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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2925-2934
Epstein-Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes for the
Treatment of Patients With EBV-Positive Relapsed Hodgkin's Disease
By
Marie A. Roskrow,
Nobuhiro Suzuki,
Yan-jun Gan,
John W. Sixbey,
Catherine Y.C. Ng,
Sarah Kimbrough,
Melissa Hudson,
Malcolm K. Brenner,
Helen E. Heslop, and
Cliona M. Rooney
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.
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ABSTRACT |
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.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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.
The EBV status of the tumors was determined as described
elsewhere.18 In brief, dewaxed sections of
paraffin-embedded tissue were hybridized to digoxygenin-labeled
riboprobes specific for EBER1 (EBV-encoded small nuclear RNA) in a
standard in situ hybridization assay. Bound probe was detected by an
antidigoxygenin antibody-alkaline phosphatase conjugate (Boehringer
Mannheim, Mannheim, Germany) as suggested by the
manufacturer. For detection of latent membrane protein 1 (LMP1), the
CS1-4 mouse monoclonal antibody (Dako, Carpenteria, CA) was used in an
immunoperoxidase protocol recommended by the manufacturer (Vectastain
Elite ABC; Vector, Burlingame, CA). Of 35 cases analyzed, 13 were
positive for either EBER1 or LMP1 or both, and were considered
EBV-positive (Table 1).
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 5 or lower were
used to produce patient LCL.
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
To increase the rate of expansion when large cell numbers were required
or when the cell line was growing poorly, the third feeding consisted
of a "superexpansion cocktail" that was a variation of one
reported by Riddell and Greenberg.21 This mitogenic mixture contained 10-15 U/ml of IL-2, a submitogenic (0.01 µg/mL) dose of
anti-CD3 monoclonal antibody (Ortho Diagnostics, Raritan, NJ), irradiated autologous LCLs (T cell:LCL ratio, 4:1), and irradiated PBMC
from cytomegalovirus-negative donors who met our institutional requirements for blood donors (T cell:PBMC ratio, 8:1). To avoid stimulating allospecific clones, this cocktail was never used less than
21 days after initiation of the CTL line. This step was rarely required
for the generation of CTLs from the normal bone marrow donor controls.
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
To determine the efficiency of transduction, DNA was extracted from an
aliquot of the transduced cells by using an anion-exchange column
(Qiagen, Chatsworth, CA). The DNA concentration was measured with a
fluorimeter (Hoeffer, San Francisco, CA), and 1 µg was added to a
polymerase chain reaction (PCR) to detect the neo gene, using
primers and conditions described previously.17 The
amplification products were analyzed by Southern blotting using a
radiolabeled, neo probe. Positive control DNA was prepared from
G1Na-transduced K-562 cells (one integrant per cell) diluted with
nontransduced cells to give mixtures containing 0.1%, 1%, and 10%
neo-positive cells.
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).
To determine whether EBV-specific CTL lines generated in this way would
be able to recognize the EBV antigens expressed by Hodgkin's cells, we
depleted patient CTLs of nonspecific (CD16+ and
CD56+) killer cells using a FACStar (Becton Dickinson, San
Jose, CA). A dermal fibroblast line was prepared from a normal donor,
who shared HLA-A,B antigens with the patient. These fibroblasts were treated for 24 hours with interferon- 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).22 A total of 106
fibroblasts was infected with each construct and the incubation took
place in the presence of 0.1 mCi 51Cr. The target cells
were then washed five times and plated at 5 × 103 cells
per well with effector cells to give effector:target ratios of 40:1,
20:1, 5:1, and 2.5:1. After 5 hours, supernatants were harvested and
chromium release measured to determine specific cytotoxicity.
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- , CD16, CD56, CD19 (Becton Dickinson), or TCR (T-Cell
Diagnostics, Cambridge, MA). Cells were analyzed on a FACScan flow
cytometer.
To simultaneously stain T cells for CD3 (a cell-surface marker) and TCR
zeta (an intracytoplasmic antigen), PBMC were isolated and washed in
phosphate-buffered saline (PBS). The cell pellet was resuspended in 10 µL of a CD3-FITC monoclonal antibody (Dako) and incubated for 10 minutes at room temperature in the dark. The cells were then washed
once in PBS and fixed by incubating them in 0.01% formaldehyde (in
PBS) for 20 minutes on ice. After four washes with ice-cold PBS, the
cells were resuspended at 5 × 106 cells/mL in 10 µg/mL
digitonin (Sigma Chemicals, St Louis, MO; in PBS) and incubated for 5 minutes on ice. The cells were pelleted, resuspended in 10 µL of a
zeta-chain antibody (IgG1 unconjugated; Dako) or an isotype-matched
control, and incubated for 10 minutes at room temperature. After
washing twice in 0.05% Tween-20 (in PBS), the cells were resuspended
in 10 µL rat antimouse IgG1-PE (Dako) and placed in the dark for 10 minutes. The cells were then resuspended in PBS containing 1%
formaldehyde and analyzed by flow cytometry. For LMP1 staining of
biopsy materials, fixed sections were incubated with the CS1 to CS4
antibodies as described.18
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  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.
