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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3103-3111
Detection of Infectious Simian Immunodeficiency Virus in B- and T-Cell
Lymphomas of Experimentally Infected Macaques
By
Maria Teresa Maggiorella,
Francesca Monardo,
Martin Luther Koanga-Mogtomo,
Livia Cioè,
Leonardo Sernicola,
Franco Corrias,
Carlo David Baroni,
Paola Verani, and
Fausto Titti
From the Laboratory of Virology, Istituto Superiore Sanità,
Rome, Italy; and II Chair of Pathological Anatomy, Department of
Experimental Medicine and Pathology, University "La Sapienza,"
Rome, Italy.
 |
ABSTRACT |
An increasing frequency of malignant lymphomas occurs among patients
infected by human immunodeficiency virus. Because of the close
similarities to human malignancies, we used a nonhuman primate model to
study the pathogenesis of simian immunodeficiency virus
(SIV)-associated malignancies. Specifically, we investigated (1) the
presence of the SIV genome in tumor cells, (2) the presence of
coinfecting viruses, and (3) the presence of a rearrangement of the
immunoglobulin and c-myc genes. We observed 5 cases of non-Hodgkin's lymphomas (4 of B- and 1 of T-cell origin) among 14 SIV-infected cynomolgus monkeys. No c-myc translocation was observed in the tumors, whereas B-cell lymphomas were characterized either by a monoclonal (in 2 of 4) or by an oligoclonal (in 2 of 4) VDJ
rearrangements of the immunoglobulin heavy chain gene. Molecular,
biological, and immunological analyses did show the presence of
infectious SIV in the tumor cells of 1 T-cell and 2 oligoclonal B-cell
lymphomas. Neither Simian T-lymphotropic nor Epstein-Barr viruses were
detectable, whereas Simian herpes virus Macaca fascicularis-1
was detectable at a very low copy number in 3 of 4 B-cell lymphomas;
however, only 1 of these also harbored the SIV genome. These results
support the possibility that SIV may be directly involved in the
process of B or T lymphomagenesis occurring in simian acquired
immunodeficiency syndrome.
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INTRODUCTION |
THE INCIDENCE of non-Hodgkin's
lymphomas (NHLs) in patients infected with the human immunodeficiency
virus (HIV) has increased since the first report describing a high
frequency of high-grade B-cell lymphomas in HIV-1-infected homosexual
men1,2; therefore, the appearance of B-cell lymphomas was
included among the diagnostic criteria of the acquired immunodeficiency
syndrome (AIDS).3 It is expected that the frequency of
lymphomas will further increase among HIV-positive patients as they
live longer following antiretroviral therapy.4,5
Immunohistological studies have proved that most of these NHLs are
high-grade B-cell lymphomas characterized by a heterogeneous morphology
and by an unusual distribution in extranodal sites. Epidemiological
cofactors have not yet been identified, and the pathogenesis of the
AIDS-related lymphomas is not yet completely understood, although the
role of Epstein-Barr virus (EBV) as well as of chromosomal
translocations involving the c-myc locus has been reported in
numerous studies.6
Nonhuman primates experimentally infected by the simian
immunodeficiency virus (SIV) have been widely used to study the
pathogenesis of AIDS. SIV infection induces in macaques a severe
immunosuppression that mimics the course of HIV infection in
humans.7 Like in human HIV infection, an increased
frequency of B-cell lymphomas has been observed by Feichtinger et al in
immunosuppressed monkeys infected with SIV.8 Because of
their clinical, morphological, and immunological characteristics, these
malignancies closely resemble the EBV-associated lymphomas in
HIV-infected patients. EBV-like herpesviruses have been isolated from
several nonhuman primate species but, with the exception of few cases
of lymphomas in a Macaca mulatta9 and in a
baboon,10 they were not usually associated with any known
disease of monkeys. However, experimental infection of cottontop tamarins with EBV gives rise to B-cell lymphomas containing multiple EBV genomes, histologically similar to the human posttransplant lymphomas.11 An EBV-like B-lymphotropic Simian herpesvirus
termed herpesvirus Macaca fascicularis-1 (HVMF-1) showing
variable homology with several regions of the EBV genome has been
recently identified12 and found to be associated with
lymphomas in SIV-infected monkeys.13 Nevertheless, like HIV
in human B-cell lymphomas, the SIV genome has never been found in tumor
cells from these monkey lymphomas.
