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IMMUNOBIOLOGY
From the Department of Pediatrics, Bone Marrow
Transplantation Service; the Department of Clinical Laboratories,
Cellular Immunology Laboratory; the Department of Epidemiology
and Biostatistics; the Department of Medicine, Infectious Diseases
Service, Memorial Hospital; and the Immunology Program,
Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New
York, NY.
Techniques for the quantitation of virus-specific and
alloantigen-reactive T cells vary in their measurement of clinically relevant T-cell effector populations, their sensitivity and
quantitative accuracy, and the time required to obtain measurable
results. We compared frequencies of Epstein-Barr virus (EBV)-specific
and major alloantigen-reactive T cells as measured by flow cytometric analysis of responding T cells producing intracellular interferon- Adoptive immunotherapy with donor leukocytes
or Epstein-Barr virus (EBV)-specific T-cell infusions is effective in
inducing durable remissions of monoclonal EBV-lymphoproliferations
complicating allogeneic bone marrow transplantation.1,2
However, infusions of peripheral blood mononuclear cells (PBMCs) or
donor-derived EBV-sensitized T-cell lines early in their
development may also contain various numbers of alloreactive T cells
capable of inducing severe graft-versus-host disease (GVHD).
Determination of antigen-specific and alloreactive T-cell frequencies
in these cell fractions before adoptive transfer can provide a means
for estimating the likelihood of a subsequent tumor response and of the
risk for GVHD.1-4
Limiting-dilution analysis (LDA) is a standard method for the
determination of cytotoxic T-cell precursor (CTLp)
frequencies.5,6 Previous reports by Lucas et
al7 and others8 have illustrated the
feasibility and usefulness of monitoring CTLp frequencies to EBV after
allogeneic marrow transplantation and after adoptive T-cell therapy of
EBV-lymphoproliferative disorders. However, LDA is labor intensive.
Twelve days are needed to generate results, and LDA underestimates the
frequencies of antigen-reactive T cells detectable by other
methods.9,10 Recently, techniques using Brefeldin A
(Sigma, St Louis, MO) to inhibit cytokine secretion and
anti-interferon- Generation of EBV-specific T cells
Alloantigen-reactive T cells were generated using the same culture
conditions, but by stimulating PBMCs with EBV BLCL generated from an
HLA-A- and -B-disparate unrelated healthy donor. Quantitation of
EBV-reactive and alloresponsive T cells by LDA and by measurements of
IFN- Quantitation of EBV-specific and alloreactive
IFN- Immediately before fixation and permeabilization, BLCL and effector
cells of the nonstimulated control tubes were combined. Aliquots of the
bulk nonstimulated and of the stimulated cultures were transferred to
tubes for staining with monoclonal antibodies. Cells were stained with
5 µL monoclonal anti-CD3 labeled with allophycocyanin (APC) and 10 µL anti-CD8 peridin chlorophyll protein (PerCP) or anti-CD4 PerCP (BD
Biosciences, San Jose, CA) and were incubated for 20 minutes at room
temperature in the dark. Cells were washed with 2 mL phosphate-buffered
saline (PBS)-bovine serum albumin (BSA)-azide (AZ) (PBS + 0.5%
BSA + 0.1% AZ). Cells were centrifuged, supernatant discarded,
and 100 µL reagent A (Fix & Perm Cell Permeabilization Reagents A & B; Caltag Laboratories, Burlingame, CA) was added to each tube to fix
the cells. These cells were then incubated for 15 minutes. Cells were
washed with PBS + BSA + AZ, and 100 µL reagent B (Caltag
Laboratories) was added for permeabilization. Intracellular staining
was performed by adding 10 µL phycoerythrin (PE)-labeled mouse
immunoglobulin G1 (IgG1) isotype control PE or CD69 PE (BD Biosciences)
and 10 µL mouse IgG1 isotype control fluorescein isothiocyanate
