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Immunology |
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21250 [B. A. P., E. M., S. O-R.]; Enerjen, Carmel, California 93921 [D. S. T.]; and Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 [S. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In most clinical situations, primary mammary tumors are cured by surgery, yet approximately 33% of women successfully treated for primary tumors die subsequently from spontaneous metastatic disease (4) . To further refine the 4T1 system and to more closely parallel clinical disease, we have now developed a postsurgical model of the 4T1 mammary tumor. In this model, mice receive inoculations s.c. in the abdominal mammary gland and the primary tumor is allowed to grow progressively, become extensively vascularized, and metastasize. The primary tumor is then surgically resected, and therapy with the cell-based vaccines is initiated.
The cell-based vaccines consist of tumor cells transfected with syngeneic MHC class II (I-Ad) and CD80 (B7.1) costimulatory molecule genes and were designed to enhance activation of tumor-specific CD4+ T lymphocytes via improved presentation of tumor-encoded class II-restricted epitopes. Although CD8+ T lymphocytes have been traditionally the focus of immunotherapy approaches, accumulating results have demonstrated that CD4+ T lymphocytes also play a critical role in effective antitumor immunity (5, 6, 7, 8, 9) . Whereas our previous vaccines showed significant reduction of established, spontaneous metastatic tumor, the antitumor response was limited to small burdens of metastatic cells and did not completely eliminate metastases (3) . In addition, we did not assess the effects of immunotherapy on survival. Furthermore, the vaccine was tested in mice with metastatic disease and carrying intact primary tumor so the model did not mimic the clinical situation in which primary tumor would have been surgically removed before initiation of immunotherapy. We now report a second-generation cell-based vaccine that is significantly more effective than the original vaccine for the treatment of spontaneous 4T1 metastatic mammary cancer and that is tested in a postsurgical model. The new vaccine incorporates a gene encoding the bacterial toxin SEB3 . SEB is a sAg that when complexed with MHC class II molecules on APCs is a potent polyclonal activator of CD4+ T lymphocytes (10 , 11) . Although CD4+ T-cell activation by SEB is not antigen specific, we reasoned that the addition of SEB to the MHC class II/CD80 vaccine will provide additional activation signals to the CD4+ T cells that have been activated in an antigen-specific fashion by the MHC class II+ CD80+ vaccinating cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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d, I-Aßd) and
mouse B7.1 have been described previously (3)
. The
SEB gene (12)
was subcloned into the
SalI/BamHI site of the pHß-Apr-1-neo expression
vector. The final construct, pHß-SEB-neo, contains the amino acid
sequence for the mature SEB protein minus the signal peptide and
confers resistance to G-418. The pZeoSV2 plasmid was purchased from
Invitrogen (San Diego, CA).
Animals, Cell Lines, and Transfectants.
Female BALB/c and BALB/c nu/nu mice were obtained from The
Jackson Laboratory (Bar Harbor, ME) and/or bred in the University of
Maryland Baltimore County animal facility and used at 8 weeks of age.
4T1, a 6-thioguanine-resistant cell line derived from a BALB/c
spontaneous mammary carcinoma (13)
, was kindly supplied
by Dr. Fred R. Miller (Michigan Cancer Foundation,
Detroit, MI) and grown in culture as described previously
(3)
. Transfectants were made to express MHC class II and
CD80, or SEB by using lipofectin (Life Technologies, Inc.) according to
manufacturer’s instructions. Cells were selected with 400 µg/ml
G-418 (Life Technologies, Inc.) or 200 µg/ml zeocin (Invitrogen),
cloned by limiting dilution, stained for surface antigen expression,
and analyzed by flow cytometry, as described previously
(3)
.
SEB Assay.
Naive BALB/c spleen cells (5–10 x 105) were cultured in serial dilutions of
transfectants’ supernatants or purified SEB (Sigma Chemical Co., St.
Louis, MO), as indicated. To demonstrate specific SEB activity, a
polyclonal rabbit antibody against SEB (Sigma Chemical Co.) was added
to cultures, as indicated. After 3 days in culture, spleen cell
proliferation was measured using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent,
as described previously (14)
.
Surgery.
