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Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-05-1514.
IMMUNOBIOLOGY
From the Center for Cell and Gene Therapy, Texas
Children's Cancer Center, Departments of Pediatrics, Medicine, and
Molecular Virology and Microbiology, Baylor College of Medicine,
Houston, TX.
The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in
EBV-positive Hodgkin disease and is a potential target for cytotoxic
T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is
low, and so far the generation of LMP1-specific CTLs has required
T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic
cells (DCs) for CTL stimulation, and we reasoned that an inactive,
nontoxic LMP1 mutant ( Immunotherapy with cytotoxic T cells (CTLs) is
increasingly used to treat malignancies and viral
infections.1,2 For example, polyclonal Epstein-Barr virus
(EBV)-specific CTLs have been used for the prevention and treatment of
posttransplantation EBV-associated lymphoma (PTLD).3
EBV-specific CTLs persisted long-term, reconstituted immunity against
EBV, and produced antiviral and antilymphoma effects. We have also used
EBV-specific CTLs to treat 13 patients with EBV-positive Hodgkin
disease and, while the results have been promising, no patient with
bulky disease has been cured.1,4 One explanation for this
failure is that current methods of EBV-specific CTL generation produce
CTL lines that are dominated by clones reactive to EBV proteins not
expressed in the malignant Reed-Sternberg cells (H-RS cells) of
EBV-positive Hodgkin disease.5 Only a limited number of
EBV-derived antigens (EBNA1, BARF0, LMP1, and LMP2) are present in H-RS
cells,6,7 and the immunodominant response of CTL lines is
against EBNAs 3A, B, and C, which are not expressed by the tumor cells.
Of the EBV proteins expressed in H-RS cells, only LMP2 and LMP1 are
potential targets for CD8+ T cells, because EBNA 1 is
mainly presented on major histocompatibility complex (MHC)
class II molecules8 and the level of BARF0 expression might be too low for CTL recognition.9 Our group and
others have recently reported the generation of LMP2-specific
CTLs10-12; however, for future clinical protocols it is
desirable to generate CTLs against more than one tumor-associated
antigen expressed in H-RS cells to reduce the risk of T-cell escape
mutants13-15 and to ensure that good CTL epitopes are
available regardless of the patient's HLA type. LMP1-specific CTLs are
rarely detected in healthy EBV-seropositive individuals,5
and reactivation of LMP1-specific CTL lines has been difficult, in part
because LMP1 is toxic when expressed at high levels.16 It
has been possible to generate LMP1-specific CTLs by cloning using
LMP1-derived peptide epitopes17 or antigen-presenting
cells infected with a recombinant vaccinia virus overexpressing
LMP1.18 The generated T-cell clones were able to lyse
targets expressing LMP1 and autologous lymphoblastoid cell lines
(LCLs), providing the rational for the development of an adoptive
immunotherapy for EBV-associated malignancies like Hodgkin disease or
nasopharyngeal carcinoma. However, the adoptive immunotherapy with
T-cell clones is not ideal, because the generation of T-cell clones is
labor-intensive, limiting its application for adoptive immunotherapy
protocols. Moreover, T-cell clones (1) do not persist in patients
without the presence of specific CD4+ T helper
cells19 and (2) carry the inherent risk that they may
be evaded by epitope escape mutants.13-15
Thus, we have taken a different approach and generated polyclonal
LMP1-specific CTL lines by stimulating peripheral blood mononuclear
cells (PBMCs) with dendritic cells (DCs) expressing a functionally
inactive, nontoxic LMP1 mutant. We used a 43-amino acid N-terminal
deletion mutant, which renders the molecule inactive and
nontoxic.20 About 90% of the molecule remains intact for antigen processing, and the 2 previously identified HLA-A2-restricted epitopes remain intact.17 Here we show that high levels of
LMP1 expression can be achieved in human dendritic cells (DCs) with little toxicity using a recombinant Blood donors and cell lines
HLA type of cell lines
Construction of recombinant adenoviruses The plasmid pSG5-LMP1 was provided by Bill Sugden, University of Wisconsin, Madison. LMP1 was subcloned into pEGFP-C1 (BD Biosciences, Palo Alto, CA) using BamHI and BglII sites. The SmaI/XbaI LMP1 fragment of pEGFP-C1-LMP1 was inserted into the XbaI and NotI (Klenow filled in) sites of pShuttleX (BD Biosciences). LMP1 was generated by
