Received 8 January 2001/Returned for modification 23 March
2001/Accepted 9 May 2001
Cell-mediated immunity appears to be critical for the prevention
and control of varicella-zoster virus (VZV) infection and complications
arising from zoster. Current assays of VZV-specific cell-mediated
immunity are cumbersome or lack sensitivity. We have developed a gamma
interferon ELISPOT assay that provides a direct measure of the number
of T cells secreting a cytokine following stimulation with antigen.
This assay is extremely sensitive and specific, with the ability to
detect gamma interferon spot-forming cells (SFC) in the range of 10 to
1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This
assay has been validated by demonstrating the following: (i) the
response detected is mediated almost entirely by CD4+ T
cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to
those from freshly isolated cells, (iii) frozen PBMCs can be shipped on
dry ice for up to 48 h without loss of activity, (iv) frozen PBMC
samples can be stored in liquid nitrogen over long periods (>22
months) without any significant change in response, and (v) the numbers
of ELISPOTs counted using a computer-based imaging system are
equivalent to those counted by humans but have lower variability. The
ability to use frozen cells is facilitated by the use of a recombinant
nuclease (Benzonase) that can prevent cell clumping when samples are
thawed. Frozen PBMC samples can be cycled through multiple changes in
storage between liquid nitrogen and dry ice without any change in
response being detected. This facilitates collection of samples at one
site and testing performed at a remote location. This VZV ELISPOT assay
provides a new versatile tool for monitoring cellular immune responses
either during a herpes zoster disease outbreak or following vaccination.
 |
INTRODUCTION |
The importance of cellular immunity
in prevention and control of varicella-zoster virus (VZV)
infection has been well documented (1-4, 10, 12).
Components of cellular immunity for memory responses following both
natural infection and vaccination have been described. This includes
detection of both CD4+ (helper)- and
CD8+ (cytotoxic)-T-cell responses specific to
numerous VZV antigens (7, 11, 13-19, 25, 28, 30, 33,
36-38). Lymphoproliferation assays are not quantitative and
measure only CD4 T-cell responses. The limiting-dilution format
responder cell frequency (RCF) assay permits some quantitation of
response yet is very cumbersome. The cytotoxic-T-cell (CTL) assays can
measure CD8 T-cell responses but are also quantitative only in the
cumbersome format of limiting-dilution analysis. Intracellular cytokine
staining can be used for both CD4 and CD8 T-cell responses, but high
background signals can limit detection of low-frequency responses. All
of these methods utilize freshly isolated cells for optimum detection
of signal. There is a need for new quantitative assays to assess these
cellular immune responses both during the course of infection and after vaccination. ELISPOT assays for the detection of cytokine-producing T
cells are becoming more widely adopted for these purposes, and the
detection of gamma interferon (IFN-
) production with this method is
especially important in monitoring TH1 (helper) and TC1 (cytotoxic) responses.
The IFN- ELISPOT assay is a method for determining the number of
individual T cells secreting a cytokine after stimulation with a
specific antigen or peptide (5, 9, 23, 26, 29, 31). The
number of spots increases proportionately with the strength of the
immune response. An important advantage of the IFN- ELISPOT response
is that it is a direct measurement of a TH1 cell-mediated immune
response. As such, it is useful for monitoring the effectiveness of a
vaccine to induce cell-mediated immunity. The ELISPOT assay utilizes
two high-affinity cytokine-specific antibodies directed against
different epitopes on the same cytokine molecule. Spots are generated
with a colorimetric reaction in which soluble substrate is cleaved,
leaving an insoluble precipitate at the site of the reaction. The spot
represents a footprint of the original cytokine-producing cell. The
number of spots is a direct measurement of the frequency of
cytokine-producing T cells. The IFN- ELISPOT assay presents
novel challenges to validation compared with traditional
standard-curve-type assays. The assay endpoint (a spot) is the result
of a complex series of events that can be broken down into three
categories: (i) the cell culture conditions leading to production of
IFN-; (ii) the antibody capture, enzyme-mediated detection system;
and (iii) the technique for spot counting.
The present report describes both the development of a new IFN-
ELISPOT assay for the quantitation of cellular responses to VZV antigen
and the steps taken to validate the assay. The results indicate that
the assay is highly sensitive and specific. Additionally, we show that
this assay can be used to evaluate responses with either fresh or
frozen peripheral blood mononuclear cell (PBMC) samples, with PBMCs
obtained from blood that was stored at 4°C overnight, and that
responses can be quantitated with or without sophisticated equipment.
This assay provides a new, versatile tool for analysis of cellular
immune function following either disease outbreak or vaccination.
| |
MATERIALS AND METHODS |
Isolation of PBMCs from whole blood.
Whole blood was
collected from donors into either heparin- or EDTA-containing
Vacutainer tubes (Becton Dickinson, Franklin Lakes, N.J.). The blood
was diluted with Hanks balanced salt solution without calcium and
magnesium (Gibco BRL, Gaithersburg, Md.) and layered on top of the frit
in Accuspin System Histopaque-1077 cell separation tubes (Sigma, St.
