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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 109-117
GENE THERAPY
Rapid selection of antigen-specific T lymphocytes by retroviral
transduction
Guenther Koehne,
Humilidad F. Gallardo,
Michel Sadelain, and
Richard J. O'Reilly
From the Bone Marrow Transplant Service, Department of Pediatrics,
Memorial Hospital; Gene Transfer and Somatic Cell
Engineering Facility; Department of Human Genetics; and Immunology
Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer
Center, New York, NY.
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Abstract |
Infusions of donor peripheral blood T cells can induce durable
remissions of Epstein-Barr virus (EBV) lymphomas complicating marrow
grafts, but they contain alloreactive T cells capable of inducing
graft-versus-host disease. EBV-specific T-cell lines or clones avoid
this problem but require 30 to 40 days of culture to establish. To
accelerate the generation of EBV-specific T cells, we tested whether
retroviral vectors, which only integrate in dividing cells, could be
used to transduce and select antigen-reactive T cells early after
sensitization to autologous EBV-transformed B cells. T cells were
transduced with a dicistronic retroviral vector, NIT, which encodes
low-affinity nerve growth factor receptor as an immunoselectable
marker and herpes simplex virus thymidine kinase as a suicide gene, at
different time points after sensitization. EBV-specific cytotoxic T
lymphocyte precursor (CTLp) frequencies in purified NIT+
T-cell fractions transduced on day 8 of culture were comparable to
those of EBV-specific T-cell lines cultured for 30 days or more.
Alloreactive CTLp frequencies were markedly reduced in the NIT+ fraction relative to the untransduced T-cell
population. NIT+ fractions transduced on day 8 possessed
more CD4+ T cells than the cell lines at day 30 and
exhibited the same selective pattern of reactivity against
immunodominant antigens presented by specific HLA alleles. In contrast,
T cells transduced with NIT 5 days after stimulation with mitogen and
interleukin-2 were relatively depleted of T cells specific for
autologous EBV-transformed cells. Thus, retroviral vectors may be used
for rapid selection of viral antigen-reactive T cells depleted of
alloreactive T cells.
(Blood. 2000;96:109-117)
© 2000 by The American Society of Hematology.
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Introduction |
Lymphomas associated with Epstein-Barr virus (EBV) are
a potentially lethal complication of marrow and organ
allografts.1-3 The risk of developing an EBV lymphoma is
increased in recipients of HLA-disparate related or unrelated
unmodified marrow grafts receiving prolonged immunosuppression or
T-cell-depleted marrow grafts.3,4 Because of the
increasing use of such transplants, the need for consistently
effective, logistically practicable approaches for the treatment of EBV
lymphomas has increased. In 1994, our group reported that infusions of
small numbers of peripheral blood mononuclear cells (PBMCs) derived
from a seropositive marrow donor could induce durable and complete
regression of EBV lymphomas emerging as a complication of related or
unrelated T-cell-depleted marrow grafts.5 This finding has
now been confirmed by several centers.6-9 Furthermore, the
role of T cells as the principle effectors of the regressions observed
has been demonstrated by Rooney et al,6 who have used
genetically marked EBV-specific T-cell lines to treat 2 patients
developing this complication and have reported that infusions of such
in vitro-derived EBV-specific T cells may prevent EBV lymphomas in
high-risk groups.
Infusions of donor-derived PBMCs, even in small numbers, may also
transfer alloreactive T cells in numbers sufficient to cause severe or
even lethal graft-versus-host disease (GVHD), particularly if the donor
and host differ at 1 or more HLA alleles.3,5,8 Conversely,
the generation of EBV-reactive T-cell lines adequately depleted of
contaminating alloreactive T cells necessitates culture of donor T
cells in the presence of irradiated autologous EBV-transformed B cells
for at least 4 weeks.10 While cloning of EBV-reactive T
cells can delete alloreactive T cells, the time required to expand
these clones to sufficient numbers for therapy remains substantial.11,12 Because EBV lymphomas progress rapidly
and are lethal in up to 50% of cases within 20 days,4
these approaches require the establishment of such T-cell lines or
clones prior to or at the time of transplantation rather than at the
time of disease presentation. This presents significant logistical and economic problems, because T-cell lines need to be established and
generated for all patients at significant risk but may be needed for
only the 10% to 15% of patients who develop this complication.
An alternative strategy proposed by Sadelain and
Mulligan13 and initially employed by Bonini et
al9 is the use of unselected T cells
transduced after mitogenic stimulation with a retroviral vector
encoding a selectable marker and a drug sensitivity gene such as
thymidine kinase. Because such T cells may contain subpopulations of
alloreactive cells capable of inducing GVHD, incorporation of herpes
simplex virus thymidine kinase (HSV-TK) permits eradication of
alloreactive T cells expressing HSV-TK and reversal of GVHD through
treatment of the host with ganciclovir.8,13,14 However, such treatment may also eliminate desired effectors such as transduced EBV-reactive T cells. Thus, the therapeutic advantage of suicide vector-modified unselected T cells rests with the balance existing between desired antigen-reactive T-cell populations and alloreactive T
cells capable of inducing severe GVHD.
In this study, we have investigated whether retroviral vectors, which
selectively integrate in dividing target cells,15,16 can be
used to preferentially transduce those T cells within a polyclonal
population that are specifically proliferating in response to an
antigen. Our results suggest that antigen-specific T cells can be
selected by this strategy early after in vitro sensitization and that
alloreactive T cells can be substantially depleted by this approach.
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Materials and methods |
Production and culture of EBV-lymphoblastic cell lines
PBMCs at a concentration of
1 × 106/mL were incubated for 24 hours after
isolation by Ficoll-Hypaque density centrifugation with the
EBV-containing supernatant of the marmoset cell line 95-8 in the
presence of 0.5 µg phytohemagglutinin
(PHA)-16 (Murex-Diagnostik, Norcross, GA) in
RPMI 1640 (GIBCO, Life Technologies, Grand Island, NY), 10%
heat-inactivated fetal calf serum (FCS), 10 U/mL penicillin, 10 µg/mL
streptomycin, and 1% L-glutamine. After 24 hours, cells were washed
and recultured in EBV-containing medium without PHA in 24-well plates
at a concentration of 1 × 106/mL. Cells were fed
with RPMI 1640, 10% FCS, L-glutamine, penicillin, and streptomycin
twice a week and expanded according to the growth and cell number. The
cells were finally characterized by fluorescence-activated cell sorter
(FACS) analysis using CD3, CD19, and CD20 monoclonal antibodies (Becton
Dickinson, San Jose, CA). Aliquots of the immortalized B-lymphoblastoid
cell lines (BLCLs) were frozen and the remaining cells
maintained in culture.