digesting pShuttleX-LMP1 with NheI and BsrGI, Klenow fill in, and subsequent self-ligation. From
pShuttleX-LMP1/-LMP1 the expression cassette containing the CMV
promoter, LMP1 or LMP1 and the BGH polyA, was cloned into the
E1/E3-deleted adenoviral backbone vector pAd5F35 using
pI-SceI and I-CeuI sites.22
The resultant plasmids were sequenced to confirm the sequence of LMP1 and LMP1 (SEQwright, Houston, TX). Recombinant adenoviruses
were generated as described in the literature.23 Plaques
positive for LMP1 and LMP1 expression by Western blot were expanded,
purified, and titered by standard procedures.22,23
Vaccinia viruses The vaccinia recombinants24 expressing EBV latency antigens EBNA-3A, -3B, and -3C were a gift from Elliot Kieff, Boston, MA, and the vaccinia-EGFP virus was constructed according to published procedures24 using the enhanced green fluorescent protein (EGFP) fragment of pEGFP-C1 and the shuttle plasmid pSC11 (gift from Bernard Moss, Bethesda, MD).Peptides for ELISPOT and cytotoxicity assays The 2 identified LMP1 HLA-A2-restricted peptide epitopes17 YLQQNWWTL (YLQ) and YLLEMLWRL (YLL) were prepared by Martin Campbell, Synthetic Antigen Laboratory, The University of Texas MD Anderson Cancer Center, Houston, TX. The YLQ peptide is conserved between EBV strains.25 The YLL epitope varies in between EBV strains, and the YLL epitope is not present in the LMP1 sequence used in this study (YLLEILWRL; group B virus).25 Therefore, the peptide served as a negative control, and no activity of LMP1-specific CTLs against YLL peptide was observed.DC generation DCs were generated by the "adherence method."26,27 Briefly, peripheral blood mononuclear cells (PBMCs) were purified by Ficoll (Lymphoprep, Nycomed, Oslo, Norway) gradient separation. A total of 4 × 107 to 5 × 107 mononuclear cells were plated in Cell Genix media (Technologie Transfer, Freiburg, Germany) containing 2 mM GlutaMAX-I in T-75 flasks for 2 hours. The nonadherent cells were removed by washing with phosphate-buffered saline (PBS), and the adherent cells were cultured in Cell Genix/GlutaMAX-I media with 800 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Sargramostim Leukine, Immunex, Seattle, WA) and 1000 U/mL interleukin-4 (IL-4) (R&D Systems, Minneapolis, MN) for 5 days. IL-4 and GM-CSF were replenished on day 2 and 4. On day 5, cells were harvested and transduced with recombinant adenovirus. DCs were cultured for 2 more days in Cell Genix/ GlutaMAX-I media containing 800 U/mL GM-CSF, 1000 U/mL IL-4 and, for maturation, 10 ng/mL IL-1 , 100 ng/mL IL-6, 10 ng/mL tumor necrosis factor-
(TNF-) (all R&D Systems), and 1 µg/mL (prostaglandin
E2) (PGE2) (Sigma, St Louis,
MO).12,28
CTL generation A total of 2 × 106 PBMCs per well of a 6-well plate were cocultured with 1 × 105 per well autologous, irradiated, Ad_LMP1-transduced DCs (DC_LMP1) in 2 mL complete
medium (45% RPMI 1640, 45% Click [Eagle Ham amino acids;
Irvine Scientific, Santa Ana, CA], 2 mM GlutaMAX-1, 10% FCS).
Cultures were restimulated on day 10 and, after that, weekly with
DC_LMP1 at a responder-stimulator ratio of 10:1. IL-2 (Proleukin, Chiron, Emeryville, CA), 40 U/mL, was first added with the third stimulation and then twice weekly.
Flow cytometry The expression of LMP1 andLMP1 was detected by
fluorescence-activated cell sorter (FACS) analysis. Briefly,
transduced DCs were fixed for 10 minutes in 4% paraformaldehyde (PFA)
and then permeablized with 1% saponin. The monoclonal LMP1 antibody
cocktail,29 CS1-4 (Research Diagnostics, Flanders, NJ),
recognizing the common C-terminus of LMP1 and LMP1, was used as
primary antibody and goat antimouse-fluorescein
isothiocyanate (FITC) (BD Biosciences) as secondary antibody.
Samples were acquired on a FACScan flow cytometer (BD Biosciences), and
the data were analyzed using CellQuest software (BD Biosciences).
Nontransduced DCs served as negative control. Expression of the surface
molecules was measured on nonfixed, nonpermeabilized DCs using
phycoerythrin (PE)-conjugated monoclonal antibodies: anti-CD3, -CD14,
-CD16, -CD19, -CD56, -CD80, -CD83, -CD86, and anti-DR peridinin
chlorophyll protein (PerCP). CTL lines were analyzed with
anti-CD8 FITC, anti-CD16 FITC, anti-T-cell receptor
(anti-TCR)  / FITC, anti-CD4 PE, anti-CD56 PE, anti-CD16 PE,
anti-TCR / PE, and anti-CD3 PerCP. All monoclonal antibodies were
obtained from BD Biosciences except anti-CD16 PE, anti-CD56 PE, and
anti-CD83 PE (Immunotech, Marseille, France).