During the course of studies in Macaca fascicularis
experimentally infected with SIV, we observed 5 cases of high-grade
non-Hodgkin's lymphoma. Four of them were large B-cell lymphomas of
the centroblastic type, and 1 was a peripheral, pleomorfic,
small-medium-sized T-cell lymphoma. In the attempt to understand the
lymphomagenic processes in the SIV/Macaca model, we have analyzed the
tumor for the presence of SIV and for coinfecting viruses such as
Simian T-lymphotropic virus type 1 (STLV-1), EBV, HVMF-1;
for the clonality of the VDJ rearrangement of the immunoglobulin heavy
chain (IgH) gene and for the chromosomal translocation of the
c-myc locus.
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MATERIALS AND METHODS |
Animals.
Adult cynomolgus monkeys (Macaca fascicularis), originated from
the breeding colony of the Istituto Superiore di Sanità, were
housed individually in stainless steel cages according to the European
guidelines (EEC, Directive No. 86-609, November 26, 1986) at a constant
room temperature (24 ± 2°C) and humidity level (60 ± 5%) on
a 12-hour-light/12-hour-dark cycle. At regular intervals of time and
after mild sedation of the monkeys (Ketamine HCl, 10 mg/kg;
Parke-Davis, Milan, Italy), weight and rectal temperature were
recorded, clinical examinations performed, and blood collected from the
femoral vein. All experimental procedures were done according to the
institutional guidelines "Care and use of laboratory animals" (D.L. publication No. 116, 27, January 1992, Italian Ministry of
Health). Animals whose conditions of life were not acceptable were
killed by intracardiac injection of Tanax (0.5 mg/kg; Hoechst, Frankfurt, Germany).
Viruses, virus isolation, and titration.
The 5 monkeys (no. 4, 8503, OD5, 208, and S1) that developed a
lymphoproliferative disease were part of a study14 in which 14 Cynomolgus monkeys of either sex were inoculated intravenously with
10 to 20 MID50 of either SIVmac251/32H15 grown
in human T cells (provided by M. Cranage and P. Greenway, PHLS Center
for Applied Microbiology and Research, Porton Down, UK, through the
Program EVA of the EC program on AIDS research directed by H. Holmes)
or SIVmac2517 grown in monkey peripheral blood mononuclear
cells (PBMC; provided by A.M. Aubertine, Institut National de la
Santè et de la Recherche Medicale, Strasbourg, France). Two
monkeys (no. 4 and 8503) had a history of immunization with a whole
formalin-inactivated SIV. The other animals (no. OD5, 208, and S1)
were naive monkeys inoculated with the same strains of SIV. In the
present study, both the vaccinated as well the naive-infected monkeys
are referred to as infected monkeys.
For virus isolation, Ficoll-Paque purified (Pharmacia, Uppsala, Sweden)
PBMC (4 × 106) were cocultured with human CEMX174 cells
(1 × 106)16 (obtained through the AIDS
Research and reference Reagent Program, Division of AIDS, NIAID,
National Institutes of Health; courtesy of Dr Peter Cresswell) for 30 to 40 days in RPMI 1640 containing 10% fetal calf serum (FCS) and
antibiotics. Cultures were fed twice a week and scored for syncytia
formation; supernatants were tested for the presence of p27 by an
antigen capture assay (SIV p27 Core Antigen; Coulter, Hialeah, FL) and
for reverse transcriptase activity as described
previously.17
Tumor tissues and the spleen of each monkey were mechanically minced
with a dounce, resuspended in complete medium, clarified by
centrifugation (6,000 rpm at 4°C for 20 minutes) and stored at
 152°C. Cell-free supernatants were used to determine the virus titer on C8166 human T cells that form syncytia upon infection with
SIV.
Detection of anti-SIV antibodies.
Antibody titers to SIV proteins were determined by end-point plasma
dilutions using an HIV-2 enzyme-linked immunosorbent assay (ELISA)
(Elavia II, Diagnostic Pasteur, Paris, France) because the coated
antigens, derived from HIV-2 infected cells, are recognized by anti-SIV
antibodies.
Lymphocyte subsets determination.