Homozygous BLCLs for the HLA-A and HLA-B alleles, generously provided
by Dr Bo Dupont, were maintained in the same medium. PHA blasts were
generated by culturing 1 × 106/mL PBMC with 0.5 µg/mL PHA-16 for 3 days. The cells were washed and further cultured
for 4 days in the presence of 5 IU/mL interleukin (IL-2) (Collaborative
Biomedical Products, Bedford, MA).
Generation and culture of EBV-specific CTLs
PBMCs were isolated by Ficoll-Hypaque density centrifugation of
anticoagulated whole blood. T lymphocytes were positively selected by
staining with an anti-CD3 phycoerythrin monoclonal antibody (Becton
Dickinson) on a MoFlo cell sorter (Cytomation, Fort Collins, CO),
achieving a purity of more than 98%. EBV-specific cytotoxic T
lymphocytes (CTLs) were generated by stimulating
1 × 106/mL CD3+ cells with
2.5 × 104/mL autologous BLCLs, which were
irradiated with 60 Gy in Iscove's modified Dulbecco's medium
supplemented with 10% heat-inactivated human AB serum
(Gemini, Calabasas, CA), 35-µg/mL transferrin, 5-µg/mL insulin,
2 × 10-5 -M ethanolamine, 1-µg/mL palmitic acid,
1-µg/mL linoleic acid, and 1-µg/mL oleic acid (all from Sigma, St.
Louis, MO) for 6 days in 25-cm2 flasks. Cells were washed,
recultured at a concentration of 1 × 106/mL, and
restimulated with 2 × 105/mL BLCL at day 7. Cells
were either prepared for gene transfer on day 8 (early gene transfer)
or kept in culture with restimulations weekly at an effector-to-target
ratio of 5:1. After the third restimulation, T cells were prepared for
gene transfer on day 23 (late gene transfer). A total of 5 IU of IL-2
(Collaborative Biomedical Products) were added for the first time at
day 10 to the cultures and 2 to 3 times weekly thereafter.
For generation of alloreactive cells, donor T cells were stimulated
with fully mismatched allogeneic EBV BLCL.
Generation of mitogen-activated cells
Nontissue culture-treated 12-well plates (Becton Dickinson Labware,
Franklin Lakes, NJ) were coated with 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28 monoclonal antibodies (PharMingen, San Diego, CA) for 3 to 4 hours at 37°C. Coated wells were incubated with 1%
human serum albumin for 20 minutes and washed twice with
phosphate-buffered saline (PBS). Freshly isolated PBMCs were plated at
a concentration of 1 × 106/mL for 3 days at
37°C and transferred to 25-cm2 flasks (Corning,
Corning, NY). IL-2 (10 IU/mL) was added to
fresh culture medium for 2 days before gene transfer.
Vector system and gene transfer
Vector system.
The construct used in this study is a dicistronic vector, termed NIT,
encoding a mutated and truncated human low-affinity nerve growth factor
receptor (LNGFR) as an inactive cell surface marker that
permits purification of transduced cells by immunoselection and the
monitoring of gene expression by FACS analysis, and HSV-TK, which
renders the cells sensitive to ganciclovir. The vector has been
previously described by Gallardo et al.17 The PG13/NIT7 supernatants were used as cell-free viral stocks with a viral titer of
multiplicity of infection (MOI) 1-2, as measured by FACS and Southern blot analyses on human A549 cells.
Gene transfer.
EBV-activated T cells (day 5, day 8, or day 23 of culture) or
 CD3/CD28-immobilized monoclonal antibody stimulated cells were
placed in fibronectin-coated wells according to the technique described
by Pollok et al18; 5 µg/mL of fibronectin fragments (TaKaRa Biomedicals, Shiga, Japan) were coated on
nontissue culture-treated plates for 2 hours at room temperature in
6-well plates. Plates were blocked with 1% human serum albumin for at
least 30 minutes and washed twice with PBS. Cells were plated at a
concentration of 1 × 106/mL for 24 hours. Fifty
percent of the supernatant was replaced with fresh medium containing
10% heat-inactivated human AB serum and 10 IU/mL IL-2. Cells were
maintained in culture at a concentration of
1 × 106/mL to 1.5 × 106/mL.
Three days later, cells were analyzed for gene expression by flow
cytometry using the cell surface marker anti-NGFR monoclonal antibody.
Proliferation assay
To determine optimal time points for gene transfer into
EBV-activated T lymphocytes, a proliferation assay was performed on days 1, 5, 7, 14, 21, and 28. Purified T cells were initially stimulated with irradiated autologous BLCLs on day 0 at an
effector-to-stimulator ratio of 40:1. One day before the assay, T cells
were restimulated at a ratio of 5:1. (For the day 5 and day 7 assays,
separate flasks were initially set up to avoid a repeated stimulation
on day 4 and day 6 for the day 7 proliferation assay.) A total of
2 × 105 cells/well were plated 1 day after
restimulation in triplicate in 96-well plates. Equal amounts of
stimulators only were plated separately to assess the background
proliferation. Tritium thymidine (18.5 kBq) (Dupont, NEN Products,
Boston, MA) was added per well for 24 hours. Cells were harvested, and
proliferation was determined using a Wallac-counter (WALLAC,
Gaithersburg, MD). Background values were subtracted from the
experimental data. A total of 2 × 106 cells of the
same bulk culture were transduced with the dicistronic vector on the
same days proliferation assays were performed, and gene transfer
efficiency was assessed by FACS analysis.
Flow cytometric analysis
Monitoring of the gene expression of NGFR of T lymphocytes was
performed by 2-color flow cytometry using FACScan (Becton Dickinson) by
labeling the cells with an anti-NGFR monoclonal antibody (20.4) (American Type Culture Collection, Rockville, MD) on ice
for 45 minutes. Goat antimouse fluorescein isothiocyanate was added for 15 minutes as secondary antibody. After blocking with normal mouse serum (ICN/CAPPEL, Aurora, OH) for 10 minutes, anti-CD3 phycoerythrin (Becton Dickinson) was added for 15 minutes. Cells were washed twice
with PBS after each step and before analysis.