ELISPOT assays The enzyme-linked immunospot (ELISPOT) assay was performed with modifications as described in the literature.30 A multiscreen 96-well plate (Millipore, Bedford, MA) was precoated with "capture" antibody against interferon- (IFN-) (Mabtech, Nacka, Sweden) overnight at
4°C. The wells were washed 3 times with phosphate-buffered saline
(PBS) and then blocked with RPMI 1640/2 mM GlutaMAX-I containing 10%
FCS for at least 1 hour. PBMCs and EBV- and LMP1-specific CTLs were
washed once and resuspended in RPMI 1640/2 mM GlutaMAX-I containing 5%
human serum (HS) (C-6 Diagnostic, Germantown, WI). Cells were
set up in triplicates in a separate 96-well plate to avoid
membrane damage of the multiscreen plate and serial diluted; for PBMCs
the undiluted well contained 1 × 105 cells and for CTLs
1 × 104 cells. The blocking medium was removed from the
multiscreen plate, the wells were washed twice with PBS, and 100 µL
of the serial-diluted cells was transferred into each well; 100 µL of
autologous, irradiated LCLs (1 × 105 cells) or peptide
(10 5 M) were resuspended in RPMI 1640/2mM GlutaMAX-I
containing 5% HS and 200 U/mL IL-2. In each assay, negative controls
included PBMCs, CTLs, LCLs, or peptide alone and, as positive controls, PBMCs or CTLs stimulated with 2.5 µg/mL phorbol myristate
acetate (PMA) (Sigma) and 1 µg/mL ionomycin (Sigma). After
16 to 20 hours, the plates were washed 6 times with PBS/0.05% Tween 20 and incubated for 2 hours with biotinylated "detection" antibody
against IFN- (Mabtech). After an additional 6 washes with
PBS/0.05% Tween 20, 100 µL of avidin-peroxidase complex (AEC;
prepared according to manufacturer instructions; Vector Laboratories,
Burlingame, CA) was added per well for 1 hour at room temperature. The
plates were washed 3 times with PBS/0.05% Tween 20, followed by 3 washes with plain PBS. AEC substrate (Sigma) was prepared by dissolving one AEC tablet in 2.5 mL dimethylformamide and adding 47.5 mL sodium
acetate buffer and 25 µL of 30% hydrogen peroxide. Prior to use the
AEC substrate was filtered through a 0.45-µM filter, and 100 µL was
added per well. After 4 minutes the reaction was stopped by washing
with deionized water, and the plates were dried overnight prior to
membrane removal. The spot number was determined in an independent
blinded fashion (ZellNet Consulting, New York, NY) using a
high-resolution automated ELISPOT Reader System (Carl Zeiss, Thornwood,
NY) using KS ELISPOT 4.3 software. In all assays the background was
fewer than 5 spot-forming cells (SFCs) per 100 000 cells. Linear
regression analysis and statistical analysis (Wilcoxon signed rank
test) was performed using GB-STAT (Dynamic Microsystems, Silver
Spring, MD).
Cytotoxicity assays The CTLs were tested for specific cytotoxicity against autologous fibroblasts either uninfected or infected with recombinant adenovirus or vaccinia virus constructs. Fibroblasts were exposed to 100 U/mL IFN- (R&D Systems) 24 to 48 hours prior to the cytotoxicity assay and infected on the day of the cytotoxicity assay with vaccinia virus recombinants containing EBNA-3A, -3B, -3C, and EGFP or 24 hours
before with adenovirus recombinants containing an empty expression
cassette, LMP1 LMP1. Recombinant vaccinia virus expressing LMP1
could not be used due to the additive toxicity of LMP1 and vaccinia
virus infection. Autologous LCLs, HLA class I-mismatched LCLs, and
autologous phytohemagglutinin (PHA) blasts were also tested.
Where indicated, PHA blasts were loaded with peptide for 1 hour after
51chromium (51Cr) labeling. A total of
1 × 106 target cells were labeled with 0.1 mCi (3.7 MBq) 51Cr and mixed with various numbers of
effector cells to give effector-target ratios of 40:1, 20:1, 10:1, and
5:1. Target cells incubated in complete medium alone or in 1% Triton
X-100 were used to determine spontaneous and maximum 51Cr
release, respectively. After 4 hours (LCLs, PHA blasts) or 5 hours
(fibroblasts), supernatants were collected and radioactivity was
measured on a gamma counter. The mean percentage of specific lysis of
triplicate wells was calculated as 100 × (experimental release  spontaneous release)/(maximum release spontaneous release).
Western blot Lysates of LCLs and fibroblasts were prepared by washing cells once with ice-cold PBS and boiling samples for 5 minutes in Laemmli buffer (Pierce Biotechnology, Rockford, IL). Fibroblasts were transduced with Ad_LMP1 or Ad_LMP1 24 hours prior to harvest as for
cytotoxicity assays. Nontransduced fibroblasts served as negative
control. Samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to polyvinylidene fluoride (PVDF) membranes (Pall Life Science, Ann
Arbor, MI). Membranes were blocked in Tris
(tris(hydroxymethyl)aminomethane)-buffered saline containing
0.1% Tween 20 (TBS-T) and 5% milk powder (MP). Membranes were washed
twice with TBS-T and incubated for 1 hour with anti-LMP1 antibody CS1-4
in TBS-T containing 2% MP. Membranes were washed 4 times with TBS-T
and incubated with a horseradish peroxidase (HRP)-conjugated secondary
antibody (Amersham Biosciences, Piscataway, NJ) for 11/2 hours
in TBS-T containing 1% MP. The membrane was washed 4 times with TBS-T,
and bound HRP was detected by enhanced chemiluminescence (ECL Plus;
Amersham Biosciences).