Lymphocyte subsets were evaluated by direct immunofluorescence using
R-phycoerithryn or fluorescein-labeled monoclonal antibodies (MoAb)
directed against human CD2, CD20, CD4, and CD8 cell surface markers
(DAKO, Glostrup, Denmark). Citrated whole blood (100 µL) was
incubated for 30 minutes at 4°C with the MoAb (10 µL each) and then
lysed with lysis buffer (Becton Dickinson, Palo Alto, CA). After being
washed twice with phosphate-buffered saline (PBS) containing 2.5% FCS,
cells were resuspended and fixed in PBS pH 7.4 containing
paraformaldehyde 1% (wt/vol). Ten thousand lymphocytes were gated from
leukocytes based on forward and 90° light scatter and analyzed for
each sample by using a FACScan cytometer (Becton Dickinson).
Histology and immunohistochemistry.
Autopsies were performed immediately after spontaneous death or
sacrifice, and tissue samples were fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned at 5 µ, and stained with
hematoxylin-eosin and Giemsa; additional paraffin sections were
processed for in situ hybridization analysis. Care was taken to ensure
that samples of lymphoma tissues did not contain significant amounts of
soft tissues or other structures. Other tissue aliquots were frozen in
liquid nitrogen and stored at 80°C. Cryostat sections were
immunohistochemically stained using mouse monoclonal primary antibodies, anti-mouse secondary antibody conjugated with biotin, and a
final avidin-peroxidase color development stage (PK 4002; Vector
Laboratories, Burlingame, CA) as described elsewhere.18 Positive and negative controls and isotype-matched antibodies were also
included in each experiment. The following antibodies were used to
establish the phenotype of the lymphomas according to the
manufacturer's specifications: CD20-L26, CD3-UCHT1, CD4-MT310, CD8-DK25, CD68-PGM1 (Dakopatt, Glostrup,
Denmark).
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Table 1.
Clinical and Immunological Status of Lymphoma-Bearing
Monkeys (4, 8503, OD5, 208, S1) Compared With That of Monkeys Which Did
Not Develop Lymphomas (C1, 8302, H8, OD3, 203)
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| Fig 1.
SIV-associated lymphomas.
Sections were stained with hematoxylin and eosin (A, B, and C),
immunostained with anti-CD3 MoAb (D) and anti-CD20 MoAb (E, F), and
hybridized with the SG5 SIV gag oligoprobe (G, H, I). Monkey S1
(A, D, and G) is a peripheral CD3+ T-cell lymphoma (D);
CD3+ cells (yellow) show a positive in situ hybridization
signal (brown) (G). Monkey 8503 (B, E, and H) is a centroblastic
CD20+ B-cell lymphoma (E) showing a negative in situ
hybridization signal (H). Monkey OD5 (C, F, and I) is a centroblastic
CD20+ B-cell lymphoma (F) showing a positive in situ
hybridization signal (I).
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| Fig 2.
Immunohistology of the retro-orbital centroblastic B-cell
lymphoma arising in monkey 4. Sections were stained with
hematoxylin-eosin (A) and immunostained with anti-CD20 MoAb (B).
Immunohistochemical and in situ hybridization (brown) double-staining
of CD20+ cells (yellow ring) (C) and of
CD68+ cells (orange ring) (D) displaying intracellular
SIV genome signals (dark brown).
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| Fig 3.
VDJ-IgH PCR analysis of DNA samples from lymphoma tissues
of SIV-infected monkeys. Lanes 1 through 4 are from monkeys 4, 208, OD5, and 8503, respectively. A spontaneous nasal non-Hodgkin's B-cell
lymphoma from uninfected monkey (lane 5), Rajii cells (lane 6), SL-691,
an in vitro established Simian Lymphoblastoid B-cell line (lane 7), and
monkey PBMC (lane 8) were also included as controls.
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| Fig 4.
PCR analysis of high-molecular-weight DNA extracted from
lymphoma tissues of infected monkeys. Primers and probes amplifying gag (A) and env (B) regions of the SIV genome were used
as described in Materials and Methods. Lanes 1 through 5 are samples of
monkeys 4, 208, OD5, 8503, and S1, respectively. DNA samples derived
from SIV chronically infected HUT 78 cells (lane 6) or HUT 78 uninfected (lane 7) cells were included as positive or negative
control, respectively.
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| Fig 5.