Phenotyping of EBV-specific CTL lines was performed by gating
lymphocytes using forward sight scatter and sideward sight scatter. Cells were stained with anti-CD3, anti-CD4, and anti-CD8 for T-cell subpopulations. Although cells had been purified initially for T
lymphocytes, the transduced cells were reanalyzed for the presence of
natural killer (NK) cells, defined as
CD3 CD16+CD56+, using
anti-CD16 and anti-CD56 monoclonal antibodies (Becton Dickinson).
Cell purification by FACS sorting
Gene-modified cells were prepared for purification using a
FACStarPlus cell sorter (Becton Dickinson). A dual-color
cell staining was performed with anti-NGFR and anti-CD3 as described
for FACS analysis. Dual-color positive cells (NIT positive fraction)
and single-color CD3 cells (NIT negative fraction) were sorted to high
purity (more than 95%). One unsorted fraction was retained. The 3 cell
fractions were subsequently used separately for cytotoxicity assays and
limiting dilution analyses as described below.
Cytotoxicity assay
Cytolytic activity of effector cells was assayed against
51Cr-labeled targets in standard 4-hour release assays.
Target cells included autologous BLCLs, HLA class I mismatched
allogeneic BLCLs, and K562 for major histocompatibility complex
(MHC)-unrestricted lysis as a parameter for NK cell lysis and PHA
blasts. For each donor HLA class I allele, a BLCL expressing the HLA-A
and HLA-B allele homozygously could be included to determine the HLA
restriction of the EBV-specific CTLs. Briefly,
1 × 106 target cells were incubated with 3700 kBq
51Cr for 1 hour, washed 3 times, and plated
in 96 wells. Cytotoxicity was analyzed using
0.8 × 105 effector cells:
4 × 103 target cells per well in a total volume of
200 µL, at an effector-to-target ratio of 20:1. All targets were
plated in triplicate.
After an incubation of 4 hours, supernatants were harvested and the
specific cytotoxicity determined using a microplate scintillation counter (Packard Instruments, Downer's Grove, IL). The
percentage of specific lysis was calculated as
100% × (experimental release  spontaneous
release)/(maximum release spontaneous release). Maximum
release was obtained by adding 100 µL of 5% Triton X-100 to the
100-µL medium containing target cells. Spontaneous release was
consistently below 15% of maximum release in all assays.
Limiting dilution analysis
To evaluate CTL precursor (CTLp) frequencies in EBV-BLCL-activated
or mitogen-activated T cells, limiting dilution analyses were performed
using a modification of the methods of Bourgault et al19
and Langhorne and Lindahl,20 as described by
Lucas et al.21 Briefly, the cultured T-cell fractions were
seeded in final volumes of 200 µL in 24 replicate wells per dilution of T cells in 96-well round bottom microplates. A decreasing number of
effector cells (2 × 104,
1.25 × 104, 7813, 4883, 3052, 1907, 1192, and 745)
were stimulated with 1 × 104 6000-cGy-irradiated
autologous BLCL. A total of 1 × 104 autologous
PBMCs, irradiated with 3000cGy, were added as feeders. On days 0, 2, and 5, 10 IU/mL of IL-2 were added to the cultures. On day 8, the
plates were split and tested against autologous BLCLs and allogeneic
targets. Wells were scored positive when 51Cr release
exceeded the average plus 3 SD of control wells. The CTLp frequencies
were calculated by the method of Taswell22 using a computer
program provided by Dr Y. Kawanishi (Medical College of Wisconsin,
Milwaukee, WI).
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Results |
Transduction conditions for selection of antigen-specific T
cells
To define conditions that maximize selection of antigen-reactive
cells, we initially examined the transduction of purified T-cell
populations at increasing time intervals after stimulation with
irradiated autologous EBV-transformed B cells. To measure vector gene
expression, we took advantage of the mutant LNGFR encoded by the
dicistronic NIT vector. Expression of this gene induces a high level of
LNGFR protein on the surface of transduced cells. Thus, by using
2-color fluorescence, the proportion of T cells expressing the LNGFR
can be accurately assessed and quantitated (Figure
1). The experiments evaluating the time
after stimulation with autologous BLCL at which transduction is
maximized are presented in Figure 2. These
results demonstrate that efficiency of transduction, under the
conditions used, paralleled the level of proliferation of the
stimulated T cells, measured by tritium thymidine
incorporation at the time of retroviral transfer. Thus, maximum
transduction of the stimulated T cells was observed after 5 days of
stimulation for donor U.S. and 7 days for donor S.B.
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| Fig 1.
Cell purification by dual-color FACSort.
(A) Monitoring of gene expression (19.3%) 3 days after transduction of
NIT. (B) CD3+ and NGFR+ T cells, termed
NIT+ fraction, and (C) CD3+ and
NGFR, termed NIT fraction, were
sorted to high purity, 97.52% and 99.54%, respectively, in this
example. Purity of sorted cell fractions was above 95% in all
experiments. Cells of the unsorted fractions were included in the
functional assays.
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| Fig 2.
Proliferation and gene transfer efficiency of purified
T-cell populations.
(A) Proliferation of and (B) gene transfer of NIT into T cells of donor
U.S. and donor S.B. were compared at different time points after
initiation with irradiated autologous EBV-transformed B cells. Cultures
were restimulated 24 hours before the assays. Background proliferation
of the irradiated BLCL was subtracted from the proliferation value of
the stimulated T cells. To standardize the gene transfer procedure,
previously frozen supernatant was used, explaining the relatively low
gene transfer efficiency in this experiment. Cells, transduced with
fresh supernatant as controls, showed a 30% to 50% higher gene
expression at all time points.
