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Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; and
Samuel C. Johnson Medical Research Building, Mayo Clinic Scottsdale, Scottsdale, AZ 85259
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC transfected with RNA derived from tumor cells have also been used as vaccines (13, 14). DC transfected with chicken OVA RNA stimulate primary anti-OVA CTL responses in vitro. Moreover, vaccination of mice with OVA RNA-transfected DC has been used to treat OVA-positive tumor metastases to lung (13). Other studies using human DC transfected with carcinoembryonic Ag RNA have demonstrated effective generation of Ag-specific CTL in vitro (14, 15). An advantage of using RNA from tumors, rather than peptides, is that RNA encodes multiple epitopes for presentation by diverse HLA alleles. In addition, DC that are transfected with RNA encoding a tumor Ag or RNA extracted from patient-derived tumor endogenously express the tumor Ag by translation of the transfected RNA. The tumor Ag is then subject to endogenous processing by the DC and presentation of peptides to induce a T cell response (16, 17, 18).
The human DF3/MUC1 Ag is aberrantly overexpressed in breast, ovarian, pancreatic, and other carcinomas (19, 20). The heavily glycosylated MUC1 protein is normally expressed by epithelial cells at apical borders lining the lumen of duct (21, 22). By contrast, in transformed epithelial cells, MUC1 is expressed at high levels over the entire cell surface. Moreover, defective glycosylation of the MUC1 protein in transformed epithelial cells has been associated with unmasking of cryptic epitopes. Immunity against MUC1 has been identified in certain patients with tumors (23). Non-MHC-restricted cytotoxic T cell responses to MUC1 have been reported in patients with breast, pancreatic, and ovarian cancers (24, 25, 26, 27, 28). In addition, Ab responses to MUC1 have been found in patients with ovarian cancer (29). These findings suggest that MUC1 represents a potential target for active specific immunotherapy of certain human tumors.
The present studies have assessed the effects of transfecting MUC1 RNA into DC as a vaccine for the induction of immunity against MUC1-positive tumors. The results demonstrate that DC transfected with MUC1 RNA (DC/MUC1 RNA) induce protection against challenge of wild-type mice with MUC1-positive MC38 carcinoma cells. We also demonstrate that vaccination with DC/MUC1 RNA and coadministration of IL-12 reverses immunologic unresponsiveness to MUC1 Ag in MUC1-transgenic (MUC1.Tg) mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female wild-type C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). C57BL/6 MUC1-transgenic mice (MUC1.Tg) were housed and maintained in microisolator cages under specific pathogen-free conditions. MUC1.Tg-positive mice were identified by PCR analysis as previously described (30, 31).
Synthesis of mRNA transcripts
An XbaI fragment containing a full-length MUC1 cDNA
with seven tandem repeats was cloned into the XbaI site of
the pcDNA3 plasmid under control of the T7 RNA polymerase promoter
(pcDNA3/MUC1) (32, 33). Clones containing the MUC1 cDNA
were isolated, and large scale preparations were generated using Maxi
Prep kits (Qiagen, Valencia, CA). The plasmid was then linearized and
transcribed using the mCap TM RNA Capping kit (Stratagene, La Jolla,
CA). The linearized DNA template was removed by digestion with DNase,
and mRNA transcripts were purified by phenol/chloroform extraction
followed by ethanol precipitation. Capped mRNA was polyadenylated by
yeast poly(A) polymerase (Amersham, Piscataway, NJ). Capped,
polyadenylated MUC1 mRNA was recovered by phenol/chloroform extraction
and ethanol precipitation. As a control, capped, polyadenylated
-galactosidase mRNA was also synthesized in vitro.
Cell culture and DC generation
Murine (C57BL/6) MC38 adenocarcinoma cells were stably transfected with MUC1 cDNA (MC38/MUC1) (34, 35). The MC38, MC38/MUC1 tumor cells, and MCF7 human breast carcinoma cells (MUC1 positive; American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. DC were generated from bone marrow of wild-type C57BL/6 mice as previously described (36). Briefly, bone marrow was flushed from the long bones and depleted of lymphocytes, granulocytes, and Ia+ cells using mAbs 2.43 (anti-CD8; American Type Culture Collection), GK1.5 (anti-CD4; American Type Culture Collection), RA3-3A1/6.1 (anti-B220/CD4SR; American Type Culture Collection), B21.1 (anti-Ia; American Type Culture Collection) RB6-85C (anti-Gr-1; PharMingen, San Diego, CA), and rabbit complement. Cells were resuspended in RPMI 1640 medium containing 15 ng/ml recombinant murine GM-CSF (Sigma, St. Louis, MO). On day 6 of culture, nonadherent and loosely adherent cells were collected and replated in 100-mm tissue culture dishes (106 cells/ml; 10 ml/dish). The nonadherent cells were removed after 30 min by washing, and medium containing GM-CSF was added to the dish. The cells were incubated for 24 h, and nonadherent cells (DC) were removed for transfection with RNA.