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 primer, 5-AACTTCAACCCACACCATCA-3; 3 primer, 5-TTCTGGACTATCTGGATCAT-3) led to amplification of a 115-bp
segment of the EBNA2 gene; the reaction volume was 100 µL and
contained 1 µg PBMC DNA. The second PCR (template, 2 µL of the
first-round product; total volume, 100 µL) used the same 5 primer,
but the 3 primer was 5-GGTGCATTGATTGGTCT-3, leading to amplification
of a 78-bp segment of the EBNA2 gene. The conditions for both rounds of
amplification were 93°C for 1 minute, 55°C for 1 minute, and 72°C
for 2 minutes for 25 cycles. We used the BL2/B95-8 cell line, which
contains two integrated copies of EBV per cell, to determine the
sensitivity of the nested PCR reaction by amplifying 0.1 µg BL2/B95-8
DNA (representing  10 to 20 copies of EBV) diluted in 1 µg DNA from
the EBV-negative cell line HSB-2.7
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RESULTS |
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
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| Fig 1.
Growth kinetics of representative EBV-specific CTL
cultures from normal donors, 2 patients with Hodgkin's disease in
remission, and 2 patients with relapsed disease. All CTL lines from
Hodgkin's patients required weekly stimulation with the four-component
cocktail of IL-2, allogeneic irradiated PBMC, LCLs, and anti-CD3 from
day 28. Lines from normal donors received only IL-2. Total cell number is illustrated as a log scale on the y-axis.
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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
+ cells seen in three of the nine lines was also seen
in five of 29 lines from normal individuals.23
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).
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| Fig 2.
Level of TCR zeta chain is abnormally low in fresh,
unstimulated T cells from patients either in remission of Hodgkin's
disease or with relapsed disease v that in T cells from normal
donors ( ). 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 ( ).
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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).
The level of LAK-activated killing varied from 0% to 77% (median,
26%) in CTL lines generated from healthy individuals and from 21% to
86% (median, 38%) in CTL lines from Hodgkin's patients (Table 2).
Lines containing high levels of LAK activity were often not sensitive
to HLA-class I blocking antibody. However, removal of CD56+
and CD16+ nonspecific cytotoxic effector cells was able to
reveal class I/II-restricted killing of autologous EBV LCLs, even when
bulk cultures were apparently major histocompatibility
complex (MHC)-unrestricted (Fig
3). This phenomenon has been
reported previously in EBV-specific CTLs generated from normal
donors.23
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| Fig 3.
Analysis of PBMC for evidence of the neo marker
and for EBV DNA. (A) Levels of neo DNA are shown in the upper
portion. They were measured by semiquantitative PCR analysis of 1 µg
total DNA at various time points after the second CTL infusion. Lower
portion shows EBV DNA in PBMC for the corresponding time points. Levels of EBNA 2 DNA were measured by nested semiquantitative PCR analysis of
1 µg total DNA. (B) Detection of marker gene in pleural fluid of
patient no. 13, 3 weeks after CTL infusion. Detection in the infused
CTL line and blood obtained on the same day are shown for comparison.
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Hence, EBV-specific CTLs generated from patients with Hodgkin's
disease in relapse or remission were similar in activity to those
generated from normal individuals, even though the lines from patients
had been in culture longer and had required exposure to the mitogenic
cocktail for expansion. Having shown that we could generate
EBV-specific CTLs with normal phenotype and cytotoxicity from
Hodgkin's patients, we tested the function of these CTLs in vivo by
gene modifying them and infusing them into three patients with multiply
relapsed disease.
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).
We next looked for evidence that these CTLs were functional.
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).
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| Fig 4.
Increase in EBV-specific CTL precursors after two
infusions of CTL in a patient with relapsed Hodgkin's disease ( ).
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 (---).
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In addition to the increase in CTLp frequency, Fig
5 shows that the CTL activity, tested after
culture with autologous LCLs, also increased in vivo. PBMC from patient
no. 1 cultured before CTL infusion showed a low level of cytotoxic
activity (1% killing of autologous LCLs at an effector:target ratio of
40:1). This level of killing increased substantially in PBMC drawn and
cultured 4 weeks and 8 weeks after infusion (28% killing of autologous LCLs at the same effector:target ratio). There was little effect on the
activity of MHC-unrestricted cytotoxic effector cells, suggesting that
an increase in classical MHC-restricted CTL activity was responsible
for the observed increase in autologous LCL target killing.