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Enrichment of EBV-reactive T cells by retroviral transduction and
selection early after in vitro sensitization
To determine the proportion of EBV-reactive T cells transduced after
different periods of in vitro stimulation, T cells from 2 seropositive
donors were transduced 5, 8, and 23 days after initial stimulation. The
frequencies of EBV-reactive and alloreactive cytotoxic T cells were
assessed in the unselected, NIT+, and
NIT T-cell populations. The T cells were
sorted by dual-color staining for CD3 and LNGFR (monoclonal antibody
20.4) (Figure 1B and 1C). As shown in Table
1, NIT+ T cells transduced
after 5 days of culture exhibited frequencies of EBV-specific and
allospecific cytotoxic T cells that did not differ significantly from
the unsorted population. In contrast, sorted NGFR+ T cells
transduced 8 days after initial sensitization contained EBV-specific T
cells at frequencies that were 4- to 6-fold greater than in the
unsorted T-cell populations and alloreactive T cells at frequencies
that were 5- to 15-fold lower than those detected in the unsorted
fraction. Furthermore, the frequencies of EBV-specific and allospecific
cytotoxic T cells in the NIT+ T cells transduced at day 8 of culture were comparable to those detected in unsorted populations of
EBV-stimulated T cells in culture for 30 days after initial
sensitization.
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Table 1.
Anti-EBV and anti-allo frequencies, including confidence
intervals of unsorted, NIT+, and NIT T
cells of donor S.B. and U.S. after gene transfer on days 5, 8, or
23 and further processing as described
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The potential of retroviral transduction at day 8 of culture to select
for EBV-specific T cells and against alloreactive T cells was assessed
in studies of an additional 3 seropositive donors. Composite results
for the 5 normal donors tested are presented in Table
2. In this table, frequencies of
EBV-specific and allospecific T cells in the unsorted,
NGFR+, and NGFR populations are
presented together with confidence intervals for the limiting dilution
analyses performed. In 4 of 5 donors studied, there was a significant
enrichment of EBV-specific T cells in the NIT+ transduced
T-cell population. This enrichment ranged from 1.5- to 6.1-fold over
the unsorted fraction. Similarly, in each case there was a significant
reduction in the frequency of alloreactive T cells, ranging from
2- to 15-fold, in the transduced and selected NIT+ T-cell fraction.
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Table 2.
Anti-EBV and anti-allo frequencies, including confidence
intervals of unsorted, NIT+, and NIT T
cells of 5 donors after gene transfer on day 8
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To determine whether this strategy of retrovirus transduction and
selection could also permit early enrichment of T cells reactive
against other determinants, we concurrently sensitized T cells with
irradiated allogeneic EBV-transformed B cells and assessed T cells
transduced 8 days after sensitization for allospecific cytotoxicity in
51Cr cytotoxicity assays and for the frequencies of
allospecific cytotoxic cells in unsorted and
NIT+-transduced T-cell populations. As shown in Figure
3, unsorted T cells from donor S.B.
sensitized to fully allogeneic EBV BLCL from donor M.A. exhibit low but
appreciable specific reactivity against EBV BLCL or PHA blasts from
donor M.A. In contrast, the NIT+-transduced T-cell fraction
sorted on the basis of NGFR expression exhibits strong and specific
reactivity against the PHA blasts and EBV BLCL of donor M.A. In
limiting dilution analyses, the NIT+ T-cell fractions
exhibited a 13-fold higher frequency of T cells reactive against the
allogeneic EBV BLCL stimulating cells and, concurrently, a 3- to 8-fold
depletion of EBV-specific T cells reactive against autologous
EBV-transformed B-cell targets (Table 3).
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| Fig 3.
Cytotoxicity of transduced and unmodified T cells after
activation with allogeneic EBV BLCL.
Purified T cells of donor S.B. were stimulated with fully
MHC-mismatched allogeneic BLCLs of donor M.A. MA-BLCL, not recognized
by the NIT+ T-cell fraction after stimulation with
autologous BLCLs of S.B., exhibited strong reactivity against EBV BLCL
and PHA blasts of M.A., after initiation and restimulation with the
allogeneic BLCLs and gene transfer on day 8 as described, showing the
preferential selection of anti-allogeneic T cells. The corresponding
anti-allo frequencies are described in Table 3.
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Table 3.
Anti-EBV and anti-allo frequencies of donor S.B. after
sensitization of T cells with fully allogeneic BLCLs in the unsorted
and NIT+ cell fraction
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Representation of T-cell subsets in NIT-transduced
T-cell populations
Extended culture of T cells reactive against EBV-transformed B cells
has been shown to favor expansion of CD8+ EBV-specific
cytotoxic T-cell populations. However, CD4+ T cells may
provide important signals for expansion of virus-specific T-cell
populations. For this reason, we were interested in comparing the
representation of CD4+ and CD8+ subsets in
T-cell populations transduced early and late postsensitization in vitro.
T cells isolated from normal seropositive donors were sensitized in
vitro to irradiated autologous EBV BLCL and transduced with the NIT
vector supernatant at day 8. Three days later, the unsorted populations
were assessed by immunocytofluorometry for NIT+ and
NIT cells in the T-cell populations. A
representative FACS analysis is presented in Figure
4. Thirteen percent of the isolated T cells were transduced; the distribution of CD4+ and
CD8+ T cells in the transduced NIT+ fraction
(7.5% and 4.5%, respectively; CD4/CD8 ratio, 1.6) did not differ from
the relative proportions of CD4+ and CD8+ T
cells in the nontransduced NIT fraction (55% and
33%, respectively; CD4/CD8 ratio = 1.6).
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| Fig 4.
FACS analysis obtained 3 days after gene transfer.
Cells were stained with anti-CD3, anti-CD4, or anti-CD8 and anti-NGFR
(monoconal antibody 20.4). The results indicate an equal transvection
of CD4 and CD8 lymphocyte subsets, reflected by the CD4/CD8 ratio of
1.6 in both the transduced NIT+ T cells and the
nontransduced NIT T-cell fraction.
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We analyzed the representation of NIT+ T cells in cultures
of EBV BLCL-sensitized T cells transduced 8 or 23 days after initial sensitization in vitro. These results indicate that the proportional representation of CD4+ and CD8+ T cells in
transduced populations closely parallels that detected in the
sensitized T-cell population as a whole and that CD4+ T
cells are represented in a significantly higher proportion of the T
cells transduced early after sensitization (Figure
5).
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| Fig 5.
FACS analyses of lymphocyte subsets in the unsorted and
sorted T-cell fractions.