All surgical supplies and surgical equipment were purchased from Henry
Schein Veterinary & Medical Supply Catalogue (Melville, NY) and Roboz
(Rockville, MD), respectively, unless indicated otherwise. Before
surgery, animals were weighed and anesthetized with i.p. injections
(0.02 ml/g body weight) of 2.5% recrystalized avertin
(2,2,2-tribromomethanol; Sigma Chemical Co.). Once the animals were
unconscious, the tumor-bearing abdominal area was prepared and
sterilized for surgery by shaving with Oster Finisher Trimmer, followed
by 2 washes each with diluted Nolvasan surgical scrub (chlorhexidine)
and isopropanol. Tumors were resected with sterilized surgical
instruments, removing the smallest amount of skin tissue as possible.
Wounds were closed with either Nexaband liquid or stainless steel 9-mm
wound clamps with a Mikon autoclamp applier, as necessary. Wound clamps
were removed 10 days after surgery with a Mikon autoclamp remover. Mice
were monitored for survival, and those that died from surgery (within
1–4 days after surgery, survival rate of 67–80%) were not included
in the experiment. All mice were autopsied at the time of death to
confirm the presence of lung metastases as well as recurrence of the
primary tumor.
Tumor Challenges, Metastases Assays, and in Vivo
Depletions.
Mice were challenged s.c. in the abdominal mammary gland with
(7 x 103/50 µl) parental 4T1
tumor cells. Primary tumor growth and spontaneous metastases were
measured as described previously (3)
. Depletions of
CD4+ and CD8+ T cells were
performed as described previously (15)
. Splenocytes of all
depleted mice were checked by immunofluorescence for depletion at the
conclusion of the experiment. Mice depleted for
CD4+ or CD8+ T cells had
<4% or 7% of CD4+ T cells or
CD8+ T cells, respectively.
Statistical Analyses.
To determine the statistical significance of the data, the Tukey’s
Honestly Significant Difference Test was performed at a P
set at 0.05. The Tukey’s test is a multicomparison test that
determines the statistical significance of data sets of size 3 or
greater and allows for unequal sample size (n) and sample
variances (16)
. To determine the statistical significance
of the effects of immunotherapy on primary tumor growth, the Student’s
t test for unequal variances (Microsoft Excel, version 5.0)
was performed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 1
, 4T1 is also similar to human mammary carcinoma in that morbidity is
due to outgrowth of spontaneous micrometastatic tumor cells that
migrate to distant organs relatively early (week 2) during primary
tumor growth. Groups of female BALB/c mice were challenged s.c. in the
abdominal mammary gland with 7 x 103 wild-type 4T1 tumor cells. Starting at 2
weeks after challenge and continuing at 1-week intervals, TDs were
measured, primary tumors were surgically removed, and mice were
followed for survival times. Because we were interested in knowing
whether primary tumor size affected survival time, the data have been
plotted as tumor size (mm) at the time of surgery versus the
number of days the mice survived after 4T1 tumor challenge. As shown in
Fig. 1
, the average survival time of 55 of 58 mice was 48.9 (±7.4)
days, whereas the remaining 3 mice, whose tumors were <3 mm in
diameter at the time of surgery, lived >90 days and did not die of
metastatic cancer. Surprisingly, all mice that died from spontaneous
metastatic disease showed approximately the same mean survival time
regardless of the size of the primary tumor at the time of surgery.
These results demonstrate that lethal metastasis is established as
early as 2 weeks after inoculation of primary tumor, that the mean
survival time is 7 weeks, and that surgical removal of primary tumor
does not change these kinetics.
