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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3242-3250
Simultaneous Ex Vivo Expansion of Cytomegalovirus and Epstein-Barr
Virus-Specific Cytotoxic T Lymphocytes Using B-Lymphoblastoid Cell
Lines Expressing Cytomegalovirus pp65
By
Qi Sun,
Karen E. Pollok,
Robert L. Burton,
Li Jun Dai,
William Britt,
David J. Emanuel, and
Kenneth G. Lucas
From the Bone Marrow Transplantation Program, University of Alabama
at Birmingham, Birmingham, AL; the Herman B. Wells Center for Pediatric
Research, Riley Hospital for Children, Indianapolis, IN; the Department
of Pediatrics, The Children's Hospital of Alabama, Birmingham,
AL; and Schering-Plough Research Institute, Kenilworth,
NJ.
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ABSTRACT |
Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced
lymphoproliferative disease are serious complications associated with
allogeneic stem cell transplantation. Immunotherapy using ex vivo
expanded, virus-specific cytotoxic T lymphocytes (CTL) has been
explored and proven to be effective in therapeutic or prophylactic
regimens for CMV and EBV infections. To generate CTL specific for both
CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines
(BLCL) to express CMV pp65 for use as antigen-presenting cells (APC).
BLCL were transduced with a recombinant retrovirus encoding pp65, the
immunodominant CMV polypeptide. Western blot analysis and
immunocytochemistry confirmed the expression of pp65 in the transduced
cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV
seropositive donors were stimulated with autologous pp65-expressing
BLCL weekly for 3 weeks. Chromium release assays showed that the
resulting CTL cultures possessed specific cytotoxicity against EBV and
CMV. Recombinant vaccinia viruses encoding individual CMV peptides were
used to demonstrate that this CMV-specific cytotoxicity was specific
for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that
CD8+ CTL mediated the pp65-specific cytotoxicity. These
CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing
HLA class I antigens, but not HLA mismatched targets. Our results
demonstrate that BLCL can be used as APC to stimulate expansion of EBV-
and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN CYTOMEGALOVIRUS (CMV) and
Epstein-Barr virus (EBV) are members of the herpesvirus family, and in
the normal host these viruses cause self-limited
disease.1,2 In immunocompromised individuals, especially
recipients of allogeneic, T-cell-depleted (TCD) stem cell transplants
(SCT), CMV and EBV are responsible for significant morbidity and
mortality.3-8 The incidence of CMV reactivation has been
reported to be between 70% to 80% after SCT.3,6,7,9 The
likelihood of developing EBV-induced lymphoproliferative disease
(EBV-LPD) can be as high as 20% after a TCD graft, depending on the
patient's extent of immunosuppression.10,11 In these
patients, cellular immunity, which plays a major role in preventing and
controlling viral infections, is compromised as a result of
immunosuppression aimed at preventing graft-versus-host disease (GVHD)
and graft rejection.10,12,13 For example, more than 65% of
patients at day 40 posttransplant are deficient in CD8+
T-cell responses to CMV.13,14 Similarly, there is a
correlation between the peak incidence of EBV-LPD and levels of
EBV-specific cytotoxic T lymphocytes (CTL) in SCT patients, who are
most susceptible to developing this complication between days 60 to 90 posttransplant.10 Thus, the restoration of cellular
immunity is necessary to prevent viral
reactivation.12,13,15,16 Although pharmacological agents exist to treat CMV infections, these medications have several side
effects, the most serious of which is
myelosuppression.7,17-19 Therefore, alternative therapies
that restore host cellular immunity to CMV and EBV are of interest.
