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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3256-3261
TRANSPLANTATION
Herpesvirus saimiri-transformed macaque T cells are tolerated and
do not cause lymphoma after autologous reinfusion
Andrea Knappe,
Gisela Feldmann,
Ulf Dittmer,
Edgar Meinl,
Thomas Nisslein,
Sabine Wittmann,
Kerstin Mätz-Rensing,
Thomas Kirchner,
Walter Bodemer, and
Helmut Fickenscher
From the Institut für Klinische und Molekulare
Virologie and the Institut für Pathologie,
Friedrich-Alexander-Universität Erlangen-Nürnberg,
Erlangen, Germany, and the Abteilung Virologie und Immunologie and the
Abteilung Tiermedizin und Primatenhaltung, Deutsches Primatenzentrum,
Göttingen, Germany.
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Abstract |
Human T cells are transformed in vitro to stable growth after
infection with herpesvirus saimiri subgroup C strain C488, and they
retain their antigen-specific reactivity and other important functional
features of mature activated T lymphocytes. The virus persists as
nonintegrating episomes in human T cells under restricted viral gene
expression and without production of virus particles. This study
analyzes the behavior of herpesvirus-transformed autologous T cells
after reinfusion into the donor under close-to-human experimental conditions. T cells of 5 macaque monkeys were transformed to stable interleukin-2 dependent growth and were intravenously infused into the
respective donor. The animals remained healthy, without occurrence of
lymphoma or leukemia for an observation period of more than 1 year.
Over several months virus genomes were detectable in peripheral blood
cells and in cultured T cells by polymerase chain reaction. In naive
control animals, a high-dose intravenous infection rapidly induced
pleomorphic peripheral T-cell lymphoma. In contrast, monkeys were
protected from lymphoma after challenge infection if they had
previously received autologous T-cell transfusions. High levels of
antibodies against virus antigens were detectable after challenge
infection only. Taken together, herpesvirus-transformed T cells are
well tolerated after autologous reinfusion. This may allow us to develop a novel concept for adoptive T-cell mediated immunotherapy.
(Blood. 2000;95:3256-3261)
© 2000 by The American Society of Hematology.
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Introduction |
Herpesvirus saimiri is a  2-herpesvirus
of the squirrel monkey (Saimiri sciureus), which is the
naturally infected host and does not show signs of disease related to
this virus. However, when herpesvirus saimiri is experimentally
transferred to other New World primate species, such as the cottontop
tamarin (Saguinus oedipus) or common marmoset (Callithrix
jacchus), the animals develop acute T-cell lymphoma or leukemia
within a few weeks after infection. This disease is associated with
massive virus replication. Herpesvirus saimiri can easily be recovered
by cocultivation of peripheral blood mononuclear cells (PBMCs) from
leukemic monkeys with permissive owl-monkey kidney
cells.1-3
Herpesvirus saimiri subgroup C strains, especially strain C488, are
capable of transforming human T cells to a stable
interleukin-2-dependent (IL-2-dependent) growth in culture, which is
largely independent of their T-cell subtype.4 The virus
genome persists as nonintegrating episomes under restricted virus gene
expression. The transformed human T cells are not permissive to the
virus, and it has not been possible to induce reactivation of virus
replication.4-6 The transformed T cells show the phenotype
of mature activated T lymphocytes including expression of CD4 or CD8
and activation markers. Most importantly, the antigen-specific
reactivity of parental nontransformed T cells is retained after
transformation. After stimulation, herpesvirus saimiri-transformed T
cells are cytotoxic and secrete T-helper-1-type cytokines such as
IL-2, interferon- (IFN-), granulocyte macrophage-colony
stimulating factor (GM-CSF), and tumor necrosis factor-
(TNF-).7 Thus, herpesvirus saimiri
provides a convenient means to amplify functional human T lymphocytes
to large cell numbers in culture.