LMP1 expression is toxic in dendritic cells Different adenoviral vectors have been tested for their efficiency in transducing human DCs.31 The most efficient vector identified has a serotype 5 capsid containing the short-shafted fiber protein of serotype 35 (Ad5F35). Recombinant Ad5F35 vectors containing LMP1 andLMP1 were generated according to Yotnda et al,22 and the resultant constructs are shown in Figure
1. To evaluate the effect of LMP1 and
LMP1 expression, DCs were transduced with increasing multiplicities
of infection (MOIs) of Ad_LMP1 and Ad_LMP1. Forty-eight
hours after transduction, the number of viable and LMP1-positive cells
was determined by FACS analysis. A total of 32% of DCs were positive
for LMP1 expression after transduction with Ad_LMP1 at an MOI of 3. Higher MOIs led to no significant increase in transduction efficiency
but resulted in more than 60% cell death (Figure
2). In contrast, more than 80% of DCs
were positive for LMP1 at all MOIs tested. There was an adenoviral dose-dependent decrease in DC viability, but 90% of DC were
viable at an MOI of 3, indicating that high levels of LMP1
expression could be achieved with minimal toxicity (Figure 2). The
marked cytopathic effects of LMP1 expression in DCs prevented the
further use of Ad_LMP1-transduced DCs for CTL generation.
LMP1 was functionally inactive in DCs, we
measured their production of TNF- and IL-10, 2 cytokines that are
known to be induced by LMP1.32 Immature DCs were
transduced with Ad_EGFP, Ad_LMP1, and Ad_LMP1, and 48 hours after
transduction the concentration of the 2 cytokines was determined
by FACS using the Cytokine Cytometric Bead Array kit. DC-expressing
LMP1 produced high amounts of TNF- and IL-10, while LMP1- and
EGFP-expressing DCs did not, indicating that LMP1 is functionally
inactive (Figure 3A).
The CTLs generated with LMP1 (DC_LMP1). The phenotype of DC_LMP1-induced
CTLs was similar for all 3 donors, with more than 95% being
CD3+. More than 80% of the CD3+ T cells were
positive for CD8, 11% to 20% for CD4, and more than 95% were
TCR-+. Less than 5% of cells were positive for
natural killer cell markers (CD3 and
CD56+).
To determine if stimulation of PBMCs with DC_
The frequency of YLQ-specific CTLs was less than 0.01% (undetectable)
in EBV-specific CTLs generated using LCLs as antigen-presenting cell
lines (APCs), indicating that only DC_ LMP1-specific CTLs recognize LCLs Having established that the DC_LMP1-activated CTLs recognize
the LMP1-derived YLQ peptide epitope, it was important to determine if
the generated CTLs recognize autologous LCLs, in which LMP1 is
naturally expressed by EBV. Depending on the donor, 2% to 8.5% of
CTLs recognized LCLs as judged by IFN- secretion in ELISPOT assays
(Figure 5), in contrast to less than
0.7% in PBMCs prior to stimulation. The frequency of CTLs recognizing
LCLs in EBV-specific CTL lines for all 3 donors was 38% to 49% (data
not shown).
Cytotoxic activity of LMP1-specific CTLs To determine if the generated LMP1-specific CTLs not only secreted IFN- after YLQ peptide or LCL stimulation but also had cytotoxic
activity, CTLs were tested against a panel of targets in cytotoxicity
assays. LMP1-specific CTLs lysed autologous PHA blasts loaded with YLQ
peptide, whereas PHA blasts alone or with an unrelated
HLA-A2-restricted peptide were resistant to killing (Figure
6A). In addition, LMP1-specific CTLs
lysed autologous fibroblasts expressing LMP1 and LMP1 from the
adenoviral vector (Figure 6B). No lysis of targets transduced with an
adenovirus without a transgene was observed, indicating that the
stimulation protocol did not lead to the expansion of
adenovirus-specific CTLs. LMP1-specific CTLs of all 3 donors were
tested for their ability to lyse autologous LCLs in which LMP1 is
solely expressed by EBV. Only CTL from donor A, who had the highest
frequency of reactive CTLs against YLQ peptide and LCLs, lysed
autologous LCLs (Figure 6C). Decreased susceptibility to CTL lysis
might be explained either by sequence variation of LMP1, differences in
the level of LMP1 expression in fibroblasts and LCLs, or by the low
frequency and/or affinity of LMP1-specific CTLs.30,33,34
The LMP1 sequence of B95-8 EBV used to generate LCLs is almost
identical (99.1% homology) to the LMP1 used for CTL generation,
excluding epitope variation that would result in LCL resistance to CTL
lysis. The level of LMP1 and LMP1 expression in fibroblasts after
adenoviral transduction was much higher than in LCLs (Figure
7), indicating that the lysis of target
cells depends on the level of antigen expression. Thus, the frequency
and/or affinity of LMP1-specific CTLs in donor B and C only allows for
killing of targets that express LMP1 at higher levels than
LCLs. In an attempt to increase the frequency and/or affinity
of LMP1-specific CTLs for donor B, we combined initial DC_LMP1
stimulations with subsequent autologous LCL stimulations. The resultant
LMP1-specific CTL line had a 2-fold higher frequency of YLQ-specific
CTLs in comparison to stimulations with DC_LMP1 alone (Figure
8). In addition, killing of autologous fibroblasts expressing LMP1 as well as autologous LCLs was observed (Figure 9A). To exclude that
stimulation with LCLs after initial DC_LMP1 stimulations led to an
expansion of CTLs recognizing immunodominant EBV proteins, cytoxicity
assays were performed using autologous fibroblasts expressing EBNA-3A,
-3B, and -3C. No lysis of targets was observed, in contrast to
LCL-activated EBV-specific CTL, which killed EBNA-3B- and
-3C-expressing targets (Figure 9). Moreover, EBV-specific CTLs did not
lyse LMP1-expressing targets, confirming the ELISPOT assay results that
only DC_LMP1 and not LCLs direct the expansion of LMP1-specific CTLs
(Figure 9B).
