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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2925-2934
By
From the Departments of Virology and Molecular Biology, Infectious
Diseases, and Hematology/Oncology, and Division of Bone Marrow
Transplantation, St Jude Children's Research Hospital, Memphis, TN;
and the Department of Pediatrics and Pathology, University of Tennessee
College of Medicine, Memphis, TN.
Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T
lymphocytes (CTLs) is effective prophylaxis and treatment of
EBV-positive immunoblastic lymphoma in immunocompromised patients. In
50% of patients with Hodgkin's disease, the tumor cells are EBV
antigen-positive and may therefore also be suitable targets for
treatment with virus-specific CTLs. However, Hodgkin's disease may
produce several inhibitory effects on immune induction and effector
function in vivo, which may preclude the generation or effector
function of CTLs reactive against EBV viral proteins, including those
expressed by the tumor cells. We have investigated whether EBV-specific
CTLs could be generated ex vivo from 13 patients with Hodgkin's
disease: nine with active relapsed disease and four who were in
clinical remission after a first or subsequent relapse. CTL lines were
successfully generated from nine of 13 patients (five active disease,
four remission). Although these lines had an abnormal pattern of
expansion comparable to EBV-specific CTLs generated from normal donors,
their phenotype was normal except for reduced expression of the zeta
chain of the T-cell receptor (TCR). Their cytotoxicity was also
compared to EBV-specific lines generated from normal donors and
included activity against LMP2a, one of the three weakly immunogenic
viral antigens expressed by Hodgkin's tumor cells. To assess the
activity of the CTLs in vivo, they were gene-marked and infused into
three patients with multiply relapsed disease. The CTLs persisted for
more than 13 weeks postinfusion and retained their potent antiviral
effects in vivo, thereby enhancing the patient immune response to EBV. This approach may therefore have value in the treatment of EBV-positive Hodgkin's disease.
ALTHOUGH THE ETIOLOGY of Hodgkin's
disease remains unclear, approximately 50% of cases in North America
and Europe are associated with Epstein-Barr virus (EBV).1
In South America, Kenya, and parts of Asia, there is a 90% to 100%
association.2,3 EBV is a ubiquitous gamma-herpes virus that
has been associated with several malignant diseases in addition to
Hodgkin's lymphoma. These include nasopharyngeal carcinoma, Burkitt's
lymphoma, and immunoblastic lymphoma as seen in the immunocompromised
host.4-6
Recent work from our group has shown that EBV-specific cytotoxic T
lymphocytes (CTLs) generated from normal donors can be adoptively
transferred to patients who have received T-cell-depleted allogeneic
stem-cell transplants. These CTLs persist long-term in vivo,
reconstitute the immune response to EBV, and are effective as
prophylaxis and treatment of immunoblastic lymphoma.7,8 Implementing such an approach for EBV-positive Hodgkin's disease would
have considerable appeal; although 80% or more of patients are cured
with conventional therapy, more than half of those who relapse fail to
respond to salvage chemotherapy or relapse a second time have a poor
long-term prognosis. Furthermore, the unacceptably high level of
therapy-related secondary malignancies (18% at 5 years) and other
serious medical complications in those who are treated successfully
also underscores a need to improve current therapeutic
options.9
A number of obstacles may diminish the effectiveness of EBV-specific
CTLs in Hodgkin's disease. Many patients with Hodgkin's disease may
have T-cell abnormalities, such as low expression of the zeta chain of
the T-cell receptor (TCR).10,11 In addition, Hodgkin's
cells may secrete interleukin-10 (IL-10), a cytokine inhibitory to the
induction of a cytotoxic T-cell response.12 Other
CTL-inhibitory mechanisms may also operate.13,14 Finally, the malignant cells of Hodgkin's disease express a restricted set of
viral genes, namely, EBNA1 in the nucleus and LMP1 and LMP2 in the
plasma membrane.15 EBNA1 cannot enter the human leukocyte
antigen (HLA) class I processing pathway, because of its gly/ala
repeat, which inhibits its binding the TAP-transported proteins.
Although a minority of normal donors have CTLs against LMP1 and LMP2,
these antigens are weakly immunogenic in the context of most HLA
types.16
To assess the feasibility of using EBV-specific CTLs as therapy for
Hodgkin's disease, we generated EBV-specific CTLs from the peripheral
blood of patients with Hodgkin's disease, hoping that they could be
expanded in vitro, in the absence of in vivo immunosuppressive effects.
We then compared them with CTLs generated from normal
donors.17 To discover whether autologous EBV-specific CTLs
could persist and have antiviral activity in vivo, we genetically marked them and adoptively transferred them to three patients with
relapsed disease.
