Article

Challenge with mammary tumor cells expressing MHC class II and CD80 prevents the development of spontaneously arising tumors in MMTV-neu transgenic mice

Institut de Sciences et Technologies du Medicament de Toulouse, CNRS-Pierre Fabre, Toulouse, France.
Cancer Gene Therapy (Impact Factor: 2.42). 12/2006; 13(11):1002-10. DOI: 10.1038/sj.cgt.7700974
Source: PubMed

ABSTRACT

The HER-2/Neu oncogene has been implicated in human and mouse breast cancer. Indeed, transgenic MMTV-neu mice expressing this oncogene from the mammary tumor virus long terminal repeat develop spontaneous mammary tumors and die within 1 year of life. We have expressed the class II transactivator (CIITA) and/or the costimulatory molecule CD80 (B7.1) in a mammary carcinoma cell line (MCNeuA) derived from these mice. Class II transactivator directs the expression of MHC class II and the machinery for antigen processing and presentation by this pathway. When injected into MMTV-neu mice, tumor cells expressing CD80 or CD80 and CIITA, were rejected completely. In addition, following the rejection of dual expressing cells, 75% of the mice were protected against the development of subsequent spontaneous tumors. Cells expressing only CD80 or CIITA were not as effective as antitumor vaccines in preventing the development of spontaneous tumors. Thus, converting cancer cells into antigen presenting cells could represent an effective immunotherapy for breast cancer.

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Available from: Michael Campbell, Apr 14, 2014
ORIGINAL ARTICLE
Challenge with mammary tumor cells expressing MHC class II
and CD80 prevents the development of spontaneously arising
tumors in MMTV-neu transgenic mice
N Jabrane-Ferrat
1
, MJ Campbell
2
, LJ Esserman
2
and BM Peterlin
3
1
Institut de Sciences et Technologies du Medicament de Toulouse, CNRS-Pierre Fabre, Toulouse, France;
2
Department of Surgery, University of California, San Francisco, CA, USA and
3
Department of Medicine,
Rosalind Russell Medical Research Center, University of California, San Francisco, CA, USA
The HER-2/Neu oncogene has been implicated in human and mouse breast cancer. Indeed, transgenic MMTV-neu mice expressing
this oncogene from the mammary tumor virus long terminal repeat develop spontaneous mammary tumors and die within 1 year of
life. We have expressed the class II transactivator (CIITA) and/or the costimulatory molecule CD80 (B7.1) in a mammary carcinoma
cell line (MCNeuA) derived from these mice. Class II transactivator directs the expression of MHC class II and the machinery for
antigen processing and presentation by this pathway. When injected into MMTV-neu mice, tumor cells expressing CD80 or CD80
and CIITA, were rejected completely. In addition, following the rejection of dual expressing cells, 75% of the mice were protected
against the development of subsequent spontaneous tumors. Cells expressing only CD80 or CIITA were not as effective as antitumor
vaccines in preventing the development of spontaneous tumors. Thus, converting cancer cells into antigen presenting cells could
represent an effective immunotherapy for breast cancer.
Cancer Gene Therapy (2006) 13, 1002–1010. doi:10.1038/sj.cgt.7700974; published online 7 July 2006
Keywords: breast cancer; immunity; costimulation; MHC class II; CIITA
Introduction
One of the major goals of tumor immunotherapy is the
induction of tumor-specific immune responses that
recognize and eradicate neoplastic cells. Insights into the
mechanisms of T-cell activation have provided the basis
for developing such new approaches. Both antigen-
dependent and -independent interactions are necessary
for the generation of specific effector T cells and for the
establishment of T-cell memory. Activation of T cells by
antigen presenting cells (APCs) requires two signals. The
first signal is provided by the recognition of antigenic
peptides presented by major histocompatibility (MHC)
class I or class II molecules. A second costimulatory
signal is provided by the ligation of CD28 on the surface
of T cells by a B7 family member (B7.1 or CD80 and/or
B7.2 or CD86) on these APCs.
1–3
Importantly, in some
cases, T cells that have encountered antigen in the absence
of a costimulatory signal undergo a state of unrespon-
siveness or anergy that cannot be overcome easily by
subsequent costimulation.