We demonstrate that human DCs, expressing functionally inactive
LMP1, efficiently activate and expand LMP1-specific CTLs from PBMCs of
healthy, HLA-A2, EBV-seropositive donors. Polyclonal LMP1-specific CTL
lines recognized an HLA-A2-restricted LMP1 peptide, YLQ, and lysed
target cells expressing wild-type LMP1 in cytotoxicity assays. In
contrast, expression of wild-type LMP1 was toxic in DCs, preventing CTL
generation, and no LMP1-specific CTLs were detected in LCL-activated
EBV-specific CTLs, indicating that only DC_ Different methods have been developed to deliver antigens to DCs for
CTL activation and expansion, such as (1) loading with peptides,
proteins, or tumor cell lysate, (2) fusion of DCs with tumor
cells, and (3) gene transfer with DNA, RNA, or recombinant viral
vectors.35,36 Genetic modification of DCs offers several advantages including persistent expression of full-length antigen, allowing for the presentation of multiple/undefined epitopes and the
ability to genetically modify antigens to make them more potent or To separate the functional properties of LMP1 from its potential
epitopes for antigen presentation, we constructed recombinant adenoviral vectors expressing LMP1 or The DC_ If LMP1-specific CTLs are to be of therapeutic benefit, they must
recognize LMP1 expressed at physiologic levels from its natural
promoter. Therefore, we determined the frequency of CTLs recognizing
LCLs in LMP1-specific CTL lines. The frequency of LCL-reactive CTLs in
IFN- LMP1-specific CTL lines killed PHA blasts loaded with YLQ peptide and
autologous fibroblasts expressing wild-type LMP1 or No adenovirus-specific killing was observed in the present study. This finding is in accordance with results of other investigators using similar DC-based CTL stimulation protocols.10,12,39,40 The absence of adenovirus-specific CTLs may be protocol-specific because in most stimulation protocols adenoviral-transduced DCs are cultured for 2 days to allow for maturation and transgene expression. This most likely results in loss of adenoviral-derived epitopes, because adenoviral-specific CTLs could be reactivated when DCs were cultured with PBMCs immediately after adenoviral transduction.53 In summary, we described the generation of LMP1-specific CTLs for future adoptive immunotherapy protocols for patients with LMP1-positive malignancies, like EBV-positive Hodgkin disease or nasopharyngeal carcinoma. The generation of LMP1-specific CTLs was possible only using an inactive, nontoxic LMP1 mutant. The strategy of rendering a protein inactive to allow antigen presentation may be applicable to other tumor-associated antigens that are cytotoxic, immunosuppressive, or oncogenic in their native form.
We thank Malcolm K. Brenner for helpful discussion and advice.
Submitted May 23, 2002; accepted October 7, 2002.
Prepublished online as Blood First Edition Paper, October 31, 2002; DOI 10.1182/blood-2002-05-1514.
Supported by National Institutes of Health grants RO1 CA61384 and RO1 CA74126; peptide synthesis was supported by Core grant CA-16672. S.G. is the recipient of a Doris Duke clinical scientist development award. H.E.H. is the recipient of a Doris Duke distinguished clinical scientist award.
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.
Reprints: Stephen Gottschalk, Center for Cell and Gene Therapy, Baylor College of Medicine, 6621 Fannin St MC 3-3320, Houston, TX 77030; e-mail: smg{at}bcm.tmc.edu.
1. Gottschalk S, Heslop HE, Rooney CM. Treatment of Epstein-Barr virus-associated malignancies with specific T cells. Adv Cancer Res. 2002;84:175-201[Medline] [Order article via Infotrieve]. 2. Riddell SR, Greenberg PD. T-cell therapy of cytomegalovirus and human immunodeficiency virus infection. J Antimicrob Chemother. 2000;45(suppl T3):35-43[Abstract].
3.
Rooney CM, Smith CA, Ng CYC, 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 4. Heslop H, Rooney C, Brenner M, et al. Administration of neomycin resistance gene-marked EBV-specific cytotoxic T-lymphocytes as therapy for patients receiving a bone marrow transplant for relapsed EBV-positive Hodgkin disease. Hum Gene Ther. 2000;11:1465-1475[CrossRef][Medline] [Order article via Infotrieve]. 5. Rickinson AB, Moss DJ. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol. 1997;15:405-431[CrossRef][Medline] [Order article via Infotrieve].
6.
Herbst H, Dallenback F, Hummel M, et al.
Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells.
Proc Natl Acad Sci U S A.
1991;88:4766-4770
7.
Oudejans JJ, van den Brule AJ, Jiwa NM, et al.
BHRF1, the Epstein-Barr virus (EBV) homologue of the BCL-2 protooncogene, is transcribed in EBV-associated B-cell lymphomas and in reactive lymphocytes.
Blood.
1995;86:1893-1902
8.
Leen A, Meij P, Redchenko I, et al.
Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses.
J Virol.