Patients and EBV status of the tumors.
Patients were enrolled onto a study that was approved by the
hospital's institutional review board and by the Food and Drug Administration. All of these children and adolescents (Table
1) had histologically proven EBV-positive
Hodgkin's disease. Samples were collected from the patients while
their disease was in clinical remission after a first or subsequent
relapse, or while they had active relapsed disease provided they had
received no chemotherapy for a minimum of 4 weeks.
Normal donors.
EBV-specific CTL lines were generated from the peripheral blood of
normal bone marrow donors, for the prophylaxis and treatment of EBV
lymphoma in the marrow recipients.17 Data from these CTL
lines have been collected over the past 4 years.
EBV-transformed B-cell lines.
Samples of peripheral blood (20 to 40 mL) were collected from patients
with EBV-positive Hodgkin's disease or normal bone marrow donors and
used to generate both a B-lymphoblastoid cell line (LCL) and a CTL
line. To establish spontaneously transformed or B95-8-transformed LCL,
peripheral blood mononuclear cells (PBMC) were plated at
106 cells per well in flat-bottomed 96-well plates
containing RPMI 1640 medium (GIBCO Life Technologies, Grand Island, NY)
supplemented with 10% heat-inactivated fetal calf serum, 1%
L-glutamine, 1% penicillin/streptomycin, and 1 µg/mL
cyclosporine A (Sandoz Pharmaceuticals Inc, Washington,
DC). Concentrated supernatant (10 µL, see later) from
B95-8 cultures, a human type I EBV-transformed marmoset B-cell line,
was added to parallel samples.19,20 Once B95-8-infected LCL were established, they were expanded into 25-cm2
flasks. Aliquots of these long-term cultures were frozen.
Concentrated supernatant from B95-8 cells.
We used a B95-8 virus-producer cell line that was negative for
mycoplasma (Gen-Probe, San Diego, CA), for squirrel monkey retrovirus
(by Southern and Western blotting), and for viruses, other than EBV,
detectable by electron microscopy. Virus stocks were made from a master
cell bank that was grown for 7 to 9 days in supplemented RPMI 1640. Supernatants were harvested by centrifugation, filtered (0.45-µm
pores), and then concentrated 50-fold by ultrafiltration through a
pressurized concentrator (Cole Parmer, Niles, IL) containing a disk
with a molecular weight cutoff of 500,000 kD. Aliquots of virus were
frozen in liquid nitrogen, then tested for their ability to transform
peripheral B cells from EBV-negative donors. Virus stocks that
transformed B cells at dilutions of 10 EBV-specific CTL lines.
EBV-specific CTLs were activated from the peripheral blood of patients
and normal donors by coculturing 2 × 106 PBMC per well of
a 24-well (1.5-cm diameter) plate with 5 × 104 autologous
LCLs that had been irradiated with 40 Gy from a cesium source. This
radiation dose entirely prevented LCL outgrowth in control cultures.
After 10 days, the cells were harvested on Ficoll gradients (ICN,
Irvine, CA), subcultured in 24-well plates at 5 × 105 per
well, and restimulated with 1.25 × 105 irradiated LCLs
per well. After 4 days, the cultures were fed with 20 U/mL of IL-2
(Proleukin; Cetus, Emeryville, CA). Thereafter, the cultures were fed
three times weekly, twice with 20 U/mL of IL-2 and the third time with
10 to 15 U/mL of IL-2 supplemented with irradiated LCLs (T cell:LCL
ratio, 4:1). CTL lines were successfully established from nine of the
13 patients: seven on the first attempt and two on the second attempt.
CTL lines could not be established on the only attempt from four
patients, all with relapsed disease. Lines from patients no. 1, 2, and
13 were infused into patients for the treatment of their relapsed
disease. In contrast, CTL lines were successfully established from
greater than 95% of normal EBV-seropositive donors.17
CTL reactivations.
To assess the contribution of the infused CTLs to the patient's immune
response to EBV postinfusion, EBV-specific CTL lines were reactivated
from patients no. 1 and 13, 1 and 2 months postinfusion using the
autologous LCLs as stimulators as described earlier. After 4 weeks of
culture, the cytotoxic specificity of the line was analyzed and DNA was
extracted to compare the level of gene marking of the reactivated line
with that of the infused line (results not shown).
Gene marking of EBV-specific CTLs.
