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*
Department of Pathology and Comprehensive Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016; and
Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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16+ CD4+ T cells,
thereby acquiring T cell help necessary for RCS growth. Other strains
of B cell lymphoma-prone mice include Mtv29+
C57L and MA/MyJ, and the Mtv29-
Mtv7+-recombinant inbred strain, SW x
J-1. The lymphomas of these mice produce similar mouse
mtv-vSAg-encoding mRNA, as characterized by Northern blotting, PCR, and
RNase protection. A 1.8-kb mRNA in C57L/J and MA/MyJ lymphomas
hybridized with an Mtv29-specific oligonucleotide,
whereas SW x J-1 lymphomas produced 1.8-kb transcripts
hybridizing with an Mtv7-specific oligonucleotide.
Similar META-env-initiated transcripts were absent from LPS-activated B
cells from any strain examined but were detected in Peyer’s patch RNA
from SJL mice. Like typical SJL-derived RCS, all these lymphomas
stimulated syngeneic CD4+ T cells and V16+ T
hybridoma cells. Immunohistochemical staining of primary tumors showed
the presence of peanut agglutinin binding (PNA+) highly
mitotic lymphoblasts, suggesting their GC derivation. The findings
indicate that this novel mRNA for Mtv29 is present in B
cell lymphomas from several Mtv29+ mouse
strains. Additionally, this is the first description of the ability of
Mtv7 to produce transcripts that are controlled and
spliced identically to those of Mtv29 and that are
expressed in SW x J-1, I-As+, lymphomas that also
stimulate V16+ T cells. Our results suggest an important
role for mouse mtv-vSAgs and V16 T cell stimulation in the
development of GC-derived murine B cell
lymphomas. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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16+ T cells via
expression of an endogenous mouse mammary tumor virus
Mtv29-encoded viral superantigen, vSAg29 (2, 3). The cytokines produced by these activated
CD4+ T cells promote growth of the lymphomas
(4, 5). In fact, in the absence of
CD4+ T cell help, SJL lymphomas are unable to
grow in vivo (6, 7). More recently, Ponzio and coworkers
found that the lymphomas that arise in another
Mtv29+ mouse strain, C57L/J, have
properties similar to SJL-derived RCS, including the requirement for
help from CD4+ T cells for growth of a
transplantable line developed from a primary lymphoma (8).
Moreover, a third conventional inbred
Mtv29+ strain, MA/MyJ, also was found to
develop B cell lymphomas (W. J. Simmons, N. M. Ponzio,
V. K. Tsiagbe, W. Beamer, G. Inghirami, and G. J. Thorbecke,
manuscript in preparation). In addition, some of the SW x J recombinant inbred (RI) strains, bred from SJL and SWR mice, develop B cell lymphomas by the age of 14 mo, including strains SW x J-1 and SW x J-7 (W. J. Simmons, N. M. Ponzio, V. K. Tsiagbe, W. Beamer, G. Inghirami, and G. J. Thorbecke, manuscript in preparation). Although like SJL, SW x J-7 possesses only Mtv29 and Mtv8 in its genome, SW x J-1 has Mtv7 and 8, but does not have Mtv29 (9). Therefore, in the present study we have determined whether the lymphomas of SW x J-1 produce mouse mammary tumor virus (MMTV)-encoded vSAg(s) and, like those derived from SJL mice, stimulate the proliferation of syngeneic CD4+ T cells.
Well-characterized vSAg mRNAs, such as those for Mtv8 and Mtv9, initiate in the 5' long terminal repeat (LTR), splice out the pol, gag, and env transcripts, and continue in the 3' LTR (10, 11, 12). However, the mRNA for Mtv29 encoding the vSAg in SJL lymphomas initiates in the env (META) region, undergoes splicing in the 3'-env region, and continues through the 3' LTR (13). The present studies suggest that a similar Mtv29-encoded product, characterized by Northern blotting, PCR, and RNase protection assays, is produced in the B cell lymphomas arising in C57L/J and MA/MyJ mice. Moreover, in the lymphomas from the SW x J-1 strain, which lack Mtv29, a similar env-initiated mRNA for Mtv7 is identified.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J (Mtv8 and Mtv29 positive), male SWR (Mtv7, Mtv8, Mtv14, Mtv17, and Mtv31 positive), female DBA/2 (Mtv1, Mtv6, Mtv7, Mtv8, Mtv11, Mtv13, Mtv14, and Mtv17 positive), C57L (Mtv8, Mtv9, Mtv11, Mtv17, and Mtv29 positive), LAF1 (Mtv6, Mtv8, Mtv9, Mtv11, Mtv13, Mtv17, Mtv23, and Mtv29 positive) MA/MyJ (Mtv8, Mtv9, Mtv17, Mtv29 and Mtv43 positive), and female NOD mice (Mtv3 and Mtv17 positive; Ref. 14, 15) were purchased from The Jackson Laboratory (Bar Harbor, ME). (SJL x SWR)F1 mice were bred in the animal facilities of New York University School of Medicine (New York, NY). Lymphoma cell lines were derived from primary tumors that developed in aging mice of different strains and designated as follows: SJL-derived, cRCS-2, cRCS-3, cNJ117, and cNJ101; C57L-derived, cNJ123 and cNJ120; MA/My derived, cNJ126 and cNJ124. In addition, primary tumors were studied from SW x J-1, lymphomas 326 and 340. The SW x J-1 strain was derived at The Jackson Laboratory, and the mice were generously donated by Dr. W. Beamer. These mice were studied previously for genetic markers (9, 16, 17). The lymphomas examined here were detected in another study in which the lymphoma incidences of the various SW x J RI mouse strains were examined (W. J. Simmons, N. M. Ponzio, V. K. Tsiagbe, W. Beamer, G. Inghirami, and G. J. Thorbecke, manuscript in preparation). Unlike other SJL-derived lymphomas, NJ101 arose in a 2-year-old SJL mouse that was receiving chronic treatment with anti-CD4 mAb (18). It differs from other SJL tumors by its phenotype (IgM+), absence of vSAg29 expression, and an inability to stimulate syngeneic SJL T lymphocytes (2, 3).
Hybridomas with TCR V8.1, TCR V8.2, TCR V11, TCR V12, TCR
V13, and TCR V15 were generously provided by Dr. D. Woodland (St.
Jude Children’s Research Hospital, Memphis, TN); the hybridoma with
TCR V17a was kindly provided by Drs. P. Marrack and J. W. Kappler
(National Jewish Center of Immunology, Denver, CO); hybridomas with TCR
V16 were derived in our laboratory (2, 3).
DNA preparation and PCR
High-m.w. DNA was isolated from cells by using the genomic DNA isolation kit of Promega (Madison, WI) according to the protocol provided by the manufacturer. PCR was performed with reagents provided in the PCR kit from Promega with a DNA thermal cycler 480 (Perkin-Elmer, Foster City, CA). Reaction conditions were as follows: buffer containing 20 mM Tris-HCl, pH 8.4, 2.5 mM MgCl2, 50 mM KCl; 30 cycles of 45 s of denaturation at 94°C, annealing for 1 min at 60°C, and extension for 30 s at 72°C, with a final extension step for 10 min at 72°C. Some PCR products were cloned into the PCR TM 2.1 vector (Invitrogen, San Diego, CA) and sequenced.
RNA preparation and RT-PCR
Total RNA was isolated from cells by using RNA Stat-60 (Tel Test, Friendswood, TX). Pretreatment of RNA with DNase 1 was performed to exclude DNA contamination. cDNA was synthesized by using the Superscript preamplification system (Life Technologies, Gaithersburg, MD). Briefly, 2.5–5 µg of total RNA was mixed with 1 µl (0.5 µg) of oligo(dt) 12–18 and incubated for 10 min at 70°C. The mixture then was placed on ice after addition of 2.5 mM MgCl2, 10 mM DTT, 0.5 mM deoxyribonucleotide triphosphate, reverse transcription buffer (1x), and 200 U of Superscript II reverse transcriptase. cDNA synthesis was performed in 20 µl at 42°C for 50 min. The cDNAs were treated with RNase H for 20 min and 2–5 µl was used for PCR.
Oligonucleotide primers used for PCR were obtained from Gene Link (Thornwood, NY). Their sequences were: RLTR: 5'-CTTACTTAAACCTTGGGAACCGCAAG-3'; R7, 5'-CGAAGCCAACGCGACCCCCATGAG-3'; R8, 5'-CATTATAGCTCATGCCAAAAGTATC-3'; R17, 5'-TTATTATACCTTATGTCAAAGGTATCC-3'; R29, 5'-GCAATTTATCCATGCCAAGTTTACC-3'; L1, 5'-TATCATCACAAGAGCGGAACGGACTC-3'; L2, 5'-TACAGACCCCATTAGAGTTCTGAC-3'; L3, 5'-GAGACGAGTCTGCTCCTCCACGG-3'; META-2: 5'-GAAGGGGTGATAAAAGGCGTATGTG-3'; and META-3, 5'-CCCCTTGGTCAGGGAATGCGCAAGG-3'.
The positions of these primer sequences within the MMTV genome are
indicated in Fig. 1
. The sequence homology between Mtv8 and
Mtv11 (15) and between Mtv7 and
Mtv43 (19) makes it unlikely that PCR performed
with these primers can distinguish between these MMTVs when both are
present in the mouse.
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The LTR-ORF probe was cloned into pBluescript as described
(3, 13). Plasmid DNA was isolated by using a maxi prep kit
purchased from Life Technologies and double digested with
EcoRI and BamHI to recover the LTR-ORF fragment,
separated through 1% agarose gel. The META probe was prepared from
pGEM-3 vector as described in the protocol for RNase protection assay.
The META fragment was recovered from the plasmid by double digestion
with EcoRI and HindIII and separation through 1%
agarose gel. Equal amounts (20 µg) of total RNA samples were
electrophoresed under denaturing conditions in 1.2%
agarose/formaldehyde gels. RNA was transferred onto a nylon membrane
(
probe, GT; Bio-Rad, Richmond, CA) and covalently cross-linked to
the membrane by UV irradiation. The membrane was prehybridized for
3 h at 42°C in prehybridization/hybridization solution (50%
formamide, 2.5% Denhardt’s solution, 6x SSC phosphate/EDTA, 200
µg/ml salmon sperm DNA, 1% SDS) followed by overnight hybridization
at 42°C with random-primed 32P-labeled LTR
probe. The hybridized membrane was washed three times for 15 min each
in 0.1x SSC/0.1% SDS at room temperature followed by a wash for 30
min at 55°C. The washed filter was exposed to x-ray film (Fuji Film,
Tokyo, Japan) at -70°C. To strip the probe, the filter was boiled in
0.5% SDS for 10 min, cooled to room temperature for 10 min, and rinsed
in 2x SSC to recharge the membrane. The stripped filter was hybridized
with random-primed 32P-labeled META probe.
LTR-ORF-specific oligonucleotide probes, 31 and 30 mers for
Mtv29 and Mtv7, respectively, were purchased
from Gene Link (Thornwood, NY). Their sequences were as follows:
Mtv29 probe, 5'-GTAAAGTGCAATTTATCCATGCCAAGTTTAC-3';
Mtv7 probe, 5'-GATCGAAGCCAACGCGACCCCCATGAGTAT-3'. These
oligonucleotide probes were 5'-end labeled with
[
-32P]ATP by polynucleotide T4 kinase and
used for probing the RNA transcripts. The membranes were
prehybridized for 3 h at 55°C in prehybridization/hybridization
solution (6x SSC, 10 mM EDTA, pH 7.5, 2x Denhardt’s solution, 100
µg/ml sheared and denatured salmon sperm DNA, and 1% SDS). This was
followed by overnight hybridization at 55°C with
32P-labeled oligonucleotide probe. The filter was
washed twice for 15 min at 45°C in prewarmed 6x SSC/0.1% SDS
followed by two washes for10 min in 2x SSC/0.1% SDS at room temp. The
filter was exposed to x-ray film at -70°C.
RNase protection assay
PCR was performed with the META-2 (7136–7160) and META-3 (7315–7339) primers with SJL liver DNA and the 204-bp product obtained was cloned into the pGEM-3 vector (Promega). The plasmid was linearized with HindIII, and radiolabeled RNA probe representing positions 7136 through 7339 was synthesized by using [32P]UTP and T3 RNA polymerase. The RNase protection assay was performed according to the manufacturer’s protocol with the RNase protection kit of BD PharMingen (San Diego, CA). Briefly, 10–20 µg of total RNA was hybridized with 4 x 104 cpm of RNA probe overnight at 56°C. Nonhybridized probe was digested with 10 µg/ml RNase A and 65 U/ml RNase T1 for 30 min at 30°C. After extraction with phenol-chloroform and ethanol precipitation, the protected RNA fragments were separated on a 6% polyacrylamide/8 M urea gel and then subjected to PhosphorImaging (Molecular Dynamics, Sunnyville, CA).
Preparation of LPS-activated B cells
Spleen cell suspensions at 106 cells/ml in 20-ml Falcon flasks were stimulated with 50 µg LPS/ml (Escherichia coli 055:B5; Difco Laboratories, Detroit, MI) for 48 h in IMDM (BioWhittaker, Walkersville, MD) containing 10% FCS (Life Technologies) and 0.05 mM 2-ME.
Mixed lymphocyte culture
MLC were prepared in flat-bottom 96-well plates (Costar,
Cambridge, MA) in RPMI 1640 medium (Mediatech, Springfield, NJ),
supplemented with 10% FCS (HyClone, Logan, UT), 2 mM glutamine, 100
IU/ml penicillin, 100 µg/ml streptomycin, and 0.05 mM 2-ME, as
described (20). Responder cells (2 x
105/well) were mixed at various
responder/stimulator ratios with -irradiated lymphoma cells
(5,000–15,000 rad) in 0.2 ml. Cultures were incubated for a total of
96 h at 37°C, the last 16–18 h with 1 µCi
[3H]TdR/well (spec. activity 2 Ci/mmol;
DuPont/NEN, Boston, MA). Cells from triplicate cultures were harvested
onto glass fiber filters (No. 934-AH; Whatman , Maidstone, U.K.) and
counted in a scintillation counter. Data for MLC cultures are expressed
as 
cpm, calculated as cpm of MLC - (cpm of responder cells
alone + cpm of stimulator cells alone).
Stimulation of T cell hybridomas
Under similar culture conditions as for MLC, lymphoma cells were
cocultured with an extensive panel of T-T hybridoma cell lines
(105 cells/well), each expressing a different TCR
V. Plate-bound anti-TCR mAb (H57-597) or anti-CD3 mAb
(145-2C11), and/or PHA (5 µg/ml), were used as positive control
stimuli. Supernatants from replicate cultures were harvested 24 h
after initiation of culture and stored at -20°C. IL-2 concentrations
were measured with an ELISA kit, with the recombinant mIL-2 standard
provided (MiniKit KM-IL-2; Endogen, Woburn, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RT-PCR products from various lymphomas are represented in
Table I. Some 3'-primers were specific
for Mtv29 (R29), for Mtv7 (R7), for
Mtv8 (R8), and for Mtv17 (R17), whereas the RLTR
represents a common region of the 3' LTR sequence (see Fig. 1
). The 5'-primers (L1, L2, and L3) were
common for these MMTVs. The results in Table I show that the detected
transcripts that initiated in the 5' LTR (1.8 kb, detected with L1),
for both Mtv29 and Mtv7, were all 0.7 kb longer
than would be expected for mRNA encoding a traditional vSAg. In
contrast, Mtv8 and Mtv17 each showed typical vSAg
1.1-kb transcripts when L1 was used as the 5'-primer. Mtv8
vSAg mRNA was amplified in lymphomas of C57L and MA/My origin, but not
in SJL RCS lymphomas nor in lymphomas from SW x J-1 (Table I).
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, A
and B). The same is true for the 1.8-kb transcripts of
Mtv7 in the RNA prepared from SW x J-1 lymphomas 326
and 340 (Fig. 2, A and B). Thus, all the 1.8-kb
transcripts in lymphoma cells contain part of the env
region, in agreement with initiation in the env rather than
in the 5' LTR. In contrast, the 1.8-kb band of the RNA from
LPS-activated B cell blasts from the Mtv7+
strain, DBA/2, hybridizes with the LTR, but not with the
META-env probe (Fig. 2). The Mtv29-LTR-specific
oligonucleotide probe hybridizes with each of the 3 Mtv
transcripts in the SJL lymphomas cNJ-117 and cRCS-X and the C57L
lymphoma cNJ123.5 (Fig. 2C), but not with RNA from lymphomas
340 and 326 (not shown), whereas the Mtv7-LTR -specific
oligonucleotide probe hybridizes with RNA from the SW x J-1
lymphomas but not with RNA from cRCS-X (Fig. 2D). Thus, the
SW x J-1 lymphomas exhibit the characteristic mRNAs for
Mtv7 of 4.1-, 2.9-, and 1.8-kb lengths.
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92 bp, starting at the initiation
site at position 7247 (13) and ending at the splice donor
site (SD2, 7338; see Fig. 1). The protected bands of 204 bp most
likely detect env transcripts initiating in the 5' LTR.
Consistent with the presence of transcripts initiated at position 7247
and spliced at position 7338 is the detection of a 1.1-kb RT-PCR
product when the L3 primer is used in combination with either R29 or R7
(Table I). The L3 primer binds within the env, downstream
from the initiation site of the vSAg mRNA identified for
Mtv29 (13). The unspliced 2.3-kb RT-PCR
products, amplified with these primer pairs, contain the complete
3'-env region and are not likely to encode the vSAg
mRNA.
vSAg mRNA initiating immediately downstream from the
META-env region was detected by RNase protection assay (Fig. 3A). Except for cNJ101 (the
atypical IgM+ SJL lymphoma that does not
stimulate T cells; Refs. 3 and 18) and one
MA/My lymphoma (cNJ124), all lymphomas examined contained at least one
Mtv that used this initiation site. Comparison of the
results for RNase protection and RT-PCR shows that in SJL lymphomas and
in the C57L lymphoma cNJ123, Mtv29 was using this site.
However, considering the detection of more than one 1.1-kb RT-PCR
product with L3 in another C57L lymphoma (cNJ120) and in the MA/My
lymphoma (cNJ126), it was not certain whether Mtv29 or one
of the other Mtvs, Mtv8, Mtv9, and/or
Mtv17, was responsible for the use of the
META-env initiation site.
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A) and 326, where Mtv7 is the only candidate, as
judged by the RT-PCR products detected. These results clearly indicate
that the Mtv7 transcripts in the lymphomas from SW x
J-1 mice are analogous to those for Mtv29 in the other
lymphoma prone strains studied here. Characterization of MMTV transcripts in normal B cells
Normal LPS-stimulated B cells never used the env
initiation site for Mtv transcripts (Fig. 2B) and
also failed to show a 5' LTR-initiated vSAg mRNA, as judged by the
RT-PCR product for either Mtv29 or Mtv7 (Table II). However, the 5' LTR-initiated vSAg
mRNA (1.1-kb product with L1 and R8) was seen for Mtv8 in
LPS-activated B cells from some strains, including DBA/2 and
LAF1, but not from SJL mice (Table II).
LPS-stimulated B cells from NOD mice showed an unidentified vSAg mRNA
that gave a 1.1-kb product on RT-PCR with L3 and RLTR but not with any
other reverse primer used, in agreement with the known absence of these
MMTVs in this strain. RNase protection assays did not show META
env-initiated vSAg transcripts in the RNA from LPS blasts.
However, it is interesting that the RNA isolated from Peyer’s patches
taken from SJL mice aged 3–6 mo, i.e., several months before the
typical lymphomas arise in this strain, already showed use of this
initiation site (Fig. 3B). Indeed, although normal lymphoid
tissue from other sites in SJL mice, such as spleen and brachial lymph
nodes, failed to show these transcripts, Peyer’s patches consistently
contained them.
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It was shown previously that unprimed syngeneic T cells are
stimulated to proliferate in vitro by -irradiated cells from SJL
lymphomas (21, 22). The same appears to be true for
lymphomas of C57L, MA/My, and SW x J-1 origin (Table III). In the case of SJL lymphomas, this
was shown previously to be primarily attributable to the specific
stimulation of V16+CD4+
T cells and inhibited by anti-I-AS
(2). Therefore, the ability of C57L and MA/My lymphoma
cell lines to stimulate T hybridoma cells bearing various individual
V-chains in their TCRs was compared with that of the SJL lymphoma
line cNJ117 (Table IV). The in vitro C57L
lymphoma line, cNJ123.5, strongly stimulated
V16+ T hybridomas, as did cNJ117, whereas weak
stimulation of the V8.1- and the V11-bearing T hybridomas by
cNJ123.5 also was observed. This finding suggested the presence of
vSAg29 in both cNJ117 and cNJ123.5, as well as that of an additional
vSAg (Mtv8, Mtv9 and/or Mtv17) in the
C57L line that was absent from the SJL line. The MA/My line, cNJ126,
also caused detectable stimulation of V16+-T
hybridoma cells, and strongly stimulated
V11+-, V12+- and
V17a+-T cells (Table IV).
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16+ T hybridoma cells examined, all responded
strongly, whereas no stimulation of V6+ T
cells was obtained (Table IV). The stimulation of
V16+ T hybridoma cells by both tumors 326 and
340 was abolished by inclusion of anti-I-As
in the medium. Stimulation of V6+ T cells by
vSAg7 is known to be strongly dependent on the expression of I-E in the
presenting cells (23). Thus, in view of the fact that
SW x J-1 is H2s, it seemed likely that
vSAg7 could not properly be presented by the lymphoma cells
(24). Indeed, when the abilities of LPS-stimulated B cells
from SWR (H2q, Mtv7+)
and BALB.D2 (H2d,
Mtv7+) to stimulate these
V6+ T hybridoma cells were compared, the SWR
cells failed to stimulate (< 0.15 pg IL-2/ml), whereas the BALB.D2
cells did (1.55 pg IL-2/ml). Histological features of the SW x J-1 lymphomas
In previous studies, the characteristics of C57L lymphomas were
described as very similar to those of SJL primary lymphomas
(25). Like the GC cells from which they are derived, the
majority of the primary tumor cells in both SJL and C57L mice also
stain with PNA-peroxidase (see also Ref. 1). Therefore, in
the present study we examined sections of primary lymphomas from
SW x J-1 mice. Again, a very similar morphology and
PNA+ staining of the tumor cells in both Peyer’s
patches and mesenteric lymph nodes was seen, entirely comparable to SJL
primary lymphomas (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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16+ CD4 T cells also is expressed. This
observation is supported by the presence of the typical RT-PCR
products, use of the env initiation site, and the presence
of prominent Mtv29+ 1.8-kb mRNA. However,
what is more striking is the observation that the RNA from lymphomas
arising in the Mtv29- strain, SW x
J-1, exhibits identical RT-PCR products, bands hybridizing with the LTR
probe on Northern blots, and META-env initiation of
Mtv7+ transcripts. These transcripts
clearly come from Mtv7, because Mtv8, the only
other MMTV in that strain, does not show any RT-PCR products in either
of the two lymphomas examined in detail. In a previous study in which a
quantitation was made of RsaI-digested RT-PCR products
obtained from 5' LTR initiated (presumably vSAg) transcripts from
Mtv6, Mtv7, Mtv8, and Mtv9 in normal B and T cells from
BALB-D2.Mls-1a mice, such transcripts for Mtv7 were found to
be highly expressed, particularly in B cells (26). These
LTR-initiated transcripts were thought to encode vSAg7, which is known
to be highly expressed in B cells and to cause efficient deletion of
Mtv7-responsive V6+,
V7+, V8.1+, and
V9+ T cells in I-E+
strains that possess Mtv7 in their genome
(27, 28, 29). However, the present results suggest that
LPS-activated B cells do not produce a typical LTR-initiated vSAg for
Mtv7, neither in DBA/2 nor in SWR x SJL
F1 B cells, but that they also fail to produce
META-env-initiated transcripts. Therefore, these data do not
describe the mRNA encoding the potent vSAg7 in normal B cells. It is
likely that the 3'-env promoter (P2env), described by Arroyo
et al. (30), is used for vSAg7-encoding transcripts in
Mtv7+ LPS blasts. We have not yet examined
whether the lymphoma cells studied here also use, in addition to the
META, the P2env promoter for vSAg7-encoding transcripts; the prominence
in Northern blots of an additional smaller mRNA for Mtv7 in
lymphoma 340 suggests this possibility. The sequence of Mtv29 differs by only a single base in the regions found important for promoter activity in the 3'-env of Mtv7 (30). However, with respect to the possibility that Mtv29 might also use a 3'-env promoter and initiation site in LPS blasts, it should be mentioned that in previous studies we have not found syngeneic T cell stimulation by LPS blasts from SJL mice. Moreover, RNA prepared from LPS blasts from SJL mice does not exhibit a 1.8-kb band on probing with an LTR probe, suggesting that, unlike the SJL lymphomas, they fail to express vSAg29 (or vSAg8).
The early histology associated with lymphoma development strongly
indicates that SJL lymphomas usually arise in GCs from gut-associated
lymphoid tissue, as originally shown by Siegler and Rich
(31) and, more recently, by Secord et al. (1)
in both normal and Bcl2-transgenic SJL mice. Similarly, all
the primary lymphomas from the other strains studied here have a
similar morphology, frequently involve Peyer’s patches, and are
PNA+. If the META-env-initiated
transcripts of Mtv29 play a causal role in the induction of
these lymphomas by eliciting a response from
V16+ CD4 T cells that then causes a chronic
stimulation of B cells, one would expect to see such Mtv29
transcripts in Peyer’s patches before the lymphomas arise. As shown in
the present study (Fig. 3B), this was indeed found. At this
point, it cannot formally be excluded that some lymphoma cells are
already present in the Peyer’s patches from SJL mice months before
significant lymphadenopathy occurs and that this early expression of
mRNA for vSAg29 is attributable to them rather than to normal GC cells.
In preliminary studies, it was found that Peyer’s patch cells from
6-mo-old but not from 3- mo-old SJL mice or from spleen were able to
stimulate syngeneic lymph node cells. A more complete study of the
stimulatory ability of these cells is underway. We also observed that
similar transcripts are found in Peyer’s patches from SJL x
BALB/c mice that lack V16+ T cells and do not
develop lymphomas. This suggests that the expression of vSAg29 is in
normal B cells from Peyer’s patches rather than in lymphoma cells.
Expression of Mtv8 may be regulated differently in various
strains of mice (32). Therefore, it is of interest that
Mtv8, which encodes a relatively potent vSAg in many
strains, exhibiting vSAg8 transcripts that clearly initiate in the 5'
LTR, does not exhibit such transcripts in either SJL lymphomas or
LPS-induced B cell blasts from SJL mice. Lymphoma cells from the RI
strains SW x J-1 also lack such Mtv8 transcripts. In
contrast, the lymphoma cell lines derived from MA/My mice clearly
exhibit 5' LTR-initiated Mtv8 and Mtv17 encoded
vSAg transcripts as well as proteins, as shown by their ability to
stimulate V11, V12, and V17a bearing T hybridoma cells
(15). These lymphoma cells weakly stimulate
V16+ T hybridoma cells. In contrast, although
both vSAg29 and vSAg8 transcripts also are present in the C57L lymphoma
lines, the ability of C57L lymphomas to stimulate V16 T hybridomas
was significantly greater than their stimulatory activity for V8.1-,
V11-, or V12-bearing hybridomas. It is possible that MHC class II
molecules on the lymphoma cells influence their relative abilities to
preferentially present one or another vSAg (33, 34). It is
of great interest that vSAg7 expressed in SW x J-1 lymphomas
appears to stimulate V16+ T cells. In fact,
LPS-stimulated Mtv7+, I-E+
BALB.D2 B cells also were found to stimulate
V16+ T hybridoma cells, but to a lesser extent
than did the lymphoma cells. No previous report of vSAg7 stimulation of
V16+ T cells has been published, but this
result is perhaps not very surprising in view of the fact that a
sequence comparison of Mtv29 and Mtv7 shows
striking similarities in their 3' LTR regions, as well as some
significant differences at the 3' end (35, 36).
Whereas the present studies show that all of these lymphomas clearly
stimulate syngeneic T cells, the exact nature of the most important T
cell subset in this response needs further investigation. This is
particularly true for the I-E+ MA/My mice, where
V16+ T cells (our unpublished
observation), in addition to V5.1+,
V11+, and V12+ T
cells, are deleted. Moreover, although the need for CD4 T cells in
promoting in vivo growth of SJL and C57L lymphomas was clearly
established in previous studies (7, 20, 25, 37), this
information is not yet available for the MA/My or the SW x J-1
lymphomas studied here.
It is possible that differential methylation of the LTR and env promoter regions of various Mtvs in different strains, as well as in different tissues of mice, influences the transcription patterns seen in the present study. Preliminary data indicate that significant differences exist between SJL liver and lymphoma DNA in the degree of methylation present in the META-env promoter region of Mtv29 (R. M. Thomas, N. Sen, D. J. Zhang, V. K. Tsiagbe, and G. J. Thorbecke, manuscript in preparation). Further studies are needed to evaluate whether such variations in DNA methylation patterns and/or differences in the presence and recruitment of transcription factors may explain the production of META-env controlled transcripts of Mtv29 in GC cell-derived lymphomas (and perhaps also normal GC cells), but not in LPS-activated B cells.
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Acknowledgments |
|---|
6-bearing
T- hybridoma cells. We also thank Dr. J. Bluestone (Ben May
Institute, Chicago, IL) for a gift of anti-CD3-producing cells. Dr.
Hans Acha-Orbea (Ludwig Institute for Cancer Research, Institute of
Biochemistry, University of Lausanne, Lausanne, Switzerland) kindly
provided breeding pairs of BALB.D2 mice. The excellent technical
assistance by Kamran Haleem is gratefully acknowledged. |
Footnotes |
|---|
2 N.S. and W.J.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. G. J. Thorbecke, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016.
4 Abbreviations used in this paper: RCS, reticulum cell sarcoma; GC, germinal center; Mtv, mammary tumor virus; RI, recombinant inbred; MMTV, mouse mtv; META, MMTV-env promoter/enhancer with associated transcription initiation site; LTR, long terminal repeat; ORF, open reading frame.
Received for publication December 18, 2000. Accepted for publication February 21, 2001.
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References |
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  W. J. Simmons, M. Koneru, M. Mohindru, R. Thomas, S. Cutro, P. Singh, R. H. DeKruyff, G. Inghirami, A. J. Coyle, B. S. Kim, et al. Tim-3+ T-bet+ Tumor-Specific Th1 Cells Colocalize with and Inhibit Development and Growth of Murine Neoplasms J. Immunol., February 1, 2005; 174(3): 1405 - 1415. [Abstract] [Full Text] [PDF]
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 P. R. Etkind, A. F. R. Stewart, T. Dorai, D. J. Purcell, and P. H. Wiernik Clonal Isolation of Different Strains of Mouse Mammary Tumor Virus-Like DNA Sequences from Both the Breast Tumors and Non-Hodgkin's Lymphomas of Individual Patients Diagnosed with Both Malignancies Clin. Cancer Res., September 1, 2004; 10(17): 5656 - 5664. [Abstract] [Full Text] [PDF]
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R. M. Thomas, K. Haleem, A. B. Siddique, W. J. Simmons, N. Sen, D.-J. Zhang, and V. K. Tsiagbe Regulation of Mouse Mammary Tumor Virus env Transcriptional Activator Initiated Mammary Tumor Virus Superantigen Transcripts in Lymphomas of SJL/J Mice: Role of Ikaros, Demethylation, and Chromatin Structural Change in the Transcriptional Activation of Mammary Tumor Virus Superantigen J. Immunol., January 1, 2003; 170(1): 218 - 227. [Abstract] [Full Text] [PDF]
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 N. M. Ponzio G. Jeanette Thorbecke, 1929-2001 Am. J. Pathol., June 1, 2002; 160(6): 1917 - 1920. [Full Text] [PDF]
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-32P]UTP and T3 RNA
polymerase. RNA-RNA hybrids were digested by RNase A and RNase T1, and
double-stranded RNase-resistant fragments were separated on a 6%
denaturing polyacrylamide sequencing gel. Arrows indicate the
predominant fragments; the lengths of the fragments in nucleotides are
indicated. The shorter fragments, seen with RNA from the RCS cell lines
cRCS-X and cNJ117 but not NJ101, the C57L cell line, cNJ123.5, the
MA/My cell line cNJ126 but not cNJ124, and the RI strain 1 lymphoma 326
(A), are the products associated with the initiation
sites within env, the longer ones seen with all the
lymphomas and also with RNA from LPS-stimulated B cells
(B) are the products protected by the env
mRNAs that initiate in the 5' LTR. The shorter fragment also is
detected in RNA from SJL Peyer’s patches (B).