In theory, this method for in vitro expansion of T cells could be a
useful tool for adoptive immunotherapy. However, it has been unclear as
to which consequences this type of viral transformation would lead to
under in vivo conditions. The observed growth transformation could be a
mild phenotype that is only observed in culture. Alternatively, it
could be a strong type of oncogenic transformation leading to
tumorigenesis in vivo. Therefore, the behavior of transformed autologous T cells after retransfusion into the donor remained to be
determined. New World primates, such as S oedipus, are not suitable for this purpose because transformed marmoset T cells express
all viral genes tested and produce considerable amounts of virus
particles. We addressed this question in macaque monkeys (Macaca
mulatta and Macaca fascicularis) because herpesvirus
saimiri-transformed T cells from macaques closely resemble their human
counterparts.8-10 Moreover, macaques are the closest
relatives to humans that are available for animal experiments.
Herpesvirus saimiri-transformed autologous macaque T cells were
retransfused into the donor animals. The transfused animals did not
develop signs of disease, although virus DNA could be demonstrated in
PBMCs by polymerase chain reaction (PCR) over several months. After
autologous transfusion, the animals were protected from acute disease
caused by a herpesvirus saimiri challenge infection. In summary,
transformed autologous T cells did not induce leukemia or lymphoma and
were clinically well tolerated in the recipients.
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Materials and methods |
Virus and cell culture
The propagation of herpesvirus saimiri strain C488, as well as the
transformation and cultivation of transformed T cells, was done
according to published protocols.7,10 To enhance the
sensitivity of herpesvirus saimiri isolation experiments, potentially
existing virus particles were sedimented from large volumes (at least
35 mL) of T-cell culture supernatants. The concentrates were tested on
owl-monkey kidney cells for the induction of cytopathic changes. Virus
isolation tests from monkey blood were performed on fresh owl-monkey
kidney cell layers (25 cm2) with either 0.5 mL plasma or
106 PBMCs.7 To circumvent the problem of foamy
virus reactivation,9 the fresh monkey T cells were cloned
by limiting dilution in microtiter wells in the presence of
105 irradiated human feeder PBMCs (120 Gy) and 5 µg/mL
phytohemagglutinin (PHA) (Murex/Diagnostica, Burguedel,
Germany). The primary T-cell lines were further
amplified by restimulation with irradiated human feeder cells and
mitogen, followed by the addition of 20 U/mL recombinant IL-2 (Roche
Biochemicals, Mannheim, Germany) after 24 hours. The cell lines were
finally used for the standard transformation procedure in the presence
of IL-2.7
Animal experiments
The animal experiments were performed at the German Primate Center,
Göttingen, Germany. Based on an approval according to the German
animal protection regulations, 5 healthy macaque monkeys (2 M
mulatta and 3 M fascicularis) were transfused twice with 80 × 106 washed transformed autologous T cells per
kg of body weight (Table 1). Under
short-term anesthesia, blood samples were taken at increasing intervals
(Figure 1). PBMCs were prepared by Ficoll density gradients (Biochrom, Berlin, Germany) and subjected to DNA-PCR,
virus isolation, and long-term T-cell culture. After 1 year of
observation, 4 animals and 2 naive control monkeys were intravenously
infected by herpesvirus saimiri C488 with 106
plaque-forming units in 1 mL. After necropsy, detailed histological analysis was performed. Sections of formalin-fixed, paraffin-embedded tissue were stained with hematoxylin/eosin and subjected to
immunohistochemistry by using a streptavidin-biotin-complex peroxidase
detection system (StreptABComplex/AP; Dako, Glostrup, Denmark).
Immunostaining of deparaffinized sections was performed with a mouse
antihuman CD20 monoclonal antibody (mAb) (L26, Dako) or with an
affinity-purified rabbit antihuman CD3 mAb (M756, Dako), which proved
to react with lymphocytes of rhesus and cynomolgus
monkeys.
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| Fig 1.
Time course of the animal experiments.
In a pilot experiment, the animal Mm5574 received 2 transfusions (Tr1
and Tr2) of autologous T cells at a 10-week interval. At necropsy (N)
in week 25, pathological changes were not observed. Subsequently, the
animals Mm7067, Mf6311, Mf6490, and Mf6698 received a first autologous
transfusion (Tr1) and then a second transfusion (Tr2) after 20 weeks.
At week 55, these animals and 2 naive control monkeys (Mf406 and Mf409)
were subjected to intravenous challenge infection (Inf). Whereas the
control animals died within 2 weeks from peripheral T-cell lymphoma,
the other animals survived without pathological changes at necropsy at
week 68. In 2 cases, the transformed T cells were successfully
recultivated from peripheral blood, as indicated by (R).