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, combined with our previous studies
(3)
, demonstrate that the 4T1 system is comparable with
human stage IV breast cancer. Human breast cancer at stage IV is
characterized by several diagnostic factors: (a) the
presence of edema and ulcerations of the skin in and around the tumor
burden; (b) extension of the primary tumor to the chest
cavity lining; (c) presence of metastatic cells in the
draining lymph nodes; and (d) presence of metastases in
distant organs (4)
. This postsurgical 4T1 system exhibits
all of these characteristics. Metastases are present in the draining
lymph nodes and distant organs as early as week 2 and progress into
more advanced metastatic disease with time (3)
. All of the
resected 4T1 tumors, regardless of their size at the time of surgery,
were highly vascularized. The primary tumors displayed edema when there
was a TD 
4 mm and ulcerations of the skin in approximately 70% of
tumors regardless of size (data not shown). Most tumors extended to the
lining of the peritoneal cavity, whereas invasion through the
peritoneal lining was less frequent (<5%) and only occurred when
primary TD was large (5–6 mm; data not shown). Therefore, at the time
of surgery, the mouse 4T1 tumors are comparable with stage IV human
breast cancer and are a much more rigorous animal model for the
development of effective therapies than other experimental systems
reported in the literature.
Postoperative Treatment of Mice with Transfectants Expressing MHC
Class II, CD80, and SEB Increases Survival.
Previously, we have shown that therapy with transfectants expressing
MHC class II or CD80 reduced metastatic disease in a model where the
primary 4T1 tumor remained in situ and had been established
for 9–14 days. The success of this treatment, however, was limited to
small tumor burdens and did not completely eliminate spontaneous
metastases (3)
. A potential problem with this earlier
therapy is that the transfectants did not coexpress MHC class II and
CD80, and previous data using a mouse sarcoma showed that coexpression
of these molecules is synergistic (15)
. We have,
therefore, generated 4T1 transfectants that coexpress MHC class II and
CD80 as detected by indirect immunofluorescence staining (data not
shown) to test this hypothesis. To further increase the potency of the
vaccine, we have combined the MHC class
II+/CD80+ double
transfectants (4T1/Ad/B7.1) with
SEB+ transfectants (4T1/SEB), reasoning that SEB
may provide additional proliferation signals to the tumor-specific T
cells activated via the MHC class II/CD80 interaction.
SEB expression was tested by coculturing supernatants of transfectants
with naive BALB/c spleen cells and monitoring lymphocyte proliferation.
To determine a relative amount of SEB secretion, splenocytes were also
cultured with soluble SEB. As shown in Fig. 2
, supernatants from two independent clones (4T1/SEB-12 and 4T1/SEB-14)
stimulated splenocyte proliferation as efficiently as soluble SEB at a
concentration of 2 µg/ml. This activity was reduced on the addition
of a polyclonal anti-SEB antibody, demonstrating that the spleen cell
proliferation was due to SEB expression by the 4T1 transfectants.
Supernatants from parental 4T1 cells as well as supernatants from 4T1
cells transfected with empty vector (4T1/neo) did not induce
proliferative responses (Fig. 2
and data not shown). Therefore, the
4T1/SEB transfectants secrete SEB, which induces splenocyte
proliferation comparable with proliferation induced by soluble
exogenously added SEB.
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and our previously published
results (3)
established that at this stage and size of
primary tumor the mice have very extensive disseminated metastases. At
the time of surgery (3 weeks after primary tumor challenge), the
primary TD in each treatment group ranged between 3.5 mm and 5.7 mm.
The extent of spontaneous metastatic cancer at this time is
significantly more advanced than that tested in other immunotherapy
experiments. Therapeutic injections of irradiated vaccine cells
(1 x 106 total) were started 1
week after surgery (i.e., 4 weeks after initial tumor
challenge) and were administered every 3–5 days for the duration of
the experiment. Because we are interested in determining whether
primary tumor size affects vaccine efficacy, the results in Fig. 3
are plotted as the survival time versus the size of the
primary tumor at the time of surgery. A line
denoting the average survival time of the 4T1-treated control group (45
days) is included to demonstrate the effects of the vaccine
on survival. The survival time in days for 4T1-treated (Fig. 3A
), 4T1/SEB-treated (Fig. 3B
),
4T1/Ad/B7.1-treated (Fig. 3C
), and
4T1/SEB+4T1/Ad/B7.1-treated (Fig. 3D
)
animals was 35–52, 40–59, 47–54, and 41–74 days, respectively.