The infusion of ex vivo expanded, EBV-specific CTL has been proven to
be safe and efficacious for the treatment and prophylaxis of
EBV-LPD.15,16 To expand donor-derived, EBV-specific CTL, peripheral blood mononuclear cells (PBMC) are cocultivated ex vivo with
autologous B-lymphoblastoid cell lines (BLCL), which are latently
infected with EBV. Similar protocols have been developed to generate
CMV-specific CTL cultures by cocultivating donor PBMC with CMV-infected
autologous skin fibroblasts (SF), the only cell type supporting CMV
replication in vitro.20-22 CD8+, CMV-specific
CTL used for patient infusion are cloned from bulk CTL cultures due to
the high allogeneic cytotoxicity in the latter.14 Walter et al23 and Riddell et al24
demonstrated that the administration of CMV-specific CTL clones
resulted in the restoration of CMV-specific cellular immunity in SCT
patients at risk for CMV disease. Current strategies for
posttransplant, antiviral adoptive immunotherapy may be limited by the
need for cultivating separate CTL for each important target antigen.
BLCL can be readily established in the laboratory by EBV
immortalization. These lines phenotypically resemble the transformed B
cells in posttransplant EBV-LPD and express immunogenic EBV peptides,25 making them ideal stimulators for expanding
EBV-specific CTL. Among the more than 150 polypeptides encoded by CMV,
several are known to be immunogenic.26 Those targeted by
CTL include the immediate-early protein, virion envelope glycoprotein
B, and the internal matrix proteins pp65 and pp150.27-31
CMV pp65 has been identified as the immunodominant antigen, targeted by
70% to 90% of CMV-specific CTL.29,32,33 Because SCT
patients are at risk for both EBV and CMV disease, a CTL preparation
specific for both EBV and CMV would be of potential benefit, especially if CMV/EBV-specific CTL could be expanded simultaneously in the same
culture. To this end, we developed a novel system, using BLCL as
antigen-presenting cells (APC) to present both EBV and CMV epitopes.
BLCL were transduced with a recombinant retrovirus encoding CMV pp65
and were then used to prime autologous PBMC. We demonstrated that (1)
CTL cultures stimulated with pp65-expressing BLCL had CMV- and
EBV-specific cytotoxicity and (2) this cytotoxicity was mediated by
CD8+ CTL.
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MATERIALS AND METHODS |
Donors.
Four normal CMV-seropositive donors (AE, RB, SQ, and ST) and 2 seronegative donors (AD and SB) provided PBMC, SF, and serum for this
study under protocols approved by the Institutional Review Board of the
University of Alabama at Birmingham (UAB). CMV serostatus was
determined by the UAB Core Immunology Laboratory with an IgG enzyme-linked immunosorbent assay (Abbott Laboratories, Chicago, IL).
Major histocompatibility complex (MHC) class I and II typing was
performed by the UAB Histocompatibility Laboratory.
Expression vectors and viruses.
To construct the pp65-encoding recombinant retrovirus MSCVpp65, an
1,800-bp EcoRI-BamHI fragment containing the entire CMV pp65 coding sequence (a gift from J.A. Zaia,34 City of Hope Medical Center, Duarte, CA) was subcloned into the
EcoRI-BglII linearized retroviral vector MSCV2.1 (a
gift from R.G. Hawley,35 University of Toronto, Toronto,
Canada). CMV pp65 was placed under the control of the long terminal
repeat (LTR), while the internal human phosphosglycerole kinase
promoter drove the expression of the selection marker bacterial
neomycin phosphotransferase.
To generate replication-defective recombinant retroviruses,
CsCl-purified construct DNA was electroporated into psi-cre cells, an
ecotropic packaging cell line.36 Culture medium from the transfected psi-cre cells was used to infect the amphotropic producer cell line GP+envAM12.37 The producer
cells were grown in Dulbecco's modified Eagle's medium (DMEM)
(Mediatech, Herndon, VA) with 10% fetal bovine serum (FBS; Hyclone,
Logan, UT), and were selected with G418 (Geneticin, BRL-Life
Technologies, Gaithersburg, MD) at 600 µg/mL for 7 days. The
virus-containing media was collected after the producer cell line was
grown to confluence, and this supernatant was used to infect either
human SF or BLCL. Retrovirus titer was determined by G418-selectable
colonies in human SF and ranged from 5 × 105 to
106/mL.