(FITC) or IFN- Stained and fixed cells were subsequently analyzed and quantitated using a FACSCalibur flow cytometer with dual lasers for 4-color capability (BD Biosciences), using CELLQuest software. Cells were first identified by forward and side light scatter and then by gating the CD3+ cells in a CD3 APC versus side scatter dot plot. Ten thousand events were acquired in the combined gate. For further identification of the cells, gating on the CD3+CD8+ or CD3+CD4+ cells was performed. Quadrant markers were established based on analysis of the nonstimulated control and isotype control tubes. Limiting-dilution analysis To evaluate CTLp frequencies in EBV-BLCL-activated T cells, LDA was performed on days 0, 7, 14, 21, and 28 of cultured T-cell lines according to a modification of the methods of Bourgault et al5 and Langhorne and Lindahl,6 as described by Lucas et al.7 Briefly, the T cells were seeded in final volumes of 200 µL in 24 replicate wells per dilution of T cells in 96-well round-bottomed microplates. Decreasing effector cell concentrations were stimulated with 1 × 104 6000 cGy-irradiated autologous BLCL. Autologous PBMCs (1 × 104) irradiated with 3000 cGy were added as feeders. On days 0, 3, and 7 of each assay, 10 IU/mL IL-2 was added to the cultures. On day 10, the plates were split and tested against autologous BLCL and allogeneic targets. Wells were scored positive when chromium Cr 51 release exceeded the average plus 3 SD of control wells. CTLp frequencies were calculated by the method of Taswell11 using a computer program provided by Dr Y. Kawanishi (Medical College of Wisconsin, Milwaukee).Comparisons of sensitivity and precision of assays To measure the precision of this assay and compare it with the assay measuring IFN- -producing cells, 5 LDAs were performed simultaneously on replicate samples of T cells obtained at the same
time from the same cultured T-cell line. To assess and compare the
sensitivity of the LDA with the sensitivity of the flow cytometric analysis of producing IFN- in response to antigen, simultaneous assays were performed on samples from the same EBV-specific T-cell line
diluted 1:2, 1:4, 1:8, 1:16, and 1:160 of the number of effector cells
plated in the standard assays.
Determination of peptide-specific CD8+ T cells HLA-A2 and HLA-B8 tetrameric complexes were generated with human 2-microglobulin and an HLA-A2 binding EBNA 3c (LLDFVRFMGV) or an
HLA-B8 binding peptide of EBNA 3a (QAKWRLQTL) as previously described
and provided by the MSKCC Tetramer Core Facility.12,13 T
cells from an HLA-A*0201+ donor and an
HLA-B*0801+ donor, sensitized for 3 weeks against
autologous EBV BLCLs, were prepared for FACS analyses by staining the
cells with 0.25 mg/mL PE-labeled tetrameric complex, 5 µL monoclonal
anti-CD3 APC, 10 µL anti-CD8 PerCP, and 10 µL anti-CD69L FITC (BD
Bioscience). Cells were incubated for 20 minutes, washed, and analyzed
with a FACSCalibur flow cytometer as described above.
To compare the results of quantitation of antigen-reactive T
cells by tetramer assays or IFN- Purification of viable IFN- -secreting T cells were isolated using
the IFN- secretion assay (Miltenyi Biotec), according to the
principles and instructions of the manufacturer.14,15 In
brief, EBV-sensitized T cells were stimulated for 14 hours with
autologous EBV BLCLs at an effector-stimulator ratio of 5:1.
Alloantigen-sensitized T cells were stimulated identically with the
allogeneic EBV BLCLs. Cells were washed in cold buffer (PBS containing
0.5% BSA and 2 mM EDTA) and were resuspended in cold medium, and 20 µL IFN- catch reagent per 107 cells was added for 5 minutes on ice. Cells were diluted in warm medium (37°C) at a
concentration of 1 × 106/mL for 45 minutes. After
washing in cold buffer, 20 µL IFN- detection antibody (PE) per
107 cells was added. In addition, CD8 FITC or CD4 FITC was
added, and cells were incubated for 15 minutes on ice. Cells were
washed, supernatant was completely removed, and
CD8+IFN-+ and
CD8+IFN- populations or
CD4+IFN-+ and
CD4+IFN- cells were purified using a MoFlo
cell sorter (Cytomation, Fort Collins, CO). Dual-color cell staining
with anti-CD8 or anti-CD4 and anti-IFN- monoclonal antibodies
allowed purification of double-color positive cells
(CD8+IFN-+ or
CD4+IFN-+) and single-color CD8 or CD4 cells
(CD8+IFN- or
CD4+IFN-) to high purity (greater than
95%). Cell fractions were subsequently used for cytotoxicity assays as
described below.
Cytotoxicity assay Cytolytic activity of purified CD8+IFN-+ and
CD8+IFN- or
CD4+IFN-+ and
CD4+IFN- effector cells was assayed
against 51Cr-labeled targets in standard 4-hour release
assays. Target cells included autologous BLCLs and HLA class I
mismatched allogeneic BLCLs. Briefly, 1 × 106 target
cells were incubated with 100 µCi (3.7 MBq) 51Cr
for 1 hour, washed 3 times, and plated in 96 wells. Cytotoxicity was
analyzed using 4 × 104, 2 × 104, and
1 × 104 effector cells to 4 × 103 target
cells per well in a total volume of 200 µL. All targets were plated
in triplicate.
