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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 241-248
IMMUNOBIOLOGY
Application of the ELISPOT assay to the characterization of
CD8+ responses to Epstein-Barr virus antigens
Jie Yang,
Victor M. Lemas,
Ian W. Flinn,
Chris Krone, and
Richard F. Ambinder
From the Oncology Center, Johns Hopkins University School of
Medicine, Baltimore, MD 21231.
 |
Abstract |
CD8+ cells have an important role in controlling
Epstein-Barr virus (EBV) infection. We adapted the interferon-
ELISPOT assay to the quantitative analysis of EBV-specific
CD8+ cells. Using peripheral blood mononuclear cells
(PBMCs) from healthy donors, we measured both the aggregate response to
the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as
stimulators, and the specific responses to 2 A2-restricted peptide
epitopes: the subdominant latency membrane protein-2 (LMP2) peptide
CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8+ cells were detected in all
EBV-seropositive donors (range 954 to 37 830 spots/106
CD8+ cells). LMP2 peptide-responsive CD8+
cells were detected in 10 of 11 healthy seropositive A2 donors (range
11 to 83 spots/106 PBMC). BMLF1 peptide-responsive
CD8+ cells were detected in all seropositive A2 donors
examined (range 13 to 943 spots/106 PBMC).
Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive
CD8+ cells and a lesser degree of expansion of BMLF1
peptide-responsive CD8+ cells. Finally, we applied the
ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive
CD8+ cells could be detected after infusion. Thus, the
ELISPOT assay can be applied to the analysis of CD8+
responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and
in PBMCs from patients treated with ex vivo expanded CTL
lines. (Blood. 2000;95:241-248)
© 2000 by The American Society of Hematology.
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Introduction |
CD8+ T cells have an important role in
controlling viral infection by recognizing small peptides derived from
intracellular pathogens presented on the surface of infected cells by
major histocompatibility complex (MHC) class I molecules.1
The present study concerns the CD8+ response to
Epstein-Barr virus (EBV), a ubiquitous herpesvirus associated with
several human malignancies including Burkitt's lymphoma,
nasopharyngeal carcinoma (NPC), Hodgkin's disease, and posttransplant
lymphoproliferative disease (PTLD).2-4 Studies suggest that
tumor cells in NPC, Hodgkin's disease, and PTLD are sensitive or
should be to CD8+-mediated killing.5-9
Furthermore, adoptive cellular immunotherapy is effective in treating
or preventing PTLD in some settings.10 With the ability to
manipulate CD8+ responses rapidly advancing, the importance
of assays to measure EBV-specific T-cell responses increases.
Standard methods of detecting and characterizing EBV antigen-specific
CD8+ T cells that rely on initial in vitro expansion in
response to stimulation with EBV-immortalized lymphoblastoid cell lines
(LCLs) have inherent limitations. The amplified repertoire of T cells may be skewed, responses to subdominant antigens obscured by responses to dominant antigens,6 and responses to lytic cycle
antigens missed altogether inasmuch as these are generally not
expressed by LCLs.
The ELISPOT assay for the detection of antigen-specific T cells offers
an alternative approach.11-14 This assay relies on the visualization of cytokine secretion by individual T cells following in
vitro stimulation with antigen. The assay is more sensitive than
enzyme-linked immunosorbent assay15,16 and does not require in vitro expansion of specific T cells before testing. In the present
study we adapted the interferon- (IFN-) ELISPOT assay to the
quantitative detection of EBV-specific CD8+ cells. We
assessed the aggregate CD8+ response to EBV latency
antigens expressed by LCLs, to a peptide derived from the subdominant
latency membrane protein 2 (LMP2), and to a peptide derived from the
lytic cycle antigen BMLF1. Responses were assayed in both unstimulated
peripheral blood mononuclear cells (PBMCs) and in polyclonal cytotoxic
T-lymphocyte (CTL) lines. Finally, we used the assay to monitor
adoptive cellular immunotherapy in vivo.