4
A common defect in the recognition and eradication of
tumor cells by T lymphocytes is the lack of appropriate
antigen processing and presentation (APP) and/or the
absence of costimulation by the tumor cells themselves.
As the expression of CD80 is mostly limited to profes-
sional APCs, the weak immunogenicity of tumor cells
could reflect the lack of appropriate costimulation of
naı
¨
ve T cells. Indeed, tumor cells engineered to express
costimulatory molecules can induce antitumor responses
in several animal models of cancer.
5–15
The coexpression
of cytokine molecules with costimulatory molecules
has also been shown to induce tumor immunity.
16–20
However, in most cases, the therapeutic efficacy of this
strategy is limited, possibly due to lack of T-helper cells,
whose activation depends on the expression of MHC class
II.
21,22
One limitation of this approach is the suboptimal
APP by MHC class I on many tumor cells, such that the
CD80- or CD86-transduced cells deliver only a costimu-
latory signal to T cells.
Although several attempts have been made to restore
the expression of MHC determinants on tumor cells, the
full restoration of APP has been more difficult, especially
since it requires additional accessory molecules. The
identification of the class II transactivator (CIITA),
which is the master coactivator for the transcription of
genes required for APP, offered new insights and
possibilities for cancer immunotherapy.
23,24
Class II
transactivator not only induces the expression of the
Received 19 December 2005; revised 10 February 2006; accepted 22
April 2006; published online 7 July 2006
Correspondence: Professor BM Peterlin, Department of Medicine,
Box 0703, UCSF, 3rd and Parnassus Aves, San Francisco, CA
94143-0703, USA.
E-mail: matija.peterlin@ucsf.edu
Cancer Gene Therapy (2006) 13, 10021010
r
2006 Nature Publishing Group All rights reserved 0929-1903/06 $30.00
www.nature.com
/
cgt
Page 1
polymorphic structural a- and b-chains of MHC class II
molecules but also induces monomorphic DOA and
DOB, as well as the DMA, DMB, and invariant chain
(Ii) genes. In some cells, CIITA can also increase the
expression of MHC class I genes.
25–27
Thus, engineering
tumor cells to express CIITA can reconstitute APP by
both the MHC class I and class II compartments, and
with the addition of costimulatory molecules may trans-
form them into APCs.
Despite this promise, results of such manipulations of
tumor cells have been variable. In some tumors, the
expression of CIITA and CD86 paradoxically increased
the growth of the tumor.
28
In others, the absence of Ii led
to a greater immunogenicity of tumors, presumably
because of better loading of self-peptides onto the
unprotected structural heterodimers during their bio-
synthesis.
29
Finally, in other studies, CIITA had ther-
apeutic effects even in the absence of costimulation.
30
Variable levels of expression of transduced genes and/or
the extinction of MHC class II on tumor cells might
explain some of these findings. Alternatively, some of
these solid tumors might express endogenous costimula-
tory molecules. In this study, we examined whether high
levels of expression of MHC class II and CD80 molecules
could create an effective antitumor vaccine against the
development of mouse mammary tumors. We expressed
these molecules in MCNeuA cells, which were derived
from MMTV-neu mice.
31
We describe the impact of
expressing CIITA and CD80, alone or in combination,
on the ability of these cells to form tumors in MMTV-neu
mice as well as their effects on the development of
spontaneous mammary tumors in these mice.
Materials and methods
Retroviral constructions
cDNAs encoding the N-terminal Flag epitope-tagged
human CIITA (hCIITA) and mouse B7.1 (mCD80)
proteins were subcloned into the EcoR1 site of the
pWZLblast2 bicistronic retrovirus, which also encodes
resistance to the antibiotic blasticidin. Recombinant
amphotropic retroviruses were generated by transient
transfection of Phoenix A packaging cells (PA317), as
described previously.
32,33
Transgenic mice and mammary carcinoma cell line
The transgenic mice used in this study, FVB/N-TgN
(MMTV-neu) N202 (denoted MMTV-neu), carries the
wild-type rat neu transgene under the control of the
mouse mammary tumor virus (MMTV) long terminal
repeat (LTR).
