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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2090-2101
By
From the National Cancer Institute, Basic Research Laboratory,
Bethesda, MD; the Leukaemia Research Fund Virus Centre, Department of
Veterinary Pathology, University of Glasgow, Glasgow, UK; the Advanced
Biotechnology Laboratory, Rockville, MD; the Department of Comparative
Medicine and the Washington Regional Primate Research Center,
University of Washington, Seattle, WA; and the Frederick Cancer
Research Development Center, National Cancer Institute, Frederick, MD.
Epstein-Barr virus (EBV) infection of humans has been associated
with the development of lymphoid malignancies mainly of B-cell lineage,
although occasionally T-cell lymphomas have been reported. We describe
here the characterization of a novel EBV-like virus (HVMNE)
isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II)
seronegative pigtailed macaque (Macaca nemestrina) with a
cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin
lesions demonstrated that the infiltrating cells were of the
CD3+/CD8+ phenotype. Two primary
transformed CD8+ T-cell lines were obtained from cultures
of peripheral blood mononuclear cells (PBMC) and skin, and, with time,
both cell lines became interleukin-2-independent and acquired the
constitutive activation of STAT proteins. Polymerase chain reaction
analysis of the DNA from the cell lines and tissues from the
lymphomatous animal demonstrated the presence of a 536-bp DNA fragment
that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In
situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell
lines as well as in the infiltrating CD8+ T cells of
skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from
the polymerase gene of HVMNE placed this virus within the
Lymphocryptovirus genus and demonstrated that HVMNE
is a distinct virus, clearly related to human EBV and other EBV-like
herpesviruses found in nonhuman primates.
MYCOSIS FUNGOIDES (MF) is a rare
cutaneous T-cell lymphoma (CTCL) that may involve lymph nodes and
viscera as well as skin.1,2 The Sézary syndrome (SS)
is the erythrodermic variant of MF characterized by the presence of
circulating tumor cells. The tumor cells in MF/SS are usually of the
CD4+ mature-cell lineage, although CD8+ lineage
has been described in a few cases.2 The diagnosis of MF is
based on clinical features and histopathological findings that include
infiltration of the dermis with lymphocytes with hyperconvoluted nuclei
and Pautrier's microabcesses.1 MF and SS are the most
frequent primary lymphomas involving the skin. Genetic predisposition,
alteration in cytokine profile, and viruses such as herpesviruses I and
II (HSV-I and HSV-II), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human T-cell lymphotropic virus type I (HTLV-I) have been
suggested as possible causative agents.1
HSV-I/II-specific antigens and DNA have been found in lesions of
CTCL.3,4 One report describes the finding of HHV-6 in 1 of
30 patients with CTCL.5 EBV DNA has been found in patients with cutaneous lymphomas6-12 and, in several cases, viral
RNA expression has been demonstrated in the neoplastic tissue. Higher incidence of EBV seropositivity in CTCL patients with the consistent emergence of EBV in MF/SS-cultured lymphocytes has also been
reported.10 However, a direct causative role of EBV in CTCL
has been difficult to prove.13 In contrast, the importance
of EBV in the development of B-cell malignancies in human
immunodeficiency virus-infected individuals or iatrogenically
immune-suppressed patients is more broadly accepted.14
Although humans are the only known natural host for EBV, EBV-like
agents have been described in Old World nonhuman primates, including
chimpanzee,15-17 baboon,16,18-21 African green
monkey,22 gorilla,23 and macaque
species.24-29 Although the relative prevalence of these
viruses in animals in captivity or in the wild is
unknown,30 several studies suggest that it may be as high
as that in EBV in humans. In fact, the inability to generate models of
EBV-associated lymphomas in Old World monkeys with the human EBV
has been ascribed to the presence of cross-reactive immunity against
EBV in these species. In contrast, at least 2 New World monkey species,
including the cotton-top tamarin (Sanguinus oedipus
oedipus)31 and the owl monkey (Aotus
trivirgatus),32 develop B-cell lymphoma upon human EBV
exposure. We report here the occurrence of a rare case of
CD8+ T-cell MF in a pigtailed macaque and the isolation of
a novel EBV-like virus from 2 transformed CD8+ T-cell lines
obtained from the blood and the skin of the lymphomatous macaque.