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Materials and methods |
PBMCs and isolation of specific cell populations
The PBMCs from healthy platelet donors and laboratory personnel with
known HLA type and known EBV antibody status were studied under a
protocol approved by a human investigations committee. PBMCs were
isolated by density gradient centrifugation using Ficoll-Hypaque 1.077 (Biochrom, Berlin, Germany) and cryopreserved immediately. CD8+, CD4+, or CD19+ cells were
positively selected from cryopreserved PBMCs using immunomagnetic beads
(Dynal, Oslo, Norway). Beads were detached from isolated cells by using
respective DetachaBead (Dynal). CD56+ cells were isolated
with CD56 MicroBeads (Miltenyi Biotec, CA).
Establishment of LCL
The PBMCs (5 × 106) were incubated with 2 mL
infectious supernatant from the B95.8 cell line for 2 hours at
37°C. An equal volume of RPMI-FBS (RPMI 1640, 2 mM
L-glutamine, 10 mM HEPES, 100 IU/mL penicillin, 100 µg/mL
streptomycin, 10% vol/vol fetal bovine serum [FBS]) was added
directly to PBMCs and the culture was supplemented with 25 U/mL
interleukin 2 (IL-2) (Proleukin, Cetus, Emeryville, CA) and 5 µg/mL
phytohemagglutinin (PHA) (Sigma). Thereafter cells were maintained in
RPMI-FBS.17
Generation of Staphylococcus aureus-activated B cells
Staphylococcus aureus Cowan I strain (SAC; Calbiochem, San
Diego, CA) activated B-cell blasts were used as controls in some ELISPOT assays. For generation of SAC-activated B-cell blasts, CD19+ B cells were isolated from cryopreserved PBMCs and
cultured at 2 × 106/mL in RPMI-FBS medium
supplemented with 0.01% SAC and 100 U/ml IL-2 for 3 days. The
activated B cells were then depleted of SAC and dead cells by density
gradient centrifugation using Ficoll-Hypaque 1.077.
Synthetic peptides
Peptides (Macromolecular Resources, Fort Collins, CO)
used in our study corresponded to LMP2 residues 426-434 (CLGGLLTMV),18 and BMLF1 residues 280-288 (GLCTLVAML).19 Both peptides are HLA-A*0201-restricted CTL
epitopes. A peptide corresponding to LMP2 residues 409-417 (ILTEWGSGN)
known not to be recognized by CTLs from A*0201
individuals18 was studied as a negative control. All
peptides were dissolved in dimethyl sulfoxide (Sigma) at a concentration of 10 mg/mL and further diluted in appropriate assay media for individual experiments.
ELISPOT assay
The 96-well multiscreen HA filtration plates (MAHA S4510, Millipore,
Bedford, MA) were coated with 50 µL of mouse antihuman IFN- mAb
(1598-00, R&D System, Minneapolis, MN; 4 µg/mL in PBS). After
incubation overnight at room temperature, wells were washed 4 times
with PBS. Remaining protein-binding sites were blocked by incubating
plates with 200 µL/well RPMI-AB medium (RPMI 1640, 2 mM
L-glutamine, 10 mM HEPES, 100 IU/mL penicillin, 100 µg/mL streptomycin, 10% vol/vol heat-inactivated human AB-serum (Sigma) for
2 hours at 37°C. Autologous LCL (1 × 105/well)
or peptide (10 µg/mL, final concentration) was plated with responder
cell populations. Responder cell populations were seeded across a range
of concentrations to achieve 10 to 100 spots/well so as to facilitate
accurate and reproducible counting. For LCL stimulators with PBMC
responders, the concentration used was 2 × 103 to
4 × 104 PBMC/well; for LCL stimulators with
CD8+ responders, the concentration used was
5 × 102 to 2 × 104
CD8+ cells/well; for LCL stimulators with CTL line
responders, this was 50 to 800 CTLs/well. For peptide stimulators (LMP2
or BMLF1 peptide) with PBMC responders, cells were plated at
2 × 105 PBMC/well and a total of 2 to
5 × 106 PBMC plated. CTL line responders with
peptide stimulators were plated at 1 × 103 to
4 × 104 CTLs/well and 1 × 105