Louis, Mo.). The tubes were centrifuged at 1,000 × g
for 10 min at 20°C, and the buffy layer containing the PBMCs was
removed. The cells were washed, and then the red blood cells were lysed
with ACK lysing buffer (Gibco BRL). The cells were washed twice with
Hanks balanced salt solution. Cells were counted using a Z1 dual
particle counter (Beckman Coulter, Miami, Fla.). The cells were washed
and resuspended in complete medium to the desired concentration (see
"Preparation of frozen PBMCs" below for a description of
complete medium).
If PBMCs were to be frozen, cells were instead resuspended in freezing
medium consisting of 90% heat-inactivated fetal bovine serum (HyClone,
Logan, Utah) and 10% dimethyl sulfoxide (Sigma). Cells were
resuspended to a concentration of 1 × 107
to 2 × 107 cells/ml in the freezing medium
and placed into a Nalgene 1°C cryogenic freezing container (Fisher,
Bridgewater, N.J.). The freezing container was then stored at 
70°C
overnight, and frozen cell samples were then transferred to liquid
nitrogen (vapor phase) for long-term storage.
Preparation of frozen PBMCs.
RPMI 1640 medium was
supplemented with 10% heat-inactivated fetal bovine serum (HyClone),
10 mM HEPES buffer (Gibco BRL), 1 mM L-glutamine (Gibco
BRL), 100 µg of penicillin per ml, 100 U of streptomycin (Gibco BRL)
per ml, and 5 × 10
5 M 
-mercaptoethanol
(Sigma). The medium, termed complete medium, was warmed to room
temperature. Complete medium was supplemented with Benzonase (EM
Industries, Hawthorne, N.Y.) to a final concentration of 
50 U/ml.
Frozen cells were thawed at 37°C, and Benzonase-supplemented medium
was slowly added. Cells were washed and resuspended in complete medium
supplemented with Benzonase. Cells were again washed, resuspended in
medium without Benzonase, and quantitated with a Coulter Instruments Z1
dual particle counter. Cells were washed and then resuspended in
complete medium without Benzonase at a concentration of
107 cells/ml for assay setup.
IFN- ELISPOT assay for VZV responses.
The wells of a
96-well Multiscreen-IP membrane plate (Millipore, Bedford, Mass.) were
coated with 100 µl of an anti-human recombinant IFN- monoclonal
antibody (catalog no. M-700A; Endogen, Woburn, Mass.) at a
concentration of 5 µg/ml overnight at 4°C. The wells were washed
three times with sterile phosphate-buffered saline (PBS). The plates
were then blocked by adding 200 µl of complete medium and incubating
at 37°C with 5% CO2 for 1 to 3 h. The
wells of the plate were washed once with 100 µl of complete medium.
To each well was added 50 µl of complete medium containing the
appropriate antigen. The VZV antigen was a UV-inactivated preparation
of VZV antigens derived from clarified cell culture supernatants
from VZV-infected MRC-5 cells. The control (MRC-5) antigen was produced
by the same process as the VZV antigen preparation using uninfected
MRC-5 cells. The final VZV antigen dilution used in the assay was
selected by titration experiments. The control antigen was diluted to
contain approximately the same MRC-5 cell-associated antigen content as
in the VZV antigen preparation. Phytohemagglutinin (PHA) M
(Sigma) at 5 µg/ml was incorporated as a positive control.
Next, 50 µl of PBMC cell suspension at 107
PBMCs/ml was added to each well. Assay plates were then incubated
overnight for 16 to 20 h at 37°C, 5% CO2,
and 95% humidity. Plates were washed six times with PBS containing 5%
heat-inactivated fetal bovine serum and 0.005% Tween-20 (ELISPOT wash
buffer). A biotinylated anti-human recombinant IFN- antibody
(catalog no. M-701-B; Endogen) was diluted to 1.0 µg/ml in ELISPOT
wash buffer and added at 50 µl per well. After overnight incubation
at 4°C, the plate was washed six times with ELISPOT wash buffer.
Streptavidin-alkaline phosphatase (Pierce, Rockford, Ill.) was diluted
appropriately in ELISPOT wash buffer (the specific dilution was
determined for each lot of conjugate used). To each well of the assay
plate was added 100 µl of diluted conjugate, and the plate was
incubated at room temperature for 1.5 to 2.5 h. The plate was
washed three times with ELISPOT wash buffer and then three times with
PBS. One hundred microliters of 1-Step nitroblue
tetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate (Pierce) was
added to each well, and spots were developed for 3 to 15 min at room
temperature. Substrate was emptied from the wells of the plates, and
then the wells were rinsed with water to stop the reaction. The plates
were allowed to air dry, and then spots were enumerated by counting
under a dissecting microscope or using an ImmunoSpot Image Analyzer
system (Resolution Technology Inc., Columbus, Ohio) for automated plate
scanning, imaging, and spot counting.
CD4 and CD8 cell depletion.
CD4 or CD8 T-cell populations
were depleted from PBMCs using the Dynabead (catalog no. 111.06 or
111.08; Dynal A.S, Oslo, Norway) magnetic bead system according to the
manufacturer's recommended procedures. Cells were then resuspended in
complete medium at 107 cells/ml for the ELISPOT
assay. Depletion was confirmed by fluorescence-activated cell sorter
(FACS) analysis.
Statistical analysis of assay validation data.