Statistical analyses using the Tukey’s Honestly Significant Difference
Test revealed that only the treatment with a 1:1 mixture of
4T1/Ad/B7.1+4T1/SEB cells significantly increases
the survival time of mice with established wild-type metastatic disease
(P = 0.05). Treatment with either 4T1/SEB
alone or 4T1/Ad/B7.1 alone does not significantly
increase survival. Therefore, therapy with this cell-based vaccine
requires expression of all three molecules to extend mean survival time
from 5–7.5 weeks for control-treated mice to 6–10.5 weeks for
therapy-treated mice. Although this increase in survival time is
relatively small, it is statistically significant and compelling
because the immunotherapy was started at week 4 and untreated and/or
4T1-treated mice begin to die as early as 5 weeks after tumor
challenge.
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To assess the metastatic disease, lungs from the treated mice were
removed, dissociated into single cell suspensions, and plated in
culture medium containing 6-thioguanine to determine the number of
clonogenic tumor cells. Ten days later, the number of clonogenic
metastatic cells could be enumerated because 4T1 cells are resistant to
6-thioguanine, whereas normal cells are not resistant and die. As in
Fig. 3
, we are interested in determining whether primary tumor size
effects vaccine efficacy, therefore, the results are plotted as number
of clonogenic metastatic cells in the lungs versus TD at the
start of treatment. A line denoting a level of 10,000 tumor
cells in the lungs is also included because >85% of untreated
tumor-bearing mice contain >10,000 metastatic cells in their lungs
after 42 days of primary tumor growth (3)
. As shown in
Fig. 4
, administration of the 4T1 transfectants significantly reduces the
number of lung metastases (Fig. 4B-D
) relative to
treatment with wild-type 4T1 cells (Fig. 4A
). For example,
13 of 16 (81.2%) mice treated with irradiated parental cells (Fig. 4A
) contained >104 clonogenic lung
metastases, which contrasts 12 of 23 (52.2%) mice treated with either
4T1/SEB or 4T1/Ad/B7.1 (Fig. 4 and C
) and 6 of 21 (28.6%) mice treated with a mixture of
transfectants (Fig. 4D
). After transforming the number of
clonogenic metastases to logarithmic values and analyzing these data
using the Tukey’s Honestly Significant Difference Test, we found that
only treatment with a 1:1 mixture of
4T1/SEB+4T1/Ad/B7.1 cells (Fig. 4D
)
significantly reduced the number of clonogenic lung metastases
(P = 0.05). Treatment with either 4T1/SEB
alone (Fig. 4B
) or 4T1/Ad/B7.1 alone
(Fig. 4C
) did not significantly decrease the number of
clonogenic lung metastases. Previously, we demonstrated that therapy
with MHC class II+/CD80+
vaccines statistically significantly reduced clonogenic lung metastases
in 50% of mice whose immunotherapy was initiated 9–14 days after
tumor challenge but this reduction corresponded to only a 10-fold
maximum reduction when compared with the control group
(3)
. In contrast, treatment of mice carrying 14-day
established primary and metastatic tumor with the combination therapy
of tumor cell transfectants expressing MHC class II, CD80,
and SEB genes decreases spontaneous metastases in the lung
by a maximum of 100-fold. Therefore, effective immunotherapeutic
treatment of tumor-bearing mice with extensively established
spontaneous metastases requires expression of all three molecules.
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in CD4- or CD8-depleted animals and
BALB/c nu/nu mice. As shown in Fig. 5, C and D
, depletion of CD4+
or CD8+ T cells (monoclonal antibodies GK1.5 and
2.43, respectively) eliminates the therapeutic effect of the MHC class
II+, CD80+,
SEB+ vaccine against spontaneous metastases,
whereas depletion with control ascites (Fig. 5B
) has no
effect. In addition, the combination vaccine does not reduce metastatic
disease in BALB/c nu/nu mice (Fig. 5F
).