34
Female MMTV-neu mice develop mam-
mary lesions that are similar to human breast cancer. The
MCNeuA mammary carcinoma cell line was established
from a tumor that arose in a female MMTV-neu mouse.
31
This cell line forms tumors when transplanted into
syngeneic MMTV-neu mice.
Cell culture and retroviral infection of cells
MCNeuA and PA317 cell lines were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% (v/v) heat-inactivated fetal calf serum
(FCS), 100 m
ML-glutamine and 50 mg each penicillin and
streptomycin per ml. All cells were grown in an atmo-
sphere of 5% CO2 at 371C. PA317 cells were plated 12 h
before transfection in 100 mm tissue culture dishes.
Monolayers of PA317 cells were transfected with 10 mg
of pWZLblast2 (M cells), pWZLblast2:hCIITA (MC
cells), pWZLblast2:mCD80 (MB cells) retroviral plasmid
vectors in OptiMEM media for 5 h using Lipofectamine
(Life Technologies, Grand Island, NY). Media were
changed 24 h post-transfection (no antibiotics), and cells
were allowed to grow for an additional 48 h. Twenty-four
and 48 h supernatants were collected and cellular debris
was removed by filtration. Monolayers of MCNeuA cells
in mid-log growth phase were infected by the addition of
50% fresh viral supernatant and 8 mg/ml polybrene
(Sigma, St Louis, MO) for 12 h. The infection was
repeated with supernatants collected at 48 h. Media were
changed and cells were allowed to grow for an additional
48 h before selection in 24-well dishes. Cells were selected
in DMEM with 10% FCS containing blasticidin (25 mg/
ml). A pooled population of cells expressing CIITA or
CD80 was trypsinized, expanded and subsequently cloned
by limiting dilution. To express both CIITA and CD80
(MBC cells), CIITA selected clones cells were subse-
quently infected with the CD80 retrovirus. Double
expressors were sorted by flow cytometry and single
clones were isolated subsequently by limiting dilution.
Growing tumors were also removed from the MMTV-
neu mice, washed in phosphate-buffered saline (PBS) at
41C, cut into small pieces (2–4 mm), and incubated with of
mixture of trypsin (0.25%) and collagenase in DMEM
serum-free media at 371C for 3 h. The reaction was
stopped by the addition of 10% FCS and separated cells
were filtered through a fine mesh cloth. The cell
suspension was then washed in PBS, plated in the
complete DMEM medium with serum and allowed to
grow for 24 h. Cells were analyzed as above.
Protein extracts, immunoprecipitations and Western
blotting
Cells were lysed in lysis buffer containing 20 mM Tris,
137 m
M NaCl, 5 mM ethylenediamine-N,N,N
0
,N
0
-tetraace-
tic acid and 0.5% (w/v) NP-40 and a cocktail of protease
inhibitors.
35
The amount of protein was quantified with
the BCA assay kit (Pierce Biotechnology, Inc., Rockford,
IL). Equivalent amounts of proteins were precleared with
protein A-conjugated Sepharose beads, and were sub-
jected to immunoprecipitation by using anti-Flag M2
monoclonal antibody (mAb) (Sigma-Aldrich, St. Louis,
MO). Immunoprecipitated complexes were resolved by
sodium dodecyl sulfate-polyacrylamide gels (7.5%
PAGE) and analyzed by Western blotting with the anti-
Flag M2 mAb. After washing under stringent conditions,
immune complexes were revealed by using the horseradish
peroxidase-conjugated anti-mouse IgG antibody (Amer-
sham, Biosciences, Piscataway, NJ) and visualized by
Challenge with mammary tumor cells expressing MHC class II and CD80
N Jabrane-Ferrat et al
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using an enhanced chemiluminescence, ECL-Plus, detec-
tion kit (NEN Life Science Products, Boston, MA).
Flow cytometry and immunohistochemistry
The mAbs used for these studies were: anti-CD80
(PharMingen, San Diego, CA), anti-MHC class II (anti-
IEb/IAb, which reacts with d, b, p, q, u and j haplotypes)
(provided by Dr R Accolla, Verona, Italy), and anti-
c-Neu (Ab-4, Oncogene Sciences, Cambridge, MA).
