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The Journal of Immunology, 2001, 166: 6555-6563.
Copyright © 2001 by The American Association of Immunologists

Three Different Vaccines Based on the 140-Amino Acid MUC1 Peptide with Seven Tandemly Repeated Tumor-Specific Epitopes Elicit Distinct Immune Effector Mechanisms in Wild-Type Versus MUC1-Transgenic Mice with Different Potential for Tumor Rejection1

M. Melina Soares*, Vinay Mehta{dagger} and Olivera J. Finn2,*

* Immunology Program and Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh PA 15261; and {dagger} Biology Department, Washington and Jefferson College, Washington, PA 15301


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8+ T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human mucin MUC1 is a transmembrane glycoprotein normally expressed on the apical surface of ductal epithelial cells (1). It also is one of the few well-characterized tumor Ags found to be aberrantly expressed on a wide variety of ductal adenocarcinomas, including those of the breast, pancreas, lung, colon, ovaries, and prostate (reviewed in Ref. 2). MUC1 on normal tissues is heavily glycosylated with branched sugar chains extending out from the protein backbone (3, 4, 5). However, on tumors, MUC1 is highly overexpressed, loses polarization, and is severely underglycosylated with oligosaccharides that are mostly linear and shorter because of premature termination of glycosylation (6, 7, 8, 9). Underglycosylation and loss of polarization of tumor-associated MUC1 make it accessible to effector mechanisms such as Abs and T cells and recognizable by the immune system.

Analyses of immune responses in cancer patients with various adenocarcinomas have revealed the presence of low-titer anti-MUC1 Abs (10, 11, 12, 13, 14, 15) and of low-frequency MUC1-specific CTL (16, 17, 18, 19, 20). No MUC1-specific Th cell responses have been reported to date. Taking into consideration the pivotal role that Th cells have in regulating both humoral and cellular responses, we have proposed that the lack of MUC1-specific CD4+ Th cells in patients is responsible for their inability to generate stronger immune responses and overcome the disease. Our group reported that dendritic cells (DC)3 can endocytose the aberrantly glycosylated circulating form of MUC1 that is shed from tumor cells but are unable to efficiently process and present it to either MHC class I- or MHC class II-restricted T cells (21, 22). This could account, in part, for the lack of MUC1-specific Th cells, low Ab titer of predominantly IgM isotype, and low CTL frequency. MUC1-specific Th cell responses can be generated in vitro by priming CD4+ T cells on DC loaded with the unglycosylated synthetic MUC1 peptide that is efficiently processed and presented (23). High concentrations of peptide needed to elicit these responses suggest that in addition to inefficient Ag processing, CD4+ T cell tolerance, especially of the high affinity T cells, also could contribute to the lack of MUC1-specific Th cells in vivo. In the studies we report here, we have addressed in a Tg mouse model the extent and consequences of MUC1-specific Th cell tolerance by testing several vaccination protocols for their ability to induce MUC1-specific immunity and cause tumor rejection.

Efforts already are underway by multiple groups to use MUC1-based immunotherapy in cancer patients (24, 25, 26, 27). These are predicated on studies done primarily in conventional mice (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40). Human MUC1 is a very strong immunogen in the mouse, and these studies do not provide a realistic evaluation of its immunogenic or immunotherapeutic potential. Furthermore, as these studies side-step the issues of tolerance and autoimmunity, they provide very little information about the relevance of MUC1-specific responses for tumor rejection or their safety in cancer patients. We have reported a number of studies conducted in a more relevant chimpanzee model where MUC1 is highly homologous to human MUC1 (41, 42, 43), and they have shown that the anti-MUC1 immune responses generated did not cause autoimmunity. However, the lack of a tumor model in the chimpanzee did not allow evaluation of the role of these responses in tumor rejection.