Recombinant vaccinia viruses encoding CMV pp65 or pp150 were used as
previously described.33 Briefly, virus stocks were prepared
in BSC-40 cells (American Type Culture Collection [ATCC], Rockville,
MD). After infection at a multiplicity of infection (MOI) of 0.1, the
cells were cultured for 48 hours and then harvested and underwent 3 cycles of freeze and thaw. Virus suspension was cleared of debris by
centrifugation and stored in aliquots at  20°C. Vaccinia
virus titers ranged from 1 × 107 to
108/mL as determined by plaque assays in BSC-40 cells.
AD 169 human CMV was obtained from ATCC and virus was propagated in
human SF. The initial infection was at an MOI of 0.1. Five days after
cytopathological effects appeared in more than 90% cells, the
virus-containing media were collected, passed through a 0.45-mm filter,
and frozen in liquid nitrogen as aliquots. CMV titer was between 1 × 106 and 107/mL by plaque assays in
human SF.
Cell lines, retroviral transduction, and virus infection.
PBMC were separated from heparinized whole blood by Histopaque (Sigma,
St Louis, MO) gradient centrifugation and washed with RPMI 1640 (BRL-Life Technologies). BLCL were established from PBMC by EBV
transformation and maintained in RPMI 1640 with 10% FBS, as described
previously.10 BLCL were transduced with recombinant retroviruses by a 2-hour incubation with retrovirus-containing media.
G418 selection was applied 48 hours after infection at 400 µg/mL for
14 days.
Primary human dermal fibroblast cell lines were established from skin
biopsies from forearms. Human SF were grown in F12/DMEM (Mediatech)
with 20% FBS and transduced with retroviruses by a 1-hour incubation
with retrovirus-containing media. G418 selection was applied 24 hours
after infection at 200 µg/mL for 5 days. When growth was sufficient,
the cell lines were tested for mycoplasma contamination by a mycoplasma
detection kit (BRL-Life Technologies). When used as targets in
chromium-51 (51Cr) release assays, SF were treated with 100 IU/mL interferon- for 48 hours and were infected overnight with
either recombinant vaccinia virus at an MOI of 5 or with CMV stock at
an MOI of 10.
Protein analysis.
For immunocytochemical detection, the cells were fixed in 96-well
plates with a 50% mixture of acetone and methanol for 20 minutes. A
monoclonal antibody against human CMV-pp65,38 205, was used
to stain the cells for 60 minutes at a dilution of 1:25. Immunoperoxidase staining was performed with a kit as instructed by the
manufacturer (Immunopure Ultra-sensitive ABC Peroxidase Staining Kit;
Pierce, Rockford, IL).
For immunoblotting, 0.2 × 106 cells were lysed in a
buffer containing 4% sodium dodecyl sulfate (SDS), 0.1 mol/L Tris pH
6.8, 0.2% Brilliant Blue G250, 280 mmol/L 2-mercaptomethanol, and 20% glycerol. After boiling for 5 minutes, the samples were separated on an
8% SDS-polyacrylamide gel and transferred to a piece of Hybond-P
membrane (Amersham Lifescience, Arlington Heights, IL). The blots were
blocked with 5% nonfat milk in PBS-T (0.1 mol/L phosphate buffer,
pH7.4, 200 mmol/L NaCl, 0.05% Tween 20), and probed with the
monoclonal antibodies against pp65 (65-8)39 at 1:100
dilution and/or EBNA2 (PE2)40 at 1:500 dilution (Dako, Carpenteria, CA) for 60 minutes at room temperature. A horseradish peroxidase-labeled sheep antimouse IgG antibody (Amersham) was used as
the secondary antibody at a dilution of 1:2,000. Enhanced chemiluminescence (ECL) detection was performed with ECL detection agents (Amersham) and recorded on Hyperfilm-ECL film (Amersham).
Ex vivo expansion of cytotoxic T lymphocytes.