After an incubation of 4 hours, supernatants were harvested, and the
specific cytotoxicity was determined using a microplate scintillation
counter (Packard Instruments, Downers Grove, IL). Percentage
specific lysis was calculated as 100% × (experimental release Statistical analysis The number of EBV-specific T cells per 105 T lymphocytes was recorded using intracellular IFN- and LDA assays.
Coefficient of variation, defined as the standard deviation divided by
the mean, was used to measure the precision of 5 replicates of these positive-value variables.
Data for both assays were recorded at different titration levels. The
intracellular IFN-
Determination of antigen-specific T cells by intracellular IFN- -producing cells in specific T-cell subpopulations detectable by FACS analysis. An example, in which a culture was used after one
restimulation with autologous EBV BLCL is demonstrated in Figure
1. Forward scatter and side scatter were
used for gating the lymphocytes. Staining with CD3 APC and CD8 PerCP
allowed a second gate on the double-positive
CD3+CD8+ lymphocytes. Subsequent analysis of
the CD3+CD8+ cell fraction with CD69 PE, an
activation marker, and IFN- FITC was used to determine the
percentage of IFN--producing CD3+CD8+
lymphocytes activated in response to stimulation with EBV BLCL. In all
our experiments, we used nonstimulated controls to determine background
staining and subtracted the obtained percentage from the results of the
stimulated samples. Figure 1C shows a background of 0.02% in the
nonstimulated controls, which was subtracted from the 2.94% initially
obtained in the culture. As a result, Figure 1D thus shows 2.92% of
IFN--producing activated CD3+CD8+
lymphocytes in this example.
To examine the specificity of IFN-
Kinetics of EBV-specific and alloreactive CD8+
IFN- -producing activated EBV-specific
CD3+CD8+ T cells from 0.71% on day 0 to
24.59% after 4 stimulations with autologous EBV BLCL on day 28. As can
be seen in Figure 4, the percentage of alloreactive
CD3+CD8+ T cells in the same culture decreased
gradually from 1.10% on day 0 to 0.19% on day 28 over the same 4-week
period. These figures clearly demonstrate an increase in the number of
EBV-reactive T cells and a concurrent, but less dramatic, reduction in
the number of alloreactive T cells in the autologous EBV BLCL
sensitized T-cell cultures over time when analyzed by intracellular
cytokine staining. In the unsensitized culture on day 0, the number of alloreactive T cells in this donor was slightly higher than the number
of EBV-reactive T cells, with a ratio of anti-EBV T cells to
antialloreactive T cells of 0.6. This ratio increased in the 4-week
period to 129, achieved by day 28 of culture, by which time the
proportion of EBV-reactive T cells increased 34-fold while the
proportion of alloreactive T cells decreased 5.7-fold.
Comparison of EBV-specific and alloreactive T cells by intracellular cytokines and LDA Table 1 presents the anti-EBV and antialloreactive frequencies, determined by LDA and by flow cytometric quantitation of IFN--producing CD3+ cells of T cells
from 2 healthy seropositive donors generated over the course of 28 days
in response to autologous EBV BLCL or allogeneic BLCL. Results are
normalized to the number of T cells detected by each assay per
105 cells analyzed. As can be seen in Table 1 (donor A),
anti-EBV CTLp frequencies as measured by LDA rose from 28 per
105 cells on day 0 to 333 per 105 cells on day
28 of culture, whereas frequencies of alloreactive CTLp decreased
steadily from 36 per 105 cells on day 0 to 0.9 per
105 cells on day 28. Frequencies detected by flow
cytometric quantitation of CD3+ T cells exhibiting
intracellular IFN- staining were 700 per 105 cells for
anti-EBV and 1100 per 105 cells for alloreactive cells on
day 0, and they were 23 100 per 105 cells for anti-EBV and
200 per 105 cells for alloreactive T cells on day 28 of
culture. Results of assays performed during the generation of
EBV-specific T cells from donor B are also presented in Table 1. The
anti-EBV frequency of 10 per 105 cells and the
antialloreactive frequency of 8 per 105 cells on day 0, assessed by LDA, compares with 900 per 105 cells and 600 per 105 cells of anti-EBV and antialloreactive
CD3+ T cells, respectively, on day 0 obtained using the
intracellular cytokine staining assay. This demonstrates a 90-fold and
a 75-fold higher frequency of EBV-specific and alloreactive T cells
when assessed by staining for intracellular IFN-. The anti-EBV CTLp frequency of 132 per 105 T cells on day 28 of culture that
corresponds to a frequency of 5200 per 105 CD3+
T cells detected by intracellular IFN- production again demonstrates a 39-fold higher frequency of anti-EBV reactive T cells when assessed by cytokine staining. The frequency of alloreactive CD3+ T
cells assessed by detection of intracellular IFN- production was
0.0% at this time point and could not be compared with the results
obtained by LDA.