The variances
attributable to the different factors (assay run, reader, and bleed)
were estimated using SAS Proc Varcomp. (SAS Institute, Cary, N.C.). For
the human readers, the mathematical model compatible with the design of
the study used to estimate the three factors (assay run, reader, and
bleed) recognized assay run as being nested with donor and reader as
being nested with both assay run and donor. The component attributable
to bleed was estimated for the residual mean square. For the computer
readings, the mathematical model is simplified to one where assay run
is nested with donor and bleed was estimated for the residual mean square. The effects of run plus bleed and the sum of the three factors were constructed from the sum of the individual
component mean squares. The degrees of freedom were estimated for the
individual and sum effects using a Satterthwaite approximation
(24). The confidence limits shown in Table 5 were
established using chi-square percentiles with these estimated degrees
of freedom.
| |
RESULTS |
Optimization of assay parameters.
Pairs of coating and
detecting antibodies were compared to qualitatively select those giving
the lowest background (with no stimulation) and the highest signal with
positive control (mitogen) stimulation (data not shown). The antibody
pair selected for assay use was then tested in a cross titration over a
range of concentrations to select appropriate levels for incorporation
into a standardized assay protocol. The capture antibody was effective
over the range of concentrations tested from 2.5 up to 10 µg/ml. The
detecting antibody produced a good signal with no reduction in spot
count over the range from 0.1 to 2.5 µg/ml in combination with all
capture antibody levels. There was a slight reduction in both spot
count and signal quality when the capture antibody was used at 2.5 µg/ml in combination with the detecting antibody at 0.1 µg/ml.
A titration of VZV and MRC-5 antigen dilution levels from 1:40 down to
1:640 was performed. Responses were similar for most VZV antigen
dilutions, with no decrease in response observed even when the VZV
antigen was tested at a 1:320 dilution (data not shown). Additionally,
background responses to the MRC-5 cell lysate control antigen were
similar over the dilution range tested.
The kinetics of antigen incubation required for spot formation was
analyzed with the VZV antigen at a 1:80 dilution. There was an increase
in the number of spot-forming cells (SFC) detected at 16 h
compared with 6 h of incubation. The total number and quality of
spots in the assay were similar over an incubation range of 16 to
21 h. Beyond 21 h of incubation there was no increase in the
number of spots detected, while there was a reduction in the
qualitative measures (more diffuse and overlapping spots, making
enumeration more difficult).
For the final standardized assay protocol, the capture antibody was
applied at 5 µg/ml and detecting antibody was added at 1 µg/ml. The
VZV antigen was added at a dilution of 1:80, MRC-5 control antigen was
added at a dilution of 1:160 (providing MRC-5 protein levels equivalent
to those of the VZV antigen preparation), and an incubation time of 16 to 20 h was used. Responses were evaluated in terms of adjusted
VZV spot counts. The adjusted VZV spot count represents the mean spot
count for VZV antigen-stimulated wells minus the mean spot count for
MRC-5 control-stimulated wells.
Titration of cells per assay well.
The PBMC numbers in each
assay well were titrated over a range from 1 × 105 to 6 × 105
cells/well. The results are shown in Fig.
1. When responses were adjusted to SFC
per 106 PBMCs, the responses for individual PBMC
samples were similar over the range of cell concentrations from 2 × 105 to 6 × 105
cells/well. Responses for donor PBMCs with the highest SFC numbers overall remained fairly linear over the range of 2 × 105 up to 6 × 105
cells per well. As the donor response decreased there was a reduction in the normalized response (SFC/106 PBMC) when
the assay well concentration was at or below 2 × 105 cells/well. This was especially noted when
samples from an individual having fewer than 100 SFC per million PBMCs
were analyzed.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 1.
Effect of cell concentration on IFN- VZV ELISPOT
response. SFC represent the adjusted VZV ELISPOT response, i.e., the
mean response to VZV antigen (n = 3) minus the mean
response to MRC-5 control antigen (n = 3).
|
|
The final, standardized operating procedure for the VZV ELISPOT assay
was designed with the addition of 5 × 105
cells per assay well.
Prevention of cell clumping using a recombinant nuclease
(Benzonase).
Testing freshly isolated PBMCs is cumbersome when
monitoring large clinical trials or when monitoring patient responses
to natural infection over time. Testing frozen PBMCs offers a more practical alternative; however, when frozen PBMCs were initially utilized, problems with cell clumping upon thawing were sometimes encountered. This appears to be related to both the donor and the blood
handling. PBMCs from certain donors were found to clump more than cells
from other donors, even for bleeds separated by time. In addition,
clumping occurred more frequently when blood was stored (e.g.,
overnight) before isolation of PBMCs. To address this problem, we added
a recombinant nuclease (Benzonase) to the assay medium for the first
two wash steps during the thawing procedure for frozen cells. As a
demonstration of the utility of this method, an experiment was
performed in which blood was collected from donors and then frozen on
the day it was collected (fresh) or stored overnight at 4°C before
processing and freezing the PBMCs. At a later time, frozen PBMCs were
thawed with and without Benzonase and tested in the ELISPOT assay. As
shown in Fig. 2, the results demonstrate
no difference in response for frozen PBMCs isolated from fresh
blood when processed either with or without Benzonase. However, the
spot count results for overnight-stored blood processed without
Benzonase showed a dramatic decrease in three of the four donors
compared with the VZV responses for the corresponding PBMCs isolated
from fresh blood. The greatest decreases were detected for samples in
which the highest degree of cell clumping was observed. The SFC results
from the overnight blood PBMCs processed with Benzonase more closely
approximate the results obtained with cells isolated from fresh blood.