Southern blot analysis of the c-myc locus in
SIV-associated lymphomas. DNA samples were digested with EcoRI
and hybridized with the pMC41-3DC probe covering the 3rd exon of the
c-myc locus. Lanes 1 through 5 are samples of monkeys 4, 208, OD5, 8503, and S1, respectively. The Raji cells (lane 6) carrying the
germ line band (12.8 Kd) and a higher rearranged band (<12.8 kd) and
monkey PBMC (lane 7) were included as controls.
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| Fig 6.
Nested HVMF-1 DNA-PCR analysis of monkey lymphomas. Lanes
1 through 5 are samples of monkeys 4, 208, OD5, 8503, and S1,
respectively. Furthermore, samples from a spontaneous nasal
non-Hodgkin's B-cell lymphoma from uninfected monkey (lane 6), from
SL-P1 and SL-691 cells (in vitro established Simian Lymphoblastoid cell
lines; lane 7 and 8, respectively), and from HUT 78 human T-cell line (lane 9) were included as controls.
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DNA-polymerase chain reaction (PCR) analysis of SIV, STLV-1, EBV,
and HVMF-1.
Genomic DNA was extracted from tissues following the phenol-chloroform
method by precipitation with 3 mol/L sodium acetate and cold ethanol.
DNA was then amplified with primers (PCO3/PCO4) specific for a 110-bp
sequence of human  -globin gene to assess its competence for
PCR.19 To detect the SIV genome, DNA PCR was performed
using primers amplifying a 202-bp region of the gag gene
(Gagf3, nt 1297-1316, 5 -GGTGCATTCACGCAGAAGAG-3, Gagr4, nt 1498-1478, 5-GTTCTCGGGCTTAATGGCAGG-3) or a 368-bp region of the env gene
(EnvLfl, nt 8153-8176, 5-ATAAAAGGGGTCTTTGTGCTAG-3; EnvLr2, nt
8519-8496, 5-TCAACCTTTCGCTCCCACTCTTGC-3) of the SIVmac251 genome
(GenEMBL access number M19499). PCR was performed in 100 µL of
reaction mixture containing 1 µg of DNA, 200 µmol/L of each of the
four deoxynucleotide triphosphates, 60 pmol/L of each primer, 2.5 U Taq
polymerase (Amplitaq, Perkin Elmer, Norwalk, CT), 50 mmol/L of KCl, 10 mmol/L of Tris-HCl (pH 8.3), and 1.5 mmol/L of MgCl2. After
a denaturation step at 94°C for 5 minutes, DNA was amplified by using
the DNA thermal cycler 9600 (Perkin Elmer Cetus) for 35 cycles, each at
95°C for 90 seconds, at 55°C for 90 seconds, and at 72°C for 2 minutes with a final extension step at 72°C for 10 minutes.
Conserved pol region of the human T-lymphotropic virus type 1 genome, which shares 90% genomic identity with the pol region of STLV, was chosen for PCR amplification using primers and a probe
described elsewhere.20 PCR started with 5 minutes
denaturation step at 94°C followed by 30 cycles each at 94°C for 90 seconds, at 50°C for 90 seconds, and at 72°C for 2 minutes and by a
final extension step at 72°C for 10 minutes.
EBV was analyzed using oligonucleotide primers common for EBNA2 type A
and B sequences (EBVf, nt 48170-48189, 5-AGGCTGCCCACCCTGAGGAT-3; EBVr, nt 48339-48320, 5-GCCACCTGGCAGCCCTAAAG-3; EBVp, nt 48262-48280, 5-GTTGCCGCCAGGTGGCAGC-3) derived from human EBV sequences (GenEMBL access number V01555, K03333, and K3332). PCR was performed in a
100-µL reaction mixture with the same composition as it was for the
SIV gag PCR, started with a denaturation step at 95°C for 4 minutes followed by 30 cycles each at 94°C for 45 seconds, at 62°C
for 45 seconds, at 72°C for 90 seconds with a final extension step at
72°C for 10 minutes.