These results thus demonstrate that at each stage of culture, the
frequencies of both EBV-specific and alloreactive T cells are 25- to
220-fold higher by quantitation of intracellular IFN- We also examined the frequencies of EBV-specific and alloreactive
CD8+IFN- Limiting-dilution assay measures clonogenic cells of either
CD4+ or CD8+ type, which likely represent only
a small fraction of the antigen-reactive T cells capable of generating
cytokines at the initiation of culture. Because CD8+
EBV-specific T cells tend to increase disproportionally in the course
of development of a T-cell line, however, we were interested to
determine whether the disparities in T-cell frequencies detected by the
2 techniques reflected differences in the subpopulations of T cells
expanded after in vitro sensitization over the course of culture. As
shown in Figure 5A-D, alterations in
frequencies of EBV-specific and alloreactive
CD3+IFN-
Comparison of precision and sensitivity of EBV-specific T cells by intracellular cytokines and LDA To determine and to compare precision of the quantitation of T cells generating IFN- by intracellular cytokine staining and frequencies detected by LDA, we generated an autologous EBV-specific T-cell line for 13 days and performed each of the assays 5 times independently using the same T-cell line. The results, shown in Table
2, present the absolute numbers of
intracellular IFN--producing CD3+ T cells per
105 T lymphocytes obtained by the intracellular cytokine
assays, from 3220 to 3700 CD3+IFN-+ cells
per 105 T lymphocytes of the EBV-reactive T-cell line. T
cells of the same culture were used to perform 5 LDAs simultaneously.
As demonstrated in Table 2, the results of CTLp frequencies obtained
varied between 1.7 and 11 per 105 T lymphocytes. To assess
the precision of intracellular IFN- assay and the LDA, the 5 independent samples of the number of EBV-specific T cells per
105 T lymphocytes were recorded as the coefficient of
variation for the intracellular IFN- assay and LDA and were 0.06 and
0.6, respectively, demonstrating the greater precision for the
intracellular IFN- assay.
We were further interested in evaluating the sensitivity of the assays.
For this reason we performed linear titrations of effector cells,
without changing any other variable, and we performed the assays using
the same T-cell culture with reduced numbers of effector cells. To
evaluate the percentage of IFN-
As shown in Table 2, when the serially titrated T cells were examined
by LDA, the CTLp frequencies at the first 3 dilutions were 4.4, 1.2, and 0.8 EBV-specific T cells per 105 T lymphocytes and
could not be determined for the last 3 concentrations because there was
no demonstrable cytotoxicity at the lower titration levels. For this
reason the results for the LDA assay, shown in Figure 6B, were based on
the 3 values of undiluted, 1:2 and 1:4 concentrations of the titrated T
cells. Varying the undiluted value between 1.7 and 11 EBV-specific T
cells per 105 T cells produced an R2
range between 0.0 and 0.96. In comparison, the range of
R2 values based on the same 3 titrations for the
intracellular IFN- Evaluation of the EBV-specific cytotoxic activity of
CD8+ and CD4+ IFN- could in part reflect differences in the
clonogenic potential of IFN--producing T cells and variations in
the capacity of cytokine-producing T cells to lyse susceptible targets.
To evaluate the function of the IFN--secreting and -nonsecreting CD8+ and CD4+ lymphocytes, we used the IFN-
secretion assay, which allows labeling of surface IFN- with a
PE-conjugated anti-IFN- monoclonal antibody. Staining with a second
FITC-labeled anti-CD8 or anti-CD4 monoclonal antibody allows
purification of CD8+IFN-+ and
CD8+IFN- or
CD4+IFN-+ and
CD4+IFN- T lymphocytes by FACS analysis.