These increases in response for overnight blood PBMCs were greatest for
the two samples (donors 1015 and 4963) that had the highest degree of
cell clumping when processed without Benzonase. However, these
responses were still lower than those detected with the PBMCs frozen
from freshly collected blood. The incorporation of Benzonase did not
result in detectable changes either in cell viability upon thawing or
in the expression of cell surface markers for CD4, CD8, CD38, or CD62L
(data not shown).
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 2.
Comparison of results with frozen PBMCs isolated from
freshly collected blood versus blood stored overnight (4°C). SFC
represent the adjusted VZV ELISPOT response, i.e., the mean response to
VZV antigen for replicate wells minus the mean response to MRC-5
control antigen for replicate wells.
|
|
Comparison of fresh versus frozen PBMC samples.
The response
of freshly isolated (not frozen) PBMCs was compared with the response
of frozen PBMCs, isolated from the fresh blood (fresh frozen), in the
VZV ELISPOT assay. The blood was collected into EDTA anticoagulant,
PBMCs were isolated and aliquoted, and some aliquots were frozen.
Frozen PBMCs were thawed following the procedure with incorporation of
Benzonase-supplemented medium. As shown in Fig.
3, the adjusted VZV responses of frozen
PBMCs were comparable to the responses observed with the freshly
isolated PBMCs. This was consistent for both low- and high-responding
donors.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 3.
Comparison of IFN- VZV ELISPOT responses with fresh
versus frozen PBMCs. SFC represent the adjusted VZV ELISPOT response,
i.e., the mean response to VZV antigen for replicate wells minus the
mean response to MRC-5 control antigen for replicate wells.
|
|
Characterization of T-cell response in ELISPOT assay.
Fresh-frozen PBMC samples were tested in the ELISPOT assay after
depletion of CD4+- or
CD8+-T-cell subsets. Following subset depletion,
PBMCs were tested in the ELISPOT assay along with the undepleted cell
population for responses to both VZV antigen and tetanus toxoid. As
shown in Table 1, depletion of the
CD4+ T cells resulted in a complete loss of
response to both VZV and tetanus toxoid antigens in the assay. The
response to mitogen (PHA) remained very strong with these samples
(scored as too numerous to count [data not shown]). The loss
of response signal was seen even in the absence of complete depletion
of the CD4+-T-cell population (approximately 10%
CD4+ T cells remained following depletion as
assessed by FACS staining analysis [data not shown]). When the
CD8+-T-cell population was depleted, there was no
observed reduction in VZV- or tetanus toxoid-specific response. In
fact, there was a slight increase in the measured response. The
adjusted VZV responses went from 227 to 277 SFC for donor 1481 and from
191 to 207 for donor 2515, when comparing the undepleted response with
response following CD8+-T-cell depletion. The
CD8+ depletion was complete, with <1%
CD8+ T cells detected by FACS analysis (data not
shown). The increase in detected response for these samples most likely
resulted from enrichment of the CD4 population concomitant with the
depletion of CD8 T cells.
Effect of storage conditions on frozen PBMCs.
Aliquots of
frozen PBMCs were exposed to different combinations of liquid nitrogen
(vapor phase) and dry ice storage. The samples were then tested in an
ELISPOT assay (Table 2). There were no
significant differences in the adjusted VZV SFC for PBMC aliquots
following any of the storage conditions tested, with the exception of
the 2515 sample, which was cycled from liquid nitrogen to dry ice for
48 h and then back to liquid nitrogen. This PBMC sample had a
response of 108 SFC, compared with 185 SFC for the sample that was
stored only in liquid nitrogen. This decrease in response was not
observed with PBMC samples from other donors exposed to the same
storage conditions.
We have also tested frozen PBMC samples over an extended period (>22
months) of liquid nitrogen storage. A large number (>100) of PBMC
aliquots were prepared and frozen at a single time from two different
donors. These PBMC aliquots were then monitored over time for cell
yields, viability on thawing, and ELISPOT response. As shown in
Fig. 4, ELISPOT responses from
these donor cells remained relatively consistent for up to 22 months of
storage. Table 3 contains a summary of
the mean cell yields and adjusted VZV spot counts for assays over this
22-month period. The cell yield and spot count results were determined
from a total of 68 events for donor 1481 and >40 events for donor
2515. There were no changes in either cell yield (per frozen aliquot)
on thawing or cell viability (data not shown) detected over this
storage period. The cell yields per vial had standard deviations that
were less than 10% of the total cells obtained on thawing. The
viability of the frozen aliquots is consistently around 95% (by trypan
blue dye exclusion) with up to 22 months of frozen storage. The
response (SFC) variability for PBMC aliquots from these donors (percent
relative standard deviations [RSDs] of 20.27 and 31.20) falls within
the assay variability attributable to the combination of reader
(23.3%) and run (17.8%) variabilities for testing fresh-frozen PBMC
samples in the ELISPOT assay (see below).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
IFN- ELISPOT response of frozen cells from a positive
control cell bank over a 22-month period. The dates shown for the open
and closed circles indicate when PBMCs were collected and frozen.