2001;75:8649-8659 9. Kienzle N, Sculley TB, Poulsen L, et al. Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression. J Virol. 1998;8:6614-6620. 10. Gahn B, Siller-Lopez F, Pirooz AD, et al. Adenoviral gene transfer into dendritic cells efficiently amplifies the immune response to the LMP2A-antigen: a potential treatment strategy for Epstein-Barr virus-positive Hodgkin's lymphoma. Int J Cancer. 2001;93:706-713[CrossRef][Medline] [Order article via Infotrieve]. 11. Su Z, Peluso MV, Raffegerst SH, Schendel DJ, Roskrow MA. The generation of LMP2a-specific cytotoxic T lymphocytes for the treatment of patients with Epstein-Barr virus-positive Hodgkin disease. Eur J Immunol. 2001;31:947-958[CrossRef][Medline] [Order article via Infotrieve].
12.
Ranieri E, Herr W, Gambotto A, et al.
Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus latent membrane protein 2B: a new modality for vaccination.
J Virol.
1999;73:10416-10425
13.
Gottschalk S, Ng CYC, Smith CA, et al.
An Epstein-Barr virus deletion mutant that causes fatal lymphoproliferative disease unresponsive to virus-specific T cell therapy.
Blood.
2001;97:835-843
14.
Furukawa Y, Kubota R, Tara M, Izumo S, Osame M.
Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia.
Blood.
2001;97:987-993 15. Goulder PJ, Brander C, Tang Y, et al. Evolution and transmission of stable CTL escape mutations in HIV infection. Nature. 2001;412:334-338[CrossRef][Medline] [Order article via Infotrieve].
16.
Hammerschmidt W, Sugden B, Baichwal VR.
The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels.
J Virol.
1989;63:2469-2475 17. Khanna R, Burrows SR, Nicholls J, Poulsen LM. Identification of cytotoxic T cell epitopes within Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2 supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes. Eur J Immunol. 1998;28:451-458[CrossRef][Medline] [Order article via Infotrieve].
18.
Sing AP, Ambinder RF, Hong DJ, et al.
Isolation of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes that lyse Reed-Sternberg cells: implications for immune-medicated therapy of EBV+ Hodgkin's disease.
Blood.
1997;89:1978-1986
19.
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
20.
Martin J, Sugden B.
Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.
J Virol.
1991;65:3246-3258 21. Rooney CM, Smith CA, Ng C, et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation. Lancet. 1995;345:9-13[CrossRef][Medline] [Order article via Infotrieve]. 22. Yotnda P, Onishi H, Heslop HE, et al. Efficient infection of primitive hematopoietic stem cells by modified adenovirus. Gene Ther. 2001;8:930-937[CrossRef][Medline] [Order article via Infotrieve]. 23. Davis AR, Meyers K, Wilson JM. High throughput method for creating and screening recombinant adenoviruses. Gene Ther. 1998;5:1148-1152[CrossRef][Medline] [Order article via Infotrieve].
24.
Murray RJ, Kurilla MG, Brooks JM, et al.
Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies [abstract].
J Exp Med.
1992;176:157-168
25.
Fielding CA, Sandvej K, Mehl A, Brennan P, Jones M, Rowe M.
Epstein-Barr virus LMP-1 natural sequence variants differ in their potential to activate cellular signaling pathways.
J Virol.
2001;75:9129-9141 26. Thurner B, Roder C, Dieckmann D, et al. Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J Immunol Methods. 1999;223:1-15[CrossRef][Medline] [Order article via Infotrieve]. 27. Romani N, Reider D, Heuer M, et al. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods. 1996;196:137-151[CrossRef][Medline] [Order article via Infotrieve].
28.
Reddy A, Sapp M, Feldman M, Subklewe M, Bhardwaj N.
A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells.
Blood.
1997;90:3640-3646
29.
Rowe M, Evans HS, Young LS, Hennessy K, Kieff E, Rickinson AB.
Monoclonal antibodies to the latent membrane protein of Epstein-Barr virus reveal heterogeneity of the protein and inducible expression in virus-transformed cells.
J Gen Virol.
1987;68:1575-1586
30.
Yang J, Lemas VM, Flinn IW, Krone C, Ambinder RF.
Application of the ELISPOT assay to the characterization of CD8(+) responses to Epstein-Barr virus antigens.
Blood.
2000;95:241-248
31.
Rea D, Havenga MJ, van Den AM, et al.
Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells.
J Immunol.
2001;166:5236-5244 32. Vockerodt M, Haier B, Buttgereit P, Tesch H, Kube D. The Epstein-Barr virus latent membrane protein 1 induces interleukin-10 in Burkitt's lymphoma cells but not in Hodgkin's cells involving the p38/SAPK2 pathway. Virology. 2001;280:183-198[CrossRef][Medline] [Order article via Infotrieve]. 33. Riker AI, Kammula US, Panelli MC, et al. Threshold levels of gene expression of the melanoma antigen gp100 correlate with tumor cell recognition by cytotoxic T lymphocytes. Int J Cancer. 2000;86:818-826[CrossRef][Medline] [Order article via Infotrieve]. 34. Lethe B, van der Bruggen P, Brasseur F, Boon T. MAGE-1 expression threshold for the lysis of melanoma cell lines by a specific cytotoxic T lymphocyte. Melanoma Res. 1997;7(suppl 2):S83-S88[Medline] [Order article via Infotrieve]. 35. Steinman RM, Dhodapkar M. Active immunization against cancer with dendritic cells: the near future. Int J Cancer. 2001;94:459-473[CrossRef][Medline] [Order article via Infotrieve]. 36. Jenne L, Schuler G, Steinkasserer A. Viral vectors for dendritic cell-based immunotherapy. Trends Immunol. 2001;22:102-107[CrossRef][Medline] [Order article via Infotrieve]. 37. Jenne L, Hauser C, Arrighi JF, Saurat JH, Hugin AW. Poxvirus as a vector to transduce human dendritic cells for immunotherapy: abortive infection but reduced APC function. Gene Ther. 2000;7:1575-1583[CrossRef][Medline] [Order article via Infotrieve].