The results of virus isolation assays (V) and DNA-PCR
(P) from peripheral blood leukocytes are summarized as follows: open
squares indicate negative results and filled squares represent positive
results for virus isolation (V) or PCR detection (P), respectively.
While infectious virus was rarely isolated from peripheral blood, virus
DNA was detectable in peripheral blood cells for long periods.
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DNA and protein methods
Virus DNA from PBMCs or cultivated T cells was analyzed by PCR.
Primer pairs specific for the virus genes stpC and
vIL-17 (gene 13) were applied.6 The PCR products
were separated on 1.5% agarose gels, transferred onto nylon membranes,
and hybridized with phosphorus 32 (32P) radiolabeled probes
for stpC or vIL-17 genes. PCR for the cellular  -globin gene served as an internal control. The semiquantitative PCR
assays for the virus genes stpC and vIL-17 detected low
copy numbers of viral genomes, corresponding to 10 (stpC) or 1 (vIL-17) genome equivalents. This is based on previous
estimations that 1 transformed T cell contained up to 100 episomes.4 Increasing numbers of transformed human T cells
were mixed with nontransformed T cells as a control for PCR validation.
At a mix ratio of 1 in 106 cells, a single virus genome
would then be present in the reaction if 1% of the lysate of
106 cells were used for PCR. The signal intensities from
fresh PBMCs after autologous transfusion correlated to about 1 transformed T cell in 104 to 106 cells. This
semiquantitative estimation was supported by the observation that
signals corresponding to about 1 herpesvirus saimiri genome copy per
reaction were positive only in a fraction of repetitive tests. In such
cases, the PBMC samples were classified positive if at least 50% of
the attempts yielded specific PCR products.
Viral nonintegrated episomal DNA was demonstrated by
Gardella in situ lysis gel electrophoresis and
subsequent Southern blot hybridization with an stpC specific
probe.7 The surface phenotype of macaque T cells was
analyzed by standard flow cytometry (FACStrak; Becton Dickinson,
Heidelberg, Germany). Murine mAbs against the following human antigens
were used for flow cytometry (names of hybridoma clones in
parentheses): CD2 (clone TS1/18.1.1), CD4 (SK3), CD8 (SK1),
CD18/LFA1 (TS1/18.1.2.11.4), CD25 (2A3), CD56 (My31), CD58/LFA3
(TS2/9.1.1.4.3), CD69 (L78), MHC-I (W6/32), and MHC-II/HLA-DR (L243)
(Becton Dickinson and American Type Culture Collection, Manassas,
Virginia). The rhesus CD3-specific mAb FN1811 was applied
together with a secondary antibody against mouse immunoglobulins (Dianova, Hamburg, Germany) in indirect staining reactions. Antibody sets for the human cytokines IFN-, TNF-, and IL-6 (Genzyme, Rüsselsheim, Germany) were applied in enzyme-linked immunosorbent assays (ELISAs). Control tests with supernatants of
phorbolester-stimulated transformed T cells from humans and macaques
showed that these antibodies were cross-reactive with the respective
macaque cytokines (data not shown).
Antiviral immune response
ELISAs were performed with 1:200 diluted plasma samples collected at
various time points during the experiment.
Gradient-purified virus particles7 (400 ng protein per
well) were coated onto 96-well plates (Maxisorp; Nunc, Roskilde,
Denmark). Unspecific reactions were blocked with rabbit serum. A
peroxidase-conjugated secondary antihuman antibody (Dianova) was
applied for the ELISA detection reaction, which was monitored as an
optical density at 450 nm. Western blot analyses used antigen derived
from cultivated transformed T cells or from gradient-purified virion
particles.7 The proteins were separated under reducing
conditions on sodium dodecyl sulfate/polyacrylamide (10%) gels,
transferred onto nitrocellulose membranes, and tested with 1:200
diluted plasma samples. A peroxidase-labeled secondary antihuman
antibody (Dianova) was used for detection.