Secondary Abs used were goat anti-mouse IgG-conju-
gated to either fluorescein isothiocyanate or phycoery-
thrin. Single-cell suspensions or monolayer cells grown on
tissue culture chamber slides were washed in PBS
containing 1% mouse serum, were stained with primary
and secondary Ab, and then analyzed. Flow cytometry
was performed on a fluorescence-activated cell sorting
calibur (Becton Dickinson Immunocytometry Systems,
San Jose, CA). Dead cells were excluded by their uptake
and staining with propidium iodide. Cells attached to
tissue culture slides were analyzed by using fluorescence
microscopy (Nikon, Japan).
Tumorigenicity of cell lines
Cells at 50–80% confluency were harvested by trypsiniza-
tion, washed, and resuspended at 2.5 10
7
cells/ml in
PBS. Two hundred microliters (5 10
6
cells) were injected
subcutaneously (s.c.) into the hindleg of 6- to 8-week-old
female MMTV-neu mice. Mice were then monitored for
tumor growth every other day starting at one week
postinjection. Tumor measurements (length and width)
were recorded by using a caliper. Tumor volume was
calculated as (length width2)/2.
Lymphocyte purification and growth
Single-cell suspensions were prepared from harvested
spleens of 8- to 10-week-old MMTV-neu female mice
using a 40 m
M nylon cell strainer (Becton Dickinson).
After lysis of red blood cells using ACK lysis solution
(Biofluids, Rockville, MD), splenocytes were washed in
PBS, layered onto Ficoll–Paque solution (Pharmacia
Biotech, Piscataway, NJ) and were centrifuged for
20 min at 2000 r.p.m. at 41C. Live cells were washed and
nonadherent mononuclear leukocytes were collected after
depletion of adherent cells by incubation for 3 h at 371Cin
tissue culture flasks. The enrichment of each subset of T
cells was assessed by flow cytometry.
36
MCNeuA-derived
cells were plated to 80–90% confluency 24 h before the
assay in 96-well flat-bottom plates (Costar, Gaithersburg,
MD), and were irradiated with 3000 Rads 3–4 h before
use as APC. Purified lymphocytes were treated for 3 h
with 0.5 m
M phorbol myristate acetate plus 0.1 mM
Ionomycin and then were plated at 5 10
4
cells/well onto
a monolayer of irradiated mammary cells in a final
volume of 200 ml/well. Media were changed every 2–3
days for a total of 6 days. Cultures were pulsed with 1 mCi
of [
3
H]thymidine and were harvested 16 h later onto glass
filters by using a 96-well plate harvester. Incorporated
radioactivity was measured by using a liquid scintillation
counter. Results are presented as mean thymidine uptake
7s.e.m.
Statistical analyses
Comparisons of tumor sizes were performed by using the
Student’s t-test. Tumor incidence curves were analyzed by
Wilcoxon’s two-sample test.
Results
Expression of CD80 and/or CIITA in MCNeuA cells
To stably express high amounts of CD80 and MHC class
II molecules in MCNeuA cells, we constructed replica-
tion-defective retroviral expression plasmids, which en-
coded the cDNAs of interest and the blasticidin resistance
gene. The mouse CD80 and human Flag epitope-tagged
CIITA open reading frames were inserted in the EcoR1
site of the pWZLblast2 retroviral vector. In pWZLblast2,
the genes of interest, followed by the internal ribosomal
entry site (IRES) from the encephalomyocarditis virus
and the blasticidin resistance gene, are transcribed from
the MLV LTR.
37,38
Amphotropic retroviruses were
produced and used to infect MCNeuA cells. Resistant
colonies were drug-selected, expanded, and isolated by
limiting dilution cloning. With each retroviral transduc-
tion, at least three independent clones were assayed for
the expression of the appropriate genes. Cells expressing
the highest amounts of the transduced genes were
characterized further and used throughout the study.
MCNeuA cells expressing only blasticidin resistance (M),
CD80 (MB), CIITA (MC) or both CD80 and CIITA
(MBC) were used for all further experiments (Figure 1A).