A nested PCR was used to detect the EBV-like HVMF-1 genome. The outer
set of primers (Ws and Was-3) has already been published by Li et
al.12 The inner set of primers (Ws-4,
5-CAGTTATTCTGCTCAGCCCAC-3; Was-2, 5-CCAGACTAGACCCCAGGTTCC-3), and
the probe (Wdp, 5-AAGGTGCAGGCACAACAGCC-3) were chosen from the
nucleotide sequence data reported in the GenEMBL access number X77781
HVMFINA5. The first round of PCR was performed by using the same
mixture composition and cycling conditions as were for the SIV
gag PCR. The final product of the first PCR (10 µL) was
subjected to a second round of amplification by using the same buffer
composition. PCR started with a 5-minute denaturation step at 94°C,
followed by 30 cycles each at 94°C for 30 seconds, at 55°C for 30 seconds, at 72°C for 1 minute, and a final extension step at 72°C
for 10 minutes.
Ten microliters of the PCR products were electrophoresed on 1.5% to
2% agarose gel, transferred to filter (Nytran-N; S&S, GmbH, Germany),
and hybridized with 5-end 32P-labeled-specific
oligoprobes.
Rearrangement of the c-myc locus and of the VDJ region of IgH gene.
To analyze the c-myc gene, 20 µg of DNA from lymphoma tissues
were digested with HindIII and EcoRI restriction
enzymes, separated onto agarose gel, transferred to Nytran filters, and
hybridized with a 32P-labeled 1.6-kilobase (kb) pMC41-3RC
probe21 representing the third exon of the
c-myc locus. The c-myc translocation could be identified when bands other than the germ line c-myc
configuration (12.8 kb) were present.
A seminested DNA-PCR amplification of rearranged VDJ
fragments of IgH gene was performed by using primers
corresponding to the human complementarity determining region 3 (CDR 3)
as described elsewhere.22 Final PCR products (20 µL) were
electrophoresed onto a 12% polyacrylamide gel, and then DNA was
stained by ethidium bromide and visualized by ultraviolet light.
In situ hybridization and immunostaining.
Paraffin sections were deparaffinized for 20 minutes at room
temperature with xilene, rehydrated with ethyl alcohol, and washed with
distilled water for 5 minutes and with PBS for 10 minutes. Standard in
situ hybridization was done by using a probe homologous to the
gag region of the SIV genome (SG5p, nt 1470-1502 5-AATAGGTGGTAACTATGTCCACCTGCCATTAAG-3) or a SIV-unrelated oligo probe
(SK19, nt 1134-1174 gag, HIV-1; GenEMBL access number K02013) that was
used as the negative control. After protease digestion (10 µg/mL for
15 minutes at 37°C), the slides were treated for 5 minutes with cold
paraformaldehyde 4% (wt/vol) and then sequentially washed with cold
PBS and 20 × SSC (1 × SSC = 0.15 mol/L of NaCl, 15 mmol/L of
sodium citrate) for 10 minutes at room temperature. The excess of
liquid was soaked out, and 50 µL of the prehybridization solution
(4 × SSC, 50% formamide, 1× Denhardt's, 5% dextran sulfate,
0.5 mg/mL salmon sperm) was added and the slides covered with a cover
slip. The samples were then incubated for 2 hours at 42°C (Omnigene
Temperature Cycler, Hybaid Lt, Middlesex, UK). The probe was labeled
with digoxigenin (dig)-dUTP using the 3-tailing method according to the manufacturer's instructions (3-labeling DNA kit; Boehringer Mannheim, Indianapolis, IN). The dig-labeled probe was added to the
same solution and, after a denaturation step at 90°C for 10 minutes,
the hybridization was performed at 42°C overnight. After hybridization and washing, the slides were incubated with a sheep anti-dig MoAb conjugated with alkaline phosphatase (dilution 1:400) for
2 hours at room temperature. The probe/target complex was visualized by
incubation with chromogen (NBT/Xphosphate; Boehringer Mannheim). The
hybridization signal was evident as a dark brown precipitate. For
immunohistochemistry, after the final wash of the in situ hybridization
procedure, the slides were overlaid with PBS containing 10% FCS and
the immunoreaction was performed as described above.
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RESULTS |
Clinical and immunological status of SIV-infected monkeys developing
lymphomas.