Transfection of DC
DC were collected on day 7 and washed twice in serum-free
Opti-MEM medium (Life Technologies, Grand Island, NY). DC were
resuspended in Opti-MEM at 1 x 107 cells/ml
in 50-ml Falcon tubes (Becton Dickinson, Franklin Lakes, NJ). MUC1 RNA
(25 µg/500 µl of Opti-MEM) was added to 500 µl of Opti-MEM
containing
N-[1-(2,3-dioleoyloxypropyl]-N,N,N,-trimethylammonium
methylsulfate (DOTAP) (50 µg/500 µl of Opti-MEM; Roche, Mannheim,
Germany). After 20 min at room temperature, the mixture was added to a
50-ml tube containing 1 x 107 DC and
incubated at 37°C with occasional agitation for 2.5 h. The DC
transfected with MUC1 RNA (DC/MUC1 RNA) and DC transfected with
-galactosidase RNA (DC/-gal RNA) were washed twice and
resuspended in PBS (13). The mature DC/MUC1 RNA were
obtained by addition of 0.1 µg/ml LPS (Escherichia coli;
Sigma) for 20 h after DC transfected with MUC1 RNA.
Flow cytometry analysis
DC, DC/MUC1 RNA, or DC/-gal RNA were washed with PBS and
incubated with mAb DF3 (anti-MUC1), M1/42/3.9.8 (anti-MHC class
I; American Type Culture Collection), M5/114 (anti-MHC class II;
American Type Culture Collection), 16-10A1 (anti-B7-1), GL1
(anti-B7-2), or 3E2 (anti-ICAM; PharMingen) for 30 min on ice.
After washing, the cells were incubated with FITC-conjugated
anti-hamster, anti-rat, or anti-mouse IgG for 30 min on
ice. After washing with PBS, cells were fixed and analyzed by FACScan
(Becton Dickinson).
MLRs
The DC, DC/MUC1 RNA, and DC/-gal RNA were incubated at a 1:20
ratio with syngeneic (C57BL/6) or allogeneic (BALB/c) T cells in
96-well U-bottom plates for 4 days. The T cells were prepared by
passing spleen suspensions through nylon wool columns, incubating for
90 min in 100-mm culture dishes to remove the residual APCs, and
collecting the nonadherent cells. Stimulation of T cells was assessed
by pulsing with 1 µCi/well
[3H]thymidine (New England Nuclear, Boston, MA)
for 6 h and monitoring for tritium incorporation.
Immunohistochemistry
Immunohistochemical staining with mAb DF3 (anti-MUC1) and M5/114 (anti-MHC class II) Ab was performed on acetone-fixed cells. Lymph nodes were collected from the mice immunized with DC or DC/MUC1 RNA. Frozen sections prepared on slides were incubated with mAb DF3 for 40 min at room temperature. The slides were washed and incubated with biotinylated horse anti-mouse IgG for an additional 30 min. Red was generated by staining with avidin-biotinylated peroxidase complex solution (Vector Laboratories, Burlingame, CA). The slides were then incubated with M5/114 Ab for 30 min followed by alkaline phosphatase-labeled anti-rat IgG. Alkaline phosphatase/avidin-biotin-peroxidase complex solution (Vector Laboratories) was used to generate a blue counterstain.
Humoral immune responses
Sera were obtained from mice immunized with DC, DC/MUC1 RNA, or
DC/-gal RNA. Microtiter plates were precoated overnight at 4°C
with 5 U/well of purified MUC1 Ag. The wells were washed with PBS
containing 5% horse serum albumin and then incubated for 1 h with
a 4-fold dilution of mouse sera. After washing and incubation with goat
anti-mouse IgG conjugated to HRP (Amersham), Ab complexes were
detected by development with o-phenylenediamine (Sigma) and
measurement in an ELISA Microplate Reader 550 (Bio-Rad, Hercules, CA)
at an OD of 490 nm.