Besides immunofluorescence (Figure 1A), cells were
examined for the expression of CIITA by Western
blotting (Figure 1B, lanes 1–4) and of MHC class II by
flow cytometry (Figure 1C, white peaks in M and MBC
panels). All cells expressed high amounts of c-Neu
(Figure 1A, panels b, d, f and h). MB and MBC cells
also expressed abundant CD80 (Figure 1A, panels c
and g) whereas MC cells did not express CD80
(Figure 1A, panel e). As MC and MBC cells contained
CIITA (Figure 1B, lanes 3 and 4), they expressed high
amounts of MHC class II on their surface (Figure 1C,
white peak, MBC panel). Although only MBC cells are
presented in Figure 1C, similar results were obtained with
the MC cells. Of note, the amounts of MHC class II
expressed on these transfectants, as measured by im-
munofluorescence, were three logs over the background
fluorescence, which is similar to amounts of MHC class II
found on activated B cells (data not presented). We
conclude that MCNeuA cells were transduced efficiently
with the retroviral vectors and expressed high levels of the
introduced genes.
MB, MC and MBC cells stimulate lymphocyte
proliferation in vitro
To examine functional consequences of expressing CD80
and/or CIITA in MCNeuA cells, we performed prolifera-
tion assays with nonadherent syngeneic mouse spleno-
cytes, cocultured with M, MB, MC or MBC cells in vitro.
As shown in Figure 2, MB (white squares), MC (white
diamonds) and MBC (black stars) cells were able to
increase the proliferation of lymphocytes as measured by
Challenge with mammary tumor cells expressing MHC class II and CD80
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[
3
H]thymidine incorporation. The coexpression of CIITA
and CD80 in MBC cells led to the greatest response
(Figure 2, black stars). However, M cells (white circles)
even at the highest responder to stimulator cell ratio
(20:1) did not stimulate lymphocyte proliferation (Fig-
ure 2, white circles). We conclude that T cells recognize
these new antigens on MCNeuA cells and that two
activation signals have the greatest effect.
Suppressed growth of MC, MB and MBC cells in
MMTV-neu mice
To determine whether the expression of CD80 and/or
CIITA in MCNeuA cells effects tumor growth in MMTV-
neu mice, groups (n ¼ 8–14) of 6- to 8-week-old MMTV-
neu female mice were inoculated s.c. with 1 10
6
modified
tumor cells and monitored for a period of 40 days. Mice
in the control group were inoculated with M cells. These
M cells are highly malignant and tumors arose 12 days
after inoculation. Indeed, all these mice had to be killed
by day 40 (Figure 3, white circles). In contrast, expression
of CIITA in MC cells slowed tumor growth (Figure 3,
white diamonds). Although seven out of eight mice in this
group developed tumors, these tumors were significantly
smaller compared to those in the MMTV-neu mice that
received M cells (Figure 3, white diamonds). The
expression of either CD80 alone or CD80 and CIITA in
MCNeuA cells resulted in complete inhibition of tumor
growth in MMTV-neu mice that received MB and MBC
cells (Figure 3, white squares and black stars). We
repeated these experiments with two independently
derived MB, MC and MBC clones and obtained similar
results (data not presented). We conclude that expression
of the costimulatory CD80 molecule alone or together
with MHC class II prevents the growth of mammary
tumors in MMTV-neu mice. These findings correlated
with the in vitro proliferative responses presented in
Figure 2.