The individual histories of the monkeys with lymphomas (4, 8503, OD5,208, and S1) compared with those of monkeys without lymphomas (C1,
8302, OD3, 203, and H8) are summarized in Table 1. Because the strains
of viruses used to infect the animals were almost identical in their
pathogenic potential, the monkeys have been matched according to the
treatment (4 and 8503 v C1 and 8302; OD5 and 208 v OD3
and 203; S1 v H8). All but 1 monkey (monkey S1) had a mean
survival time longer than that observed in a comparable group of 4 monkeys that did not develop lymphomas (109.5 weeks v 92 weeks
after infection, respectively). Monkey S1 did not seroconvert although
virus was frequently recovered from PBMC. This monkey appeared healthy
before SIV inoculation; however, soon after this, a rapidly increasing
mass in the right-side ocular bulb was observed.
Monkeys 4, 8503, OD5, and 208 showed a marked depletion of
CD4+ T cells and an altered CD4/CD8 ratio similar to that
observed in the comparable tumor-free SIV-infected monkeys (C1, 8302, OD3 and 203; Table 1). Immunodeficiency was not observed in monkeys S1
(with lymphoma) and H8 (without lymphoma), likely due to the very short
period of time between infection and death. Monkey OD5 had an abnormal
level of CD2+ and CD8+ cells, likely due to the
splenectomy performed at 1 year after infection or to the presence of
cutaneous inflammatory necrotic lesions.
Immunohistology and VDJ-IgH clonality analysis of lymphomas.
The tumor masses histologically diagnosed according to the Kiel
classification23 were classified as large-cell NHL
centroblastic type (monkeys 8503, OD5, 208, and 4; Fig 1B through C and
Fig 2A; monkey 208 not shown) and peripheral, pleomorphic,
small-medium-sized cell NHL (monkey S1; Fig 1A). As shown in Table 2,
lymphomas displayed a different localization independent of the
phenotype. Four out of the 5 tumors (monkeys 8503, OD5, 208, and 4)
stained positive for the B-cell marker CD20 and were therefore
classified as B-cell lymphomas (Table 2, Fig 1E through F, and Fig 2B;
monkey 208 not shown), whereas the tumor tissue from monkey S1 was
positive for the CD3 cell-surface marker and was classified as a T-cell lymphoma (Table 2; Fig 1D).
A molecular study was performed on SIV-associated NHLs specimens to
determine the clonality of the B-cell lymphomas. Figure 3 shows the
single band that represents the monoclonal rearranged VDJ
region, which was detected in tumor samples of monkeys 208 and 8503. These were therefore classified as monoclonal lymphomas. Two bands plus
an additional band below 70 bp and at least three other bands (size 70 to 120 bp) were observed in the PCR products of amplified DNA of
monkeys OD5 and 4, respectively, that were classified as oligoclonal
lymphomas.
Detection of SIV in B- and T-cell lymphomas.
The presence of the SIV genome was demonstrated in 3 of 5 lymphomas
(monkeys 4, OD5, and S1) by DNA-PCR analysis, using primers amplifying
fragments of the gag and env regions of the SIV genome (Fig 4). In one case (monkey 8503), a faint band was observed when the
gag gene was analyzed.
Paraffin sections were then analyzed by in situ nucleic acid
hybridization for the SIV genome. A clear SIV-specific signal was found
in cells of lymphoma tissues of monkeys S1, OD5 (Fig 1G and I,
respectively) and 4 (Fig 2C) but not in those of monkeys 8503 (Fig 1H)
and 208 (not shown). In the case of the T-cell lymphoma (monkey S1) the
SIV-bearing tumor cells were numerous (about 60% to 80%) and
homogeneously distributed throughout the section, whereas in the two
cases of B-cell lymphomas (monkeys 4 and OD5), they appeared to have a
less homogeneous distribution. In addition, the number of SIV-positive
areas appeared to be more numerous in the tissue of monkey 4 than in
that of monkey OD5.
To verify the phenotype of the SIV-positive cells, an
immunohistochemical analysis was performed at the end of the in situ hybridization procedure. SIV-positive cells were stained positively for
the CD3 marker in monkey S1 (Fig 1G) or for the CD20 marker in monkeys
OD5 and 4 (Fig 1I and Fig 2C, respectively). Double-staining experiments performed on tissue sections of monkey 4 showed the presence of a low number of CD68-positive cells displaying also positivity for the SIV genome (Fig 2D).