Both pp65-expressing BLCL and SF were used as stimulators for ex vivo
CTL expansion. PBMC were cocultivated with autologous pp65-expressing
BLCL in 24-well plates (Falcon; Becton Dickinson Labware, Franklin
Lakes, NJ) in RPMI 1640 supplemented with 10% FBS and 50 µmol/L
2-mercaptoethanol (Sigma). BLCL were exposed to 100 Gy of gamma
irradiation before use as stimulator cells. The CTL cultures were
primed weekly following a regimen of decreasing responder:stimulator
ratios from 40:1 at day 0, 20:1 at day 7, and 5:1 on day 14 over a
period of 3 weeks. pp65-expressing SF were used as stimulators
following conditions described previously using CMV-infected
SF.13,20 Briefly, PBMC were dispensed at 10 × 106 cells/well in 6-well plates containing 0.5 × 106 pp65-expressing SF in RPMI 1640 with 10% human CMV
seronegative AB serum and 50 µmol/L 2-mercaptoethanol (Sigma). Weekly
the CTL cultures were collected, washed with RPMI 1640, and replated
with the same number of fresh SF stimulators at a ratio of 20:1 using autologous gamma-irradiated PBMC as feeder cells. For both BLCL and SF
stimulators, interleukin-2 (IL-2) (Collaborative Biomedical Products,
Bedford, MA) was added 10 days after the initial stimulation to a final
concentration of 2.5 IU/mL, and medium was then changed every 3 days by
replacing one half of the supernatant with fresh medium.
Chromium release assays.
Chromium release assays were performed as previously
described.10 Target cells included the following: BLCL and
BLCL transduced with MSCVpp65 or the backbone MSCV; SF, SF transduced
with MSCVpp65 or the backbone MSCV, SF infected with CMV, and SF
infected with recombinant vaccinia viruses encoding either pp65 or
pp150. Target cells were labeled with 51Cr (New England
Nuclear, Boston, MA) overnight (100 µCi/106 cells) and
labeled cells were harvested, either by trypsinizing for SF or by
centrifugation for BLCL. Cells were then washed in phosphate-buffered
saline (PBS) and dispensed in triplicates into 96-well V-bottom plates
(ICN, Costa Mesa, CA) at 4 × 103 cells/well. The
effector:target (E:T) ratios were 12.5:1, 5:1, and 2.5:1. Spontaneous
and total release for each target were used to calculate percent
specific release using the following formula:
CD4+ and CD8+ cell isolation.
To isolate CD4+ and CD8+ populations of
polyclonal CMV/EBV-specific CTL, bulk culture CTL were depleted of
either CD4+ or CD8+ cells and natural killer
(NK) cells using monoclonal antibodies specific for CD4/CD56 or
CD8/CD56 (Pharmingen, San Diego, CA). Briefly, cells were placed in
RPMI 1640 at 4°C at a concentration of 5 × 106/mL, and the monoclonal antibody was added for a final
concentration of 20 µg/mL. After a 30-minute incubation at 4°C,
the cells were washed twice in RPMI 1640, and Dynabeads (Dynal, Lake
Success, NY) were added following the manufacturer's instructions.
After 30 minutes incubation at 4°C, the cell-bead suspension was
placed on a magnet and the supernatant was removed, according to the manufacturer's guidelines.
Cytofluorometry.
The cell-surface phenotype of EBV/CMV CTL was determined by staining
with directly conjugated monoclonal antibodies and analyzing on a
cytometer (FACScan; Becton Dickinson, San Jose, CA). These antibodies
(Becton Dickinson) were specific for the following: CD3, CD4, CD8, and
CD16/56 (simultest). Cells were washed with PBS supplemented with 3%
FBS and 0.01% sodium azide. Dilutions and incubations for the
antibodies followed the manufacturer's instructions.
| |
RESULTS |
APC presenting both EBV and CMV antigens.