Aliquots were stored in vapor phase liquid nitrogen.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Historical IFN- VZV ELISPOT responses and
frozen-aliquot cell yields for long-term storage control PBMC samples
|
|
ELISPOT assay variability using fresh-frozen PBMCs.
A
validation study was performed with the final, standardized VZV ELISPOT
protocol testing fresh-frozen PBMC samples. This protocol was designed
to assess the assay repeatability and variability for monitoring
patient responses over time. PBMCs were collected from four donors on
at least four different days. The PBMCs were isolated and frozen from
blood on the day it was collected (fresh-frozen cells). Aliquots of
frozen PBMCs from four different bleeds for each donor were assayed
together within a run. This was repeated for three separate assay runs.
The spots on the assay plates were then counted by five
different human readers and were also imaged and counted five times
with the ImmunoSpot image analysis computer system. The variability of
the ELISPOT assay was assessed for determination of significant
differences between bleeds from the same individual. The adjusted VZV
spot count (SFC) results for the individual blood donors, determined by
both human and computer readings, are presented in Table
4. The assay variability (percent RSD)
determined by the statistical analysis of these validation data is
presented in Table 5. The overall
variability for the assay has been broken down into three areas: assay
run, bleed, and (in the case of human readers) reader. The spot counts
obtained by the computer are similar in magnitude, and without change
in rank order, to those obtained with the human readers (Table 4). The
counts obtained with the ImmunoSpot computer system are at least as
reproducible as the human counts, with percent RSDs that are either
equivalent to or smaller than those associated with the comparable
human counts. The assay variabilities (percent RSD), shown in Table 5,
associated with bleed (PBMCs collected at different times) were similar
for human readers (20.13) and the computer (21.03). The variability for
assay run (run to run) was determined to be higher when the assay was
quantified with human readers (17.78) than when counted with the
computer-assisted imaging system (8.55). The combined run-plus-bleed
variability of the assay, indicating overall assay variability when
counted by the same reader, was higher for human readers (30.45) than for the computer-read assays (23.09). The largest single factor for
variability associated with the VZV ELISPOT assay was attributed to the
human reader (23.30).
| |
DISCUSSION |
We have developed a VZV IFN- ELISPOT assay applicable for
assessing cellular immune responses following either natural infection or vaccination. The traditional methods used for assessment of cellular
responses to VZV have proven to be both cumbersome and labor-intensive.
In general, they also lack desired levels of sensitivity and are not
very versatile. The use of a lymphoproliferation assay, the RCF assay,
has been described and extensively employed for quantifying T-cell
responses (8, 14-16, 21, 33). However, this assay has
disadvantages in that it requires the use of freshly isolated PBMCs, is
very labor-intensive, requires clonal expansion of responding cell
populations over an extended incubation period, and incorporates
labeling and detection with radionucleotide. CTL assays have also been
previously employed to look at CD8+-T-cell
responses (13, 30). These assays have many of the same
encumbrances as the RCF assay and have the additional constraint of not
being quantitative without incorporation of the even more laborious
limiting-dilution format. The limited data for CTL responses following
VZV immunization have indicated low frequencies for these populations,
and the specific role of CD8+ T cells in the
control of varicella infections is not well described to date
(13, 28, 30). More recently, the method of intracellular cytokine staining for detection of antigen-specific IFN- production has been employed (6). This method permits monitoring of
both CD4+ and CD8+
responding cells within the same assay. Additionally, this assay allows
a quantitative analysis of responding cell frequencies without the need
for clonal expansion and large numbers of cells being analyzed with
limiting dilution. The limitations with intracellular cytokine staining
may lie with the background responses detected. The signal-to-noise
ratios presented for VZV antigen stimulation may not allow adequate
resolution of changes in low-frequency responses of samples and
individuals. Additionally, this assay utilizes either fresh
blood or freshly isolated cells and requires expensive instrumentation
and trained personnel for sample analysis. However, this is a new
methodology with much promise and the opportunity for further refinement.
The ELISPOT assay that we have developed is extremely sensitive, with
the ability to detect fewer than 10 SFC per million PBMCs.
Additionally, background responses to medium or MRC-5 cell lysate
controls in this ELISPOT assay are very low, generally less than 10%
of the antigen-specific responses. The assay is very sensitive for
measuring changes in VZV-specific responses between two bleed points
for an individual donor. The ability to use a computer-assisted image
analysis for spot quantitation also reduces the most labor-intensive
and variable aspect of the assay. The ImmunoSpot image analysis system
utilized in this study produced spot counts statistically similar to
the counts obtained by human readers, with reduced variability
attributed both to reader and total assay (reader and run variabilities combined).
Only CD4+-T-cell responses were detected with
this assay. Evidence for low-frequency
CD8+-T-cell responses has been presented for
immunized individuals (13, 30). However, assessment of
memory responses to VZV with this ELISPOT assay has not detected any
CD8+-T-cell component with the antigens tested to
date, consistent with the observations recently published for
intracellular cytokine staining (6). It may be that
CD8+ memory populations are undetectable without
clonal expansion. It remains to be determined if this assay can be
adapted (i.e., by using peptide antigens, testing cells during acute
infection, or longer incubation) for detection of any
CD8+-T-cell responses to VZV that may exist.