autologous PBMCs were added to each well to serve as antigen presenters. The culture medium for the ELISPOT assay was RPMI-AB supplemented with 100 U/mL IL-2 (final volume 200 µL/well). After undisturbed incubation for 24 hours at 37°C, plates were washed 4 times with PBS containing 0.05% Tween 20 (PBS-Tw). Wells were incubated with 100 µL polyclonal rabbit antihuman IFN- antibody (IP-500, R&D System; 1:250 dilution in PBS-Tw) at 4°C overnight and
washed 4 times with PBS-Tw. Then 100 µL polyclonal
peroxidase-conjugated goat anti-rabbit IgG antibody (P-0448, DAKO,
Carpinteria, CA; 1: 625 dilution in PBS-Tw) was added and
incubated for 3 hours at 37°C. Wells were washed 3 times with
PBS-Tw and 3 times with PBS. Peroxidase substrate was prepared by
dissolving 20 mg 3-amino-9-ethycarbazol (Sigma) in 2 mL
dimethylformamide (Sigma). The solution was diluted 1:30 in 14.5 mL of
50 mmol/L sodium acetate buffer at pH 5.0. Immediately before use, 7.5 µL 30% H2O2 was added. The substrate solution was filtered through a 0.22 filter and added to wells at 100 µL/well. After incubation at room temperature for 8 minutes, the
reaction was stopped by discarding the substrate solution and washing
the plates under running water. The plates were then air dried and
colored spots counted using a stereomicroscope.
Data analysis
Frequencies of antigen-specific IFN--secreting cells were
calculated based on the numbers of responder cells and the number of
spots per well after subtraction of background. The procedure for
estimating background was tailored to the particular combination of
stimulator and responder. For ELISPOT assays with LCL stimulators, background was the sum of IFN- spots associated with LCLs alone and
those associated with responders alone. For ELISPOT assays with peptide
stimulators and PBMC responders, background was obtained by incubating
PBMC in the presence of a control peptide. For ELISPOT assays with
peptide stimulators and polyclonal CTL responders, background spots
include those from incubating CTL with control peptide and those from
incubating autologous PBMC with the peptide of interest. Correlation
analysis of ELISPOT and 51Cr-release assays was carried out
using Microsoft Excel software.
Establishment of EBV-specific polyclonal CTL lines
Polyclonal CTL lines were established according to previously
published methods.20 Briefly, 2 × 106
PBMC were co-cultured with -irradiated (8000 rad) autologous LCLs at
a responder/stimulator ratio of 40:1 in wells of 24-well plates. These
were restimulated on days 7 and 14. On day 21, CTL cultures were
restimulated again but at a new responder/stimulator ratio (4:1), and
IL-2 was added to a final concentration of 25 U/mL. CTLs were harvested
on day 14 or day 28, cryopreserved, and stored at  135°C
until being tested in ELISPOT or 51Cr-release assays. All
CTL cultures were maintained in lymphocyte expansion medium, LyEM (45%
RPMI 1640, 45% EHAA, 2 mM glutamine, 10 mM HEPES, 100 IU/mL
penicillin, 100 µg/mL streptomycin, 10% vol/vol FBS).
51Cr-release assays
PHA blasts were generated by culturing PBMCs at a concentration of
2 × 106/mL in the presence of 5 µg/mL PHA (Sigma)
in RPMI-FBS for 3 days. PHA blasts were washed 4 times and then
cultured for at least 4 more days in RPMI-FBS containing 100 U/mL IL-2
before being used in 51Cr-release assays. LCLs or PHA
blasts were incubated with 51CrO4 for 90 minutes. Labeled cells were washed and resuspended to
1 × 105/mL in RPMI-FBS, and 50 µL was added to
wells of 96 V-bottom plates containing 50 µL/well RPMI-FBS with or
without 40 µg/mL of the LMP2 or BMLF1 peptide and incubated for 2 hours at 37°C. Then cryopreserved CTLs were thawed and added to
each well (1.5 × 106/mL, 100 µL/well, E/T ratio
30:1) for the subsequent 5-hour incubation. The concentration of
peptide in the final assay volume (200 µL) was 10 µg/mL.