The lymphomatous tissues of monkeys 4, OD5, and S1 that resulted
SIV-positive by DNA-PCR and by in situ hybridization analyses did
reveal a viral load higher than that found in the spleen of the same
monkey. In particular, in line with the number of the SIV-positive
cells, the titer of the virus present in tumor samples of monkey S1 was
about from two to four log higher than that detected in tissues of
monkeys 4 and OD5 (Table 3).
C-myc translocation and detection of STLV, EBV, and HVMF-1 genomes in
monkey lymphomas.
Genomic DNA extracted from the lymphomatous tissues of all monkeys was
analyzed by Southern blot hybridization for the presence of the
translocation of the c-myc locus. In all cases, no bands other
than the 12.8 kb of the germ line were observed (Fig 5).
We next examined the possibility that coinfecting viruses could be
present in the lymphomatous tissues of the monkeys. All monkeys were
negative for STLV-1 and EBV-like sequences as determined by DNA-PCR
analysis (data not shown), whereas 3 of 4 B-cell lymphomas were
positive by nested PCR for HVMF-1 sequences (Fig 6).
| |
DISCUSSION |
Lymphoid neoplasms, the most frequent tumors observed in nonhuman
primates,24-26 have evoked a great interest because their anatomic and physiological features closely resemble those observed in
humans. In this study we have described 5 cases of NHL in 14 monkeys
experimentally infected with SIV. The immunodeficiency was a hallmark
of monkeys affected by B-cell lymphomas, whereas CD4+
T-cell depletion was not observed in the monkey developing the T-cell
lymphoma. Interestingly, SIV sequences were present at levels
detectable by in situ hybridization within the cells of 2 cases of the
B- and in one case of the T-cell lymphoma. To the best of our knowledge
this is the first demonstration that B- and T-cell simian lymphomas can
harbor SIV sequences in the tumor cells, although SIV was also detected
in some infiltrating macrophages. Several attempts have been made to
detect HIV genome in AIDS-associated B-cell malignancies, but the
results were not conclusive.27,28 Furthermore, the
phenotype of the "infected cells" was not determined. Similarly,
in neoplasms from SIV-infected monkeys, some non-neoplastic cells,
mainly macrophages and multinucleated cells, were occasionally found
positive for the presence of p27 SIV protein. Moreover, even in in
vitro cell lines derived from monkey lymphomas, no reverse
transcriptase activity has been detected.8 In addition, we
observed that the SIV titer in lymphomatous tissues was higher than
that found in the spleen, a macrophage-rich tissue known to be a
relevant source of the virus. However, this does not exclude the
contribution of infected cells, other than "transformed" B cells
or T cells, in the SIV viral load of tumor tissues.
It has been suggested that monoclonal or oligoclonal NHLS in
human29 and simian AIDS30 as well as in murine
retrovirus-induced immunodeficiency syndrome lymphomas31
progress from an initial polyclonal expansion of activated B
cells.32 Although lymphomagenesis in vivo might depend on a
complex cascade of events also involving a complex network of cellular
interactions,33-38 it is likely that "transformation"
in some cases can be initiated through chronic antigen stimulation,
heightening the probability of chromosomal alterations or genetic
lesions.39 Indeed, in vitro studies on human B lymphocytes
have suggested that HIV or its transactivating protein Tat may have
transforming properties because they may mediate c-myc
expression that, in some cases, seems to be closely linked to the
development of B-cell neoplasia.40-43 Nevertheless, as
already reported for a group of human AIDS-associated
lymphomas,44 c-myc rearrangement was not detected
in the lymphomas we have observed in infected monkeys, including those
harboring the SIV genome. Thus, it seems that alternative molecular
pathways may be involved in the transformation processes.
Interestingly, we have observed that only B-cell lymphomas positive for
the presence of the SIV genome had an oligoclonal rearrangement of the
VDJ region of the IgH gene. Taking into consideration the oligoclonal
expansion of the lymphocytes, one could suppose that a B cell at one
time became susceptible to SIV infection45-47 with
resultant further proliferation. In line with this hypothesis, we
observed that only some B cells were infected by SIV. The presence of
the SIV genome in simian tumor cells might suggest a possible transforming potential of SIV through insertional mutagenesis, as
already described in AIDS-related T-cell lymphoma48,49 and in some virus-infected T-cell neoplasms in rhesus.50,51
Transformation may occur also by a fusion process between lymphocytes
and infected macrophages as shown by the CD3 and CD14 double
immunostaining of tumor cells of AIDS-related lymphoma.52
Although such analysis was not performed on our samples, we observed
giant cells doubly positive for CD3/SIV, CD20/SIV, and CD68/SIV in the
case of T-cell lymphoma and in two cases of B-cell lymphomas,
respectively.
