotoxic T lymphocyte (CTL) response is elicited in
healthy individuals.2 This response is believed to play an
important role in controlling the virus both during primary infection
and in the long-term carrier state whereby EBV persists for life in a
subset of B cells.
An increased understanding of the mechanisms by which T lymphocytes
recognize virus-specific antigens has stimulated much interest in the
use of CTLs as adoptive immunotherapy for EBV-associated disease. An
early candidate for such treatment was EBV-associated lymphoproliferative disease (LPD), which is seen in patients receiving allogeneic bone marrow transplantation from mismatched family members
or unrelated donors.3,4 The incidence is between 5% and
30%, particularly if marrow was depleted of T cells to prevent
graft-versus-host disease.5
Unfractionated populations of lymphocytes from the peripheral blood of
donors6,7 or EBV-specific CTL lines from donor lymphocytes
have been used for treatment and/or prophylaxis of posttransplant
EBV-associated LPD.8,9 However, the utility of
unfractionated lymphocyte infusion is limited by potentially fatal
communications that arise from alloreactive T cells also present in the
infusion. To overcome this problem, infusions of EBV-specific CTL lines
from donor lymphocytes have been successfully used, especially as
prophylaxis against EBV-associated LPD in transplant recipients
considered at high risk.10 Recently, adoptive immunotherapy
(using unfractionated lymphocytes or EBV-specific CTLs) has been
applied for treatment or prophylaxis of EBV-associated LPD in patients
with solid organ transplants,7,11 severe chronic active EBV
infection,12 or EBV-positive Hodgkin's
disease.13
In any cases of immunotherapy for EBV-associated diseases, to confirm
the effects of the treatment, demonstration of elevation of
EBV-specific cellular immunity in the patients is necessary. For this
purpose, bulk CTL activities in peripheral blood mononuclear cells
(PBMCs) stimulated in vitro with an autologous EBV-infected lymphoblastoid cell line (LCL) or limiting dilution analysis (LDA) for
CTL precursors that lysed the LCL have been frequently
used.7-13 The disadvantage is that it takes at least
approximately 10 days to raise bulk CTL, and the assay is not very
quantitative. LDA is one of the established methods for quantitative
detection of CTL precursor frequencies in PBMCs of healthy seropositive
individuals14 and of patients receiving allogeneic bone
marrow transplants.15 However, it again takes 2 to 4 weeks
to obtain the results, and the procedures are particularly laborious.
In addition, both experiments need -irradiating radioisotopes, which
are potentially hazardous for the examiners.
Recently, Waldrop et al16 reported that human
cytomegalovirus-specific CD4+ T-cell frequencies can be
detected by flow cytometry. Kern et al17 showed that
CD8+ peptide-specific T-cell frequencies can be measured in
samples whose HLA is known. Both methods are based on multiparameter
flow cytometric assays that detect rapid intracellular accumulation of
cytokines after in vitro antigen stimulation in the presence of
intracellular transport blockers such as Brefeldin A.
We report here a highly efficient method to detect whole EBV-specific
CD8+ T-cell frequencies irrespective of HLA typing. PBMCs
are thereby incubated with an autologous LCL in the presence of
Brefeldin A for a short period (5 hours), and the rapid intracellular
accumulation of interferon (IFN) is detected by multiparameter
flow cytometric analysis. Using these methods, we found approximately
1% of peripheral CD8+ T cells of seropositive individuals
to be EBV-specific. The method is more sensitive and takes less time
than LDA for CTL precursors and enzyme-linked immunospot (ELISPOT)
assays. In addition, it can provide more information on
cytokine production of EBV-specific CD8+ T cells upon
natural stimulation when combinations of anticytokine monoclonal
antibodies (MoAbs) are used.
| |
MATERIALS AND METHODS |
Blood donors.
The blood donors consisted of (1) long-term healthy carriers of EBV
defined as having both EBV-viral capsid antigen (VCA)-IgG and EBV
nuclear antigen (EBNA) antibodies and (2) EBV-seronegative individuals
defined as having no EBV-VCA-IgG antibodies.1 Some donors
were tested for their HLA class I typing of PBMCs with classical
serological methods. Study design and purpose were fully explained to
all donors. Peripheral blood was obtained after informed consent was confirmed.