Patients and CTL infusion protocol
Two patients developed PTLD after solid organ transplantation. They
received EBV-specific CTL infusions from HLA partially matched donors
at a dose of 5 × 107 CTL/m2. Blood was
drawn before and at different time points after infusion. CD8+ cells were used as responders in ELISPOT assays with
the patient's LCLs as stimulators. Patient 1 had active PTLD and
patient 2 was in remission at the time of infusion. The donor
EBV-specific CTL lines were generated by weekly restimulation with
irradiated donor LCLs for 4 weeks.
| |
Results |
Detection of LCL-responsive CD8+ cells
To characterize the cellular sources of LCL-stimulated IFN-
production, PBMCs from a healthy seropositive donor were fractionated into CD8+, CD4+, CD56+, and
CD8 CD4CD56
populations by positive selection with immunomagnetic
beads. ELISPOT assays with autologous LCL stimulators were performed on
each population. Spots were found in CD8+,
CD4+, and CD56+ populations but not in the
CD8CD4CD56
population (Figure 1). The sum of the
numbers of spots from CD8+, CD4+, and
CD56+ populations was similar to that produced by the
corresponding number of unfractionated PBMCs tested in parallel.
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| Fig 1.
Cellular sources of IFN- secretion in response to
autologous LCLs.
PBMCs from a healthy EBV-seropositive donor FT were sequentially
fractionated into CD8+, CD4+,
CD56+, and
CD8CD4CD56
cell populations using CD8 and CD4 Dynal-beads and CD56 Microbeads.
Each cell population was incubated with autologous LCL stimulators for
the detection of IFN- secretion in an ELISPOT assay. Each bar
represents the mean ± SD of triplicate wells. The numbers of
spots/1 × 106 cells of the respective cell
population are shown on the Y axis.
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To measure the aggregate CD8+ response to EBV latency
antigens, we used isolated CD8+ cells from a series of
healthy donors with autologous LCL stimulators. In seropositive donors
the frequency of LCL-responsive IFN--producing cells ranged from 954 to 37 830 spots/106 CD8+ cells (mean 13 489)
(Figure 2). This corresponded to 0.1% to 3.8% (mean 1.3%) of the CD8+ population. In 3 seronegative donors, the frequency of LCL-responsive cells ranged from
680 to 1231 spots/106 CD8+ cells (mean 908, 0.1% of CD8+ population). Background produced by
CD8+ cells alone was < 7 spots/106
CD8+ cells (mean 1). Background associated with LCLs alone
varied from 0 to 16 spots/105 LCL (mean 3.2).
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| Fig 2.
Frequency of LCL-responsive
CD8+ cells.
Isolated CD8+ cells from 13 healthy EBV-seropositive and 3 seronegative donors were incubated with autologous LCL stimulators in
ELISPOT assays. Each bar represents the mean ± SD as determined in
2 separate experiments; data from each experiment represent the average
of triplicate wells.
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To determine whether the CD8+ responses to LCLs were EBV
specific or responses to the activated B-cell phenotype, SAC-activated B-cell blasts generated from 3 seropositive donors and 1 seronegative donor were used as negative controls in ELISPOT assays. No
CD8+ response to SAC-activated B blast cells was found
(data not shown).
Detection of peptide-responsive CD8+ cells
The LCL-responsive CD8+ cells consist of a mixed
population of peptide-specific CD8+ cells. Responses to
individual epitopes were assayed by using synthetic peptides as
stimulators. We studied 2 peptides: CLGGLLTMV from the LMP2 and
GLCTLVAML from the early lytic protein BMLF1. Recognition of both
peptides is restricted through HLA-A*0201.18 Peptide
stimulators elicited IFN- spots in the CD8+ population
and in unfractionated PBMCs, but not in the CD8+-depleted
population (data not shown). Therefore, in further experiments, unfractionated PBMCs were used as responder cells for the detection of
peptide-responsive CD8+ cells. We assayed PBMCs at various
peptide concentrations. No differences in the number of IFN- spots
were observed at concentrations of peptide ranging from 1 to 100 µg/mL, so 10 µg/mL was used in further experiments.