The proportional representation of CD4+ and
CD8+ cells in the NIT+ T-cell fraction
parallels the unsorted T-cell population after early and after late
transduction. (A) The results after gene transfer on day 8 showed a
proportional higher representation of CD4+ T cells in the
cell populations, whereas in panel B, after gene transfer on day 23 of
cultures, T cells consisted of 80% CD8+ cells and 20%
CD4+ T lymphocytes.
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HLA restriction patterns of EBV-reactive T cells transduced early
after in vitro stimulation with autologous EBV LCL
An advantage hypothesized for suicide vector-modified T cells
transduced early after activation is the potentially broad spectrum of
antigen-reactive cells represented in such T-cell populations, as
compared with the more restricted reactivities represented in T-cell
lines.9 To examine this hypothesis, we analyzed the patterns of HLA restriction exhibited by unsorted and sorted
LNGFR+ T cells from in vitro cultures transduced with the
NIT vector on day 8 after sensitization with EBV BLCL and compared them
with the patterns of HLA-restricted cytotoxicity exhibited by EBV
BLCL-sensitized T cells from the same donor after 30 days of in vitro
culture and expansion. In these experiments, the different
EBV-sensitized T-cell fractions were assayed for their capacity to lyse
autologous EBV LCL and EBV LCL derived from allogeneic donors
homozygous for HLA-A, -B, and -Dr alleles shared by or allogeneic to
the donor. Results of analyses of T-cell fractions from 2 donors are presented in Figures
6 and 7. As
seen in Figure 6, unsorted T cells from cultures transduced with NIT on
day 8 induced 33% lysis of autologous EBV LCL (HLA A*0301,
B*5101/A*6801, B*4403) and were also cytotoxic for targets sharing HLA
B*4403 (53%) and HLA A*0301 (51%) and HLA B*5101 (28%) but exhibited
little reactivity against targets expressing HLA A*6802. In contrast,
after 30 days of culture, cytotoxicity against EBV LCL-expressing HLA
B*4403 was dominant (48%), with less activity detected against targets bearing HLA A*0301 (26%) and minimal reactivity against targets bearing HLA B*5101 and HLA A*6802. Strikingly, an almost identical pattern of a dominant cytolytic response against an HLA B*4403 target
was also observed in testings of the LNGFR-expressing T cells
transduced with NIT 8 days after sensitization. A similar enrichment of
T cells reactive against EBV antigens presented by an immunodominant
allele (ie, HLA B*3502) was also observed in the sorted
LNGFR-expressing fraction of T cells from donor J.K., transduced on day
8 and in the unsorted EBV-specific T cells tested after 40 days of in
vitro culture. This pattern contrasts with the broader pattern of HLA
restriction exhibited by the unsorted T cells tested after transduction
at day 8. These results suggest that immunodominant EBV-reactive T
cells, ie, T cells that recognize immunodominant EBV antigens that are
presented by specific HLA alleles, are preferentially transduced early
after in vitro stimulation with autologous EBV BLCL.
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| Fig 6.
HLA restriction pattern of EBV-reactive T cells of donor
U.S.
The pattern is shown (A) after 12 days in culture, (B) after 30 days in
culture, and (C) in the NIT+ fraction after gene transfer
on day 8 and FACSort on day 11. In the 51Cr release assay,
each donor MHC class I allele could be paired with a BLCL, expressing
that allele homozygously. HLA-B*4403 is the allele, presenting
predominantly EBV peptide to the T cells in the late culture, as
opposed to the broader HLA spectrum of T cells cultured for 12 days
only. The more restricted pattern to HLA-B*4403 can be demonstrated in
the NIT+ fraction after early selection.
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| Fig 7.
HLA restriction of EBV-reactive T cells of donor J.K.
(A) The unsorted EBV-reactive T cells. (B) After 40 days in culture.
(C) The NIT+ fraction on day 12 of donor J.K.
HLA-B*3502 is the dominantly presenting allele in the purified
NIT+ T-cell population and the established
EBV-specific T-cell line after 40 days of culture.
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Assessment of EBV-specific and alloreactive T cells in
mitogen-activated NIT-transduced T-cell populations
Published reports of clinical trials using suicide vector-modified
T cells for treatment of EBV lymphomas or leukemic relapse after
transplantation have employed T cells transduced after nonspecific stimulation with the mitogen PHA. Accordingly, we sought to
characterize and quantify EBV-reactive and alloreactive T cells in
T-cell populations transduced after stimulation with PHA or immobilized
CD3 and CD28. In Figure 8A, we
compared the cytotoxic activities of unsorted and NIT-transduced
LNGFR-expressing T cells with autologous and allogeneic EBV BLCL and
with allogeneic uninfected PHA blasts and K562 targets. As can be seen,
the transduced cells exhibit negligible activity against any of these
targets. This lack of reactivity was confirmed in limiting dilution
analyses. The frequencies of CTLp reactive against autologous or
allogeneic EBV BLCL were less than
3 × 105 in the
mitogen-stimulated unsorted and the sorted NIT-transduced LNGFR-expressing T cells derived from donors S.B. and U.S. These frequencies are markedly lower than the frequencies of EBV-specific or
alloreactive T cells detected in the unsorted or transduced T-cell
population specifically sensitized with autologous or allogeneic EBV
BLCL.
View larger version (35K):
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| Fig 8.
Cytotoxic activity of NIT+ T cells transduced
after mitogen stimulation.
T cells of donor S.B. were stimulated with CD3/CD28-immobilized
monoclonal antibodies for 3 days and subsequently with 10 IU/mL IL-2
for 2 days in 6-well plates before gene transfer. NIT+
cells were purified as described and used for functional assays. (A)
The cytotoxic activity of the NIT+ T cells is less than
10% against autologous EBV BLCL and allogeneic targets. Equally
activated but unmodified T cells were used as controls, showing no
difference between the 2 T-cell populations. (B) The result of the same
NIT+ T cells 8 days later after secondary stimulation with
autologous BLCLs. The cells lysed both autologous and allogeneic EBV
BLCL with comparable activity.
|
|
Given the low frequency of EBV-reactive and alloreactive T cells in the
mitogen-stimulated and NIT-transduced populations, we were interested
to determine whether and to what degree EBV-specific T cells could be
generated from these transduced fractions after secondary stimulation
with autologous EBV BLCL. Results of such analyses for 1 of 3 donors
tested are presented in Figure 8B. As can be seen, cytotoxicity of 20%
against autologous EBV BLCL and 18% against allogeneic EBV BLCL was
detected 8 days following stimulation of these LNGFR+ cells
with autologous BLCLs. We were unable to sustain these cells beyond an
additional 14 days in vitro. Similarly activated cells from 2 additional donors failed to generate populations that exhibited
significant cytotoxicity to autologous or allogeneic EBV-transformed
targets after 8 days of sensitization to autologous EBV-transformed B cells.