These findings may help in the development of an animal model for
EBV-like virus-induced malignant proliferation of T cells.
Establishment of macaque blood and skin T-cell lines.
Peripheral blood mononuclear cells (PBMC) were isolated by
density-gradient centrifugation on lymphocyte separation medium (LSM;
Organon Teknika Corp, Durham, NC) from anticoagulated blood obtained
from pigtailed macaque J94356 before death. The cell layers were washed
twice in Dulbecco's phosphate-buffered saline (DPBS; GIBCO BRL,
Gaithersburg, MD) and were suspended in RPMI (GIBCO BRL) with 10%
heat-inactivated (HI) fetal bovine serum (FBS; GIBCO BRL) with
penicillin/streptomycin (500 U/mL and 500 g/mL, respectively; GIBCO
BRL) and L-glutamine (2 mmol/L; GIBCO BRL) and stimulated with
phytohemagepletrinic (PHA; 5 g/mL; Murex Diagnostics, Norcross, GA). At
72 hours, the cells were washed twice in DPBS and resuspended in fresh
RPMI with 10% HI FBS, penicillin/streptomycin, L-glutamine, and
recombinant interleukin-2 (IL-2; 20 U/mL; Boehringer Mannheim,
Indianapolis, IN). Fresh skin biopsy tissues from diseased areas were
minced to release single cells. These were banded on LSM and placed in
culture as described above. All cells were incubated at 37°C with
5% CO2; fresh media were added once or twice per week, as
needed, to maintain adequate cell growth. After 6 weeks in culture,
both the PBMC and skin-derived cells showed evidence of increased
proliferation and clustering and the cells have been cultured for more
than 1 year. The concentration of IL-2 in the media in both cultures
was decreased in a stepwise manner from an initial concentration of 20 U/mL. Each cell line was maintained at a dose of IL-2 until it was
clear that cell growth was unimpaired. At the end of 5 months, both
J94356PBMC and J94356SKIN cells were growing
well, albeit at a slower rate than their IL-2-dependent counterparts.
DNA extraction, polymerase chain reaction (PCR) amplification, and
DNA sequence analysis.
Pellets of PBMC obtained by Ficoll gradient separation were incubated
for 1 hour at 37°C in a buffer containing 0.5% sodium dodecyl
sulfate (SDS), 10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA (pH 8.0), and
RNAse (20 g/mL; Boehringer Mannheim) before the addition of Proteinase
K (100 µg/mL; Boehringer Mannheim). After an overnight incubation at
37°C, DNA was extracted from the lysates with phenol and
chloroform. Frozen tissues, stored at Southern blot analysis.
DNA (10 µg) of each sample was digested with Sau3AI or
Pvu II, electrophoresed in 0.8% agarose gels, and blotted onto
Nylon membranes (Nytran plus; Schleicher & Schuell, Keene, NH). The membranes were hybridized overnight at 42°C with the PCR-amplified EBV-like probe labeled using random-primer reaction (Boehringer Mannheim). Hybridization, washing, and detection were performed according to the manufacturer's instructions.
Phylogenetic analysis.
Phylogenetic analysis of the novel HVMNE pol-gene
fragment (465 bp) was performed using sequences from an analogous
strain from Macaca arctoides29 (HVMA) and the
following related strains published previously: human EBV: V01556;
retroperitoneal fibromatosis herpesvirus (RFHV) Electrophoretic mobility shift assay (EMSA).