The PBMCs from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-A2, were tested for their reactivity to the LMP2 peptide (Figure
3). Ten of the 11 EBV-seropositive HLA-A2
donors showed a response to the peptide (range 11 to 83 spots/106 PBMC; mean 33). In contrast, the 2 EBV-seropositive non-HLA-A2 donors generated few IFN- spots (1 and 3 spots/106 PBMC, respectively). Responses to the BMLF1
peptide were measured in the same 13 seropositive donors (Figure
4). All donors who were HLA-A2 showed a
response to the BMLF1 peptide (range 13 to 943 spots/106
PBMC, mean 276). Donors who were EBV-seropositive but not HLA-A2 showed
no response (0 spots/106 PBMC). Background reactivity for
peptide stimulators was determined by incubating PBMC in the presence
of a control peptide corresponding to LMP2 residues 409-417 known not
to elicit a CD8+ response. No difference in the number of
spots was observed when PBMCs were incubated alone or with the control
peptide. The number of spots ranged from 0 to 33 spots/106
PBMC (mean 9).
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| Fig 3.
Frequency of LMP2 peptide-responsive
CD8+ cells.
PBMC from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-HLA-A2, were incubated with 10 µg/mL of the LMP2 peptide
(residues 426-434) in ELISPOT assays. Each bar represents the mean ± SD as determined in 2 separate experiments; data from each
experiment represent the average of more than 12 wells.
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| Fig 4.
Frequency of BMLF1 peptide-responsive
CD8+ cells.
PBMC from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-HLA-A2, were incubated with 10 µg/mL of the BMLF1 peptide
(residues 280-288) in ELISPOT assays. Each bar represents the mean ± SD as determined in 2 separate experiments; data from each
experiment represent the average of triplicate wells.
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To get a sense of the relative strength of responses to LCLs, to the
subdominant LMP2 peptide, and to the lytic BMLF1 peptide, we compared
the frequencies of CD8+ cells responsive to these
stimulators (Table 1). In EBV seropositive HLA-A2 donors, CD8+ cells specific for the LMP2 peptide
accounted for a minority (0.2% to 15.0%, mean 4.6%) of
LCL-responsive CD8+ cells. This was consistent with the
notion that LMP2 is a subdominant antigen. Responses to the lytic BMLF1
peptide were markedly higher than those to the LMP2 peptide in 10 of 11 HLA-A2 donors (range 2- to 35-fold, mean 13-fold).
Detection of EBV-specific CD8+ cells in polyclonal CTL
lines
Polyclonal LCL-stimulated EBV-specific CTL lines are being used in
ongoing clinical studies of adoptive cellular immunotherapy at our
center and others. We sought to evaluate these T-cell expansions using
the ELISPOT methodology. EBV-specific polyclonal CTL lines were
generated from 4 healthy HLA-A2 donors by weekly restimulation with
irradiated autologous LCLs for 4 weeks. The phenotype of these CTL
lines as determined by flow cytometry was predominantly CD8+ (on average 73.2% CD8+, 23.2%
CD4+, and 0.8% CD56+). EBV-specific
CD8+ responses were measured in unstimulated PBMCs and the
polyclonal CTL lines they yielded (Figure
5). Compared with unstimulated PBMCs,
polyclonal CTL lines showed an average 314-fold increase in
LCL-responsive CD8+ cells. This corresponded to 11% to
22% of the CTL population (106 383 to 223 214 spots/106
cells in the CTL line). The LMP2 peptide-responsive and BMLF1 peptide-responsive CD8+ cells showed an average increase of
160-fold (275 to 8673 spots/106 cells in the CTL line)
and 28-fold (700 to 12 755 spots/106 cells in the CTL
line), respectively, compared with PBMCs.
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| Fig 5.
Increases in the number of EBV-specific
CD8+ cells over the course of polyclonal
CTL line generation.
EBV-specific polyclonal CTL lines from HLA-A2 donors ES (A), SB (B), MS
(C), and TZ (D) were established. Cells were harvested on day 28 and
cryopreserved. On the day of the ELISPOT assay, aliquots of CTL lines
were thawed and incubated with stimulators in multiscreen HA plate.
Stimulators for ELISPOT assay included autologous LCLs
(1 × 105/well), and autologous PBMCs
(1 × 105/well) in medium containing the LMP2 or
BMLF1 peptide (10 µg/mL, final concentration). The fold-increase in
the number of LCL-responsive (filled diamonds), LMP2 peptide-responsive
(open circles), and BMLF1 peptide-responsive CD8+ cells
(open triangles) is indicated to the right of the day 28 data point.