Preparation of PBMCs, LCLs, and CD8+ EBV-specific CTL
lines.
PBMCs were obtained by centrifuging heparinized blood over
Ficoll/Hypaque (Pharmacia Biotech AB, Uppsala, Sweden). LCLs were prepared by transforming PBMCs with B95-8 cell culture supernatant as
previously described.12 LCLs were cultured in Iscove's
modified Dulbecco's medium (GIBCO, Grand Island, NY) supplemented with 2 mmol/L L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, 5 × 10 5 mol/L  -mercaptoethanol, and 10%
heat-inactivated fetal calf serum (FCS; Hyclone, Logan, UT; referred to
as culture medium). EBV-specific T-cell lines were initiated by
culturing 2 × 106 PBMCs and 2 × 105
autologous irradiated LCLs in 2 mL of culture medium in each well of
24-well plates. Ten days after the stimulation, CD8+ cells
were isolated using immunomagnetic beads (Dynabeads M-450 CD8; Dynal,
Oslo, Norway) following the manufacturer's instructions. Detachment of immunomagnetic beads from isolated cells was achieved using Detachabeads (Dynal). Isolated cells were greater than 95% CD8+ according to flow cytometric analysis. These
CD8+ T cells were further stimulated with autologous
irradiated LCLs weekly in the presence of 50 U/mL recombinant
interleukin-2 (IL-2).
CTL assay.
CTL assays were performed using 51Cr-release as previously
described.12 Briefly, CTLs were suspended in fresh culture
medium at the desired cell concentration and seeded in wells of
V-bottomed 96-well plates (Costar, Cambridge, MA) containing
51Cr-labeled LCLs (2,500 cells/well). Each assay was
performed in triplicate. After 5 hours of incubation, the supernatants
were harvested and radioactivity was counted with a -counter. The percentage of specific lysis was calculated as follows: percentage of
specific lysis = (experimental lysis  minimum lysis) × 100/(maximum lysis minimum lysis). Minimum lysis was
obtained by incubating the target cells with the culture medium alone.
Maximum lysis was obtained by exposing the target cells to 1%
Nonidet-P40.
Detection of CTL precursor frequency by LDA.
LDA for cytotoxicity was performed essentially as previously
reported.14,15 Isolated CD8+ cells were diluted
in 96-well U-bottom plates (24 replicates/dilution). Irradiated
autologous LCLs (2 × 104), PBMCs (103),
and IL-2 (40 U/mL) were added to each well. On day 7, IL-2 was added to
40 U/mL. CTL assays were performed on day 12. To assay CTL precursor
frequency, 100 µL of cell suspension from each well was transferred
into a well of 96-well V-bottom plates containing
51Cr-labeled 2,500 LCLs. After 5 hours of incubation in a
humidified 5% CO2 incubator at 37°C, the supernatant
was harvested and radioactivity was counted with a -counter. Wells
were scored as positive for CTL recognition if the level of specific
lysis exceeded 3 standard deviations above the mean spontaneous release
from the target cells. The frequency of CTL precursors was estimated at
which 37% of the wells were negative from the slope of a regression plot of the log percentage of negative versus input cell numbers.
Detection of IFN producing CD8+ T cells in response
to LCLs by flow cytometry.
For determination of CD8+ antigen-specific T lymphocyte
frequency, intracellular cytokine assessment using flow cytometry was performed as previously described, with slight
modifications.16,17 Briefly, PBMCs or EBV-specific CTLs
were resuspended at a concentration of 1 × 106/mL in
culture medium. Autologous or HLA-disparate LCLs were resuspended at a
concentration of 1 × 106/mL in the culture medium.
Aliquots of the responder cells (1 mL) and LCL (1 mL) were mixed in 16 × 125 mm culture tubes in the presence of 10 µg/mL Brefeldin A
(Sigma Chemical Co, St Louis, MO) and incubated in a humidified 5%
CO2 incubator at 37°C for 5 hours. As a control, the
same numbers of responder cells and LCLs were separately incubated in
the presence of the Brefeldin A and mixed before staining with MoAbs.