The VZV antigen response was shown to be linear over a narrow range of
input cell numbers (from 2 × 105 to 6 × 105 cells/assay well). Accessory cells play an
important role in antigen processing and presentation in association
with major histocompatibility complex class II molecules for
CD4+-T-cell responses. Dilution of cell numbers
in the assay well reduces both responding cells and antigen-presenting
cells, which also may affect contact between the two populations. It
has been shown in a mouse ELISPOT assay system that the ratio of spots (response) to primed cells remained linear only when feeder cells were
added to keep the total number of cells per well constant (26). There is a critical level reached where the
concentration of antigen-presenting cells and
CD4+ T cells is inadequate for detection of a
response. This presents an even greater detection problem for donors
whose responses are low, even when tested at higher cell
concentrations. These lower responses became more difficult to detect
when seeding only 2 × 105 cells/assay well.
The ability to use cryopreserved PBMCs in this IFN- ELISPOT
lends itself to monitoring patient responses over time. The ability to
cryopreserve PBMCs for long periods without a significant reduction over time in cell recovery percentage, viability, and
CD4+ lymphocyte levels has previously been
demonstrated (20). Cryopreserved PBMCs have been shown to
respond to mitogen (PHA) in an ELISPOT assay similarly to fresh PBMCs
(32). We have demonstrated that the VZV antigen-specific
responses detected with cells frozen at the time of blood collection
(fresh blood) accurately represent responses measured with the freshly
isolated cells. These cryopreserved cell aliquots can be stored over
long periods in liquid nitrogen without any significant change in the
antigen-specific response being detected. Additionally, the
cryopreserved samples can be exposed to certain changes in storage
conditions without reduction in the assay signal. This allows sample
collection at one site with testing performed at a remote site without
compromising the responses detected.
Cryopreserved PBMCs isolated from blood that was stored overnight can
also be tested with this assay. However, in most cases the responses
detected are substantially reduced compared with those from
cryopreserved PBMCs isolated from fresh blood. Cell clumping was
observed during the thawing of many frozen PBMC samples isolated from
blood stored overnight. Incorporation of DNase has been utilized to
minimize cell clumping in other systems (22, 27).
Extraction of lymphocytes from tissue using high DNase levels (1 mg/ml)
over a prolonged incubation period (3 h) has been shown to have no
effect on levels of CD4, CD25, or CD38 cell surface markers, but did
reduce detection of CD8 and adhesion molecules such as L-selectin, by
FACS analysis (34). We have chosen to incorporate an
ultrapure nuclease (Benzonase), to eliminate the concern about any
contaminating proteinases, at very low levels (50 U/ml) with short
exposure periods. Incorporation of Benzonase in the thawing procedure
eliminated the cell clumping. In some cases, responses detected for
overnight blood PBMC samples processed with Benzonase were similar to
those detected with the PBMCs isolated from fresh blood. However, the
ELISPOT responses for most PBMC samples from overnight blood were still
not representative of responses detected with fresh blood. We observed
no differences in cell surface detection by FACS of CD4, CD8, CD38, or
CD62L with or without the incorporation of Benzonase. Whereas most
cryopreserved PBMCs from fresh blood did not exhibit cell clumping,
infrequently there was a sample that did clump on thawing. Benzonase
prevented cell clumping without any negative effects on assay results
for cryopreserved PBMCs isolated from fresh blood. Based on all of these results, we have adopted the use of Benzonase in all thawing procedures for cryopreserved PBMCs from either fresh or overnight blood.
The use of cryopreserved cells presents an additional advantage for the
ELISPOT assay over the RCF assay. RCF assays require testing of PBMCs
immediately after they are isolated from freshly collected blood
(14, 35). The use of cryopreserved PBMC samples allows
direct comparison, within a single assay run, of PBMC samples from
multiple time points during the course of patient study. This reduces
the variability associated with sample testing, as only intra-assay
variability need be applied and the components of interassay
variability (run-to-run or reader variability) in response comparisons
can be eliminated. The use of the computer-assisted ImmunoSpot image
analyzer can also reduce the assay variability associated with readers
and assay runs, while providing sample response values and variability
estimates that are consistent with those of human readers. This new,
validated VZV IFN- ELISPOT assay provides a sensitive, versatile,
and quantitative tool for assessing the cellular immune responses to VZV.
We thank Paul Keller for providing the VZV and control antigens
used in these studies, and we thank Rupert Vessey for helpful comments
on the manuscript.
| 1.
|
Abendroth, A., and A. Arvin.
1999.
Varicella-zoster virus immune evasion.
Immunol. Rev.
168:143-156[CrossRef][Medline].
|
| 2.
|
Arvin, A. M.
1992.
Cell-mediated immunity to varicella-zoster virus.
J. Infect. Dis.
166:S35-S41.
|
| 3.
|
Arvin, A. M.
1987.
Clinical manifestations of varicella and herpes zoster and the immune response to varicella-zoster virus, p. 67-130.
In
R. Hyman (ed.), The natural history of varicella-zoster virus. CRC Press, New York, N.Y.
|
| 4.
|
Arvin, A. M.,
C. M. Koropchak,
B. R. G. Williams,
F. C. Grumet, and S. K. H. Foung.
1986.