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To test whether the number of EBV-specific CD8+ cells as
measured by ELISPOT correlates with the function of these cells,
cytotoxic activities of the polyclonal CTL lines were measured by
standard 51Cr-release assays. A positive correlation
(R = 0.9) was found between the frequency of EBV antigen-responsive
CD8+ cells and the cytolytic activity of CTL lines (Figure
6).
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| Fig 6.
Positive correlation between the number of
IFN--secreting cells and cytolytic activity in CTL lines.
EBV-specific CTL lines from 4 HLA-A2 donors were generated by weekly
restimulation with irradiated autologous LCLs for 2 to 4 weeks. The CTL
lines were then assayed as responders/effectors in a paired
ELISPOT/51Cr-release assay. Stimulators for the ELISPOT
assay or targets for 51Cr-release assay included autologous
LCLs for both the ELISPOT and 51Cr-release assays (filled
diamonds), autologous PBMCs in medium containing peptide (LMP2 or BMLF1
peptide) as stimulators for ELISPOT assays, and autologous PHA blasts
in medium containing peptide as targets for 51Cr-release
assay (open diamonds).
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Application of ELISPOT assay to the monitoring of changes in
LCL-responsive CD8+ cells following adoptive CTL infusion
In an ongoing clinical trial, patients with EBV-associated tumors
were infused with in vitro expanded EBV-specific CTL products from HLA
partially matched donors (Ambinder et al, in preparation). We sought to
determine whether changes in the frequency of LCL-responsive CD8+ cells could be detected by ELISPOT assay following
adoptive CTL infusion. Two patients receiving an infusion of
5 × 107 CTL/m2 from unrelated donors
were included in the pilot experiments. A transient increase in
LCL-responsive CD8+ cells was detected after infusion in
both patients examined (Figure 7).
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| Fig 7.
Application of the ELISPOT assay to the monitoring of
changes in the number of LCL-responsive
CD8+ cells following adoptive CTL
infusion.
Two patients with PTLD received EBV-specific polyclonal CTL infusion
from partially matched donors. The HLA types of patients and donors
were: patient 1 (A1, 24, B7, 8), donor for patient 1 (A1, 33, B8, 17);
patient 2 (A2, 32, B13, 27), donor for patient 2 (A2, 3, B7, 27). Blood
was drawn immediately before and at the indicated time points after
infusion. CD8+ cells were used as responders in ELISPOT
assays with the patient's LCLs as stimulators. Each bar represents the
mean ± SD of triplicate wells.
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Discussion |
We have adapted the ELISPOT assay to the quantitative detection of
EBV antigen-specific CD8+ cells. This assay is sensitive
and does not require prior expansion of specific T cells. In its
various formats the assay is suitable for the analysis of the aggregate
CD8+ response to EBV-infected lymphocytes (see Figure 2),
and responses to individual peptide epitopes including subdominant
antigens such as the LMP2-derived peptide (see Figure 3) and lytic
cycle antigens such as the BMLF1-derived peptide (see Figure 4).
Finally, it appears well suited for monitoring expansion of
EBV-specific T cells for adoptive cellular immunotherapy in vitro
and in vivo.
We used whole cell (LCL) stimulators in the ELISPOT assay described
here. Several features of LCLs make them suitable as stimulators. First, LCLs are phenotypically activated B cells with high surface expression of MHC class I and other molecules involved in various immune processes and are thus potent antigen presenters. Second, LCLs
express the full spectrum of EBV latent cycle antigens, namely, the
Epstein-Barr nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C,-LP, and the
LMPs 1, 2A, and 2B.21 A comparable aggregate assessment of
EBV-responsive T cells would require a mixture of many peptides tailored to each individual's HLA type. Third, insofar as the spectrum
of viral antigen expression in LCLs corresponds generally with that in
PTLD, assessment of response may be directly relevant to the
pathogenesis or treatment of this disease.