For blocking experiments, an anti-class I MoAb (clone W6/32; Cedarlane,
Hornby, Ontario, Canada) or isotype-matched monoclonal
mouse IgG antibodies were used at a final concentration of 47 µg/mL.
After the incubation, the cell suspensions were fixed with 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at
room temperature. After washing with PBS, cells were permeabilized with
IC Perm (BioSource International, Camarillo, CA) and
stained with PC5-labeled anti-CD8 (Coulter, Miami, FL),
phycoerythrin (PE)-labeled anti-CD69 (Coulter), and fluorescein isothiocyanate (FITC)-labeled antihuman IFN
(Becton Dickinson, San Jose, CA) MoAbs. In some experiments, T cells
were stimulated with 50 ng/mL of phorbol myristate acetate (Sigma) and
500 ng/mL of ionomycin (Sigma) and stained with PE-labeled anti-IL-4
or anti-IL-13 MoAbs (Becton Dickinson). Stained cells were analyzed by
FACScan (Becton Dickinson) using the LYSIS II software. Live-gating of
CD8high lymphocytes was performed, and up to 20,000 events
were acquired for each analysis.
ELISPOT assay.
An ELISPOT assay was performed as previously described, with slight
modifications,18,19 using 96-well MultiScreen-HA plates with a nitrocellulose base (Millipore, Bedford, MA), coated with 10 µg/mL of anti-IFN MoAb (Genzyme, Cambridge, MA). Isolated CD8+ T cells were added in graded numbers of 500, 250, and
125 per well containing autologous irradiated LCLs (1 × 105) and IL-2 (50 U/mL). Each dilution was seeded in
triplicate. The plates were incubated in 5% CO2 incubator
at 37°C for 28 hours and extensively washed with PBS containing
0.05% Tween-20. A polyclonal rabbit anti-IFN antibody (Genzyme) was
added to individual wells and left for 90 minutes at room temperature,
followed by peroxidase-conjugated goat antirabbit IgG (Genzyme) for an
additional 90 minutes. For visualization of IFN-specific spots, 100 µL of 0.1 mol/L sodium acetate buffer (pH 5.0) containing
3-amino-9-ethylcarbasole (Sigma) and 0.015%
H2O2 was added to each well. After 30 minutes,
the reaction was stopped by washing with water and the plates were dried. The bottom membranes were photographed and spots were counted. Only diffuse large spots were considered specific, because wells containing only LCLs gave tiny spots. The percentage frequency of
antigen-specific CD8+ T cells was calculated as follows:
percentage frequency = numbers of spots × 100/input cell numbers.
| |
RESULTS |
We applied a strategy using tricolor analysis for detection of
EBV-specific IFN-producing T cells. First, the PC5-labeled anti-CD8
MoAb was used for gating the population. Second, the PE-labeled
anti-CD69 MoAb was used for enhancement of precise detection of
responding T cells. CD69 is upregulated on activated T cells before
cytokine production and thus allows more definitive clustering of the
true responding fraction.20 As an unstimulated control, we
used responder cells and LCLs that had been separately incubated in the
same medium with Brefeldin A and mixed and stained after fixing.
Frequency of EBV-specific CD8+ T cells in PBMCs of
EBV-seropositive and -seronegative individuals.
We first tested PBMCs of long-term healthy EBV carriers for the
frequency of EBV-reactive IFN-producing CD8+ T cells.
When PBMCs from seropositive individuals were used, 0.87% and 1.32%
of total CD8+ cells produced IFN in response to
autologous LCLs (Fig 1B and D). We defined
the specific frequency (SF) as follows: percentage of a sample
stimulated with LCLs percentage of the same sample unstimulated
(note that the unstimulated samples were also incubated with Brefeldin
A and mixed with LCLs after fixation for comparable staining
conditions). The SFs of donors 1 and 2 were 0.85% (Fig 1A and B) and
1.29% (Fig 1C and D), respectively. Those of the 2 other healthy
EBV-carriers were 0.63% and 1.08% (graphic data not shown). In
contrast, the SF of antigen-specific T cells in PBMCs of an
EBV-seronegative donor was 0.03% (Fig 1E and F), showing the
specificity of the assay. Those of the 2 other EBV-seronegative donors
were 0.02% and 0.00%, respectively (graphic data not shown). Thus,
clustering of IFN-producing CD8+ T cells was never found
using PBMCs of EBV-seronegative donors. When stimulated with completely
HLA class I-mismatched LCLs, IFN-producing CD8+ T cells
from either seropositive or seronegative individuals ranged between
0.21% and 0.55% (graphic data not shown). Thus, such alloreactive
frequency was lower than EBV-specific frequency in PBMCs of
seropositive individuals and higher than that of seronegative individuals as far as we tested.