Collectively, these data demonstrate that the three transfected genes
of the cell-based vaccines are working cooperatively to optimally
activate both CD4+ and CD8+
T lymphocytes and that these lymphocyte populations are essential for
the therapeutic effect.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The modified tumor cells may function directly as APCs for the initial
activation of tumor-specific CD8+ and
CD4+ T cells following immunization. Previous
studies demonstrate that both CD8+ and
CD4+ T lymphocytes are involved in immunity
induced by MHC class II/CD80 vaccines (15)
and that MHC
class II/CD80 modified tumor cells function directly as APCs for the
initial activation of tumor-specific CD4+ T cells
(24)
. Direct presentation of antigen by tumor cells is
possible because tumor cell expression of MHC class II molecules in the
absence of invariant chain allows for presentation of endogenously
synthesized tumor antigens by MHC class II molecules (24
, 25)
. Because the vaccines express MHC class I, class II, CD80,
and SEB molecules, antigen-specific and costimulatory signals will be
efficiently delivered to CD8+ and
CD4+ T cells. Likewise, because the activated
CD8+ and CD4+ T cells are
in close proximity to each other, there should be an efficient transfer
of cytokines between CTLs and T helper cells (see Fig. 6
, right).
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versus
B). Furthermore, other mouse tumor models have demonstrated
that both a class I- and class II-restricted tumor-encoded antigen can
be processed and presented indirectly by host-derived APCs (24
, 26
, 27)
. Taken together, it is likely that host-derived APCs,
capable of migrating to lymph nodes, coordinately present SEB and tumor
antigen to both CD8+ and
CD4+ T cells (see Fig. 6
, left). SEB may also enhance vaccine efficacy because it induces an inflammatory response that stimulates immunity (28) . Gene transfer techniques have demonstrated that in vivo expression of various sAg (SEA, SEB, and TSST-1) DNAs induces intense inflammatory responses (29) . Although systemic administration of sAg (doses >500 µg) typically triggers T-cell release of cytokines such as tumor necrosis factor and lymphotoxin that lead to cachexia (11) , we did not see any adverse side effects in SEB-treated mice.
When a sAg, such as SEB, is coexpressed by the MHC class II/CD80 vaccine, additional activation and/or proliferation signals may be delivered to the specifically activated CD8+ and CD4+ T cells. Because SEB binds to the sides of MHC class II molecules and the T cell receptor while antigenic peptide binds within the MHC class II cleft (30 , 31) , it is feasible that the sAg, tumor antigen-specific, and costimulatory signals are simultaneously received by the T cells. Whereas it is also possible that those signals are not coincident, several studies have shown that activation of T cells by SEB is facilitated or enhanced by B7/CD28 signaling (32, 33, 34, 35) . Controversy exists over the ability of costimulation to inhibit sAg-induced apoptosis, but one report demonstrates that lipopolysaccharide activation of B cells prevents sAg-induced deletion (36) . Regardless of the precise kinetics in which the various activation signals are delivered, coordinate delivery of the three signals improves the efficacy of the vaccines to reduce spontaneous metastatic tumor growth. As a result, T-cell activation may be exceptionally efficient because both direct and indirect antigen presentation occur, thus yielding larger numbers of precisely those CD8+ and CD4+ tumor-specific cells that mediate tumor cell destruction.
New immunotherapies are routinely tested in experimental animal tumor systems. Although such experiments may provide "promising" therapeutic results, tumor regression in animal models does not necessarily predict successful treatment of tumors in human patients. There may be significant physiological and biochemical differences between animals and humans that preclude direct comparison of results, and testing in humans, therefore, will always be required. However, the inability to translate therapies developed in experimental animal models to humans may also be because many of the mouse tumor systems used in immunotherapy studies do not closely model human cancers and, hence, the immunotherapies are not being tested in clinically relevant settings. For example, many immunotherapies are tested in so-called "metastatic" settings, however: (a) the extent of metastatic disease is minimal; (b) the metastases have not arisen spontaneously; and/or (c) the metastases have not been established for significant amounts of time. Furthermore, many commonly used mouse models: (a) are not spontaneously metastatic (e.g., CMS-5 fibrosarcoma, RENCA renal cell carcinoma, CT-26 colon adenocarcinoma, SaI sarcoma, and so forth); (b) rapidly loose their metastatic potential when cultured in vitro (e.g., K1735 melanoma); (c) metastasize poorly unless the primary tumor is excised (e.g., B16 melanoma, line 1 carcinoma); or (d) rapidly invade the local environment, such that animals die from primary tumor before metastatic disease is established (e.g., B16 melanoma). In contrast, the 4T1 mammary carcinoma is spontaneously metastatic and metastasizes to many of the organs to which human breast cancer metastasizes (e.g., lung, liver, and brain). Also, similar to human mammary carcinoma, 4T1 metastases spread and progress while primary tumor is in place. In addition, following inoculation of a small number of tumor cells (7 x 103) in the mammary gland, lethal metastatic disease develops early (within the first 2–3 weeks) and progresses over several weeks so that immunotherapies can be tested against early or very advanced stage disease. The 4T1 tumor, therefore, is an excellent model for testing experimental immunotherapies. In contrast to our earlier studies with the 4T1 tumor in which relatively early metastases were treated and primary tumor was left in place (3) , the studies reported here address very advanced metastatic disease in a postsurgical setting. Although the statistically significant extension of survival time following surgery and administration of immunotherapy was small, we find no comparable studies in the literature in which the efficacy of an immunotherapy is demonstrated in such a clinically relevant model of advanced stage metastatic disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the U.S. Army Research
and Development Command (DAMD17-94-J-4323) and the NIH (RO1 CA52527).