After selection with G418, BLCL and SF transduced with either the
retrovirus MSCVpp65 (BLCLpp65 and SFpp65, respectively) or MSCV
(BLCLmscv or SFmscv, respectively) continued to proliferate, whereas
uninfected cells did not survive. Western blot analysis (Fig 1) of the transduced cells showed that
the pp65-specific monoclonal antibody 65-8 detected a single band from
BLCLpp65 and SFpp65 (lanes 4 and 6), but not in the MSCV backbone
vector-transduced or the untransduced parental cells (lanes 1, 3, and
5). A band of similar size was also observed in the SF infected with
CMV (lane 2), confirming the expression and the physical integrity of
pp65 in the cells transduced with MSCVpp65. In addition, using a
monoclonal antibody specific for EBV nuclear antigen 2 (EBNA2), a
specific band was detected in the two BLCLpp65 tested (lane 8, from AD;
lane 9, from RB), both of which also expressed pp65. Because EBNA2 is 1 of the immunogenic EBV polypeptides expressed in BLCL, the detection of
both pp65 and EBNA2 from BLCLpp65 was consistent with our expectation
that BLCLpp65 would present epitopes derived from both CMV and EBV.
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| Fig 1.
Immunoblot analysis for the expression of CMV pp65 and
EBV EBNA2. Lanes 1 through 6, probed against CMV pp65; lanes 7 through
9, probed against both EBV EBNA2 and CMV pp65.
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To examine the expression of pp65 at the single cell level, a CMV
pp65-specific monoclonal antibody (205)38 was used to stain transduced cells immunocytochemically. The specific
immunoperoxidase staining was localized to the nuclei in both SFpp65
and BLCLpp65 (Fig 2b and d). In addition to
a diffuse staining pattern, some cells (most clearly in the SFpp65)
displayed the characteristic inclusion body-like structure seen in
CMV-infected cells.39 While almost all of the SFpp65 showed
positive staining, the percentage of G418-resistent BLCLpp65 cells
positive for immunoperoxidase staining varied from approximately 50%
to 90%. This may be related to intrinsic differences in these two
types of cells and their level of differentiation. Contrary to the
relatively homogeneous nature of SF cultures, BLCL are regarded as
heterogeneous, proliferating B cells at different stages of
differentiation. The expression of the pp65, which is controlled by
MSCV LTR, might be subject to the differentiation status of BLCL, as it
has been reported that the promoter activity of MSCV LTR is associated
with the differentiation status of hematopoietic cells.41
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| Fig 2.
Immunoperoxidase staining using a pp65-specific
monoclonal antibody with (a) SF transduced with MSCV; (b) SF transduced
with MSCVpp65; (c) BLCL transduced with MSCV; and (d) BLCL
transduced with MSCVpp65.
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Generation of CTL cultures.
The transduced cells were cocultivated with autologous PBMC ex vivo to
stimulate preferential expansion of EBV- and pp65-specific CTL. Before
coculturing, the stimulators were confirmed for pp65 expression by
immunoperoxidase staining. Pilot experiments included BLCLpp65 and
SFpp65 as stimulators. Approximately 3 days after cocultivation,
colonies composed of irregularly shaped cells were observed, most
abundantly in the BLCLpp65-primed cultures. Although pp65-specific
cytotoxicity was detected from CTL cultures using either cell line as
an APC, SFpp65 were discontinued to be used as stimulators due to our
limited access to human CMV seronegative AB serum, which the SF-based
protocols called for.13,20,42
Cytotoxic specificity of the BLCLpp65-stimulated CTL cultures.
51Cr release assays were used to test CTL cultures against
a panel of autologous and allogeneic target cells. Figure 3 shows that
CTL cultures from all 6 donors lysed specific lysis of BLCL, as
expected from their positive EBV-serology.
In addition, CTL cultures from the 4 CMV-seropositive donors (AE, RB,
SQ, and ST), but not the 2 CMV seronegative donors (AD and SB), also
displayed specific cytotoxicity against MSCVpp65-transduced cells.
Similar results were obtained for CTL cultures from day 14 and day 35 (data not shown). Although the stimulator BLCLpp65 should express the
neomycin phosphotransferase, as indicated by the G418 selectibility, there was no specific killing against SFmscv in 51Cr
release assays. The failure of ex vivo expansion of CTL specific for
this bacterial protein, in contrast to the viral pp65, may be due to
its weak immunogenecity, and/or a very low precursor frequency.