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| Fig 1.
EBV-specific IFN-producing CD8+ T cells
in PBMCs of EBV-seropositive (A through D) and EBV-seronegative (E and
F) individuals. PBMCs were stimulated with autologous LCLs (B, D, and
F) at a responder stimulator ratio of 1. After fixation and
permeabilization, the cells were stained for CD8, CD69, and IFN.
CD8+ cells were gated and analyzed by flow cytometry.
Unstimulated PBMCs were also incubated, fixed, and then mixed with
autologous LCLs before staining (A, C, and E). The frequency of
CD8+/CD69high T cells that produced IFN is
shown as a percentage of the total number of CD8 cells.
|
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Comparison of 4 different assays for quantification of EBV-specific
CD8+ T-cell frequencies.
Next, we compared the sensitivities of 4 different assays, namely the
standard CTL assay, intracellular IFN production assay, ELISPOT
assay, and standard LDA for CTL precursors, using 2 CD8+
EBV-specific CTL lines.
CTL activities of the lines are shown in
Fig 2. The effector target ratio that
yielded the same percentage of target lysis was 1.5 to 2 times higher
for CTLs from donor YI than for those from donor KK. Flow cytometric
analysis of EBV-specific T cells for SFs of CTL lines from donors KK
and YI were 39.2% and 25.2%, respectively
(Fig 3). With the ELISPOT assay, the
frequency of CTLs from donor KK was approximately 2 times higher than
that from donor YI (Fig 4A and B). The mean
(and standard deviation) frequencies of all the wells tested were
11.2% (3.5%) and 5.7% (1.7%), respectively
(Table 1). LDA analysis gave values of
2.3% and 0.23% for CTL precursor frequencies
(Fig 5A and B). Thus, the values with the 4 assays roughly correlated, except for LDA, which gave a 10-fold
difference between the 2 samples (Table 1). The CTL line from donor YI
was growing more slowly than that from donor KK, and this might reflect
the discrepancy of the data of LDA. Flow cytometric analysis of
EBV-specific T-cell frequencies using intracellular IFN production
gave the highest values.
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| Fig 3.
Flow cytometric analysis of IFN production in 2 CD8+ EBV-specific CTL lines from donor KK (A and B) and
YI (C and D). (A and C) Unstimulated CTLs; (B and D) CTLs stimulated
with autologous LCLs at a responder stimulator ratio of 1. After
fixation and permeabilization, the cells were stained for CD8, CD69,
and IFN. CD8+ cells were gated and analyzed by flow
cytometry. The frequency of CD8+/CD69high T
cells that produced IFN is shown as a percentage of the total number
of CD8 cells.
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| Fig 4.
ELISPOT assay for detecting IFN-producing cells in 2 CD8+ EBV-specific T-cell lines from donors KK (A) and YI
(B). The mean and standard deviation are shown for each dilution.
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Class I-restricted production of IFN from EBV-specific
CD8+ CTLs.
We further examined the class I restriction of the response.
EBV-specific CTLs were stimulated by autologous (HLA-A24/A26, B52/B62,
and C3) and various allogeneic LCLs (Fig
6). Approximately 27% of CTLs produced IFN upon stimulation of
autologous LCLs (Fig 6A and B). When LCLs sharing HLA-A26, B62, and C3
class I molecules were used as stimulators, 8.8% of CD8+ T
cells produced IFN (Fig 6C). LCLs sharing A24 and B62 with the CTL
stimulated 2.8% of the CD8+ T-cell population (Fig 6D).