Cytotoxicity assay
CTL assays were performed by assessing
51Cr release. Briefly, 1 x
106 target cells were labeled for 1 h with
50 µCi of Na51CrO4.
Spleen cells were obtained from mice immunized with DC, DC/MUC1 RNA, or
DC/-gal RNA and were harvested on a Ficoll density gradient before
use in a CTL assay. CTL activity was determined at the indicated E:T
cell ratios after incubation for 4 h. The supernatants were then
harvested and analyzed for 51Cr release. Specific
cytotoxic activity was determined with the formula: percent specific
release of 51Cr = (experimental release
- spontaneous release)/(maximum release - spontaneous
release) x 100.
In vivo studies
C57BL/6 wild-type mice were immunized twice with 1 x
106 DC, DC/MUC1 RNA, or DC/-gal RNA on days 0
and 7. The mice were challenged s.c. with 5 x
105 MC38 or MC38/MUC1 tumor cells on day 14. The
mice were also inoculated s.c. with 5 x 105
MC38 or MC38/MUC1 cells in the back. On days 3 and 10, the mice were
treated by s.c. injection of 1 x 106 DC,
DC/MUC1 RNA, or DC/-gal RNA at the same site of tumor cell
injection. MUC1.Tg mice were immunized s.c. with 1 x
106 nontransfected DC, DC/MUC1 RNA, and
DC/-gal RNA on days 0 and 7; IL-12 (500 ng) was administered by i.p.
injection on days 3, 6, 9, and 12. At 1 wk after the second
immunization, the mice were challenged with 5 x
105 MC38 or MC38/MUC1 cells. Tumor volumes were
measured in centimeters by caliper, calculated as: tumor volume =
length x (width2)/2 (30), and
then analyzed by Student’s t test. Statistical significance
was determined at the p < 0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MUC1 and -gal RNAs were generated by in vitro synthesis (Fig. 1
A). Murine bone
marrow-generated DC were transfected with the MUC1 RNA or -gal RNA
using the cationic liposome (DOTAP) (13). To assess
expression, DC transfected with MUC1 RNA (DC/MUC1 RNA), -gal RNA
(DC/-gal RNA), and DC alone were examined by immunoblot and flow
cytometric analyses. There was no detectable expression of MUC1 in DC
transfected with -gal RNA (Fig. 1B). By contrast, MUC1
expression was detectable in 10–20% DC/MUC1 RNA at 24 h by flow
cytometry (Fig. 1B) and immunoblot analysis (Fig. 1C). Transfected DC exhibited a similar cell surface level
of MHC class II, B7, and ICAM expression as nontransfected DC (Fig. 1B). Both transfected and nontransfected DC, after
stimulation by LPS, increased the expression of MHC classes I and II,
B7-1, and B7-2 (Table I). -Gal
expression on DC/-gal RNA was detected by X-gal staining (data not
shown). These findings confirmed the expression of MUC1, MHC
classes I and II, B7, and ICAM by DC transfected with MUC1
RNA.
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To determine the function of transfected DC, T cells stimulated
with nontransfected DC, DC/MUC1 RNA, or DC/-gal RNA were compared in
MLR assays (Fig. 2A). There
were no differences between DC transfected with MUC1 RNA or -gal RNA
and nontransfected DC in terms of stimulating allogeneic and syngeneic
T cell proliferation (Fig. 2A). To determine whether the
transfected DC migrates to lymph nodes, mice were injected s.c. with
transfected or nontransfected DC at the base of the tail. After 24
h, the inguinal lymph nodes were collected and examined by
immunohistochemical staining. The lymph nodes from mice immunized with
DC/MUC1 RNA exhibited dual expression of MUC1 and MHC class II on cells
in T cell areas (Fig. 2B, right). By contrast,
MHC class II, but not MUC1, was detectable in lymph nodes from mice
injected with control nontransfected DC (Fig. 2B,
left). These findings indicate that DC/MUC1 RNA are
functional in both T cell activation in vitro and migration to regional
lymph nodes in vivo.
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To determine whether vaccination with DC/MUC1 RNA induces an
anti-MUC1 immune response, wild-type C57BL/6 mice were immunized
twice s.c. with 1 x 106 nontransfected DC,
DC/MUC1 RNA, or DC/-gal RNA. Humoral and CTL responses against MUC1
were evaluated to assess the effectiveness of immunization.