To determine if MC cells continued to express MHC
class II after prolonged growth in MMTV-neu mice, these
tumors were explanted 27 days after the initial inocula-
tion. Cell suspensions prepared from the tumors were
analyzed for MHC class II expression by flow cytometry
as described in Figure 1c. Representative data of
explanted tumor cells are presented in Figure 4. As
shown, a significant proportion of MC tumor cells
explanted at day 27 have lost the expression of MHC
class II. This finding most likely reflects not only the
presence of normal cells but also the ability of tumor cells
Figure 1 Analysis of transduced cells. MCNeuA cells were transduced with pWZLblast2 bicistronic retroviruses that are either empty (M) or
expressing CIITA (MC), B7.1 (MB) or both proteins (MBC). Cells were selected for blasticidin resistance. (A) All cells express c-Neu, but only MB
and MBC cells also express CD80. Single clones were isolated and immunostained for CD80 and c-Neu by using anti-CD80 mAb and polyclonal
anti-Ab4 antibodies. Cells in panels a, c, e and g were stained for CD80 (red fluorescence); cells in panels b, d, f and h were stained for c-Neu
(green fluorescence). Identities of cells are given above and below the panels. (B) MC and MBC cells express CIITA. The expression of CIITA
was determined by immunoprecipitation, followed by immunoblotting using the anti-Flag epitope-tag mAb. Identities of cells are given to the left of
þ signs. The arrow indicates the position of CIITA on the western blot. (C) MB and MBC cells express high amounts of MHC class II on their
surface. Presented is the expression of MHC class II proteins on M and MBC cells. White histograms represent cells stained with an anti-MHC
class II mAb. Gray histograms represent basal fluorescence obtained with an irrelevant isotype-matched control mAb. Values are given as
relative fluorescence intensity.
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bearing low levels of MHC class II to survive better in
MMTV-neu mice. As MC and MBC cells did not grow in
MMTV-neu mice, the evolution of CD80 could not be
followed in parallel. Thus, although the presence of MHC
class II delayed the progression of MC tumors (Figure 3),
the subsequent loss of MHC class II molecules may
explain the eventual outgrowth of these tumors.
Immunizations with MB and MBC cells reduces the
incidence of spontaneous mammary tumors in
MMTV-neu mice
To determine the effects of expressing CD80, or CD80
plus CIITA, in MCNeuA cells on the development of
spontaneous mammary tumors, MMTV-neu mice were
given a single injection of MB or MBC cells at 6 weeks of
age and followed over time for tumor growth. The
injected tumor cells were rejected completely. In addition,
only half of the mice that were inoculated with MB cells
(Figure 5, white squares) and 25% of the mice that
received MBC cells (Figure 5, black stars) developed
spontaneous tumors by 60 weeks of age. In contrast,
control mice began to develop spontaneous mammary
tumors around 30 weeks of age and by 50 weeks of age,
100% of these mice (n ¼ 10) had developed tumors
(Figure 5, first solid line). Immunization with MB or
MBC cells resulted in significant prolonged tumor-free
period as compared to nonimmunized control mice
(Po0.002). Of note, the challenge with MBC cells rather
than MB cells delayed the onset of any tumors by
additional 20 weeks (Figure 5, compare black stars to
white squares). Of note, MC cells could not be studied in
parallel. When inoculated into MMTV-neu mice, MC
Figure 2 Syngeneic mouse splenocytes proliferate to MB, MC and
MBC but not M cells in vitro. Lymphocytes were purified from MMTV-
neu mice and used as responders. M (white circles), MB (white
squares), MC (white diamonds) and MBC (black stars) cells were
plated in flat-bottom 96-well plates at the density of 10
4
cells per well,
cultured for 48 h, irradiated and used as APCs. Nonadherent cells
were added to irradiated APCs for 3–5 days at the final density of 10
5
cells/well. They were pulsed with [
3
H]thymidine during the last 18 h of
coculture. Results are presented as mean c.p.m. 10
3
from four
replicate cultures; standard errors of the mean are represented by
error bars.
Figure 3 The expression of CIITA or CD80 on M cells leads to
decreased tumor growth. 1 10
6
cells were transplanted into the
hindlimbs of 6- to 8-week-old MMTV-neu female mice on day 0.
Individual mice were monitored for tumor growth over a period of 40
days. The volume for individual tumors was calculated for each
mouse as (length width)/2. Each curve represents the mean tumor
volume calculated for each group of 6–8 mice. Standard errors of the
mean are represented by error bars.
Figure 4 In vivo loss of MHC class II proteins on tumors. MMTV-
neu mice were injected with MC cells. Growing tumors were
harvested 27 days after transplantation. Single-cell preparation from
explanted cells, as well as input cells, were analyzed for the surface
expression of MHC class II proteins by flow cytometry. The white and
gray histograms are as in Figure 1c.