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| Fig 3.
CTL cultures at 21 days after ex vivo stimulation with
autologous BLCLpp65 were tested for specific cytotoxicity against
EBV-transformed BLCL, CMV-infected SF, and
MSCVpp65-transduced cells in chromium release assay. (A through
D) CTL activity from 4 CMV seropositive/EBV seropositive individuals.
(E through F) CTL activity from 2 CMV seronegative/EBV seropositive
individuals. (E:T ratio, 12.5:1).
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CTL cultures showing pp65-specific cytotoxicity also lysed autologous
SF targets infected with CMV, suggesting that the pp65-specific cytotoxicity was effective in a setting close to natural CMV infection. However, a legitimate concern against this interpretation would be that
residual CTL specific to other CMV antigens may be the cause of, or
contribute to, the killing of the CMV-infected cells. To exclude this
possibility, SF used as targets were infected with recombinant vaccinia
viruses encoding either pp65 or pp150. CMV pp150 is another CMV matrix
protein that is often targeted by specific CTL.31 Although
all 3 CTL cultures lysed the pp65-vaccinia infected SF, none lysed
pp150-vaccinia infected targets (Fig 4), indicating that the CMV-specific cytotoxicity was against pp65.
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| Fig 4.
Cytotoxicity against CMV-infected cells by
BLCLpp65-primed CTL cultures was mediated via pp65. CTL cultures lysed
autologous fibroblasts infected with a recombinant vaccinia virus
encoding CMV pp65, but not the one encoding CMV pp150. (E:T ratio,
12.5:1)
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Effector cells of the CMV/EBV-specific cytotoxicity.
To discern the nature of the EBV/CMV-specific cytotoxicity, bulk CTL
cultures from donor ST were selectively depleted of either CD4+/CD56+ cells or
CD8+/CD56+ cells. Resulting cells were greater
than 96% positive for CD8+ and greater than 80% positive
for CD4+ cells, respectively. Cell fractions were tested in
51Cr release assays against autologous SFpp65, BLCL, or
SFmscv (Fig 5). CD8-enriched cells lysed
both SFpp65 and BLCL, but not SFmscv (Fig 5B). A similar cytotoxicity
profile was observed from bulk culture CTL (Fig 5A). In contrast, the
CD4-enriched fraction failed to lyse any of these targets (Fig 5C).
Furthermore, bulk culture and CD8-enriched CTL sustained specific
cytotoxicity against BLCL and SFpp65 at 3 E:T ratios (12.5:1, 5:1, and
2.5:1). The CD4-enriched fraction failed to show any lysis of the BLCL
or SFpp65 at any E:T ratios. These data indicated that the
CMV/EBV-specific cytotoxicity was mediated mostly, if not entirely, by
CD8+ CTL.
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| Fig 5.
Cytotoxicity against EBV-transformed BLCL and
MSCVpp65-transduced SF was mediated by CD8+ CTL. Bulk,
CD8-enriched, or CD4-enriched CTL culture from donor ST were tested at
different E:T ratios in chromium release assay. CD8-enriched CTL (B)
retained the cytotoxicity displayed by the bulk CTL (A), while the
CD4-enriched CTL (C) did not.
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The differential cytotoxicity towards the autologous and allogeneic
pp65-expressing targets by BLCLpp65-stimulated CMV/EBV CTL was
consistent with a pattern of HLA-restricted cytotoxicity (Fig 3). We
confirmed this by comparing specific cytotoxicity against a panel of
targets consisting of autologous, partially matched allogeneic, or
completely mismatched allogeneic cells (Fig
6). CTL from donor ST were used, which shared A2,B44 with AD, a CMV
seronegative donor of which the CTL culture showed no cytotoxicity
against autologous pp65-expressing target cells. Bulk and CD8-enriched
CTL from donor ST lysed all the 3 pp65-expressing targets from AD
almost as well as the autologous counterparts (ST). The same CTL
cultures failed to lyse the completely mismatched targets from donor SQ
(Fig 6) or targets from 2 other mismatched individuals (data not
shown). Note the EBV and CMV targets from SQ were lysed by the
autologous CMV/EBV CTL (Fig 3D). This suggested that the pp65-specific
cytotoxicity was HLA A2 and/or B44 restricted. The CMV/EBV-specific CTL
from donor ST also showed cytotoxicity against the partially matched
allogeneic BLCL, although the activity was lower than that for the
autologous BLCL. This may be explained by the fact that BLCL express at
least 8 EBV antigens, and it is possible that not all of the epitopes
were presented by the 2 shared HLA alleles.