| |
Discussion |
As seen in our study population and also recorded by other groups,
the frequencies of CTLp reactive against EBV in seropositive adults are
similar to the frequencies of CTLp directed against HLA alloantigens in
normal individuals.19,21,23 Current approaches involving
adoptive cell therapy with EBV-specific T-cell lines require 28 to 40 days of in vitro expansion to achieve a comparable depletion of
alloreactive T cells capable of inducing severe GVHD. Unfortunately,
EBV lymphomas evolve rapidly. Indeed, the median time from diagnosis to
death due to EBV lymphomas developing early after transplantation has
been reported in 1 international compilation to be as short as 20 days.4 For this reason, such T cells must be generated
prior to or at the time of transplantation to be available for
treatment during the 2- to 8-month period posttransplant during which
such patients are at maximal risk of developing an EBV
lymphoproliferative disorder.3,4
In this study, we examined whether the ability of retroviral vectors to
selectively integrate in dividing cells could be exploited as a method
for early selection of virus-specific or allospecific T cells
replicating in response to irradiated EBV-transformed B cells. To
facilitate in vitro separation of transduced T cells, we employed a
dicistronic vector, termed NIT, which encodes a mutant nerve growth
factor receptor, LNGFR, that is expressed on the surface of transduced
cells at levels permitting their isolation byimmunoadsorption, and
HSV-TK, to permit depletion of transduced cells in vivo in the event
that transduced cells infused induced clinically severe acute GVHD. Our
experiments indicate that such a strategy does indeed permit
early selection and enrichment of virus-specific T cells while at
the same time markedly reducing populations of alloreactive T cells in
the transduced fraction. EBV-specific CTLp frequencies in populations
transduced 8 days after in vitro stimulation with autologous irradiated
EBV BLCL and selected by immunoadsorption were comparable to those detected in cultures of T cells sensitized to autologous EBV BLCL and
expanded in vitro for 30 days. Furthermore, early transduction and
selection also resulted in depletion of alloreactive T cells to
concentrations similar to those detected in T-cell lines cultured for
30 days. Thus, this approach reduces the time required to generate
virus-specific T cells depleted of alloreactive T cells by more than 3 weeks. For the logistics of a program of adoptive cell therapy, this
may represent significant advantages. Furthermore, it provides a
treatment option for patients who present with this complication who do
not have prepared T cells.
The number of EBV-specific T cells required to induce remissions of
widespread lymphoma is small. For example, our previous studies
demonstrated that total doses providing as few as
1 × 103 CTLp could be infused and
would expand rapidly to induce durable regression of disease. Based on
our results, the sensitization and early selection of NIT+
T cells could provide doses of NIT+ EBV CTLp that do not
require further expansion in vitro prior to infusion. For example,
among the donors we studied, PBMCs from donor U.S. had an EBV CTLp
frequency of 15 × 103 at day
0. A dose of donor leukocytes providing 106 T cells/kg from
this seropositive donor (doses of 0.5 × 106 to
1.0 × 106 have induced durable regressions in each
patient in our series) would thus provide 70 kg × 106 T
cells/kg × 15 × 103 = 4666
EBV CTLp. If 1 × 108 T cells were
isolated from about 150 mL of blood from this donor, sensitized with
autologous EBV BLCL, and then transduced with the vector on day 8, by
day 11, the time when we isolate the NIT+ cells, the total
T-cell population would be about
3 × 108. Assuming 20% are transduced
(6 × 107 T cells) and 50% of the
NIT+ cells are recovered after isolation by
immunoadsorption, the yield would be
3 × 107 NIT+ T cells. As
noted in Table 2, the EBV CTLp frequency in the NIT+ T-cell
fraction of donor U.S. is 1 in 755. Thus, the total dose of
NIT+ EBV CTLp that would be provided, were all of these
cells infused, would be 4 × 104 EBV
CTLp or 8.5-fold greater than the dose that would have been provided
with the conventional 106 T cells/kg dose used by us in
dosing donor leukocytes. Were we to use the dose of T cells used by
Rooney et al6,28 in their trials of adoptively transferred
EBV-specific T-cell lines (3 × 105 T cells/kg), the
dose of CTLp provided by the NIT+ T cells isolated from
1 × 108 starting T cells would be
1.5-fold the dose used by Rooney, assuming CTLp frequencies are
equivalent. Thus, the technique proposed does not necessitate further
expansion of the NIT+ T cells to achieve a dose adequate
for treatment.
Although retroviral vector-mediated selection of antigen-reactive T
cells may markedly reduce the time required to generate EBV-specific
T-cell populations appropriately depleted of alloreactive cells, it
does not alter the time required to generate autologous EBV-transformed
B cells for T-cell sensitization. The use of mitogens rather than
EBV-transformed B cells to stimulate the T cells prior to transduction
circumvents this limitation and has been hypothesized to have the
advantage of fostering transduction of a large array of
antigen-reactive T cells.9 In fact, our experiments suggest that mitogen stimulation may induce a striking depletion of both EBV-specific and allospecific T cells. Indeed, the frequencies of CTLp
reactive against either EBV or allogeneic targets were markedly lower
than those detected in unstimulated donor T-cell populations. This
finding may in part explain the delayed onset of acute GVHD noted by
Contassot et al in murine allograft recipients treated with
mitogen-stimulated T cells transduced with a vector encoding HSV-TK and
neomycin phosphotransferase.24
Our findings that the frequencies of alloreactive CTLp in the isolated
NIT+-transduced T cells are as low as those detected in
EBV-reactive T-cell lines cultured for 30 to 40 days suggests the
possibility that such cells, administered at doses of
1 × 107/m2 to
5 × 107/m2 may contain numbers of
alloreactive T cells that are lower than the threshold dose required to
initiate GVHD in HLA-matched unrelated or HLA single allele disparate
hosts, because administration of such doses by Rooney et al derived
from T-cell lines cultured for 30 to 40 days has not been associated
with clinically significant GVHD.25 However, because the
transduced NIT+ populations contain CD4+ T
cells well in excess of those that would be detected in T-cell lines
derived for 30 to 40 days, it is also possible that these transduced T
cells would have a greater potential for GVHD induction, necessitating
the inclusion of the HSV-TK gene as a safety element. While unmodified
donor-derived PBMCs can be used to induce durable regression of EBV
lymphomas emerging in HLA-matched recipients, infusion of as few as
106 T cells/kg in the PBMC mixtures can induce significant
GVHD in up to 30% of cases if administered less than 6 months
posttransplant.3,5 Thus, this approach may also have
advantages for such patients as well.