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cells proliferated (Figure 4) and these mice had to be
killed early because of transplanted rather than sponta-
neous tumors.
Histologic analyses were performed on mammary
tissues derived from 35 week-old mice that were
inoculated with MBC cells. As presented in Figure 6,
wild-type MMTV-neu mice developed massive mammary
tumors composed of poorly differentiated, densely packed
tumor cells (left panels, low and high power views). In
contrast, mice inoculated with MBC cells did not develop
tumors and had histologically normal mammary glands
(Figure 6, right panels, low and high power views). The
only exception was the appearance of dense mononuclear
infiltrates near the ducts in the mice that received the
MBC cells 9 months earlier (Figure 6, right panel, m). It is
likely that these infiltrates represent antitumor immune
responses in these manipulated MMTV-neu mice. These
histological findings suggest that the absence of mammary
tumors is due to beneficial immune responses in the
immunized MMTV-neu mice.
Discussion
In this report, we demonstrated that expression of CD80
or CD80 and CIITA molecules in mouse mammary tumor
cells abrogates their tumorigenicity. Furthermore, these
cells when administered as a single injection to 6-week-
old MMTV-neu transgenic mice, conferred significant
Figure 5 Spontaneous formation of tumors in MMTV-neu mice that
rejected MB or MBC tumors. MMTV-neu mice, 6- to 8-week-old,
were injected at day 0 with PBS or 5 10
5
live MB or MBC cells. The
incidence of spontaneous tumors was monitored for more than than
70 weeks. Control mice (MMTV-neu), and those injected with MB (39
mice, white squares) and MBC (14 mice, black stars) were followed.
Inoculation with MB or MBC cells resulted in a significantly prolonged
tumor-free period as compared to nonimmunized parental mice
(Po0.002).
Figure 6 Hematoxylin and eosin-stained sections of mammary tissues from MMTV-neu mice and those injected with MBC cells. Sections were
derived at 35 weeks of age from MMTV-neu mice (left panels) or mice that were transplanted with MBC cells at 6–8 weeks of age (right panels).
Sections were stained with hematoxylin and eosin. Left two panels depict a typical large invasive malignant tumor (t) and mammary ducts (d) as
visualized by light microscopy at low and high power. Right two panels contain mammary tissues from an age-matched MMTV-neu mouse that
was injected with MBC cells and contains normal ducts (d) and extensive mononuclear infiltrates (m).
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protection from the subsequent development of sponta-
neous mammary tumors. These findings suggest that
mammary tumor cells can become efficient APCs that
immunize against the development and growth of breast
cancer. As such, they may serve as an effective antitumor
vaccine.
Although expression of CIITA alone delayed tumor
growth, it did not completely inhibit it (as compared to
CD80 or CD80 plus CIITA). This finding could be due to
the need for the coexpression of a costimulatory molecule
such as CD80. Alternatively, the lack of complete tumor
inhibition may have been due to a loss of MHC class II
expression over time in vivo (see Figure 4). Loss of MHC
class II expression may also explain the observation that
mice immunized with CD80 plus CIITA expressing tumor
cells were not protected completely against spontaneous
tumor growth (Figure 5). Thus, repeated immunizations
with cells expressing CIITA and CD80 should circumvent
this problem.
In vivo animal immunotherapy models using tumor
cells transfected with CIITA have been reported for a
mouse sarcoma, SaI,
29
lung carcinoma, Line 1,
28
and
mammary adenocarcinoma, TS/A.
30
As in our study,
transfection of CIITA resulted in the expression of MHC
class II molecules in each of these models. However,
effects of CIITA expression on tumor growth in vivo
varied. For example, SaI tumor cells expressing CIITA or
only structural MHC class II determinants were not or
were rejected when injected into mice, respectively. In
contrast to these two studies, the expression of CIITA in
TS/A mammary carcinoma cells significantly reduced
their tumorigenicity and mice that rejected TS/A-CIITA
cells were resistant to a subsequent challenge with
parental TS/A tumor cells.
The differences in results between these four studies
may be due to differences in the types of tumors used. TS/A
and MCNeuA are mammary carcinomas as compared
to the SaI sarcoma and the Line 1 lung carcinoma.