38.
Kruse M, Rosorius O, Kratzer F, et al.
Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity.
J Virol.
2000;74:7127-7136 39. Zhai Y, Yang JC, Kawakami Y, et al. Antigen-specific tumor vaccines. Development and characterization of recombinant adenoviruses encoding MART1 or gp100 for cancer therapy. J Immunol. 1996;156:700-710[Abstract]. 40. Reed DS, Romero P, Rimoldi D, Cerottini JC, Schaack J, Jongeneel CV. Construction and characterization of a recombinant adenovirus directing expression of the MAGE-1 tumor-specific antigen. Int J Cancer. 1997;72:1045-1055[CrossRef][Medline] [Order article via Infotrieve].
41.
Wherry EJ, McElhaugh MJ, Eisenlohr LC.
Generation of CD8(+) T cell memory in response to low, high, and excessive levels of epitope.
J Immunol.
2002;168:4455-4461
42.
Dukers DF, Meij P, Vervoort MB, et al.
Direct immunosuppressive effects of EBV-encoded latent membrane protein 1.
J Immunol.
2000;165:663-670 43. Haraguchi S, Good RA, Cianciolo GJ, Engelman RW, Day NK. Immunosuppressive retroviral peptides: immunopathological implications for immunosuppressive influences of retroviral infections. J Leukoc Biol. 1997;61:654-666[Abstract].
44.
Fiebiger E, Meraner P, Weber E, et al.
Cytokines regulate proteolysis in major histocompatibility complex class II-dependent antigen presentation by dendritic cells.
J Exp Med.
2001;193:881-892
45.
Pai S, O'Sullivan BJ, Cooper L, Thomas R, Khanna R.
RelB nuclear translocation mediated by C-terminal activator regions of Epstein-Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells.
J Virol.
2002;76:1914-1921 46. Mosialos G. Cytokine signaling and Epstein-Barr virus-mediated cell transformation. Cytokine Growth Factor Rev. 2001;12:259-270[CrossRef][Medline] [Order article via Infotrieve]. 47. Koehne G, Leiner I, Williams RY, Ferguson TL, Pamer EG, O'Reilly RJ. Functional characterization of EBV peptide-specific T lymphocytes isolated with MHC tetramers. Biol Blood Marrow Transplant. 2002;8:88. 48. Tussey L, Speller S, Gallimore A, Vessey R. Functionally distinct CD8+ memory T cell subsets in persistent EBV infection are differentiated by migratory receptor expression. Eur J Immunol. 2000;30:1823-1829[CrossRef][Medline] [Order article via Infotrieve].
49.
Pittet MJ, Zippelius A, Speiser DE, et al.
Ex vivo IFN-
50.
Kim SH, Shin YK, Lee IS, et al.
Viral latent membrane protein 1 (LMP-1)-induced CD99 down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Sternberg phenotype.
Blood.
2000;95:294-300 51. Pallesen G, Hamilton-Dutoit SJ, Zhou X. The association of Epstein-Barr virus (EBV) with T cell lymphoproliferations and Hodgkin's disease: two new developments in the EBV field. Adv Cancer Res. 1993;62:179-239[Medline] [Order article via Infotrieve].
52.
Munz C, Bickham KL, Subklewe M, et al.
Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1.
J Exp Med.
2000;191:1649-1660
53.
Smith CA, Woodruff LS, Kitchingman GR, Rooney CM.
Adenovirus-pulsed dendritic cells stimulate human virus-specific T-cell responses in vitro.
J Virol.