As shown in Figure 7, before sorting,
3.2% of the CD8+ T cells also stained for anti-IFN- PE
(Figure 7B) after they were stimulated overnight with autologous EBV
BLCL, whereas among the nonstimulated control cells IFN--secreting
T cells could not be detected (Figure 7A). Stimulated T cells could
thereafter be sorted into CD8+IFN-+ (purity,
98%) and CD8+IFN- (purity, 99%)
fractions (Figure 7C-D). We then performed a standard 51Cr
release assay to analyze the cytotoxic activity of the separated CD8+IFN-+ and
CD8+IFN- or
CD4+IFN-+ and
CD4+IFN- T lymphocytes. Figure
8 presents results obtained with purified CD8+IFN-+ and
CD8+IFN- subpopulations of donor A. Figure
9 presents the results acquired with the
sorted CD8+IFN-+ and
CD8+IFN- and the sorted
CD4+IFN-+ and
CD4+IFN- populations of donor B. In both
experiments, the CD8+IFN-+ T cells induced
significant lysis of the autologous EBV BLCL at low effector-target
cell ratios, whereas these cells did not display lysis of the
allogeneic targets. The CD8+IFN-
subpopulations of donor A did not induce significant lysis of the
autologous or the allogeneic targets, whereas those from donor B
exhibited a low level of cytotoxicity against the autologous targets.
Of importance, neither the CD4+IFN-+ nor the
CD4+IFN- cells induced significant lysis
of the autologous EBV BLCL of donor B. These results indicate that the
IFN--secreting CD8+ T-cell fraction is enriched for
virus-specific cytotoxic effector cells, whereas the
CD8+IFN- and the CD4+
subpopulations are largely depleted of cytotoxic effectors.
In a separate experiment, T cells from donor B were sensitized to
allogeneic EBV BLCL, and the CD4+ and CD8+
fractions were again separated and sorted into IFN-
Comparison of EBV peptide-specific T cells by intracellular cytokines and tetramer analysis We were also interested in determining the relative frequencies of T cells reactive against immunodominant peptides of EBV in the T cells sensitized to EBV BLCL over 3 weeks of culture. Accordingly, the sensitized T cells from 2 donors, one expressing HLA-A*0201, the other HLA-B*0801, were evaluated for CD8+ T cells binding tetramers of HLA-A*0201 bearing the EBNA 3c immunodominant peptide, LLDFVRFMGV, or HLA-B*0801 bearing the immunodominant EBNA 3a peptide, QAKWRLQTL. At the same time, these T cells were stimulated overnight with autologous irradiated PBMCs pulsed with these peptides or with autologous EBV BLCL and thereafter were analyzed for CD8+IFN+ T cells. As shown in Figure
11A, 4.3% of the CD8+ T
cells from the HLA-A*0201 donor bound the HLA-A*0201-EBNA 3c peptide
tetramers, with most (3.44%) of these cells CD62L low (Figure
11B). Only 0.05% of these cells bound the HLA-B*0801-EBNA 3a peptide
tetramers (Figure 11C-D). Similarly, as shown in Figure 11E-F, 4% of
the CD8+ T cells produced IFN- after secondary
stimulation with EBNA 3c peptide-pulsed PBMCs compared with 0.05% of T
cells secondarily stimulated with the EBNA 3a peptide-pulsed PBMCs. In
studies of lymphocytes from a second HLA-A*0201+ donor
sensitized to autologous EBV BLCL for 2 weeks, 0.4% of the T cells
bound the EBNA 3c tetramer (Figure
12A). Similarly, 0.3% generated
IFN- after a secondary sensitization with the EBNA 3c peptide
(Figure 12B). In contrast, only 0.02% of the T cells generated IFN- |
production and growth of cytotoxic
precursors
Top
Abstract
Introduction
spontaneous release)/(maximum release × spontaneous release). Maximum release was obtained by adding 100 µL 5% Triton X-100 to the 100 µL medium-containing target cells. Spontaneous release was consistently below 15% of maximum release in all assays.
the value of
an assay at the undiluted concentration and the origin. The statistic
used to assess the closeness of the assay values to this straight line was R2, which measures the proportion of
variability in the assay about the origin explained by the line through
the origin. An R2 value of 1 means the points
fit the line exactly. Given that the recorded assay value at the
undiluted value is subject to variability, a range of
R2 values was computed based on the range of
undiluted values to assess the sensitivity of the
R2 statistic.
) and absolute numbers of
intracellular IFN-
),
CD8+ (
), and CD4+ (
) per 105
T cells in response to stimulation with autologous EBV BLCL of donor A
and donor B, respectively. (B, D) Absolute numbers of anti-alloreactive
CD3+ cells per 105 T cells by LDA (