In the present study we aimed at measuring the CD8+
responses to EBV by detection of IFN- secretion. CD8+ T
cells, CD4+ T cells, and natural killer cells have been
identified by previous investigators as producers of
IFN-.22-26 When LCLs are used as stimulators, cells from
the CD8+, CD4+, and CD56+
populations secrete IFN-, whereas PBMCs depleted of these
populations (CD8CD4CD56
cells) do not (see Figure 1). In EBV seropositive individuals, non-CD8+ responders account for 20% to 81% (mean 62%) of
PBMC responders producing spots in response to LCL stimulation (data
not shown), whereas in EBV-seronegative individuals,
non-CD8+ cells account for a higher portion of the
responses (84% to 89%, mean 86%). Previous investigators have also
reported a high frequency of LCL-reactive CD56+ cells in
cord blood and seronegative donors.27
Peptides were also used as stimulators to characterize responses to
single epitopes. The first peptide chosen in the present study was the
LMP2-derived peptide restricted through HLA-A*0201. ELISPOT assays
reproducibly detected responses to the subdominant LMP2 peptide in most
healthy EBV-seropositive HLA-A2 donors (see Figure 6). Consistent with
previous reports that LMP2 is a subdominant antigen, responses to this
peptide accounted for about 5% of the CD8+ response to
autologous LCLs. Because LMP2 is expressed in most types of EBV tumors
8,28-30 and is restricted through a common HLA
type,31 assays of responses to this antigen may provide a
window on immune responses to viral tumor antigens in patients with
these malignancies.
The second peptide tested is derived from the early lytic protein
BMLF1. Recent work by Steven et al showed that primary EBV infection is
accompanied by unusually strong responses to lytic cycle
antigens.19 Responses to the BMLF1 peptide were as strong as those to an immunodominant latency antigen epitope. Callan et al
reported a high frequency of BMLF1 peptide-responsive CD8+
cells during primary infection with tetramer staining
technique.32 Using the ELISPOT assay, we could reproducibly
detect CD8+ cells specific for this early lytic BMLF1
peptide in all healthy EBV-seropositive HLA-A2 donors tested. The
number of BMLF1-responsive cells is 13-fold higher on average than the
number of LMP2 peptide-responsive cells. Similar results have recently
been published by Tan et al.14 These authors also found
good responses to a B8 restricted epitope in the lytic BZLF1 protein.
The frequency of responses to these lytic epitopes was similar to the
frequency of responses to the immunodominant EBNA3A and EBNA3B
epitopes. This demonstrates not only that lytic cycle antigen-specific
T cells persist in the healthy carrier state, but also that they
persist at high frequencies. Other investigators have called attention
to the cellular immune response to lytic epitopes.33,34
Thus, lytic cycle antigen-specific T cells may have a role in
controlling lytic reactivation of EBV infection in the carrier state.
Inasmuch as PTLD is often associated with lytic antigen
expression,35-37 the ability to monitor these responses may
be important in understanding the pathogenesis of these disorders and
in developing new approaches to therapy.
The ELISPOT assay detects antigen-specific T cells in unstimulated
PBMCs and in CTL lines. Our study of EBV-specific CTL lines showed that
LCL-responsive CD8+ cells constitute 11% to 22% of the
total cellular population in the product used for adoptive cellular
immunotherapy. It should be noted that these data were obtained with
frozen CTL lines that were thawed immediately before the ELISPOT assay.
Limited data with fresh CTL lines have yielded higher estimates of the
frequency of LCL-responsive CD8+ cells (60% to 100%, data
not shown). In EBV-specific polyclonal CTL lines, LMP2
peptide-responsive CD8+ cells, although increased one- to
several hundred-fold, constitute only a small minority of cells,
averaging about 4% of LCL-responsive CD8+ cells. This
explains why CTL responses to this LMP2 peptide are sometimes obscured
in the analysis of cell lines but can be detected more readily by
analysis of individual T-cell clones.6,38 The lesser degree
of expansion of BMLF1 peptide-responsive CD8+ cells
supports the idea that the repertoire of EBV-specific T cells in vivo
may be skewed during in vitro expansion with LCLs toward latency
antigens.21 However, BMLF1 peptide-specific
CD8+ cells still increased significantly in all of the
polyclonal CTL lines tested, indicating that the occasional lytic
events in LCLs are capable of reactivating CD8+ cell
responses to lytic antigens. The presence of lytic antigen-specific T
cells exemplified by BMLF1-responsive CD8+ cells suggests
that polyclonal CTL products may also be effective in controlling lytic
EBV infection and the spread of virus.