Immunization with DC/MUC1 RNA induced the production of anti-MUC1
Abs compared with that found in mice immunized with the nontransfected
DC or DC/-gal RNA (Fig. 3A). Importantly, immunization
with DC/MUC1 RNA was also effective in the priming of a CTL response
against MC38/MUC1 (Fig. 3B, left), but not MC38
(Fig. 3B, right) and MCF7, targets (Table I
). By
contrast, splenocytes from the mice immunized with nontransfected DC or
DC/-gal RNA exhibited little if any CTL response against MC38 and
MC38/MUC1 cells (Fig. 3B, left and
right). Moreover, CTL from the mice immunized with mature DC
transfected with MUC1 RNA was higher in lysis of MC38/MUC1 targets than
CTL from mice immunized with their immature counterparts (Table I).
These results indicate that vaccination with DC/MUC1 RNA induces
MHC-restricted, MUC1-specific immune responses.
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To assess antitumor activity induced by the DC/MUC1 RNA, mice were
vaccinated s.c. twice with 1 x 106 DC,
DC/MUC1 RNA, or DC/-gal RNA. The mice were then challenged s.c.
with 5 x 105 MC38 or MC38/MUC1 cells.
Vaccination with DC/MUC1 RNA prevented the development of MC38/MUC1
tumors in mice (Fig. 4A and
Table II). By contrast, MC38/MUC1 tumor
growth was unaffected in the mice immunized with nontransfected DC or
DC/-gal RNA (Fig. 4A). The specificity of these responses
against MUC1 is supported by the finding that the vaccinations rendered
no protection against the MC38 tumors (Fig. 4B). To assess
the effects of DC/MUC1 RNA on treatment of established MUC1-positive
tumors, mice were inoculated s.c. with 5 x
105 MC38/MUC1 or MC38 cells on day 0 and then
treated with DC, DC/MUC1 RNA, or DC/-gal RNA on days 3 and 10.
Immunization with DC/MUC1 RNA was effective in the elimination of
established MC38/MUC1 tumors (Fig. 4C and Table II), but not
MC38 tumors (Fig. 4D). By contrast, immunization with
DC/-gal or nontransfected DC had no effect on tumor growth (Fig. 4, C and D, and Table II). These findings
demonstrate that immunization with DC/MUC1 RNA is effective in treating
MUC1-positive tumors.
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Studies have demonstrated that MUC1.Tg mice are unresponsive to
stimulation with MUC1 Ag (30, 37). To determine whether
vaccination with DC/MUC1 RNA can reverse immunologic unresponsiveness,
we immunized MUC1.Tg mice with DC/MUC1 RNA and assessed anti-MUC1
immunity. CTLs from MUC1.Tg mice vaccinated with DC/MUC1 RNA exhibited
a low level of MC38/MUC1 cell lysis (Fig. 5A). Moreover, immunization
with mature DC/MUC1 RNA failed to increase the CTL activity in MUC1.Tg
mice (Table I). By contrast, coadministration of DC/MUC1 RNA and IL-12
significantly increased the level of CTL activity against MC38/MUC1
cells (Fig. 5A). As a control, there was no detectable CTL
activity against MC38 targets (Fig. 5B). MUC1.Tg mice
immunized with DC/MUC1 RNA also exhibited no apparent protection
against challenge with MC38/MUC1 cells (Fig. 5C and Table II). However, coadministration of DC/MUC1 RNA and IL-12 to the MUC1.Tg
mice was associated with rejection of MC38/MUC1 (Table II), but not
MC38, tumor cells (Fig. 5, C and D). By contrast,
there was no rejection of either MC38/MUC1 or MC38 tumors by MUC1.Tg
mice immunized with DC or DC/-gal RNA (Fig. 5, C and
D). These results indicate that the immune response induced
by immunization of MUC1.Tg mice with DC/MUC1 RNA is insufficient to
prevent MUC1-positive tumor cell growth and that IL-12 is required to
reverse immunologic unresponsiveness to MUC1 in these mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of antitumor immunity with tumor RNA-transfected DC
represents another approach for DC-based vaccines (17, 18). RNA preparations for transfection of DC can be transcribed
from cDNA (13) or can be extracted from tumor cells
(17, 18). In the present studies, DC were transfected with
MUC1 RNA. The transfected DC expressed MUC1 Ag, costimulatory, and
adhesion molecules. Immunization with MUC1 RNA-transfected DC was
associated with induction of an MUC1-specific CTL response and resulted
in the rejection of MC38/MUC1, but not MC38, tumors in wild-type mice.