PHA-stimulated human PBMC were cultured in the presence of IL-2 (20 U/mL) for 8 days and used as a control for this assay. In starvation
experiments, 1 × 107 stimulated PBMC and
IL-2-dependent and -independent J94356PBL were
resuspended in 20 mL of RPMI 1640 with 1% FBS after washing with
1× PBS twice and incubated for 21 hours at 37°C in 5%
CO2. Protein lysates were prepared in 20 mmol/L HEPES (pH
7.9), 450 mmol/L NaCl, 0.4 mmol/L EDTA, 0.5 mmol/L dithiothreitol
(DTT), 25% glycerol, 1 mmol/L
Na3VO4, 1 mmol/L AEBSF, 20 µg/mL aprotinin, and 20 µg/mL leupeptin. The binding reaction was performed by preincubating 5 µg of nuclear extracts with 1 µg of poly(dI-dC) in
a buffer containing 5.9 mmol/L HEPES (pH 7.9), 30 mmol/L KCl, 5.9 mmol/L Tris (pH 7.4), 0.7 mmol/L DTT, 0.6 mmol/L EDTA, 8.9% glycerol,
0.1 mmol/L Na3VO4, 1 mmol/L AEBSF, 20 µg/mL
aprotinin, and 20 µg/mL leupeptin in ice for 20 minutes. A
32P-labeled probe (20,000 cpm) corresponding to the MGF
binding site in the Immunohistochemistry and EBV Epstein-Barr viral-encoded RNA (EBER)
in situ hybridization.
CD3 immunostaining was performed on formalin-fixed, paraffin-embedded
tissue using a rabbit polyclonal anti-CD3 serum (A0452; DAKO,
Carpinteria, CA) at 1:50 dilution followed by biotinylated antirabbit
secondary antibodies and ABC reagent (Vector Labs, Burlingame, CA) and
DAB substrate (Scytek Laboratories, Logan, UT). CD8 immunostaining was
performed on tissue sections frozen in OCT using a mouse anti-CD8
monoclonal antibody (clone Leu-2a; Becton Dickinson, San Jose, CA)
followed by a biotinylated antimouse Ig (ABC reagent; Vector Labs) and
DAB substrate (Scytek Laboratories). Each immunohistochemical analysis
included a test tissue assayed either using antibody against an
irrelevant antigen (from the appropriate species) or in absence of
primary antibody. Cell lines derived from peripheral blood and skin
were formalin-fixed, pelleted, and paraffin-embedded. Sections (3 to 5 µm) were cut onto glass slides and hybridized with a cocktail of
fluorescein isothiocyanate (FITC)-conjugated probes reactive with the
EBER RNAs, which are abundantly transcribed in all cells latently
infected with EBV (Novocastra Laboratories, Newcastle, UK).
Hybridization was detected using an alkaline-phosphatase-conjugated,
anti-FITC antibody (DAKO, High Wycombe, UK) and nitro-blue tetrazolium
as chromogenic substrate (DAKO). Subsequently, the EBER in situ
hybridization was performed using sections of skin biopsies obtained
from monkey skin affected with lymphoma. Hybridization using a poly-dT
probe (Novocastra) was used to confirm the integrity of the RNA in all
tissue sections.
Na-butyrate induction and electronmicroscopy (EM).
Cells (5 × 105) from the J94356PBMC cell
line were incubated in medium containing Na-butyrate (1 µmol/L) for
48 hours, fixed in 0.65% paraformaldehyde and 0.8% gluteraldehyde for
1 to 2 hours, and then incubated in 0.1 mol/L cacodylate buffer (pH 7.2 to 7.4) containing 0.1 mol/L sucrose. The cell pellet was postfixed
with 0.5% osmium fixative (pH 7.2 to 7.4) for 1 hour, processed
through an acetone gradient to 100% acetone, and then placed
sequentially in 1:1 and 1:3 acetone-resin mixes. The cells were
embedded into a pure resin capsule and cured in an oven at 60°C for
48 hours. The pellet was sliced with an Ultra Microtome (Leica
Microsystems UK, Milton Keynes, UK) at 50 to 60 nm, collected on copper
grids, and stained with uranyl acetate and lead citrate. The
preparations were analyzed on a Zeiss 109 Transmission Electron
Microscope (Cast Leiss, Welwyn Garden City, UK).