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| Fig 5.
Increased anti-EBV activity after CTL infusion.
Immediately before and at 6, 8, and 16 weeks after the first infusion
of CTL, cytotoxic T-cell lines were generated from the PBMC of a
patient with relapsed Hodgkin's disease by culturing the cells with
autologous LCLs (see Methods). The data, presented as percentage of
51Cr release from autologous LCLs at an effector:target
ratio of 20:1, demonstrate a maximum 28-fold rise in anti-EBV activity following CTL infusion (). There was no significant increase in
MHC-unrestricted activated killer activity as demonstrated by there
being no increase in the percentage of 51Cr release from
HLA-mismatched LCL targets ().
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It is possible that the increase in the EBV-specific precursor
frequency and CTL activity could be accounted for by a general improvement in immune function after cessation of chemotherapy at least
1 month previously. To determine whether the infused CTLs contributed
to this immunologic improvement, the frequency of marked CTLs in lines
reactivated from patients no. 1 and 13, 1 and 2 months after infusion,
was compared with the frequency of marked cells in the infused CTL
lines. Semiquantitative PCR analysis showed that the marking efficiency
of the infused lines was between 5% and 10% and that of the
reactivated CTL lines was between 2% and 10%. Thus, a significant
portion of the immune response to EBV was derived from the infused CTL
line, suggesting that a major portion of the immunologic improvement
and antiviral effects was due to CTL infusion.
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| Fig 6.
EBV antigen-specificity of patient no. 13 CTL line after
depletion of CD56+ and CD16+ cells. Targets
were the autologous LCLs, HH-LCL that was HLA-matched at HLA-A2, -A3,
-B35, and -DR1, and HH-derived dermal fibroblasts that had been
infected with vaccinia recombinants expressing each of the EBV
latency-associated proteins. Killing at an effector:target ratio of
20:1 is shown.
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|
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.
| |
DISCUSSION |
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.
Although the EBV-specific CTL lines generated from patients with
relapsed Hodgkin's disease are phenotypically similar to those
generated from normal donors, the expansion rate and proliferative potential of the lines from patients is much lower. This may result in
part from their suboptimal expression of the TCR zeta
chain.11 The TCR zeta chain contributes to the assembly of
the CD3 complex, and its cytoplasmic domain is involved in signal
transduction and subsequent activation and proliferation of T
cells.28 Hence, decreased expression of the TCR zeta chain
results in abnormal activation through the TCR/CD3 complex and in
depressed T-cell responses.11 Our results, showing
decreased expression of the TCR zeta chain in peripheral blood
lymphocytes from Hodgkin's patients, agree with results from Rubin et
al29 and also show that the severity of the impairment
correlates with the disease status. The TCR zeta chain defect can be
corrected in vitro by allostimulation,29 and our results
show that it may also be reversed by IL-2 when the EBV-specific CTL
lines are generated from patients in remission. However, the defect is
only partially correctable in patients with relapsed disease.
Despite this relatively limited proliferative potential, we have shown
that it is feasible to obtain substantial ex vivo expansion of
EBV-specific T cells from nine of 13 patients with advanced Hodgkin's
disease, and that these cells have normal cytotoxic activity against
autologous EBV-infected B-cell lines. Using gene marking, we were able
to compare the in vivo performance of autologous CTLs in Hodgkin's
patients with that of normal donor-derived CTLs in allogeneic stem-cell
recipients,7,8 monitoring their survival in vivo and
assessing their function directly. If EBV-positive Hodgkin's disease
resists endogenous cytotoxic T-cell activity because of a systemic
effect on CTL survival or activity, then this in vivo study of patients
with Hodgkin's disease should have shown reduced survival and
diminished antiviral activity of CTLs compared with that of CTLs in our
previous studies of bone marrow transplant patients.7,8 In
fact the marker signal in the peripheral blood of the three patients
with Hodgkin's disease persisted for 10, 12, and greater than 13 weeks
in the first three patients, which is within the range of the duration
of the marker signal observed in the peripheral blood of stem-cell
transplant recipients in our previous study.7,8 Marked
cells were also found in fluid obtained from involved pleura at levels
10-fold higher than peripheral blood, implying infused CTLs are able to traffic to sites of disease. An alternative explanation for the observed improvements in immune responses to EBV and antiviral effects
is that patient endogenous immune responses recovered after cessation
of chemotherapy, which had occurred at least 4 weeks before CTL
infusion. However, the high level of gene-marked CTLs in the lines
reactivated from two patients postinfusion suggested that a major part
of the immune response was derived from the infused CTLs.