The early immune response in healthy and immunocompromised subjects with primary varicella-zoster virus infection.
J. Infect. Dis.
154:422-429[Medline].
|
| 5.
|
Asai, T.,
W. J. Storkus, and T. L. Whiteside.
2000.
Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines.
Clin. Diagn. Lab. Immunol.
7:145-154[Abstract/Free Full Text].
|
| 6.
|
Asanuma, H.,
M. Sharp,
H. T. Maecker,
V. C. Maino, and A. M. Arvin.
2000.
Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression.
J. Infect. Dis.
181:859-866[CrossRef][Medline].
|
| 7.
|
Bergen, R. E.,
M. Sharp,
A. Sanchez,
A. K. Judd, and A. M. Arvin.
1991.
Human T cells recognize multiple epitopes of an immediate early/tegument protein (IE62) and glycoprotein I of varicella zoster virus.
Viral Immunol.
4:151-166[Medline].
|
| 8.
|
Berger, R.,
E. Trannoy,
G. Hollander,
F. Bailleux,
C. Rudin, and H. Creusvaux.
1998.
A dose-response study of a live attenuated varicella-zoster virus (Oka strain) vaccine administered to adults 55 years of age and older.
J. Infect. Dis.
178:S99-103.
|
| 9.
|
ElGhazali, G. E. B.,
S. Paulie,
G. Andersson,
Y. Hansson,
G. Holmquist,
J. Sun,
T. Olsson,
H. P. Ekre, and M. Troy-Blomberg.
1993.
Number of interleukin-4 and interferon--secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation.
Eur. J. Immunol.
23:2740-2745[Medline].
|
| 10.
|
Giller, R. H.,
S. Winistorfer, and C. Grose.
1989.
Cellular and humoral immunity to varicella zoster virus glycoproteins in immune and susceptible human subjects.
J. Infect. Dis.
160:919-928[Medline].
|
| 11.
|
Habermehl, P.,
A. Lignitz,
M. Knuf,
H. Schmitt,
M. Slaoui, and F. Zepp.
1999.
Cellular immune response of a varicella vaccine following simultaneous DTaP and VZV vaccination.
Vaccine
17:669-674[Medline].
|
| 12.
|
Hayes, F. A., and S. Feldman.
1978.
Cell-mediated immunity to varicella-zoster virus in children being treated for cancer.
Cancer
42:159-163[Medline].
|
| 13.
|
Hayward, A.,
E. Villanueba,
M. Cosyns, and M. Levin.
1992.
Varicella-zoster virus (VZV)-specific cytotoxicity after immunization of nonimmune adults with Oka strain attenuated VZV vaccine.
J. Infect. Dis.
166:260-264[Medline].
|
| 14.
|
Hayward, A. R.,
K. Buda, and M. J. Levin.
1994.
Immune response to secondary immunization with live or inactivated VZV vaccine in elderly adults.
Viral Immunol.
7:31-36[Medline].
|
| 15.
|
Hayward, A. R.,
M. Cosyns,
M. Jones,
M. J. Levin,
E. Villaneuba,
A. Weinberg, and C. Y. Chan.
1998.
Cytokine production in varicella-zoster virus-stimulated cultures of human blood lymphocytes.
J. Infect. Dis.
178:S95-S98.
|
| 16.
|
Hayward, A. R.,
G. O. Zerbe, and M. J. Levin.
1994.
Clinical application of responder cell frequency estimates with four years of follow up.
J. Immunol. Methods
170:27-36[CrossRef][Medline].
|
| 17.
|
Jenkins, D. E.,
R. L. Redman,
E. M. Lam,
C. Liu,
I. Lin, and A. M. Arvin.
1998.
Interleukin (IL)-10, IL-12, and interferon- production in primary and memory immune responses to varicella-zoster virus.
J. Infect. Dis.
178:940-948[Medline].
|
| 18.
|
Jenkins, D. E.,
L. L. Yasukawa,
C. J. Benike,
E. G. Engleman, and A. M. Arvin.
1998.
Isolation and utilization of human dendritic cells from peripheral blood to assay an in vitro primary immune response to varicella-zoster virus peptides.
J. Infect. Dis.
178:S39-42.
|
| 19.
|
Jenkins, D. E.,
L. L. Yasukawa,
R. Bergen,
C. Benike,
E. G. Engleman, and A. M. Arvin.
1999.
Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination.
J. Immunol.
162:560-567[Abstract/Free Full Text].
|
| 20.
|
Kleeberger, C. A.,
R. H. Lyles,
J. B. Margolick,
C. R. Rinaldo,
J. P. Phair, and J. V. Giorgi.
1999.
Viability and recovery of peripheral blood mononuclear cells cryopreserved for up to 12 years in a multicenter study.
Clin. Diagn. Lab. Immunol.
6:14-19[Abstract/Free Full Text].
|
| 21.
|
Levin, M. J.,
D. Barber,
E. Goldblatt,
M. Jones,
B. LaFleur,
C. Chan,
D. Stinson,
G. Zerbe, and A. R. Hayward.
1998.
Use of a live attenuated varicella vaccine to boost varicella-specific immune responses in seropositive people 55 years of age and older: duration of booster effect.