MF in animal J94356: Clinical presentation and histopathological
findings.
In May 1996, an 18-month-old pigtailed macaque (animal J94356), housed
at the Washington Regional Primate Research Center, developed a
unilateral eye infection with marked inflammation of the conjunctiva
and eyelids, which eventually spread to the opposite eye. Improvement
in the clinical manifestations was observed after treatment with
antihistamine and antibiotics, but inflammation of both conjunctivas
and eyelids recurred 8 days after treatment was discontinued. A small
unilateral corneal ulceration was found on examination and antibiotic
and antihistamine therapy was reinstated. At this time, an extensive
work-up to test for viral infections was performed. A serologic panel
of tests was run at a commercial laboratory. Tests were negative for
herpes B, HSV-1, measles, and simian immunodeficiency virus and were
positive for cytomegalovirus. In August 1996, the ulcerative lesions
recurred in the perioral area and new lesions involving the face,
trunk, forearms, and legs appeared. At this time, the serology was
negative for herpes B and STLV; however, EBV serology was positive and
the lesions improved on a 2-week course of acyclovir. Biopsy results of
skin lesions examined early in October 1996 were consistent with a multifocal cutaneous lymphoma. The animal was continued on antibiotic therapy with mild improvement of the lesions.
Detection of an EBV-like virus in the transformed CD8+
T-cell lines from animal J94356.
Peripheral mononuclear cells obtained from whole blood at the time of
necropsy were cultured with PHA and recombinant IL-2. Skin-derived
cells were also placed in culture under similar conditions and, like
the PBMC culture, proliferated continuously in response to IL-2. Both
cell lines have been in culture for approximately 2 years. Giemsa
staining of cytospin preparations of these cells at 2.5 months after
initiation of culture of the cells indicated their pleomorphism with
myeloid-like features and multiple mitotic figures at higher
magnification (lower panels of
Fig 3). Cytogenetic analyses
of the chromosomes confirmed the simian origin of both cell lines (2n = 42; data not shown).
Genetic and phylogenetic characterization of the herpesvirus from
animal J94356.
To establish the genetic relationship of the putative viral fragment
found in cells from animal J94356 to other known nonhuman primate
herpesviruses, we obtained the DNA sequence of plasmid p536 and aligned
it to the equivalent polymerase region of the human KSHV and EBV as
well as EBV-like viruses or rhadinoviruses from various animal species
(Fig 5).29,33,34,39 Both DNA sequence alignment and the phylogenetic analysis by the NJ method (Fig 6) indicated that the herpesvirus in
J94356 cells clustered with the human EBV and the nonhuman primate
herpesviruses, HVMA and HVPA,34 and was distantly related
to the known rhadinoviruses.33,39,40
JAK/STAT constitutive activation in CD8+ T cells infected
with HVMNE correlates with the acquisition of IL-2
independence.
Transforming viruses usually induce growth-factor independence by
interfering with intracellular signaling pathways,42-45 and this event occurs also in EBV-transformed B-cell lines.46
In addition, in human hematopoietic malignancies, including
Sézary syndrome, constitutive activation of JAK/STAT protein has
also been described.47-50
Detection of EBV-like DNA in the tissues of animal J94356.
To investigate whether HVMNE found in the CD8+
T-cell lines from animal J94356 was also present in the infiltrating
CD8+ neoplastic T cells from the skin lesion as well as
other organs, the DNA from various tissues was analyzed using the same
primer set used to amplify the HVMNE polymerase gene from
the DNA of the J94356 cell lines.