The cells transferred during the present study functioned normally,
producing an increase in the EBV-specific cytotoxic activity of
peripheral blood lymphocytes and a decline in the viral burden in the
peripheral blood. While it is uncertain whether supranormal levels of
EBV DNA seen in many patients with EBV-positive Hodgkin's disease is a
measure of circulating tumor cells or reflects poor control of
EBV-infected normal B cells, it is evident from our data that the
virally infected cells can be controlled by infused EBV-specific CTLs.
The infused cells must therefore be able to recognize one or more of
the EBV antigens expressed by the tumor (EBNA1, LMP1, LMP2) or by
circulating normal B cells (LMP2 only).30,31 These data
demonstrated that the EBV-infected Hodgkin's tumor cells did not
decrease survival or inhibit the function of infused EBV-specific CTLs
during the time period studied.12-14 Because the first two
patients received cytotoxic drugs after the T-cell infusion, we do not
know whether an antitumor effect was produced in addition to the
antiviral effect. However, both individuals had resolution of type B
symptoms after infusion. Patient no. 13 received no additional
intensive chemotherapy and has stable disease greater than 4 months
after CTL infusion.
Even though EBV-specific T cells can be generated from patients with
advanced Hodgkin's disease, and even though these cells survive and
have antiviral function in vivo can we anticipate that this approach
will successfully treat EBV-positive Hodgkin's disease? While we are
actively exploring this possibility, there remain a number of
mechanisms by which Hodgkin's cells may evade even a high-level,
adoptively transferred anti-EBV response. EBV-positive Hodgkin's tumor
cells express only three EBV latency proteins, EBNA1, LMP1, and LMP2.
In contrast, immunoblastic lymphoma cells, which are highly susceptible
to CTL activity, express five additional antigens, LP and EBNA2, 3A,
3B, and 3C.22,32 In the majority of cases, memory CTL
responses are preferentially directed against the highly immunogenic
EBNA3A, 3B, and 3C antigens, irrespective of the patient's HLA type.
By comparison, EBNA1 and LMP1 and LMP2 are usually only weakly
immunogenic. EBNA1 contains a repeating gly-ala amino acid sequence
that inhibits processing and presentation of the EBNA1 antigen by HLA
class I antigens.33 The reason for the limited
immunogenicity of LMP1 and LMP2 is less clear, since both contain CTL
epitopes that are presented, at least in the context of the HLA-A2.1,
-B24, -B40, -B51, and -B55 alleles.22,34 An EBV-specific
immune response may thus simply fail to recognize the EBV antigens
expressed by Hodgkin's cells. While this possibility remains a
concern, three lines of evidence suggest that CTLs from patients with
Hodgkin's disease can exhibit activity against at least one of the EBV
antigens expressed by the tumor cells. First, we demonstrated
LMP2a-specific activity in the CTL line from patient no. 13. Second,
the infused CTL lines reduced the virus burden, suggesting activity
either against the tumor cells or against EBV-infected normal B cells
in which only the LMP2 gene product can be detected.30,31
Finally LMP1- and LPMP2-specific CTLs may be isolated from patients
with Hodgkin's disease.27
While these results offer promise for CTL therapy of Hodgkin's
disease, the tumor may evade killing by other mechanisms. Many Hodgkin's tumors have mutations within the 3 region of the LMP1 oncogene, including a 30-bp deletion and a high frequency of nonrandom point mutations.35,36 These mutations may further reduce
the antigenicity of LMP1 or create epitopes that are not recognized by
CTLs specific for wild-type LMP1. Other factors, such as impaired antigen presentation37-39 or downregulation of HLA class I
molecules, may also protect Hodgkin's cells. Of note, most of the
patient-derived CTL lines described here contained HLA class
II-restricted CTLs, which may remain active against class I-negative
cells.
Many barriers may remain to prevent the successful therapy of
EBV-positive Hodgkin's disease using virus-specific CTLs. Nonetheless, our demonstration that it is possible to generate EBV-specific CTLs
from patients with advanced Hodgkin's disease, that these cells
survive and function in vivo, and that may be directed to the
subdominant viral epitopes expressed by Hodgkin's cells, provides reason to hope that such cytotoxic T-cell therapy will become a useful
adjunct to conventional treatments for EBV-positive Hodgkin's disease.
| |
FOOTNOTES |
Submitted March 12, 1997;
accepted December 2, 1997.
Supported in part by National Institutes of Health Cancer Center
Support Core Grants No. CA 21765 and CA 71426, the Assisi Foundation,
and the American Lebanese Syrian Associated Charities (ALSAC).
Address reprint requests to Cliona M. Rooney, PhD, Texas Children's
Hospital, 6621 Fannin St, MC3-3320, Houston, TX 77030-2399.
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 1784 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We acknowledge Nancy Parnell for word processing.
| |
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Abstract
Introduction
Abstract