MCNeuA cells differ from TS/A in that they were derived
from a spontaneous mammary tumor in a Neu transgenic
mouse. As such, the MCNeuA cell line overexpresses the
Neu receptor tyrosine kinase (RTK). Interestingly, the
Neu RTK activates the mitogen activated kinase signaling
cascade, which in turn can increase the transcriptional
activity of CIITA.
39,40
Variable levels of expression of the
transduced genes and/or the extinction of MHC class II
on tumor cells might also explain some of the differences
in results between these studies.
The findings from this study and others suggest that
converting tumor cells into APCs, tumor-APCs, is a
potentially useful immunotherapeutic strategy. In tumor-
APCs, the presentation of endogenous tumor antigens is
expected to improve, especially since the APP machinery
is turned on maximally even when exogenous antigens,
such as bacteria, are not encountered. Some of these
endogenous antigens might also enter tumor-APCs by
endocytosis or pinocytosis of exosomes and debris from
apoptotic cells. This MHC class II APP of exogenously
delivered antigens would not suffer from the inhibition
of early peptide loading provided by the Ii. Indeed,
differential formation of exosomes and/or levels of
apoptosis of tumor cells could well explain differences
between various studies with tumor-APCs. Importantly,
we also used live cells that provided continuous antigenic
stimulation. Additional considerations include the pre-
sence or absence of appropriate proteases that cleave
these proteins, antigens that can be recognized by the
immune system, and the abundance of regulatory T cells
(Tregs), which might blunt as vigorous an immune
response as would be required. Some of these could be
overcome by the addition of specific cytokines, such as
IFNg and/or GM-CSF, blocking effects of TGFb or
IL10, as well as the addition of optimized antigens and/or
antigenic peptides to these tumor-APCs. Indeed, with
breast cancer, most of these other approaches have been
tried in animal models and in patients, some with highly
encouraging results.
41–49
Although these results are promising, the next step is to
identify a clinically feasible method to deliver this type of
immunotherapy. Although live cells were used in our
study, the use of inactivated tumor-APCs, transduced
ex vivo with CIITA, costimulatory molecules (CD80 or
CD86), and/or cytokines or chemokines, would be a more
feasible approach and will be evaluated in our preclinical
model. Alternatively, viral or nonviral systems could be
used to deliver CIITA and CD80 expression constructs
directly to tumor cells in vivo. Since the expression of these
genes should lead to an immune response against the
tumor, the delivery system would not have to be 100%
efficient. Transduction of only a fraction of the tumor
may be sufficient to elicit an immune attack against the
untransfected cells as well.
Acknowledgements
We would like to thank Gorazd Drozina, Lewis Lanier,
Nada Nekrep and Giovanna Tosi for helpful comments
and suggestions and Talal Al Saati for help with histology
sections. This work was funded with grants from the
Breast Cancer Research Program (#6KB-0116), the NIH
(AI050770), the Treadwell Foundation, and the Breast
Cancer Research Foundation.
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  • Source
    • "DC loaded with TAAs have been also used with the aim of providing a direct source of ready-to-use MHC-II–tumor peptide complexes for optimal priming and triggering of TH cells (24, 25) and recent clinical results in melanoma patients give further hope in improving clinical responses by this approach (26). Several groups, including ours, have instead investigated the possibility to render tumor cells themselves MHC class II-positive and thus used them as potential surrogate APC for triggering tumor-specific TH cells (27–29). Within this frame, two distinct approaches have been described. "
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    Full-text · Article · Feb 2014 · Frontiers in Oncology
  • Source
    • "MHC class II positive tumor cells are also effective APCs in vivo and can present novel endogenous antigenic peptides not presented by host APCs [5]. Furthermore, transfection of tumors with class II transactivator (CIITA) elicits MHC class II expression and can restore the ability of certain tumor cells to present antigen and induce immunity [9,10]. Although cross-presentation is the major mechanism generating immunity [2,3], the above studies on tumors as APC suggest that, at least in certain tumors, direct antigen presentation could provide an alternative or additional pathway in tumor immunity. "
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    Full-text · Article · Feb 2007 · Journal of Translational Medicine
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