1996;70:6733-6740
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 |
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  E. Yvon, M. Del Vecchio, B. Savoldo, V. Hoyos, A. Dutour, A. Anichini, G. Dotti, and M. K. Brenner Immunotherapy of Metastatic Melanoma Using Genetically Engineered GD2-Specific T cells Clin. Cancer Res., September 15, 2009; 15(18): 5852 - 5860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. I. Cohen, H. Kimura, S. Nakamura, Y.-H. Ko, and E. S. Jaffe Epstein-Barr virus-associated lymphoproliferative disease in non-immunocompromised hosts: a status report and summary of an international meeting, 8-9 September 2008 Ann. Onc., September 1, 2009; 20(9): 1472 - 1482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Diekmann, E. Adamopoulou, O. Beck, G. Rauser, S. Lurati, S. Tenzer, H. Einsele, H.-G. Rammensee, H. Schild, and M. S. Topp Processing of Two Latent Membrane Protein 1 MHC Class I Epitopes Requires Tripeptidyl Peptidase II Involvement J. Immunol., August 1, 2009; 183(3): 1587 - 1597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Smith, N. Wakisaka, T. Crough, J. Peet, T. Yoshizaki, L. Beagley, and R. Khanna Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation Blood, June 11, 2009; 113(24): 6148 - 6152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Bollard, S. Gottschalk, A. M. Leen, H. Weiss, K. C. Straathof, G. Carrum, M. Khalil, M.-f. Wu, M. H. Huls, C.-C. Chang, et al. Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and T-lymphocyte transfer Blood, October 15, 2007; 110(8): 2838 - 2845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ahmed, M. Ratnayake, B. Savoldo, L. Perlaky, G. Dotti, W. S. Wels, M. B. Bhattacharjee, R. J. Gilbertson, H. D. Shine, H. L. Weiss, et al. Regression of Experimental Medulloblastoma following Transfer of HER2-Specific T Cells Cancer Res., June 15, 2007; 67(12): 5957 - 5964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Xu, T. Coleman, J. Zhang, A. Fagot, C. Kotalik, L. Zhao, P. Trivedi, C. Jones, and L. Zhang Epstein-Barr Virus Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication in Primary Effusion Lymphomas J. Virol., June 1, 2007; 81(11): 6068 - 6078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dupuis, J.-F. Emile, N. Mounier, C. Gisselbrecht, N. Martin-Garcia, T. Petrella, R. Bouabdallah, F. Berger, A. Delmer, B. Coiffier, et al. Prognostic significance of Epstein-Barr virus in nodal peripheral T-cell lymphoma, unspecified: a Groupe d'Etude des Lymphomes de l'Adulte (GELA) study Blood, December 15, 2006; 108(13): 4163 - 4169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Smith, L. Cooper, M. Burgess, M. Rist, N. Webb, E. Lambley, J. Tellam, P. Marlton, J. F. Seymour, M. Gandhi, et al. Functional Reversion of Antigen-Specific CD8+ T Cells from Patients with Hodgkin Lymphoma following In Vitro Stimulation with Recombinant Polyepitope J. Immunol., October 1, 2006; 177(7): 4897 - 4906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ishida, T. Ishii, A. Inagaki, H. Yano, H. Komatsu, S. Iida, H. Inagaki, and R. Ueda Specific Recruitment of CC Chemokine Receptor 4-Positive Regulatory T Cells in Hodgkin Lymphoma Fosters Immune Privilege Cancer Res., June 1, 2006; 66(11): 5716 - 5722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Comoli, P. Pedrazzoli, R. Maccario, S. Basso, O. Carminati, M. Labirio, R. Schiavo, S. Secondino, C. Frasson, C. Perotti, et al. Cell Therapy of Stage IV Nasopharyngeal Carcinoma With Autologous Epstein-Barr Virus-Targeted Cytotoxic T Lymphocytes J. Clin. Oncol., December 10, 2005; 23(35): 8942 - 8949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. C. Straathof, A. M. Leen, E. L. Buza, G. Taylor, M. H. Huls, H. E. Heslop, C. M. Rooney, and C. M. Bollard Characterization of Latent Membrane Protein 2 Specificity in CTL Lines from Patients with EBV-Positive Nasopharyngeal Carcinoma and Lymphoma J. Immunol., September 15, 2005; 175(6): 4137 - 4147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. N. Flynn, M. Pistello, P. Isola, L. Zaccaro, B. Del Santo, E. Ricci, D. Matteucci, and M. Bendinelli Adoptive Immunotherapy of Feline Immunodeficiency Virus with Autologous Ex Vivo-Stimulated Lymphoid Cells Modulates Virus and T-Cell Subsets in Blood Clin. Vaccine Immunol., June 1, 2005; 12(6): 736 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. C. M. Straathof, C. M. Bollard, U. Popat, M. H. Huls, T. Lopez, M. C. Morriss, M. V. Gresik, A. P. Gee, H. V. Russell, M. K. Brenner, et al. Treatment of nasopharyngeal carcinoma with Epstein-Barr virus-specific T lymphocytes Blood, March 1, 2005; 105(5): 1898 - 1904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. L. Bartlett Therapies for Relapsed Hodgkin Lymphoma: Transplant and Non-Transplant Approaches Including Immunotherapy Hematology, January 1, 2005; 2005(1): 245 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Bollard, L. Aguilar, K. C. Straathof, B. Gahn, M. H. Huls, A. Rousseau, J. Sixbey, M. V. Gresik, G. Carrum, M. Hudson, et al. Cytotoxic T Lymphocyte Therapy for Epstein-Barr Virus+ Hodgkin's Disease J. Exp. Med., December 20, 2004; 200(12): 1623 - 1633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Marshall, L. E. Christie, L. R. Munro, D. J. Culligan, P. W. Johnston, R. N. Barker, and M. A. Vickers Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma Blood, March 1, 2004; 103(5): 1755 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Comoli, R. De Palma, S. Siena, A. Nocera, S. Basso, F. Del Galdo, R. Schiavo, O. Carminati, A. Tagliamacco, G. F. Abbate, et al. Adoptive transfer of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T cells with in vitro antitumor activity boosts LMP2-specific immune response in a patient with EBV-related nasopharyngeal carcinoma Ann. Onc., January 1, 2004; 15(1): 113 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tellam, G. Connolly, N. Webb, J. Duraiswamy, and R. Khanna Proteasomal targeting of a viral oncogene abrogates oncogenic phenotype and enhances immunogenicity Blood, December 15, 2003; 102(13): 4535 - 4540. [Abstract] [Full Text] [PDF]
|
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|
|
|
|
H. E. Heslop, F. K. Stevenson, and J. J. Molldrem Immunotherapy of Hematologic Malignancy Hematology, January 1, 2003; 2003(1): 331 - 349. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
as
for LMP1