B. P. is supported by a postdoctoral fellowship from the U.S. Army
Research and Development Command (DAMD17-97-1-7152). 
2 To whom requests for reprints should be
addressed, at Department of Biological Sciences, University of Maryland
Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250.
3 The abbreviations used are: SEB,
Staphylococcal aureus enterotoxin B; sAg,
superantigen; APC, antigen-presenting cell; TD, mean tumor
diameter.
Received 11/15/99. Accepted 3/17/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. Ostrand-Rosenberg, V. K. Clements, M. Terabe, J. M. Park, J. A. Berzofsky, and S. K. Dissanayake Resistance to Metastatic Disease in STAT6-Deficient Mice Requires Hemopoietic and Nonhemopoietic Cells and Is IFN-{gamma} Dependent J. Immunol., November 15, 2002; 169(10): 5796 - 5804. [Abstract] [Full Text] [PDF]
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X. Huang, M. K. Wong, H. Yi, S. Watkins, A. D. Laird, S. F. Wolf, and E. Gorelik Combined Therapy of Local and Metastatic 4T1 Breast Tumor in Mice Using SU6668, an Inhibitor of Angiogenic Receptor Tyrosine Kinases, and the Immunostimulator B7.2-IgG Fusion Protein Cancer Res., October 15, 2002; 62(20): 5727 - 5735. [Abstract] [Full Text] [PDF]
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B. A. Pulaski, M. J. Smyth, and S. Ostrand-Rosenberg Interferon-{gamma}-dependent Phagocytic Cells Are a Critical Component of Innate Immunity against Metastatic Mammary Carcinoma Cancer Res., August 1, 2002; 62(15): 4406 - 4412. [Abstract] [Full Text] [PDF]
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E. Cretney, K. Takeda, H. Yagita, M. Glaccum, J. J. Peschon, and M. J. Smyth Increased Susceptibility to Tumor Initiation and Metastasis in TNF-Related Apoptosis-Inducing Ligand-Deficient Mice J. Immunol., February 1, 2002; 168(3): 1356 - 1361. [Abstract] [Full Text] [PDF]
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S. Ostrand-Rosenberg, M. J. Grusby, and V. K. Clements Cutting Edge: STAT6-Deficient Mice Have Enhanced Tumor Immunity to Primary and Metastatic Mammary Carcinoma J. Immunol., December 1, 2000; 165(11): 6015 - 6019. [Abstract] [Full Text] [PDF]
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L. Qi, J.-M. Rojas, and S. Ostrand-Rosenberg Tumor Cells Present MHC Class II-Restricted Nuclear and Mitochondrial Antigens and Are the Predominant Antigen Presenting Cells In Vivo J. Immunol., November 15, 2000; 165(10): 5451 - 5461. [Abstract] [Full Text] [PDF]
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,
) or supernatants from parental 4T1 (
,
), 4T1/SEB-12
(•,
), or 4T1/SEB-14 (
,
) transfectants in the presence
(open symbols) or absence (filled
symbols) of antibody specific for SEB.