T-cell proliferation tests were carried out with fresh PBMCs during the
time course experiment after the autologous transfusions. For this
purpose, 105 PBMCs per well were cultivated in 100 µL
complete culture medium with 1% human AB serum and without exogenous
IL-2. The cultures were either left untreated or supplemented
with 10 µg/mL PHA or inactivated purified virion particles at 1 µg/mL final concentration. On day 6, 0.0185 MBq (0.5 µCi)
tritium-labeled thymidine (Amersham Pharmacia Biotech)
in a volume of 10 µL was added to each well. On day 7, the
microcultures were harvested onto glass-fiber filters and measured for
tritium activity in a -counter. Control PBMCs were reactive to
mitogen treatment.
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Results |
T-cell transformation and autologous reinfusion in macaque
monkeys
Herpesvirus saimiri-transformed T-cell lines were generated from 5 macaques (Table 1): 2 rhesus monkeys (M mulatta; Mm5574 and
Mm7067), and 3 cynomolgus monkeys (M fascicularis; Mf6311, Mf6490, and Mf6698). Direct transformation of freshly isolated peripheral blood T cells from macaques was rarely successful (eg, animal Mm5574) because T-cell experiments in macaques are hampered by
the highly prevalent foamy virus infection at primate centers. In most
cases, T-cell cloning by limiting dilution using mitogenic activation
in the presence of irradiated human feeder cells was required to
generate foamy virus-free T-cell lines. The transformed T cells carried
high copy numbers of viral nonintegrated episomes, whereas linear DNA
molecules typical for virion particles were not detectable by Southern
blot analyses of Gardella gels. In contrast to their human
counterparts, most of the transformed macaque T-cell lines from this
study did release small amounts of virus particles (Table 1). The
surface phenotype of these T cells was very similar to that of
transformed human T cells including expression of CD2, CD3,
CD18/LFA1, CD25, CD56, CD58/LFA3, CD69, MHC-I, and MHC-II.
Frequently, CD4 and CD8 were coexpressed on cultivated parental and
transformed macaque T cells (Table 1).
When stable IL-2-dependent T-cell lines had been established for
several months, autologous transformed T cells
(80 × 106/kg body weight) were washed in saline and
intravenously infused into the donor animal (Table 1). After
10-20 weeks, a second infusion with identical cell numbers was
performed. Animal Mm5574 was treated prior to the others in a shorter
time course protocol in order to obtain first information from a pilot
experiment. Six months after the first transfusion, animal Mm5574 was
subjected to necropsy without pathological findings. The other animals
were frequently monitored for more than 1 year (Figure 1). There were neither signs of pathological disorders nor hints of tumor development.
Behavior of transformed T cells after autologous transfusion
During several months, blood samples were taken at weekly, biweekly,
or monthly intervals (Figure 1). Sensitive virus
isolation experiments were positive only in rare cases (PBMCs) but
never in plasma samples (Figure 1). The persistence of viral genomes was demonstrated by semiquantitative DNA-PCR. Viral genomes were detected over months in PBMCs and in cultivated T cells (Figure 1,
Figure 2). The signal intensities from
fresh PBMCs correlated to about 1 transformed T cell in 104
to 106 cells. The PCR results from PBMCs were confirmed by
testing T-cell cultures originating from the same blood samples. Using
long-term T-cell culture, herpesvirus saimiri-transformed T cells were
recultivated from the peripheral blood of animal Mf6698 in some cases
(Figure 1).
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| Fig 2.
Virus DNA in PBMCs after autologous transfusion.
Semiquantitative DNA-PCR was performed from fresh PBMCs. DNA from cell
mixtures was used as control. For this purpose, herpesvirus
saimiri-transformed human T cells were mixed with PHA/IL-2-stimulated
human T cells at ratios of 0/106, 1/106,
10/106, 102/106, and
103/106. The time points of the PBMC
preparations after the first autologous transfusion are marked for day
2 (d2), day 4 (d4), and week 1 (w1) to week 16 (w16). PCR for 2 different virus genes, stpC and vIL-17, is shown for
monkey Mm7067.
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Herpesvirus saimiri challenge infection
One year after the first autologous reinfusion, 4 of these animals
(Mm7067, Mf6311, Mf6490, and Mf6698) and 2 naive control cynomolgus
monkeys (Mf406 and Mf409) were subjected in parallel to a high-dose
intravenous challenge infection with herpesvirus saimiri C488
(106 tissue culture infectious doses in 1 mL). The 4 animals that had previously received autologous infusions did not
develop the disease. It was not possible to reisolate the virus from
PBMCs. At necropsy 2 months later, no pathological findings were
made. Using PCR with DNA from necropsy materials, only low amounts
of virus DNA were detected, similar to the levels observed in PBMCs during the time course experiment after autologous transfusion.