By contrast, there was no evidence for induction of anti-MUC1
immunity in mice immunized with nontransfected or -gal
RNA-transfected DC. These findings indicate that vaccination with MUC1
RNA-transfected DC is effective in inducing specific anti-MUC1
immunity in vivo.
C57BL/6 mice transgenic for human MUC1 (MUC1.Tg) have been developed to study immunologic responsiveness to the human MUC1 tumor-associated Ag (30). Immunizations with purified human MUC1 Ag and irradiated MUC1-positive (MC38/MUC1) tumor cells were associated with induction of an anti-MUC1 Ab response and no detectable cytotoxic T cell reactivity (44). Recent work has demonstrated that DC play a pivotal role in the regulation of immunity and tolerance (45, 46, 47, 48). In our system, DC/MUC1 RNA activated by LPS up-regulated MHC class I and II and costimulatory molecules and increased CTL activity in the wild-type mice, but not in the MUC1.Tg mice. The findings that immunization with MUC1 RNA-transfected DC was effective against challenge of MUC1-positive tumors in the wild-type mice, but failed in MUC1.Tg mice, indicated the existence of tolerance to MUC1, even immunization with mature DC/MUC1 RNA. However, coadministration of IL-12 prevented the tolerance induction.
The induction of tolerance has been reported using peptide-pulsed DC under ineffective priming conditions, including neutralization of endogenous IL-12 (49, 50). IL-12, a heterodimeric cytokine, has been found to induce stimulation of Th1 reactivity and enhancement of CTL responses (51, 52). Moreover, IL-12 has been demonstrated to up-regulate DC expression of the B7 molecule (53), inhibit anergy induction, and reverse unresponsiveness to tumor peptides (54). Coadministration of IL-12 resulted in strong proliferative response of CD8+ T cells and development of lytic effector function (55, 56). The mechanism of IL-12 activity in reversal of immunologic unresponsiveness in vivo is still unclear (54). Recent studies indicated that IL-12 acted as a third signal, along with TCR and costimulatory molecules, to reverse Ag-induced tolerance and expand Ag-specific CD8+ T cells (55, 56). In the present studies tolerance to MUC1 was reversed by coadministration of the MUC1 RNA-transfected DC and IL-12. It is possible that IL-12 functions as adjuvant to provide an optimal environment for the induction of antitumor immunity in MUC1.Tg mice (57). Our findings demonstrate that immunologic unresponsiveness to MUC1 in the MUC1.Tg is reversible when DC present MUC1 epitopes in the presence of potentiating signals induced by IL-12.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jianlin Gong, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115.
3 Abbreviations used in this paper: DC, dendritic cell; DC/-gal RNA, DC transfected with -galactosidase RNA; MUC1.Tg, MUC1 transgenic; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N,-trimethylammonium methylsulfate.
Received for publication April 24, 2000. Accepted for publication August 21, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
and syngeneic T cells (
) at a 1:20 ratio for 4 days.
[3H]thymidine uptake was measured at 6 h after a
pulse of 1 µCi/well. The stimulation index was expressed as the
mean ± SD of three experiments, each performed in triplicate.
B, Nontransfected DC (left panel) and
DC/MUC1 RNA (right panel) were injected s.c. at the base
of the tail. The inguinal lymph nodes were collected at 24 h after
immunization. Frozen sections of the lymp nodes were stained with mAb
DF3 (anti-MUC1; red color) and mAb M5/114 (anti-MHC class II;
blue color). The cells stained positively for MUC1 and MHC class II
(purple; arrow in the right panel) were observed in the
T cell area; the cells are stained positively for MHC class II (blue;
arrow in the left panel) were also observed in the T
cell area. B, B cell area. Original magnification, x25.
), DC/MUC1
RNA (
), or DC/
). PBS (•) was used as a control.
Serum from each mouse was collected at 7 days after the last
immunization, and the anti-MUC1 Abs were detected by ELISA.
B, Splenocytes were isolated from mice immunized with
PBS (•), DC (
), DC/MUC1 RNA (
) on days 0 and 7. IL-12 alone (•) was
given as a control. The mice were challenged s.c. with 5 x
105 MC38/MUC1 (C) or MC38 (D)
cells at 7 days after the last immunization. Tumor volumes are
expressed as the mean ± SD for eight mice and were analyzed by
Student’s t test. There is significant difference in
tumor volume between mice immunized with DC/MUC1 RNA and IL-12 (