CTLs were genetically marked by transduction with the G1Na retroviral
vector, which contains the Escherichia coli-derived neomycin
resistance gene (neo). Clinical grade G1Na-containing supernatant from a PA317 amphotropic packaging cell line (Genetic Therapy, Gaithersburg, MD) was incubated for 6 hours with the CTLs at a
multiplicity of infection of 10:1, in the presence of IL-2 (50 U/mL)
and protamine sulfate (4 µg/mL) in a 75-cm2
flask.17
Cytotoxicity assays.
The cytotoxicity of each CTL line was analyzed in a standard 4-hour
chromium-51 release assay using effector:target ratios of 40:1, 20:1,
10:1, and 5:1. Target cells included autologous and HLA-class
I-mismatched LCLs, and the T-cell line HSB-2, which is sensitive to
killing by lymphokine-activated killer cells. To determine whether
cytolysis was restricted by HLA class I or class II, target cells were
preincubated for 30 minutes with 20 µg/mL of W6/32 (Dako), a
monoclonal antibody that recognizes a monomorphic HLA class I
determinant or CR3/43 (Dako), which recognizes HLA-DR, -DP, and -DQ and
blocks HLA class II-restricted killing. In the minority of patients in
whom no antibody inhibition was obtained, the cell line was depleted of
CD56+ and CD16+ cells to remove nonspecific
natural killer/antibody-dependent cytotoxic cell
effectors, and then retested (see later).
Immunophenotyping.
For cell-surface phenotyping, the CTLs were incubated with combinations
of fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated
monoclonal antibodies to CD3, CD4, CD8, CD5, CD25 (Dako), HLA-DR,
TCR- Frequency of EBV-specific precursors.
To compare the precursor frequency of EBV-specific CTLs before and
after CTL infusion, patient PBMC were seeded at limiting dilution from
5 × 105 to 5 × 102 cells per well (twofold
dilutions) in 96-well plates. Each well was stimulated with
105 autologous LCLs, and supplemented with 104
irradiated autologous PBMC feeder cells if the wells contained 104 or fewer PBMC. After 6 weeks in culture, the responding
cells were split into two parts and assayed for cytotoxic activity
against autologous and HLA-mismatched LCLs; both target populations
were labeled with 51Cr. This extended duration of culture
was required to grow testable numbers of CTLs from patient
samples.8 Positive wells were scored on the basis of Detection of EBV DNA in patient PBMC.
The level of EBV DNA in patient PBMC was assessed every 2 to 4 weeks
after CTL infusion using a nested PCR to amplify a portion of the EBNA2
gene. The first pair of primers (5 CTLs from patients with Hodgkin's disease expand slowly compared with
those from healthy donors.
We compared the LCL-induced proliferation of T lymphocytes from
Hodgkin's patients (in remission or relapse) with normal donors. During the first 10 to 14 days in culture, the total cell number did
not increase significantly in lines from healthy donors or patients,
presumably due to death of unresponsive T cells. During the subsequent
expansion phase in the presence of IL-2, cell counts of cultures from
healthy donors (n = 15) typically increased by 10-fold in the first 2 weeks and then slowed down. These kinetics are illustrated for two
normal donors in Fig 1. After 16 weeks in
culture, CTLs from normal donors had expanded by a median of 1,500-fold
(range, 130- to 5,000-fold). During the same 16-week period, CTL
cultures from patients in remission expanded by a median of only
160-fold (range, 100- to 300-fold), while those from patients with
relapsed disease increased by a median of 80-fold (range, 20- to
100-fold). Despite this slower rate of expansion, addition of a
mitogenic cocktail (see Methods) allowed us to generate at least
108 CTLs from four of five patients in remission and from
five of nine patients with relapsed Hodgkin's disease. This number of cells exceeded that required for our in vivo study.7,8
Phenotype of CTL lines from patients with Hodgkin's disease.
To determine whether the phenotype of CTL lines from Hodgkin's
patients might provide an insight into the mechanism of their low
proliferative rate, we analyzed the CTL lines from patients in the two
study groups and compared them with lines previously derived from
normal individuals.23 CTL lines from normal individuals and
patients with Hodgkin's disease exhibited considerable heterogeneity with respect to the percentages of CD56+, CD8+,
and CD4+ cells and in their CD4/CD8 ratio, but were
nonetheless broadly comparable. In lines from healthy individuals,
CD8+ cells ranged from 3% to 99% (median, 77%),
CD4+ cells ranged from 2% to 98% (median, 19%), and
DR+ cells ranged from 82% to 100% (median,
98%).23 In lines from Hodgkin's patients (Table
2), CD8+ cells ranged from 2%
to 92% (median, 67%), CD4+ cells ranged from 2% to 86%
(median, 10%), and DR+ cells ranged from 73% to 100%
(median, 97.6%). The proportion of CD56+ cells was similar
in all groups and no phenotypic differences were observed in lines from
remission versus relapsed patients. The high proportion of
The level of TCR zeta chain is abnormally low in Hodgkin's patients.