Herpesviruses have been found in most animal species, and the family
Herpesviridae includes the 3 subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. Within the
Gammaherpesvirinae subfamily, 2 genuses have been distinguished, the
lymphocryptoviruses (Epstein-Barr-like viruses) and rhadinoviruses
(Saimiri-ateles-like herpesviruses).51 Only members of the
Gammaherpesvirinae subfamily have been associated with human
malignancies. EBV causes B-cell lymphoma in immunodeficient individuals
and is epidemiologically associated with Burkitt's lymphoma,
Hodgkin's disease, and nasopharyngeal carcinoma.14
Similarly, human herpesvirus 8 has been epidemiologically linked to
Kaposi sarcoma40 and rare forms of
lymphoma.52-55 EBV induces B-cell lymphoma in previously
unexposed New World monkeys but not in Old World primates, presumably
because of preexisting cross-immunity against EBV, as demonstrated by
their frequent EBV seropositivity.56 However, after simian
immunodeficiency virus infection, approximately one third of the
infected macaques develop B-cell lymphoma, and this event has been
associated with the presence of an EBV-like virus in Macaca
fascicularis.57
The authors thank Peter Biberfeld for helpful discussion and Steven
Snodgrass for editorial assistance. We thank June Freeland and Ross
Blackley, who performed the viral stimulation and EM studies.
Submitted February 17, 1999; accepted May 19, 1999.
Supported in part by National Institutes of Health Grant No. RR00166
(Washington Regional Primate Research Center, Seattle, WA).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to G. Franchini, MD, Chief, Section of Animal
Models and Retroviral Vaccines, Basic Research Laboratory, Division of
Basic Sciences, National Cancer Institute, National Institutes of
Health, 41 Library Dr, Bldg 41, Room D804, Bethesda, MD 20892; e-mail:
veffa{at}helix.nih.gov.
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TOP
ABSTRACT
INTRODUCTION
80°C until processing,
were thawed on ice and finely minced with sterile blades. The minced
tissue was resuspended in ice-cold DPBS and washed twice before lysis
and extraction as detailed above. DNAs were initially amplified with
the primer pools DFASA/GDTD1B and VYGA/GDTD1B described by Rose et
al,
F' bacterial cells using the TA cloning
kit from Invitrogen (Carlsbad, CA). White colonies were picked and
grown in LB broth in the presence of ampicillin at 37°C overnight.
Plasmids were isolated using the Promega Miniprep (Promega, Madison,
WI) and digested with EcoRI to confirm the presence of the
appropriately sized insert, and DNA sequencing was performed using the
T7 Sequenase v2.0 chain-termination method from Amersham Life Science
(Cleveland, OH) according to the manufacturer's instructions. Based on
the sequence of the DNA fragment first amplified, a primer set, EDR8/97
sense 5'-ATCTCTGTTATTCTACC-3', MGF2B antisense
5'-CCTGCAGCGTCA-3', was synthesized. An aliquot of the
first PCR products was amplified using these sequence-specific primers.
PCR amplification with the primer set was optimal at 5× buffer
(pH 8.5) containing 300 mmol/L Tris-HCl, 2.0 mmol/L MgCl2,
75 mmol/L (NH4)2SO4, 10 mmol/L
dNTP, and 2.5 U Taq polymerase. The templates were subjected to 45 cycles of amplification as described above. The nested PCR products
were analyzed by gel electrophoresis and subsequently cloned into Inv
Macaca
nemestrina: AF005478; retroperitoneal fibromatosis herpesvirus
(RFHV)
-casein gene promoter
(5'-TAGATTTCTAGGAATTCG-3') was added to the reaction
mixture and incubated on ice for 30 minutes. For the supershift assay,
STAT5 antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA) was
incubated with cell extracts on ice for 20 minutes after the addition
of radiolabeled probe. Complexes were resolved on 4.5% polyacrylamide gels.
(not shown). No
staining was observed with an irrelevant antibody (left bottom panel of
Fig 