Despite the clear enrichment of EBV-reactive T cells achieved in the
NIT+ fractions, the CTLp frequencies detected would suggest
that a large proportion of the vector-modified T cells are either not reactive against EBV antigens or are not clonogenic. Recent studies, in
which HLA tetramers presenting specific EBV peptides have been used to
quantitate EBV-reactive T cells, have suggested that the proportion of
T cells in the circulation reactive against EBV antigens may exceed the
proportion of reactive T cells detectable by limiting dilution analyses
by 20- to 100-fold.26,27 If this were also to apply to the
NIT+ populations, EBV-reactive cells would likely
constitute the major antigen-reactive populations among the transduced
cells. Nevertheless, under the conditions used in these experiments,
small numbers of T cells reactive to other antigens, including
alloantigens, were also transduced. It is possible that these T cells
are part of the small fraction of unstimulated T cells that are in
active division, or they are T cells nonspecifically activated by
culture conditions or by cytokines released by irradiated
EBV-transformed B cells. Such activation may explain the lack of
selectivity noted in T-cell populations transduced on day 5. Indeed, it
was not until 8 days of culture, when antigen-specific proliferation
had achieved a plateau, that maximal transduction of antigen-sensitized T cells was regularly observed. These observations were not unique to
the selection of EBV-reactive T cells but also held true for retroviral-mediated selection of alloantigen-specific T cells early
after sensitization in mixed leukocyte culture.
In conducting these studies, we were particularly interested to compare
the characteristics of virus-specific T cells transduced early after
sensitization with T cells generated after extended periods (30 days)
of sensitization and expansion in vitro. One striking difference is the
higher proportion of CD4+ T cells observed in the T
lymphocytes transduced early after sensitization in comparison with the
T cells transduced after extended in vitro culture. This difference
could be ascribed to differences in the proportionate representation of
CD4+ and CD8+ T cells in the
T-lymphocyte population at the time of transduction with the
NIT vector, because there was no apparent difference in the capacity of
the NIT vector to transduce CD4+ versus CD8+ T
cells. The higher proportionate representation of CD4+ T
cells in NIT+ fraction transduced early after sensitization
could constitute an advantage for such cells when used for adoptive
therapy. In clinical trials evaluating adoptive transfer of T cells
predominantly of the CD8+ phenotype derived from long-term
EBV-reactive cell lines or cytomegalovirus-specific CD8+
T-cell clones, expansion of the transformed T cells in vivo has been
limited.11,28 In contrast, infusions of fresh, unselected donor lymphocytes that contain a predominance of CD4+ T
cells have led to dramatic expansion of virus-reactive
cells.21 Similarly, in studies exploring the use of
tumor-antigen reactive T cells for adoptive cell therapy,
CD4+ T cells have been found to provide stimuli important
to the expansion of tumor-reactive T cells posttransfer and to the
clinical expression of an antitumor effect.29
It is well recognized that EBV seropositive adults maintain populations
of EBV-specific T cells in the blood, which often exhibit relatively
restricted reactivity against immunodominant EBV peptides presented by
specific HLA alleles.30,31 Strikingly, this selection of
immunodominant T cells also marks the population of T cells transduced
early after sensitization to autologous EBV BLCL. Indeed, in each
patient studied, the dominant HLA-restricting alleles targeted by the
transduced NIT+ cells mirrored these targeted by the
EBV-specific T-cell lines expanded over 30 to 40 days in vitro. These
findings suggest either that the frequency of T cells restricted by
these alleles is strikingly higher than that of other EBV reactive T
cells in the circulation or that their potential for activation by the
EBV antigens presented by these alleles is significantly greater than
that of other EBV-reactive T-cell populations. Comparisons of T cells
binding immunodominant and subdominant peptides presented on HLA
tetramers for their sensitivity to retroviral transduction after
sensitization may discriminate between these possibilities.
In summary, the results of this study suggest that retroviral vectors,
by virtue of their selective integration into dividing cells, can be
used to selectively transduce T cells activated to proliferate in
response to antigens. Use of retroviral vectors encoding a distinctive
cell surface marker such as LNGFR, which permits isolation of
transduced cells expressing the vector at high purity, permits rapid
selection of such cells early after in vitro sensitization. This
approach may significantly expedite isolation of antigen-reactive T
cells and depletion of contaminating alloreactive T cells for use in
adoptive cell immunotherapy.
| |
Acknowledgments |
We are grateful for the persistent and reliable support of Patrick
Anderson, Diane Domingo, and Tom Delohery of the Flow Cytometry Core
Facility for many FACSorts and FACS analyses. We thank Theresa Diaz-Barrientos and Linda Hirschberger for excellent technical assistance.
| |
Footnotes |
Submitted November 4, 1999; accepted February 22, 2000.
Supported in part by grants CA-59350 National Cancer Institute,
MD; CA23766 National Cancer Institute, MD; HL53752 National Heart,
Lung, and Blood Institute, MD; The Larry H. Smead Fund, The Aubrey Fund
for Pediatric Cancer Research, The Andrew Gaffney Foundation, the
Scholars Award of the McDonnell Foundation for Molecular Medicine, and
The Vincent Astor Chair Clinical Research Fund.
Reprints: Richard J. O'Reilly, Bone Marrow Transplantation
Service, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, Room
H1409, New York, NY 10021; e-mail: oreillyr{at}mskcc.org.
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.
| |
References |
1.
Bhatia S, Ramsay NK, Steinbuch M, et al.
Malignant neoplasms following bone marrow transplantation.
Blood.
1996;87:3633-3639[Abstract/Free Full Text].
2.
Witherspoon RP, Fisher LD, Schoch G, et al.