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| Fig 6.
CMV pp65-specific cytotoxicity was HLA class
I-restricted. Bulk and CD8-enriched CTL from donor ST showed
cytotoxicity against HLA class I partially matched allogeneic targets
(AD), which shared HLA A2 and B44 with donor ST. There was no specific
lysis of the complete allogeneic targets (SQ). (E:T ratio, 12.5:1)
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DISCUSSION |
CMV disease and EBV-LPD are 2 of the most serious and potentially
life-threatening viral complications after allogeneic,
TCD-SCT.5-8 Previous studies have shown that adoptive
immunotherapy with virus-specific CTL can be used to prevent
complications related to these viruses.15,24 Current
methodologies are limited by a requirement for developing CTL cultures
using separate APC. This report demonstrated the feasibility of using
retroviral technology to transduce EBV BLCL and achieve expression of
CMV pp65. These BLCL present EBV peptides, as well as CMV pp65, and
resulted in polyclonal, bispecific CTL cultures when cocultured with
autologous PBMC. These findings are of importance to centers applying
adoptive immunotherapy for viral infections in SCT patients.
Over the past decade, there has been a decrease in morbidity and
mortality caused by CMV disease in posttransplant SCT, and this is
largely attributable to the availability of pharmacological agents
effective against CMV infections.43,44 Ganciclovir has been
used to prevent CMV reactivation, either at the onset of laboratory
evidence of CMV infection (by culture, polymerase chain reaction
[PCR], or antigenemia assay) or as prophylaxis.7,17,18 However, ganciclovir-related granulocytopenia has been reported in 30%
to 60% of bone marrow transplant patients, complicating the routine
use of this drug for prophylaxis in all CMV seropositive patients.7,17-19 This side effect is of particular concern
in patients for whom slow engraftment is a potential problem and the
myelosuppressive effects of this medication could lead to graft
failure. As a result of drug-induced cytopenia, patients receiving
ganciclovir can have increased transfusion requirements. The use of
this drug has also been associated with delayed reconstitution of
CMV-specific CTL posttransplant and an increased incidence of late CMV
disease.45 The limitations of anti-CMV pharmacotherapy have
promoted interest in adoptive immunotherapy as an alternative means for
the prevention and treatment of CMV disease in SCT patients.
The application of adoptive immunotherapy has evolved as a result of an
increased understanding of the cellular immune response to viral
pathogens. Infected cells process endogenously synthesized viral
polypeptides and present viral epitopes on the cell
surface.46,47 CD8+ CTL recognize these epitopes
in an HLA class I-restricted context and are activated to proliferate
and lyse the infected cells. Thus, the prerequisite for adoptive
immunotherapy is to have suitable APC that can present relevant viral
epitopes. Regarded as professional APC, BLCL express high levels of
class I and II HLA molecules and costimulatory molecules such as
intercellular adhesion molecule (ICAM)-1, B7/BB1, and
lymphocyte function-associated antigen
(LFA)-3,48-50 in addition to key EBV
antigens. Probably due to this reason, BLCL have been used to generate
EBV-specific CTL cultures reliably by different laboratories, and as a
result, adoptive immunotherapy is now performed routinely at some
centers for EBV-LPD in TCD-SCT patients. The relative ease with which
EBV CTL are cultivated using donor BLCL led us to investigate the
possibility of using the same APC to generate CMV-specific CTL.