In contrast, the naive control animals died from lymphoma at day 13 or
14 after high-dose infection. Lymphoma cell lines were established ex
vivo from both animals, and the virus was easily reisolated from PBMCs.
Foamy virus reactivation terminated the cultures from 1 animal after
several weeks. T cell lines in the other animal were phenotypically
indistinguishable from in vitro transformed T cells. Necropsy samples
of lymph nodes, thymus, spleen, bone marrow, kidney, and brain of these
animals contained large amounts of virus DNA (data not shown). Whereas
IFN-, IL-6, and TNF- were not detectable in plasma samples after
autologous transfusions, IFN- (up to 225.1 pg/mL) and IL-6 (up to
10.2 pg/mL) were increased in plasma samples from the terminally ill
animals Mf406 and Mf409. Histopathological and immunohistochemical
analysis showed widespread infiltrates of a pleomorphic peripheral
T-cell lymphoma consisting of medium-sized and large blasts with
vesicular nuclei and prominent nucleoli (Figure
3, upper panel). The blasts were positive
for the T-cell marker CD3 (Figure 3, central panel) but negative for
CD20. The infiltrates of blasts involved the lymph nodes, spleen,
Waldeyer's ring, intestine, and pancreas of animal Mf406, and they
were detected in lymph nodes, spleen, intestine, kidney, salivary
glands, lung, and liver (Figure 3, lower panel) of animal Mf409. The
lymph nodes were enlarged in both animals and showed a widening of
sinuses and T zones by the infiltrating blasts as well as a loss of
follicular structure.
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| Fig 3.
T-cell lymphoma after intravenous infection with
herpesvirus saimiri C488 of naive cynomolgus monkeys.
The tissue sections were stained with hematoxilin/eosin. Upper panel:
Lymph node showing peripheral pleomorphic T-cell lymphoma with
medium-sized and large blasts containing vesicular nuclei and prominent
nucleoli (original magnification ×100). Central panel:
Immunostaining of the medium-sized and large blasts and some
accompanying small lymphocytes for CD3 (original magnification
×63). Lower panel: Infiltration of the portal tracts of the liver
by the blast cells (original magnification ×40).
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Antiviral immune response
To analyze the humoral immune response, plasma samples were taken
following autologous transfusions and challenge infections. The samples
were tested using both ELISA with virus particles as antigen and
Western blot strips carrying proteins from purified virions or
transformed T cells. Transformed T cells as antigen did not reveal
signals on Western blots. In contrast, gradient-purified virus
particles as antigen caused strong signals in ELISA and Western blots,
but this only occurred after the challenge infection (Figure
4A, B). The cellular immune response was
analyzed in T-cell proliferation assays. Shortly after the first
transfusion (2 weeks), in 3 of the 4 animals tested, an increased
proliferation was observed when inactivated purified virion particles
were added as antigen (Figure 4C). During the further time course of
the transfusions, this reactivity was no longer observed, which may
indicate that only small amounts of virus antigens had been exposed for
a short period after the transfusion.
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| Fig 4.
Antiviral immune response after autologous transfusions
and challenge infection.
(A) Seroreactivity of the animals was analyzed by ELISA using purified
virus particles as antigen and plasma dilutions of 1:200. Preimmune
sera (indicated at time point 0) did not show relevant background
reactivity. As examples, the time courses for the animals Mf6490 and
Mf406 are shown. The sera after autologous transfusion (Tr1 and Tr2)
were either negative or produced weak signals. In animal Mf6490, an
unstable antibody response was observed shortly after the second
transfusion. After challenge infection (Inf), the protected animals
rapidly developed strong seroreactivity. (B) To confirm the ELISA
results, Western blot strips analyses carrying virion proteins were
stained with 1:200 diluted plasma samples from monkeys Mm7067 and
Mf6490 after autologous T-cell transfusion (Tr1 and Tr2) or challenge
infection (Inf), respectively. The time points of plasma preparations
are given in months after transfusion or infection. The animals Mf6311
and Mf6698 had similar levels of reactive antibodies at equivalent time
points. (C) T-cell proliferation tests were performed during the time
course of the autologous transfusions. Two weeks after the first
transfusion (Tr1), T cells from the blood of 3 out of 4 animals showed
proliferative reactivity against inactivated virus particles as an
antigen, as demonstrated by the stimulation index of tritium thymidine
incorporation assays.