Because the cytoplasmic portion of the TCR zeta chain is involved in
signal transduction and subsequent activation and proliferation of T
cells, downregulation of this chain may result in the decreased ability
of patient T cells to make a proliferative response to autologous LCLs.
By comparison with unstimulated T cells from normal donors, those from
patients with relapsed or remission status Hodgkin's disease expressed
less TCR zeta chain (Fig 2). Normal donors had a
zeta:epsilon ratio of 0.90. In contrast, patients in remission had a
ratio of 0.75, and relapse patients had a greatly reduced ratio, 0.38 (P < .03 for comparisons with normals), which corresponded to their poor proliferative response. Evaluation of TCR
zeta-chain expression in T cells cultured for 10 days in the presence
of IL-2 showed expression levels of CTLs from remission patients had
increased versus normal levels. While zeta-chain expression also
increased in CTLs from patients with relapsed disease, the ratio
remained subnormal (Fig 2).
CTLs from Hodgkin's patients are HLA-restricted.
To verify the cytotoxic specificity of the CTL lines generated, we
tested them against autologous and HLA class I-mismatched LCLs and
HSB-2 (the LAK-sensitive EBV-negative T-cell lymphoma). Table 2 shows
the cytotoxicity of CTLs generated from patients with Hodgkin's
disease. The results are not different from CTLs from normal
donors.23 Thus, CTLs from Hodgkin's patients contained HLA-restricted components, as killing of autologous LCLs was higher than that of HLA-mismatched LCLs. In six of 10 experiments, killing of
autologous LCLs was inhibited by an anti-HLA class I monoclonal antibody, thereby confirming the presence of HLA class I-restricted EBV-specific CTL. Significant killing through class II also occurred, since inhibition was seen with antibody to class II antigens (Table 2).
Infused CTLs persist in vivo for more than 13 weeks.
To track the persistence of infused CTLs, they were genetically marked
with a retrovirus containing the neomycin resistance gene
(neo). The efficiency of gene marking of CTLs generated from Hodgkin's patients was 0.5% to 10%, identical to that obtained in
normal donor CTLs and sufficient for detection in vivo in stem-cell transplant recipients.7,8 In patient no. 1, the neo
gene was detectable in PBMC for 12 weeks after the initial dose of 2 × 107/m2 EBV-specific CTLs (Fig
3A). As the marking efficiency of the CTL
line was 2% and 0.002% of the PBMC were positive for neo, approximately one of every 1,000 circulating PBMC was derived from the
infused cell line. Patient no. 2 had a similar level of neo
signal in PBMC, persisting in this case for 10 weeks after the initial
dose, while in patient no. 13, marked cells were detected in peripheral
blood for more than 13 weeks (the duration of the study). Moreover, in
patient no. 13, who had extensive pleural involvement with Reed
Sternberg cells, marker signal was detected in the pleural fluid at a
10-fold higher level than in peripheral blood, indicating that the
infused CTLs can traffic to sites of active disease (Fig 3B).
EBV-specific CTLs activity increases after infusion of in vitro
cultured CTLs.
In patient no. 1, sufficient PBMC were available at multiple time
points after infusion to determine whether CTL infusion increased the
proportion of circulating EBV-specific cytotoxic precursor cells
(CTLp), showing an enhanced cell-mediated immune response to EBV.
Figure 4 shows that following two doses of
2 × 107/m2 EBV-specific CTLs 2 weeks apart,
the CTLp frequency increased from one in 10,000 PBMC to one in 1,000 within 3 weeks of the second CTL infusion, thereby reaching the range
found in healthy donors.24 The number of MHC-unrestricted
cytotoxic effector precursors remained constant, as shown by the
minimal change in the proportion of CTLp that killed HLA-mismatched LCL
(Fig 4).
Infused EBV-specific CTLs have antiviral activity in vivo.