CMV pp65 was chosen as CMV-specific antigen based on its immunological
and virological properties. The majority of CMV-specific CTL isolated
from CMV seropositive patients recognize pp65.29,32,33 As a
component of the virion tegument, pp65 is introduced into the host
cells during virus entry. The exogenous pp65 has been shown to be
presented by HLA class I molecules and targeted by CD8+
CTL, possibly before the viruses multiply.51 Therefore,
targeting this antigen may limit the spread of CMV infection at an
early time point of CMV reactivation. Recently, attempts have been made to elucidate CMV HLA allele-specific epitopes for developing peptide vaccines. One nonamer pp65 epitope was shown to be immunogenic when
presented in the context of HLA 0201.42 Although
peptide-pulsed APC would be a promising tool to generate CTL culture
for adoptive immunotherapy, it is limited, at least at this stage of
development, to certain HLA alleles. In contrast, BLCLpp65 have the
potential to present various pp65 epitopes, and it would be universally applicable to donors with diverse HLA alleles.
We chose a well-characterized recombinant retrovirus (MSCV) to achieve
pp65 expression in BLCL. Retroviral vectors transduce rapidly dividing
cells, such as BLCL, with high efficiency,35 making it
possible to establish pp65-expressing BLCL in a timely fashion. All of
the BLCL that we tested were readily transduced by this virus, and all
of the pp65-transduced BLCL stimulated specific cytotoxicity in the
EBV/CMV seropositive CTL cultures regardless of the heterogeneity of
pp65 expression. Furthermore, MSCV is replication-defective, and
careful selection of producer cells should eliminate the problem of
contamination with replication-competent viruses resulting from
fortuitous recombination. MSCVpp65 encoded only 2 products, the target
antigen pp65 and a selection marker that appeared not to elicit a
strong CTL reaction. A minimal number of transgenes encoded by a vector
translates into fewer antigens presented and hence fewer nonrelevant
CTL expanded.
Our method for generating CMV/EBV-specific CTL cultures has unique
advantages and is feasible in terms of both resources and time. This
approach would technically simplify adoptive T-cell transfer, as EBV-
and CMV-specific CTL are generated simultaneously in the same culture.
The use of BLCL as APC circumvents the problem of procuring skin
biopsies from the donors and the time to grow fibroblasts. In addition,
CMV/EBV CTL primed by BLCLpp65 can be cultivated in FBS, which is
readily available, does not carry the risk of exposure to human
infectious agents, and is presently in use by other investigators in
adoptive immunotherapy. The CMV/EBV CTL generated in this study were
not reactive against noninfected, allogeneic SF, suggesting that the
lengthy procedure of CTL cloning may not be necessary. Time required to
generate bispecific CTL cultures could be further shortened by
transducing BLCL with high-titer retrovirus produced by the new
generation of vector production systems.52,53 High titer
retrovirus should greatly improve the transduction efficiency and as a
result dispense with the need for G418 selection of the transduced
cells.52,53 Alternatively, retroviral transduction could be
performed at the same time as EBV transformation, eliminating the time
required to generate pp65-expressing BLCL.
It should be noted that with BLCLpp65 as stimulators, CMVpp65-specific
CTL cultures were generated only from CMV-seropositive, but not
seronegative, individuals. Similar results have also been observed by
others when CMV-infected fibroblasts were used as APC.20
These results appear to suggest that B cells, similar to fibroblasts,
are able to reactivate the memory CTL, but not to prime naïve T
cells. Thus, BLCL may not be suitable as APC to present CMV antigens
for CMV seronegative individuals. Work is ongoing in our laboratory to
determine which APC can prime in vitro expansion of CMV-specific CTL
from seronegative donors.
| |
FOOTNOTES |
Submitted March 17, 1999; accepted June 25, 1999.
Supported by Grant No. CRTG 97-043-EDT from the American Cancer Society
and Grant No. R01 CA75566-01 from the National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kenneth Lucas, MD, Bone Marrow
Transplantation Program, University of Alabama at Birmingham,
1900 University Blvd, THT 541, Birmingham, AL 35294;
e-mail: klucas{at}uabmc.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