Because lymphomagenesis is thought to be a multistep process, we
analyzed the tumors for the presence of coinfecting viruses that have
been reported to be present in human or in simian
malignancies.53-55 All lymphomas were negative for STLV and
EBV, but three of them (all of B-cell origin) were associated with
HVMF-1 infection. However, HVMF-1 sequences were not detected in one
(monkey OD5) of the two cases of SIV-positive lymphomas. Its detection
required a nested PCR analysis, thus indicating a low HVMF-1 viral load as compared with the SIV load that was detected by a single run of PCR
and in situ analysis.
In the same species of monkeys (Cynomolgus), Feichtinger et al
originally reported a high incidence of lymphomas in a group of monkeys
infected by SIVsm but not in a comparable group of monkeys infected by
HIV-2.8 Whether this observation means that
lymphoproliferative diseases depend on the pathogenic potential of the
virus strain used for infection7,56 remains to be shown. In
the same model HVMF-1 has been associated with an high incidence of
B-cell lymphomas in SIVsm-infected monkeys. However, HVMF-1 was
detected not only in tumor-bearing infected monkeys but also in
HIV-2-infected and noninfected monkeys that did not develop lymphomas.57 Thus, HVMF-1 infection per se may not be fully responsible for the development of lymphomas and may require the presence of an immunodeficient state. Consistent with this observation, human EBV-like Rhesus lymphocryptic virus (LCV) can reproduce in
monkeys the key aspects of the EBV infection in humans but was unable
to induce tumors in naive pathogen-free animals up to 24 months after
infection.58 On the other hand, AIDS-associated polyclonal
B-cell lymphomas with no evidence of c-myc rearrangement and
negative for EBV have been reported.43 Thus, according to our data, neoplasia may develop in the absence of a concomitant HVMF-1
infection in immunosuppressed monkeys. Nevertheless, we cannot provide
further evidence against or in favor of a direct involvement of HVMF-1
in lymphomagenesis because our data are based on one only case of
SIV-positive, HVMF-negative simian B-cell lymphoma. A serological and
molecular survey to asses the spread of HVMF-1 infection in our
Cynomolgus colony and the incidence of neoplasia is currently in
progress. In this framework, we have already observed some cases of
lymphomas in SIVmac-infected monkeys that were certified to be
serologically negative for STLV, type-D retroviruses, cytomegalovirus,
and EBV nuclear antigen (Shamrock Limited, England;
personal communication and unpublished results, May 1995). It is quite
clear that both simian models of SIV-associated neoplasia, although
different in some aspects, may represent the different subtypes of
AIDS-associated malignancies.59 Although from our data we
cannot definitely prove the transforming potential of SIV, it seems
reasonable to suggest that, under certain circumstances, SIV may play a
role in lymphomagenesis. Thus, monkey lymphoma models might be useful
to study the pathogenesis and treatment of immunodeficiency-associated
tumors.
| |
ACKNOWLEDGEMENTS |
The authors gratefully acknowledge Dr B. Ensoli for helpful discussion
and comments on the manuscript. We also thank S. Mochi for the
synthesis of primers and probes, to A. Carè for providing the
pMC41-3RC used to detect the c-myc translocation, and to A. Lippa and F.M. Regini for the excellent editorial assistance. We also
thank F. Varano, A. Cesolini, F. Incitti, S. Fazzitta, M. Chiodi, R. Marinelli, A. Marini, P. Di Zeo, and S. Alessandroni for handling the
Cynomolgus colony.
| |
FOOTNOTES |
Submitted October 6, 1997;
accepted February 3, 1998.
Supported by a grant from the "Animal Model Development Project"
of the Italian Ministry of Health, Istituto Superiore di Sanità,
Rome, Italy (to P.V.) and the IX AIDS Research Project, ISS No. 9403-13 (to C.D.B.).
Address reprint requests to Fausto Titti, PhD, Laboratory
of Virology Istituto Superiore di Sanità, Viale Regina Elena,
299, 00161 Rome, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract