Both T and B cells shed infectious mouse mammary tumor virus.

Copyright © 1997, American Society for Microbiology

Both T and B Cells Shed Infectious Mouse

Mammary Tumor Virus

JOHN L. DZURIS, TATYANA V. GOLOVKINA,

SUSAN R. ROSS*

Department of Microbiology/Cancer Center, University of Pennsylvania

School of Medicine, Philadelphia, Pennsylvania 19103-6142

Received 28 January 1997/Accepted 18 April 1997

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells

in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and

both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue

culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these

different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell

infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers.

Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice.

These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.

Mouse mammary tumor virus (MMTV) is a milk-borne

ret-rovirus that causes mammary tumors in mice (15). MMTV

encodes a superantigen (Sag) protein in its long terminal

re-peat (LTR) that is required for its transmission (1, 17). MMTV

first infects B cells in the Peyer’s patches of the gut, and these

infected B cells present the Sag to cognate T cells (14). B-cell

infection is required for stimulation of T cells, and infection

cannot be established in mice lacking B cells (2). Similarly, the

stimulation of cognate T cells by Sag is a requisite step in the

MMTV infection pathway, since mice lacking such T cells

cannot be efficiently infected (5, 11). Both B-cell and T-cell

amplification result from this stimulation, creating potential

reservoirs of infection-competent cells (5, 11).

Although both B and T cells are required for the milk-borne

transmission of MMTV, it is not known how this virus gets to

its target tissue, the mammary gland. Because it has been

shown that B cells are the most highly infected of all the

lymphocyte subsets, at least at early times after infection (10),

it has been suggested that these cells are ultimately responsible

for mammary gland infection (2, 10). However, lymphoid

or-gans rich in T cells also acquire integrated copies of MMTV

proviruses after milk-borne infection (4, 14) and transfer of

either B or T cells from an MMTV-infected donor to a naive

recipient results in transfer of the virus to both cell types of an

uninfected host (20, 21). Whether one or both of these cell

types carry virus to the mammary gland or even shed MMTV

virions has not been demonstrated.

We show here that both B and T tissue culture cells were

infected with MMTV and could transmit virus to other cells in

vitro. In vivo, both B and T cells were also infected with

MMTV and their level of infection was strain dependent, since

MMTV-infected C3H/HeN mice showed greater infection of B

cells than T cells, while BALB/c mice showed the opposite.

Splenic and thymic lymphocytes derived from transgenic mice

expressing a molecularly cloned MMTV and primary B and T

cells from C3H/HeN mice infected with MMTV(C3H) shed

virus when cultured in vitro. These results indicate that both B

and T cells are capable of carrying MMTV to the mammary

gland in vivo.

MATERIALS AND METHODS

Cell culture.The B-cell tumor line A20, B-cell hybridomas LBB.A, LBB.11, and LK4.5, and the T-cell tumor line BW5147 were cultured in RPMI 1640 (GIBCO/BRL, Gaithersburg, Md.) supplemented with 10% fetal calf serum and 0.5mMb-mercaptoethanol. The normal mammary gland cell line NMuMG and MMTV (HYB PRO)-transfected NMuMG (NMgCl 1) were cultured in Dulbec-co’s modified Eagle’s medium supplemented with 10% fetal calf serum and 10mg of insulin/ml. The rat hepatocyte XC cell line and the mammary gland tumor cell line Mm5MT were cultured in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal calf serum.

The NMgCl 1 cell line was generated by cotransfecting NMuMG cells with a molecular clone of MMTV [called hybrid provirus (HYB PRO), containing the

env and sag genes from MMTV(C3H)] and the MMTV-neo constructs

(de-scribed by Shackleford and Varmus [19]). Independent G418-resistant transfec-tants were isolated, and the NMgCl 1 clone was chosen because it shed high levels of MMTV virions (not shown). The MMTV-neo RNA was inefficiently packaged in this cell line (approximately 100 times lower than the HYB PRO RNA [not shown]).

Mice.C3H/HeN MTV2_{, C3H/HeN MTV}1_{, and BALB/c mice from colonies} of germ-free-derived, defined-flora animals were purchased from the National Institutes of Health, Frederick Cancer Research Facility, Frederick, Md. Trans-genic mice bearing the HYB PRO construct have been previously described (6, 7). MMTV-infected mice were examined weekly by palpation for mammary tumors.

Isolation of primary B and T cells.Primary lymphocytes were isolated from the spleen, thymi, and lymph nodes of MMTV(C3H)-infected C3H/HeN or BALB/c mice or from those of the HYB PRO transgenic mice. T and B cells were purified from the pooled lymphoid organs of two to three mice. Single-cell suspensions were prepared in Hanks balanced salt solution (HBSS) supple-mented with 20 mM HEPES. Cells were washed two times with HBSS. Eryth-rocytes were removed by 0.15 M NH4Cl lysis for 5 min at room temperature.

Cells were washed two more times with HBSS and then resuspended in RPMI 1640 medium supplemented with 20 mM HEPES. B cells were then isolated by negative selection during three rounds of panning 43108_{lymphocytes on}

150-mm-diameter petri dishes coated with anti-Thy1 antibody (100mg/ml) to remove the T cells. T cells were isolated by similar panning on plates that had been coated with anti-mouse immunoglobulin antibody (100mg/ml) to remove the B cells. Panning was carried out at 4°C for 1 h during each round. In between each pair of panning steps, the petri dishes were agitated to resuspend nonad-herent cells, which were then transferred to the next dish. B- and T-cell popu-lations were washed two times with HBSS-HEPES. The purity of the cell pop-ulations was determined by fluorescence-activated cell sorter analysis with fluorescein isothiocyanate (FITC)-labelled anti-immunoglobulin or anti-CD4 and anti-CD8 antibodies (GIBCO/BRL). Fluorescence-activated cell sorter anal-ysis of the purified cells showed that the T- and B-cell populations were 93 to 99% pure (not shown).

Virus infection of lymphocyte cell lines.Infection of the lymphocyte cell lines was done by coculturing lymphocyte cell lines (104_{cells/plate) and NMgCl 1 (10}4

cells/plate) in 60-mm-diameter tissue culture plates for 5 days, followed by

* Corresponding author. Mailing address: Department of

Microbi-ology/Cancer Center, University of Pennsylvania School of Medicine,

415 Curie Blvd., Philadelphia, PA 19103-6142. Phone: (215) 898-9764.

Fax: (215) 573-2028. E-mail: ROSSS@mail.med.upenn.edu.

6044

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transfer to 100-mm-diameter plates for an additional 5 days. For some experi-ments, coculturing was carried out with 0.4-mm-pore-size filter inserts (Falcon 3090) to prevent cell-cell contact. The cultures were supplemented with 8mg of Polybrene/ml. The adherent NMgCl 1 cells were then removed from the cultures by passaging the nonadherent lymphocytes to new T-25 tissue culture flasks four times in succession. The lymphocyte cultures were supplemented with 1mM dexamethasone. Biological clones of the MMTV-infected lymphocytes were ob-tained by limiting dilution cultures in 96-well flat-bottom tissue culture plates.

Passage of virus from the newly infected lymphocytes to uninfected NMuMG and XC cells was done by similar coculturing in the presence of 0.4-mm-pore-size filters. After the 10 days of coculturing, the lymphocytes were removed. The NMuMG and XC cells were cultured for 10 more days in the presence of 1mM dexamethasone before DNA was harvested.

PCR and Southern blot analysis.Equal amounts of genomic DNA (0.25mg) from the different cell samples were used for PCR with Taq polymerase accord-ing to the instructions of the manufacturer (Promega, Madison, Wis.). Oligonu-cleotides used to amplify MMTV(C3H) (59primer and 39primer, nucleotides [nt] 268 to 289 and 894 to 871, respectively, from the 59end of the LTR) and endogenous MMTVs (59primer, nt 507 to 527 and 39primer, nt 1203 to 1184) were described previously (7). Semiquantitative PCR, in which the amplification was still in the linear range, was carried out by 31 cycles of 1 min at 55°C, 1 min at 72°C, and 1 min at 94°C (9). After PCR amplification, a sample of the product was digested with 2 U of MunI (NEB, Beverly, Mass.) for 2 h at 37°C, as previously described (2); MunI only cuts exogenous MMTV(C3H). Genomic DNA from the Mm5MT mouse mammary tumor cell line was used as a positive control for amplification of MMTV sequences.

Southern blot analysis to determine whether cells were infected with HYB PRO was carried out as previously described (8, 19). Briefly, genomic DNAs were digested with BglII and PstI, which cut internally within the proviruses, and the blots were hybridized with an env-specific probe. Copy numbers were esti-mated by comparing the relative intensities of hybridization of the exogenous (Fig. 1, band V) and endogenous (Fig. 1, band E) MMTV bands. All of the B and T cell lines have three to six copies of endogenous MMTV.

Virus purification and RNA analysis.The B and T cell lines were plated at 106

cells/ml in the presence of 1mM dexamethasone and cultured for 3 days. Virus was isolated from 50 ml of supernatant filtered through 0.2-mm-pore-size filters by adding 0.5 volumes of 33polyethylene glycol (PEG) 6000 (25% PEG 6000, 1.5 M NaCl) and mixing at 4°C for 2 h. Virus was pelleted by centrifugation at 12,0003g for 20 min at 4°C. RNA was then extracted from the virus pellets.

Cellular and viral RNA were extracted by guanidine thiocyanate extraction and CsCl gradient centrifugation (3).

Primary lymphocytes (107_{) isolated from 6-month-old C3H/HeN MMTV}

(C3H)1_{, BALB/c MMTV(C3H)}1_{, or HYB PRO transgenic mice were plated at} 106_{cells/ml and cultured for 3 days in the presence of 10mg of concanavalin A/ml}

(T cells) or 50mg of lipopolysaccharide/ml (B cells) (both from Sigma, Inc., St. Louis, Mo.). Virus was isolated from 10 to 20 ml of filtered supernatant by centrifugation at 156,0003g for 1.5 h at 4°C. RNA was extracted from the virus

pellets.

For RNase T1protection assays, labeled RNA probes were synthesized from

a pBluescript plasmid carrying the Sau3A fragment of the MMTV(C3H) LTR with T3 RNA polymerase, as previously described (8). Either 40mg of total cellular RNA or viral RNA isolated from 50 ml of culture supernatant was used for RNase T1protection analysis, as previously described (6).

Reverse transcription (RT)-PCR was accomplished by synthesis of cDNA from RNA isolated from the B- and T-cell culture supernatants as previously

described (7). After amplification and agarose gel electrophoresis, the products were transferred to nitrocellulose and hybridized with a probe specific to the MMTV LTR (5). Relative amplification was determined by densitometric anal-ysis of the autoradiographs.

Western blot analysis.Total cellular protein was extracted from cells or from 10 ml of pelleted cell culture supernatant with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1.0% Nonidet P-40, and 1.0% Triton X-100. Cellular protein (100 mg) was separated by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis, and Western blot analysis was performed, as previously described (6), with goat anti-MMTV gp52 (SU) or goat anti-MMTV p27 (Gag) polyclonal antibodies or normal goat serum (National Cancer Institute Biochemical Carcinogenesis Branch Repository, distributed by Quality Biotech Inc., Camden, N.J.).

RESULTS AND DISCUSSION

We used two approaches to determine whether T and B cells

could both be productively infected with MMTV. First, we

established T- and B-cell tumor lines that were infected with a

molecular clone of MMTV, called HYB PRO (19). We

in-fected the B-cell tumor line A20, B-cell hybridomas LK4.5,

LBB.A, and LBB.11, and T-cell tumor line BW5147 by

cocul-turing these cells with a HYB PRO-transfected mammary

gland cell line, as described in Materials and Methods. Clonal

cell lines from each of the infected populations were isolated

and examined for the presence of integrated viral DNA by

Southern blot analysis (Fig. 1). The infected cell lines had a

diagnostic 2.3-kb band (Fig. 1, band V) that was present after

digestion of the DNA with PstI and BglII. The clones selected

for further study had from 6 to 10 copies of newly integrated

MMTV proviruses, based on the relative hybridization to the

endogenous virus- and HYB PRO-specific bands (Fig. 1).

The clones from each infected cell line were then examined

for transcription of HYB PRO-specific RNA and the

produc-tion of processed MMTV proteins. RNase T

-protection

anal-ysis with a probe specific for the U3 region of the HYB PRO

transcript was performed with RNA isolated from the various

cell lines. As can be seen in Fig. 2A, all of the infected clones

examined produced HYB PRO-specific RNA.

It had previously been reported that a T-cell lymphoma line

was not able to process virus proteins and therefore could not

produce infectious virus particles (16). To determine if all of

the cells were able to produce MMTV proteins, Western blot

analysis was performed on total cell extracts with monospecific

anti-SU (Fig. 2B, gp52) and anti-Gag (Fig. 2B, p27) sera. All of

the cell lines produced both processed viral proteins.

There-fore, there was no block to the translation and processing of

MMTV proteins in these B and T cell lines. In some cell lines

(i.e., LBB.11 and LK4.5), we detected unprocessed Gag but

not Env precursor proteins in the uninfected cells (Fig. 2B).

These most likely represent proteins produced from the

en-dogenous viruses present in these cell lines. The lack of

pro-cessed Gag and Env in the uninfected cells indicates that these

endogenous viruses must have mutations in the coding regions

for these proteins.

These results indicated that the B and T cell lines were

capable of producing MMTV virions, since they produced both

viral RNA and processed proteins. To confirm this, the

super-natants of the infected cell cultures were collected and filtered

to remove cells and the virus fraction was obtained by PEG

precipitation. RNA or protein was isolated from the pellets

and subjected to RNase protection analysis with an MMTV

(C3H) U3-specific probe or to Western blot analysis,

respec-tively. Viral RNA could be detected in the supernatants of all

of the cell lines (Fig. 3A). Moreover, gp52 (Fig. 3B) and p27

(not shown) were also found in the supernatants. These results

showed that both cultured B- and T-lymphoma and

B-hybrid-oma cell lines could produce MMTV virions after infection.

FIG. 1. Southern blot analysis of clonal isolates of the infected B- and T-cell tumor lines. Genomic DNA was isolated from clonal isolates of infected popu-lations of B (LBB.A [LA], LBB.11 [L11], LK4.5 [LK], and A20) and T (BW5147 [BW]) tumor cell lines and digested with BglII and PstI, and the Southern blots were hybridized to an env-specific probe, as previously described (8, 19). The “1” denotes infected cells, and the “2” denotes the uninfected parental cell line. E, endogenous MMTV-specific band; V, exogenous MMTV-specific band.

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To show that the virus produced by the T and B cells was

infectious, we cocultured the infected BW5147 (T) and A20

(B) cell lines with an uninfected mouse mammary gland

(NMuMG) and a rat sarcoma (XC) cell line. After coculturing

the cells for 10 days in the presence of a 0.4-

m

m-pore-size

barrier filter, we removed the lymphoma cells. DNA was

pre-pared from the cultures, and PCR with primers specific for the

U3 region of the MMTV LTR was performed. The PCR

prod-ucts were digested with MunI; this site is unique to the HYB

PRO LTR derived from MMTV(C3H). Both the XC and

NMuMG cells were infected after being cocultured with either

the infected T or B cell line (Fig. 4). Thus, there is no general

block to the production of infectious MMTV virions in B- or

T-cell tumor lines, although some cell lines may not process

the viral proteins (16).

We also examined lymphocytes infected in vivo after

milk-borne transmission of MMTV. Four-month-old BALB/c and

C3H/HeN mice nursed on C3H/HeN MMTV(C3H)

_mothers

were sacrificed, and semiquantitative PCR analysis was

per-formed on genomic DNA isolated from their various lymphoid

cells and tissues to look for newly acquired MMTV(C3H)

proviral DNA, as described in Materials and Methods. In the

case of C3H/HeN mice infected by milk-borne MMTV(C3H),

we found that B cells were more infected than T cells (Fig. 5).

In contrast, in the MMTV(C3H)-infected BALB/c mice,

infec-tion of T cells and thymi was much greater than that of B cells

or spleens (Fig. 5). Infection of C3H/HeN T cells and thymi or

of BALB/c B cells and spleens could only be detected after

Southern blotting and hybridization of the gel shown in Fig. 5;

about 10 times more MMTV DNA was seen in C3H/HeN B

and BALB/c T cells than in C3H/HeN T or BALB/c B cells

(not shown). Similar results were obtained with 7-month-old

mice (not shown).

To determine whether both cell types were productively

infected in vivo by milk-borne virus, we assayed supernatants

from equal numbers of cultured B and T cells isolated from

MMTV(C3H)-infected BALB/c and C3H/HeN mice. We also

cultured primary lymphoid tissues from HYB PRO transgenic

mice to assay for virus production. HYB PRO mice express a

molecular clone of MMTV in both lymphoid tissues and

mam-mary glands and shed an infectious MMTV into milk (7). RNA

was isolated from the filtered supernatants of these cultures

and subjected to an RT-PCR assay that was specific for the

exogenous viral RNA. MMTV-specific RNA could be detected

in the supernatants of T and B cells from infected mice (Fig.

FIG. 2. Analysis of MMTV-specific cellular RNA and protein produced by MMTV-infected B- and T-cell tumor lines. (A) RNase T1protection analysis was carried out with RNA isolated from the various infected B and T cell lines, using a probe specific for the U3 region of HYB PRO. A protected fragment of 340 nt is present in cells expressing the HYB PRO RNA. (B) Western blot analysis of cellular extracts from the same cells with monospecific SU (gp52) or anti-GAG (p27) antisera. The arrows show the Env (gp78) (upper panel) and Gag (Pr77) (lower panel) precursor proteins. Abbreviations are the same as in Fig. 1.

FIG. 3. Analysis of the virions shed by the MMTV-infected B- and T-cell tumor lines. (A) RNase T1protection analysis of virus pelleted from the super-natants of the various cell lines. Although the signal for the LK1cell line is weak, it was clearly visible upon longer exposure of the gel. (B) Western blot analysis of virus pelleted from the supernatants of the various cell lines. Monospecific polyclonal antibodies against the gp52 SU protein were used. Abbreviations are the same as in Fig. 1.

FIG. 4. T and B cell lines produce infectious virus. MMTV-infected B cell line A20 and T cell line BW5147 (BW) were cocultured with XC or NMuMG cells in the presence of a 0.4-mm-pore-size filter. DNA was isolated from the cocultured XC and NMuMG cells after an additional 10 days in culture and was subjected to PCR amplification followed by MunI digestion, as described in Materials and Methods. Mouse mammary tumor cell line Mm5MT was used as a positive control.

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6A) and in the thymi and spleens from the HYB PRO

trans-genic mice (Fig. 6B) but not in those from uninfected C3H/

HeN mice. B cells from C3H/HeN mice and T cells from

BALB/c mice appeared to shed the highest levels of virus,

approximately 2.2 and 4 times the amounts produced by C3H

T and BALB/c B cells, respectively. The ratio between the

numbers of proviruses in the C3H B and T cells or in the

BALB/c T and B cells was greater than was seen for virus

production. However, because we know nothing about the

kinetics of virus production from the various cell populations

either in vivo or in vitro, the assay for virus production is not

quantitative.

The results presented here show that both B and T cells

infected with MMTV can produce infectious virus particles

and thus are potential carriers of virus to the mammary gland.

It has been proposed that the role of the MMTV Sag is to

cause T-cell stimulation and cytokine production resulting in

B-cell proliferation and that infected B cells are ultimately

necessary for infection of the mammary gland (2, 10, 12). For

example, there is amplification of MMTV-infected B cells in

mice injected with MMTV (SW) even in the presence of

zidovudine, an inhibitor of retroviral replication (12). This

increase in the number of infected B cells has been suggested

to be an important step in the MMTV infection pathway.

However, it is also possible that this B-cell amplification only

reflects a strong humoral response to MMTV antigens and that

B cells are not the sole carriers of virus to the mammary gland.

We also found that the level of virus infection in different

lymphoid cell populations was dependent on the strain of

in-fected mice. In BALB/c mice inin-fected with milk-borne MMTV

(C3H), T cells were more highly infected than were B cells,

while B cells were more highly infected in C3H/HeN mice. We

have also found that the mammary glands of BALB/c mice are

more highly infected by MMTV(C3H) than those of C3H/HeN

mice (not shown). Moreover, the latency of mammary tumor

induction in BALB/c mice foster nursed on C3H/HeN

FIG. 5. Analysis of infected T and B cells from BALB/c and C3H/HeN mice nursed on C3H/HeN MMTV1_{mothers. (A) DNA isolated from the B cells, T}

cells, spleens, thymi, and tails (DNA) of MMTV(C3H)-infected BALB/c (lanes B) and C3H/HeN (lanes C) mice was used for PCR analysis, using primers that amplify MMTV(C3H) DNA. After amplification, the DNA was restricted with MunI, which cuts only in MMTV(C3H) and not in the endogenous proviral DNA. The Mtv-9 provirus present in BALB/c but not in C3H/HeN mice was also amplified with the primers (DNA, lanes B); however, this provirus lacks the MunI site (DNA, lane B1). The presence of the two lower-molecular-weight bands with MunI digestion in the different samples shows the presence of MMTV (C3H) DNA. These experiments were performed three times with cells and tissues isolated from different animals; a representative experiment is shown. (B) PCR analysis with primers that amplify endogenous as well as exogenous MMTVs was done as a control. The same DNAs used in A were amplified with these primers.

FIG. 6. Primary lymphoid cells from MMTV(C3H)-infected mice and HYB PRO transgenic mice shed virus. Lymphocytes isolated from HYB PRO mice and purified T and B cells from MMTV-infected C3H/HeN mice were cultured in vitro for 3 to 5 days, and their supernatants were harvested and filtered to remove cells. RNA isolated from the supernatants was subjected to RT-PCR, the amplified fragments were separated by gel electrophoresis, and Southern blotting with a LTR-specific probe was performed. (A) RT-PCR of RNA isolated from purified B and T cell supernatants was performed with or without reverse tran-scriptase. (B) RT-PCR of RNA isolated from cultured splenocytes (spleen) or thymocytes (thymus) from HYB PRO or nontransgenic (NT) mice was carried out. Mouse mammary tumor cell line Mm5MT RNA was used as a positive control.

FIG. 7. Tumor incidence in MMTV-infected BALB/c and C3H/HeN mice. The mice were foster nursed on C3H/HeN MMTV1_{mothers and were force} bred.

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MMTV

_{mothers was significantly decreased in comparison}

with that of C3H/HeN mice (Fig. 7). This indicates that the

mammary gland virus load was indeed higher in BALB/c mice.

The fact that T cells were more infected than B cells in BALB/c

mice indicates that infected T cells may be an important source

of virus in vivo for the infection of mammary gland cells.

It is unlikely that the same virus shows different tropism in

the two inbred mouse strains, so these differences may reflect

other genes that influence host response to virus. BALB/c and

C3H/HeN mice differ at their major histocompatibility locus:

BALB/c mice are H-2

_{, while C3H/HeN mice are H-2}

_{. It is}

possible that Sag presentation or immune-cell recognition of

other virus proteins differs in these two strains. For example,

Sag presentation may be more effective in C3H/HeN mice and

bystander B cell activation more vigorous, resulting in greater

infection of this subset of cells. Another possibility is that

predominantly Sag-specific T cells are infected and that these

are more slowly deleted in BALB/c than C3H/HeN mice. This

seems unlikely, given that the experiments described here were

carried out on 4-month-old mice, in which the bulk of the

Sag-cognate T cells were already deleted (13). Moreover, this

would not explain why BALB/c mice had lower B-cell infection

levels.

Similarly, C3H/HeN mice may mount a stronger humoral

response to MMTV infection than BALB/c mice, resulting in

more extensive infection of B cells due to their increased

pro-liferation. Mice infected neonatally with MMTV do mount an

antivirus response (18). If virus clearance was also poorer in

BALB/c mice, they would be expected to succumb more

rap-idly to MMTV-induced mammary tumors, as we showed here.

In summary, both B and T cells are potential MMTV

carri-ers in vivo. Clarification of whether one or the other of these

lymphocyte subsets better transmits virus to the mammary

gland and of the mechanism by which this transfer occurs

awaits the results of future studies which address these

ques-tions.

ACKNOWLEDGMENTS

We thank Marta de Olano Vela and Bernadette van den Hoogen for

expert technical assistance and Jaquelin P. Dudley for helpful

com-ments on the manuscript. We thank J. P. Dudley for the A20 and

BW5147 cells, B. T. Huber for the LBB.11 and LBB.A cells, and Y.

Choi for the LK4.5 cells.

J. Dzuris is supported by PHS grant T32 CA09140, and T. V.

Golovkina was a Cancer Research Institute fellow. This work was

supported by PHS grant CA52646.

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