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Discussion |
The adoptive transfer of human T lymphocytes is limited by the
laborious procedures to grow large numbers of T cells. Herpesvirus saimiri offers a convenient way to amplify human T cells. Easily amplifiable antigen-specific T cells, eg, directed against
tumor-specific epitopes, would form an interesting tool for adoptive
cancer immunotherapy. Alternatively, the antigen specificity of the
transformed T cells could be modified by using the transforming virus
simultaneously as a gene expression vector. However, the biological
safety of such transformed T cells has been unclear. In this report we
show that transfusions with autologous herpesvirus saimiri-transformed T cells are well accepted and even provide protection against acute
lethal leukemogenesis after experimental intravenous infection.
In this study, 80 × 106 autologous T cells per kg
of body weight were applied during transfusion, which resulted in total
cell numbers of 130-600 × 106 (Table 1). This is
approximately the same order of magnitude as that published previously
for retrovirally transduced rhesus monkey T cells, where
54-140 × 106 cells per kg were
transferred.12 In human studies, there is little experience
in the autologous situation. Early gene marking experiments with
autologous melanoma-infiltrating T cells used absolute cell numbers
between 0.2 and 43 × 106.10,13,14 Under
allogeneic conditions, 0.5-38.6 × 106 cells per kg
of retrovirally transduced T cells from bone marrow donors were
transfused.15 In other studies, dose increments of
antigen-specific donor T cells, ranging from
33 × 106 to 3.3 × 109 cells per
m2, were used.12,16-18
Retrovirally transduced human or macaque T cells were detectable
for 64 days, 12 months, or even up to 727 days, depending on the
study.12,14,15 Thus, our observations of cell numbers and
survival of the grafted cells are compatible with published transfusion
results of nontransformed T cells.
T cells of different primate species have distinct properties
concerning the replication and persistence of herpesvirus saimiri. In
contrast to the lack of virion production by herpesvirus
saimiri-transformed human T cells, transformed T lymphocytes from
marmoset monkeys release easily detectable amounts of virus particles.
Moreover, linear virion DNA and a broad transcription pattern of virus
genes have been demonstrated in herpesvirus saimiri-transformed
marmoset T cells.5,10 Unexpectedly, most transformed
macaque T-cell lines tested in this study released small amounts of
virus particles in culture when a virus isolation procedure with
enhanced sensitivity was applied. Such a release of virions from
transformed human T cells has never been demonstrated in a large test
series. It is noteworthy that the productive activity of the
transformed macaque T cells was very low because lytical linear DNA
forms had not been detected. In addition, Western blots with virion proteins remained negative after T-cell transfusion, whereas a strong
antibody response developed only after challenge infection.
Obviously, there are differences between human and macaque monkeys
regarding the control of virus replication in transformed T cells.
However, this low-level replication does not seem to be biologically
critical, as neither disease nor humoral reactivity developed after
autologous transfusion. The observed minimal virus replication in
macaque cells is relevant for the safety discussion of potential
applications in human therapy: Even if the low-level replication is not
critical for pathogenicity and even if this has not been observed for
human T cells, any unwanted side effects due to virus replication have
to be excluded for potential applications in human patients. This could
be achieved by packaging cell-dependent, replication-deficient virus
variants. Although rhesus or cynomolgus monkeys show differences to
humans with the respect of the virus persistence and replication, these
animals provide the only available and valuable close-to-human
experimental possibility to test the basic applicability of herpesvirus
saimiri-transformed T cells for therapeutic purpose.
The intravenous infection of naive macaques with herpesvirus saimiri
C488 at high doses induced peripheral pleomorphic T-cell lymphomas.