J. Infect. Dis.
178:S109-12.
|
| 22.
|
Machalinski, B.,
P. Szolomicka,
J. Kijowski,
M. Baskiewicz,
A. Karbicka,
E. Byra,
M. Majka,
S. Giedrys-Kalemba, and M. Z. Ratajczak.
1999.
Short-term storage of human haematopoietic cells. Influence of air and deoxyribonuclease I.
Ann. Transplant.
4:29-36[Medline].
|
| 23.
|
McCutcheon, M.,
N. Wehner,
A. Wensky,
M. Kushner,
S. Doan,
L. Hsiao,
P. Calabresi,
T. Ha,
T. V. Tran,
K. M. Tate,
J. Winkelhake, and E. G. Spack.
1997.
A sensitive ELISPOT assay to detect low-frequency human T lymphocytes.
J. Immunol. Methods
210:149-166[CrossRef][Medline].
|
| 24.
|
Milliken, G., and D. Johnson.
1984.
Analysis of messy data, vol. 1. Designed experiments.
Wadsworth Inc., Belmont, Calif.
|
| 25.
|
Nader, S.,
R. Bergen,
M. Sharp, and A. M. Arvin.
1995.
Age-related differences in cell-mediated immunity to varicella-zoster virus among children and adults immunized with live attenuated varicella vaccine.
J. Infect. Dis.
171:13-17[Medline].
|
| 26.
|
Power, C. A.,
C. L. Grand,
N. Ismail,
N. C. Peters,
D. P. Yurkowski, and P. A. Bretscher.
1999.
A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFN--producing cells.
J. Immunol. Methods
227:99-107[CrossRef][Medline].
|
| 27.
|
Querol, S.,
G. Capmany,
C. Azqueta,
M. Gabarro,
O. Fornas,
G. A. Martin-Henao, and J. Garcia.
2000.
Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols.
Transfusion
40:625-631[CrossRef][Medline].
|
| 28.
|
Sadzot-Delvaux, C.,
A. M. Arvin, and B. Rentier.
1998.
Varicella-zoster virus IE63, a virion component expressed during latency and acute infection, elicits humoral and cellular immunity.
J. Infect. Dis.
178:S43-7.
|
| 29.
|
Schmittel, A.,
U. Keilholz, and C. Scheibenbogen.
1997.
Evaluation of the interferon- ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood.
J. Immunol. Methods
210:167-174[CrossRef][Medline].
|
| 30.
|
Sharp, M.,
K. Terada,
A. Wilson,
S. Nader,
P. E. Kinchington,
W. T. Ruyechan,
J. Hay, and A. M. Arvin.
1992.
Kinetics and viral protein specificity of the cytotoxic T lymphocyte response in healthy adults immunized with live attenuated varicella vaccine.
J. Infect. Dis.
165:852-858[Medline].
|
| 31.
|
Skeie, G. O.,
P. T. Bentsen,
A. Freiburg,
J. A. A. Arli, and N. E. Gilhus.
1998.
Cell-mediated immune response against titin in myasthenia gravis: evidence for the involvement of Th1 and Th2 cells.
Scand. J. Immunol.
47:76-81[CrossRef][Medline].
|
| 32.
|
Sobota, V.,
J. Bubenik,
M. Indrova,
V. Vlk, and J. Jakoubkova.
1997.
Use of cryopreserved lymphocyte for assessment of the immunological effects of interferon therapy in renal carcinoma patients.
J. Immunol. Methods
203:1-10[CrossRef][Medline].
|
| 33.
|
Trannoy, E.,
R. Berger,
G. Hollander,
F. Bailleux,
P. Heimendinger,
D. Vuillier, and H. Creusvaux.
2000.
Vaccination of immunocompetent elderly subjects with a live attenuated Oka strain of varicella zoster virus: a randomized, controlled, dose-response trial.
Vaccine
18:1700-1706[CrossRef][Medline].
|
| 34.
|
Van Damme, N.,
D. Baeten,
M. De Vos,
P. Demetter,
D. Elewaut,
H. Mielants,
G. Verbruggen,
C. Cuvelier,
E. Veys, and F. De Keyser.
2000.
Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.
J. Immunol. Methods
236:27-35[CrossRef][Medline].
|
| 35.
|
Weinberg, A.,
R. A. Betensky,
L. Zhang, and G. Ray.
1998.
Effect of shipment, storage, anticoagulant, and cell separation on lymphocyte proliferation assays for human immunodeficiency virus-infected patients.
Clin. Diagn. Lab. Immunol.
5:804-807[Abstract/Free Full Text].
|
| 36.
|
Zerboni, L.,
S. Nader,
K. Aoki, and A. M. Arvin.
1998.
Analysis of the persistence of humoral and cellular immunity in children and adults immunized with varicella vaccine.
J. Infect. Dis.
177:1701-1704[Medline].
|
| 37.
|
Zhang, Y.,
M. Cosyns,
M. J. Levin, and A. R. Hayward.
1994.
Cytokine production in varicella zoster virus-stimulated limiting dilution lymphocyte cultures.
Clin. Exp. Immunol.
98:128-133[Medline].
|
| 38.
|
Zhang, Y.,
C. J. White,
M. Levin, and A. R. Hayward.
1995.
Cytokine production in varicella-zoster virus-stimulated lymphocyte cultures.
Neurology
45:S38-S40[Abstract].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