To demonstrate the in vivo antiviral activity of these circulating
cytotoxic cells, we used semiquantitative nested PCR analysis to
calculate the viral burden in peripheral blood before and after infusion. In more than 50 healthy individuals, the number of EBV DNA
genomes ranged from fewer than 20 to 2,000 copies per 106
PBMC.8,25 Before CTL infusion, the level of EBV DNA in
patient no. 1 was consistent with a genome number of 30,000 per
106 PBMC (Fig 3A). This 15-fold higher than normal level
was within the range seen for stem-cell transplant recipients with
immunoblastic lymphoma.7,8,25 As shown in Fig 3A, the level
of EBV DNA decreased dramatically after CTL infusion and was
undetectable after 4 weeks. Associated with this drop in EBV DNA, the
patient showed improvement of stage B symptoms, with increased appetite and resolution of fever and sweats; there was also stabilization of his
pulmonary disease. These effects were not due to concomitant chemotherapy, as the patient had received no other treatment for 4 weeks before or 6 weeks postinfusion. In fact, the viral load in the
peripheral blood of this patient subsequently increased coincident with
readministration of chemotherapy, at 6 weeks postinfusion (the end of
the evaluation period) (Fig 3A). Patient no. 2 had insufficient blood
counts to allow measurement of EBV DNA levels, but experienced
improvement in stage B symptoms before the institution of further
chemotherapy and disease progression. In patient no. 13, EBV burden was
at an initial level of 400 EBV genomes per 106 PBMC, but
fell to undetectable levels by 19 days after the first infusion. These
effects were again produced in the absence of intensive chemotherapy
(>4 weeks before and after).
CTLs from patients with Hodgkin's disease contain LMP2a-specific
clones.
These data, showing reduction in viral burden, suggest that the
EBV-specific CTL lines derived from patients with Hodgkin's disease
have efficacy against circulating EBV-infected cells. To discover
whether they may have activity against the EBV proteins expressed by
the tumor cells, the CTLs infused into patient no. 13 were tested for
their ability to kill HLA-matched fibroblasts that had been infected
with vaccinia recombinants and expressed each of the EBV latent cycle
proteins individually. Figure 6 shows that following
depletion of nonspecific CD56+ and CD16+
cytotoxic cells, the line contained both HLA class I and class II-restricted CTLs that killed not only the autologous and an HLA-A2,
-A3, -B35, and -DR1-matched LCL from donor HH, but also HH fibroblasts that were expressing LMP2a, confirming the presence of
effector cells able to recognize at least one of the antigens present
on Hodgkin's tumor cells.
The results presented here show the feasibility of generating and
infusing EBV-specific CTLs in patients with advanced Hodgkin's disease. Global unresponsiveness to EBV antigens clearly cannot account
for the persistence of EBV-positive tumor cells in Hodgkin's patients.
Frisan et al26 previously showed that EBV-specific CTL
lines can be generated from patients with EBV-positive Hodgkin's disease at the time of their diagnosis, and Sing et al27
showed that such lines may contain clones with specificity for LMP1 and LMP2. We have now shown that EBV-specific cellular immunity can be
detected and expanded in vitro even from patients with active disease
who have had multiple relapses, that these cell lines may be
LMP2a-specific, and that they have antiviral function in vivo.
Submitted March 12, 1997;
accepted December 2, 1997.
We acknowledge Nancy Parnell for word processing.
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Proc |
Abstract
Introduction
Abstract
5 or lower were
used to produce patient LCL.
and infected for 1 hour with 5 plaque-forming units of vaccinia virus recombinant containing one of
the EBV genes, EBNA1, EBNA2, EBNA3a, EBNA3b, EBNA3c, EBNA-LP, LMP1, or
LMP2a, or the control gene 
-galactosidase (all a gift of Elliott
Kieff, Boston, MA).
, CD16, CD56, CD19 (Becton Dickinson), or TCR (T-Cell
Diagnostics, Cambridge, MA). Cells were analyzed on a FACScan flow
cytometer.
10%
lysis of the cells; this value exceeded the mean spontaneous release of
51Cr by 3 SD. The frequency of CTL precursors was estimated
from the slope of a regression plot of the log percentage of negative wells versus the number of responder lymphocytes.
primer, 5
10 to 20 copies of EBV) diluted in 1 µg DNA from
the EBV-negative cell line HSB-2.
). Stimulation of T cells with IL-2, anti-CD3, and
HLA-mismatched PBMC resulted in upregulation of the TCR zeta-chain
expression as assessed by FACs analysis (
).
).
CTL precursor frequencies (per 106 PBMC) were measured
immediately before and for as long as 49 days after the infusion of the
T cell lines. Frequency of precursors specific for autologous LCL
increased by 10-fold. However, proportion of precursors able to kill
allogeneic LCL changed only minimally, indicating that the observed
increment in cytotoxicity was due to activity of "classical" CTL,
rather than to MHC-unrestricted killer cells (---).