Based on the histopathology, distribution, and acute onset, the
infiltrates could also be designated as a pleomorphic T
lymphoproliferative disorder. In fact, the disease shows similarities to human Epstein Barr virus-induced (EBV-induced) posttransplantation B-lymphoproliferative disorders, which can be polyclonal, oligoclonal, or monoclonal. Clonality was not investigated in our cases, but the
acute onset of disease favors a polyclonal proliferation. The observed
T-cell lymphoma is compatible with a previous brief report in which the
infection of 1 rhesus monkey with herpesvirus saimiri C488 caused an
unspecified acute lymphoproliferative disease and allowed the
establishment of a transformed ex vivo T-cell line.19
In our study, a previous transfusion with autologous transformed T
cells protected the animals from leukemic disease caused by high-dose
challenge infection. Whereas the naive animals died within 2 weeks from
lymphoma, the monkeys with previous autologous transfusions remained
healthy and controlled the systemic infection. Two months after
infection, virus DNA was not anymore detectable in PBMCs, and
histopathological changes could not be found at necropsy. Either
latently or lytically expressed virus antigens could mediate this
protection. Herpesvirus saimiri-transformed T cells of both humans and
macaques show a restricted range of virus gene expression. Only few
latently expressed viral proteins would be likely candidates for
targets of cellular and humoral immune reactivity: StpC, Tip, and
IE14/vSag.5,6 By testing monkey sera against antigens of
transformed T cells on Western blots, these proteins were largely ruled
out as either lytically or persistently expressed antigens that induce
humoral immune response.
After intravenous infection, Western blots with virion antigen revealed
signals corresponding to a protein of approximately 60 kd (Figure 4B).
This protein appears to be glycosylated, as the diffuse band shape may
suggest. It is a virion protein that is expressed upon lytic virus
replication. Most likely, the observed protection from leukemogenesis
can be explained by the generation of T cells reactive against antigens
which are produced during lytic replication. T-cell proliferation tests
with inactivated virion particles as antigen revealed a weak reactivity
early after autologous transfusions (Figure 4C). This form of immune
control may be comparable to the persistence of EBV in humans, in which cells with virus replication are efficiently eliminated by cytotoxic T
cells. The observed protective effect may even lead to an enhanced biological safety of herpesvirus saimiri-transformed autologous T
cells upon potential therapeutic applications. Because we did not
detect humoral or cellular reactivity to transformed T cells, we
speculate that nonpermissive transformed autologous T cells are
subjected to the normal immunoregulatory mechanisms and not to
antiviral immune response.
Autologous herpesvirus saimiri-transformed T cells were tolerated
after transfusion without signs of disease. Virus DNA was detected in
freshly isolated PBMCs and cultivated T cells for several months after
autologous transfusion. In contrast, virus isolations from PBMCs were
positive in rare exceptions only. It is most probable that the DNA-PCR
signals correspond to the transfused cells or their descendents.
However, it cannot be ruled out that a low-level release of virus
particles occurs in vivo. This question could be addressed in the
future by using retroviral vectors to gene mark the autologous
transformed T cells before transfusion. In the case of potential
clinical applications, any lytic replication of herpesvirus saimiri
vectors must be excluded. Establishing replication-deficient variants
and the use of prodrug activation genes for targeted
elimination20 will form the next steps for developing
herpesvirus saimiri to a T-cell vector for gene therapy. The fact
that transformed autologous T cells were well tolerated may allow us
to develop a novel concept for adoptive T-cell mediated immunotherapy.
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Acknowledgments |
The authors thank Dirk Lorenzen and Christiane Stahl-Hennig for
stimulating discussions and Gerhard Hunsmann, Franz-Josef Kaup, and
Bernhard Fleckenstein for continuous support.
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Footnotes |
Submitted October 4, 1999; accepted January 7, 2000.
Supported by grants to H.F from the Bayerische
Forschungsstiftung, Munich, Germany, and from the Wilhelm
Sander-Stiftung, Neustadt/Donau, Germany.
Reprints: Dr Helmut Fickenscher, Institut für Klinische
und Molekulare Virologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany; e-mail: fickenscher{at}viro.med.uni-erlangen.de.
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.
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References |
1.
Duboise SM, Guo J, Czajak S, Desrosiers RC, Jung JU.
STP and Tip are essential for herpesvirus saimiri oncogenicity.
J Virol.
1998;72:1308-1313[Abstract/Free Full Text].
2.
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Abstract
Introduction