Secondary cancers after bone marrow transplantation for leukemia or aplastic anemia.
N Engl J Med.
1989;321:784-789[Abstract].
3.
O'Reilly RJ, Small TN, Papadopoulos E, Lucas K, Lacerda J, Koulova L.
Adoptive immunotherapy for EBV-associated lymphoproliferative disorders complicating marrow allografts.
Springer Semin Immunopathol.
1998;20:455-491[Medline]
[Order article via Infotrieve].
4.
Curtis RE, Travis LB, Rowlings PA, et al.
Risk of lymphoproliferative disorders following bone marrow transplantation: a multi-institutional study.
Blood.
1999;94:2208-2216[Abstract/Free Full Text].
5.
Papadopoulos EB, Ladanyi M, Emanuel D, et al.
Infusions of donor leukocytes as treatment of Epstein-Barr virus associated lymphoproliferative disorders complicating allogeneic marrow transplantation.
N Engl J Med.
1994;330:1185-1191[Abstract/Free Full Text].
6.
Rooney CM, Smith CA, Ng CY, et al.
Use of gene-modified virus-specific T-lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.
Lancet.
1995;345:9-13[Medline]
[Order article via Infotrieve].
7.
Porter D.
Unrelated donor leukocyte infusions (U-DLI) to treat relapse or EBV-lymphoproliferative disease (EBV-LPD) after unrelated bone marrow transplantation (BMT) [abstract].
Blood.
1994;90:590a.
8.
Heslop HE, Brenner MK, Rooney CM.
Donor T cells to treat EBV-associated lymphoma.
N Engl J Med.
1994;331:679-680[Free Full Text].
9.
Bonini C, Ferrari G, Verzeletti S, et al.
HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.
Science.
1997;276:1719-1724[Abstract/Free Full Text].
10.
Smith CA, Ng CY, Heslop HE, et al.
Production of genetically modified Epstein-Barr virus-specific cytotoxic T cells for adoptive transfer to patients at high risk of EBV-associated lymphoproliferative disease.
J Hematother.
1995;4:73-79[Medline]
[Order article via Infotrieve].
11.
Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD.
Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones.
Science.
1992;257:238-241[Abstract/Free Full Text].
12.
Walter EA, Greenberg PD, Gilbert MJ, et al.
Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.
N Engl J Med.
1995;333:1038-1044[Abstract/Free Full Text].
13.
Sadelain M, Mulligan RC.
Efficient retroviral-mediated gene transfer into murine primary lymphocytes [abstract]. In:
Gergely J, ed.
Progress in Immunology VIII. Berlin: Springer-Verlag; 1993:34.
14.
Tiberghien P, Reynolds CW, Keller J, et al.
Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T cell depletion after bone marrow transplantation.
Blood.
1994;84:1333-1340[Abstract/Free Full Text].
15.
Miller DG, Adam MA, Miller AD.
Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.
Mol Cell Biol.
1990;10:4239-4292[Abstract/Free Full Text].
16.
Roe TY, Reynolds TC, Yu G, Brown PO.
Integration of murine leukemia virus DNA depends on mitosis.
EMBO J.
1993;12:2099-2108[Medline]
[Order article via Infotrieve].
17.
Gallardo HF, Tan C, Sadelain M.
The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes.
Gene Ther.
1997;4:1115-1119[Medline]
[Order article via Infotrieve].
18.
Pollok KE, Hanenberg H, Noblitt TW, et al.
High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments.
J Virol.
1998;72:4882-4892[Abstract/Free Full Text].
19.
Bourgault I, Gomez A, Gomard E, Levy JP.
Limiting-dilution analysis of the HLA restriction of anti-Epstein-Barr virus-specific cytolytic T lymphocytes.
Clin Exp Immunol.
1991;84:501-507[Medline]
[Order article via Infotrieve].
20.
Langhorne J, Lindhal KF.
Limiting dilution analysis of precursors of cytotoxic T lymphocytes.
Immunol Methods.
1981;2:221.
21.
Lucas KG, Small TN, Heller G, Dupont B, O'Reilly RJ.
The development of cellular immunity to Epstein-Barr virus following allogeneic bone marrow transplantation.
Blood.
1996;87:2594-2603[Abstract/Free Full Text].
22.
Taswell C.
Limiting dilution assays for the determination of immunocompetent cell frequencies. III. Validity tests for the single-hit Poisson model.
J Immunol Methods.
1984;72:29-40[Medline]
[Order article via Infotrieve].
23.
Kaminski E, Hows J, Man S, et al.
Prediction of graft-versus-host disease by frequency analysis of cytotoxic T cells after unrelated donor bone marrow transplantation.
Transplantation.
1989;48:608[Medline]
[Order article via Infotrieve].
24.
Contassot E, Murphy W, Angonin R, et al.
In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes.
Transplantation.
1998;65:1365-1370[Medline]
[Order article via Infotrieve].
25.
Rooney CM, Smith CA, Ng CY, et al.
Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.
Blood.
1998;92:1549-1555[Abstract/Free Full Text].
26.
Murali-Krishna K, Altman JD, Suresh M, et al.
Counting antigen-specific CD8+ T cells: a re-evaluation of bystander activation during viral infection.
Immunity.
1998;8:177[Medline]
[Order article via Infotrieve].
27.
Tan LC, Gudgeon N, Annels NE, et al.
A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers.
J Immunol.
1999;162:1827-1835[Abstract/Free Full Text].
28.
Heslop HE, Ng CY, Li C, et al.
Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.
Nat Med.
1996;2:551-555[Medline]
[Order article via Infotrieve].
29.
Toes REM, Schoenberger SP, van der Voort EIH, Offringa R, Melief CJM.
CD4 T cells and their role in antitumor immune responses.
J Exp Med.
1999;189:753-756[Free Full Text].
30.
Steven N, Leese AM, Annels NE, Lee SP, Rickinson AB.
Epitope focusing in the primary cytotoxic T cells response to Epstein-Barr virus and its relationship to T cell memory.
J Exp Med.
1996;184:1801[Abstract/Free Full Text].
31.
Gavioli R, Kurilla MG, de Campos-Lima PO, et al.
Multiple HLA A11-restricted cytotoxic T lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen.
J Virol.
1993;67:1572[Abstract/Free Full Text].
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Abstract
Introduction