LCLs sharing only A24 or C3 the CTL stimulated 1.2% and 0.3% of the
CD8+ T-cell population (Fig 6E and F). These data indicated
that HLA-A24, A26, and B62 are presenting EBV antigens. To confirm that
the IFN production is class I-restricted, the same CTLs were
stimulated by autologous LCLs in the presence of anti-class I MoAb (Fig
6G) or isotype-matched mouse MoAb (Fig 6H) as a control. The
IFN-producing CD8+ T-cell population was drastically
reduced (0.36%) with anti-class I MoAb, but not with control MoAb
(31%). These results indicate that the IFN in the CD8+
T cells was produced through authentic recognition of antigens presented by class I molecules.
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| Fig 6.
Class I restriction of the IFN production in an
EBV-specific CD8+ CTL line. EBV-specific CTLs were
stimulated by autologous ([B] HLA-A24/A26, B52/B62, and C3) and
various allogeneic LCLs ([C] HLA-A11/A26, B62,
and C3/C4; [D] A11/A24, B61/B62, and
C4; [E] A2/A24, B7/B61, and C7; [F] A2, B35/B46, and
C1/C3). The underlined alleles were shared by CTLs and each
LCL line. Unstimulated CTLs were also incubated, fixed, and then mixed
with autologous LCLs before staining (A). The same CTLs were also
stimulated by autologous LCLs in the presence of an anti-class I MoAb
(G) or an isotype-matched MoAb (H). The frequency of
CD8+/CD69high T cells that produced IFN is
shown as a percentage of the total number of CD8 cells.
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Dual staining for IFN/IL-4 or IL-13 in EBV-specific
CD8+ CTLs.
Recently, Nazaruk et al21 reported that EBV-specific
CD8+ T cells can be subdivided into 2 subsets: the first of
which expresses high levels of IFN, but little or no IL-4, whereas
the second subset is IFN/IL-4 or IL-13 double-positive, paralleling
the classically described Th1 and Th2 subsets of CD4+ T
cells. They used phorbol myristate acetate and ionomycin for activation
of CTLs. This artificial stimulation may give a different outcome
compared with physiological T-cell receptor engagements, because a
different signal transduction pathway is used.22 Thus, we
examined production of the 3 cytokines by EBV-specific CTLs using LCLs
as natural ligand stimulators. The results are shown in
Fig 7. When an EBV-specific CTL line was
stimulated with autologous LCLs, the 2 subsets mentioned above were
observed in both combinations of IFN/IL-4 (Fig 7B) and IFN/IL-13
(Fig 7E). The cell distribution pattern roughly resembled those after
stimulation with phorbol myristate acetate and ionomycin (Fig 7C and
F), but the proportions were different.
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| Fig 7.
Examination of production of 3 different cytokines by
EBV-specific CTLs using LCLs as natural ligand stimulators or phorbol
myristate acetate and ionomycin as chemical stimulators. (A and D)
Unstimulated CTLs; (B and E) CTLs stimulated with autologous LCL at a
responder stimulator ratio of 1; and (C and F) CTLs stimulated with
phorbol myristate acetate and ionomycin. After fixation and
permeabilization, the cells were stained for CD8, IFN, and IL-4 (A
through C) or IL-13 (D through F). CD8+ cells were gated
and analyzed using flow cytometry.
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| |
DISCUSSION |
We introduce here an efficient and rapid method for detection of
EBV-specific CD8+ T-cell frequencies both in freshly
isolated PBMCs and in vitro established CTL lines. So far, LDA has been
used for the detection of CTL precursor frequencies in healthy
individuals. The frequency of EBV-specific memory CTL precursors in
long-time carriers is thereby usually in the 0.0005% to 0.2%
range.14,23,24 We found, although in limited numbers of
samples, approximately 1% of peripheral CD8+ T cells in
long-term carriers to be EBV-specific. As Waldrop et al16
claimed, the increased sensitivity of flow cytometric assays is likely
due to a combination of factors, such as (1) the very efficient capture
of produced cytokine within the cytoplasm of the secretion-inhibited
responding cells; (2) the relatively short-term period (5 hours) that
largely precedes the onset of activation-induced apoptosis; and (3) the
higher sensitivity of fluorescence detection of cytokines by flow
cytometry than by ELISPOT assay. Alternatively, the discrepancy in
frequencies between IFN-producing CD8+ T-cell and CTL
precursors may reflect the existence of noncytolytic CD8+
EBV-specific T cells.21,25 Indeed, subpopulations with such a phenotype may increase the sensitivity of assays based on IFN production. Practically, however, the flow cytometric assay appears to
be useful, if not in all situations, for rough estimation of CTL
populations because our results demonstrated that gain of CTL activity
of EBV-specific memory T cells with in vitro stimulation by autologous
LCLs paralleled the massive increase of antigen-specific IFN-producing CD8+ T cells.