One of the potential applications of the ELISPOT assay is in monitoring
therapeutic interventions. Several investigators have reported adoptive
cellular immunotherapy with donor lymphocytes from allogeneic bone
marrow donors or with EBV-activated lymphocyte populations expanded in
vitro from allogeneic and autologous sources. Generally these infusions
have been monitored by limiting dilution assays (LDA).39-42
The ELISPOT assay with LCL stimulators presents an alternative approach
that requires fewer cells and less time for the assay. In addition, our
preliminary data show that values obtained from ELISPOT with LCL
stimulators are 2- to 10-fold higher than values obtained from LDAs
(data not shown). Other investigators also reported a 5.3-fold higher
frequency of epitope-specific CD8+ T cells as measured by
ELISPOT assays than those measured by LDAs.14 Using the
ELISPOT assay, we show that in patients with a history of PTLD,
adoptive T cell immunotherapy from HLA partially matched allogeneic
donors led to transient increase in frequency of CD8+ cells
responsive to EBV. Analysis of the fate of infused T cells in a series
of patients treated with EBV-specific T cells from partially matched
allogeneic donors is ongoing.
Other methods have also been developed for the detection of
antigen-specific T cells. Among them, direct staining of the
antigen-specific CD8+ set with multimeric (tetrameric or
dimeric) complexes of MHC class I glycoprotein with
peptide43-47 has attracted great interest. The multimer
staining technique and the ELISPOT assay using peptide have in common
high specificity that allows analysis of unfractionated PBMCs. However,
whereas the ELISPOT assay can be adopted to characterize an aggregate
response to viral antigens in fractionated cell populations as shown
here using the assay with LCL stimulators, there is as yet no
comparable approach to the detection of aggregate responses using the
multimer staining technique. The multimer staining technique and the
ELISPOT assay take advantage of different aspects of the T-cell-antigen interaction. The multimer staining technique uses the
specific physical interaction between T-cell receptor (TCR) and
peptide-bound MHC class I molecules and thus is capable of detecting
the physical presence of antigen-specific T cells without any
requirement for function and might detect anergic cells.48 The ELISPOT assay, on the other hand, relies on the ability of T cells
to secrete cytokine on activation. Our results show that IFN-
secretion correlates with specific target cell killing (see Figure 6).
Similar conclusions have been reported by previous investigators.49
Tan et al compared the ELISPOT assay with tetramer staining for the
detection of EBV epitope-specific T cells.14 They reported that on average tetramer staining yielded 4.4-fold higher frequencies than did ELISPOT assay, presumably reflecting the presence of T cells
that carried TCR of appropriate specificity, but did not produce
IFN- on exposure to peptide. Thus at higher T-cell frequencies, tetramer staining yields higher estimates than ELISPOT assay. However,
at T-cell frequencies of 20/106 PBMC or lower as measured
by ELISPOT assay, tetramer staining failed to yield signal, presumably
reflecting sensitivity limits imposed by the flow cytometry detection
method. In our hands, antigen-specific T cells at frequencies as low as
1/100 000 PBMCs could be reproducibly detected by ELISPOT assay.
In conclusion, the ELISPOT technique may be particularly useful in the
analysis of low-frequency responses. The ability to analyze aggregate
responses to whole cells suggests the possibility of a much broader
range of applications than has previously been explored, especially in
the monitoring of immunotherapeutic interventions as illustrated
here in the adoptive immunotherapy trial.
| |
Acknowledgment |
We thank Dr Hyam I. Levitsky for advice.
| |
Footnotes |
Submitted July 20, 1999; accepted August 30, 1999.
Supported by NIH grant PO1 CA15396.
Reprints: R. F. Ambinder, Johns Hopkins Oncology Center, 418 North Bond Street, Baltimore, MD 21231; email: rambind{at}jhmi.edu.
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.
| |
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Abstract
Introduction