Recently, Tan et al26 reported the frequencies of
CD8+ T cells specific for EBV antigens in long-term virus
carriers, using LDA, ELISPOT assay, and tetrameric major
histocompatibility complex-peptide complexes, focusing some CTL
epitopes. They demonstrated that values obtained from MHC-peptide
tetramer staining were 4.4-fold higher than those obtained from ELISPOT
assays, which were, in turn, 5.3-fold higher than those obtained from
LDA on the average. In our report, values obtained from IFN
production using flow cytometry were approximately 4-fold higher than
those obtained from ELISPOT assays, which were higher than those
obtained from LDA (Table 1). Thus, IFN production assay using flow
cytometry may have sensitivity comparable with that of MHC-peptide
tetramer staining.
EBV has 2 types of replication cycles, namely lytic infection, in which
infectious virions are produced, and latent infection, which is
represented by LCLs. Some of both cycle proteins are well
recognized by CD8+ T cells in PBMCs of patients suffering
infectious mononucleosis27 and also in long-term healthy
carriers.26 Because the majority of LCLs constitutively
express EBV latent cycle antigens, our system may preferentially detect
T cells specific to EBV latent cycle proteins, which are
therapeutically important to control over posttransplant EBV-associated LPD.
Another advantage of the cytokine production assay using flow cytometry
is that it is possible to assess multiple cytokines on an EBV-specific,
single-cell basis. Nazaruk et al21 demonstrated that a
subset of EBV-specific CD8+ T-cell lines produced IL-4 or
IL-13 in addition to IFN upon stimulation with phorbol myristate
acetate and ionomycin. They claimed that the subset has the ability to
activate B cells and promote EBV-associated LPD and lymphoma
development in immunocompromised individuals with impaired EBV-specific
CTL responses.21 We observed here such a subset of
EBV-specific CD8+ T cells upon stimulation with autologous
LCLs. Although the cell distribution pattern resembled those stimulated
with the drugs, the proportions were slightly different, giving more
physiological information because of the natural ligand stimulation used.
Altogether, the method presented here saves time, gives more
information, and probably is more accurate than LDA for detecting antigen-specific T cells, as is the case for human
cytomegalovirus-specific CD4+ T cells.16 The
rapid determination of EBV-specific CD8+ T-cell frequency
could have significant advantages in clinical settings in which EBV
infection is concerned. For example, the immunological effects of
adoptive immunotherapy for EBV-related disease can be monitored easily
and rapidly. In some patients with EBV-associated LPD after allogeneic
bone marrow transplantation, regression occurs in accordance with
elevation of EBV-specific cellular immunity,
spontaneously,28 or as a result of reduction of
immunosuppression.29 The flow cytometric assay as a tool for realtime monitoring of EBV-specific cellular immunity may be useful
in decision making for performing donor leukocyte
transfusion, a treatment potentially associated with fatal
graft-versus-host disease.30
| |
ACKNOWLEDGMENT |
The authors thank T. Yoshida and M. Hirata for technical assistance.
| |
FOOTNOTES |
Submitted February 22, 1999; accepted July 1, 1999.
Supported by grants-in-aid for Scientific Research from the Ministry of
Education, Science and Culture of Japan (11138268 to T.T. and 11877055 to K.K.) and partly by JSPS-RFTF 97L00703.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kiyotaka Kuzushima, MD, Laboratory of Viral
Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden,
Chikusa-ku, Nagoya 464-0021 Japan.
| |
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ABSTRACT
INTRODUCTION
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