Recently, efforts have been directed toward developing mice transgenic (Tg) for a variety of human tumor Ags such as Her 2/Neu and carcinoembryonic Ag. Such Tg mouse models have made it possible to test immunotherapeutic strategies within the context of tolerance and autoimmunity (44, 45, 46, 47, 48, 49, 50). More recently, MUC1-Tg mice have become available, and several groups have started to address some of the above questions in this model (51, 52, 53, 54, 55). We have used this model to analyze MUC1-specific immune responses that can be elicited in MUC1-Tg mice compared with WT mice. We were interested especially in knowing whether the reported Th cell tolerance in these mice (51, 53) could be broken by different vaccines based on a synthetic 140-aa MUC1 peptide containing multiple tandemly repeated tumor-specific epitopes. Another important question we addressed was whether a strong tumor rejection response, if elicited, would result in autoimmunity. Our results show that two vaccines combining MUC1 peptide with adjuvants induced only humoral responses. MUC1 peptide admixed with murine GM-CSF as an adjuvant induced IgM Abs in both WT and MUC1-Tg mice, but the switch to IgG Abs occurred only in WT mice. In contrast, MUC1 peptide admixed with the adjuvant SB-AS2 induced high-titer IgG Abs in both WT and MUC1-Tg mice. We discovered that these humoral responses were T cell independent in both MUC1-Tg and WT mice and did not result in tumor rejection. We found that the cytokines necessary for isotype switching, and in particular IFN-{gamma}, were contributed by activated NK cells and other non-T cells. The vaccine composed of MUC1 peptide-pulsed DC elicited T cell immunity and effective tumor rejection response in both WT and MUC1-Tg mice even though it was unable to break CD4+ T cell tolerance in MUC1-Tg mice. The effector cells in these mice were MUC1-specific CD8+ T cells that produced IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

MUC1-Tg mice (4–6 wk old) on a C57BL/6 background were purchased from Mayo Clinic, (Scottsdale, AZ), and conventional C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All experimental animals were housed at the University of Pittsburgh Cancer Institute Animal Facility (Pittsburgh, PA) under standard pathogen-free conditions.

Antigens

The 140-aa synthetic MUC1 peptide used for immunization corresponds to seven tandem repeats of a 20-aa sequence from the extracellular tandem repeat domain of MUC1. The amino acid sequence of one repeat is GVTSAPDTRPAPGSTAPPAH. The peptide was synthesized on a Chemtech 200 machine with N-(9-fluorenyl)methoxycarbonyl chemistry and purified by HPLC in the University of Pittsburgh Cancer Institute Peptide Facility. In some in vitro assays, a 100-aa MUC1 synthetic peptide corresponding to five tandem repeats was used instead of the 140-aa MUC1 peptide.

Vaccines and immunization protocols

Three different immunization protocols were tested in vivo. Mice were immunized with: 1) synthetic MUC1 peptide (100 µg/mouse) coadministered with soluble murine GM-CSF (2 µg/mouse; a generous gift from Immunex, Seattle, WA) injected s.c.; 2) synthetic MUC1 peptide (100 µg/mouse) coadministered with SB-AS2 (50 µl/mouse; a generous gift of SmithKline Beecham Biologicals, Rixensart, Belgium) injected i.m.; or 3) murine DC prepulsed with 20 µg/ml of synthetic MUC1 peptide in AIM-V medium (Life Technologies, Grand Island, NY) overnight (2–5 x 104 DC/mouse injected s.c.). SB-AS2 is an oil-in-water emulsion containing 3-deacylated-monophosphoryl lipid A, a detoxified form of lipid A, and purified fraction number 21 of Quillaria saponaria, known as Quil A (56, 57). The DC were generated as described previously (58), the major modification being that they were grown in serum-free medium. Briefly, they were differentiated in vitro from bone marrow precursors with murine GM-CSF (10 ng/ml) and murine IL-4 (10 ng/ml) in AIM-V medium for 7 days. On day 7, the DC were purified on a Nycoprep gradient (Nycomed, Oslo, Norway), pulsed overnight with peptide in Teflon vials, and washed before vaccination. For the DC vaccine containing soluble peptide, soluble MUC1 peptide was added to the washed peptide-pulsed DC at a final concentration of 100 µg/mouse before vaccination. The mice were immunized once and boosted twice at 3-wk intervals in the right hind flank.

MUC1-specific ELISA

Ten days after the last boost, blood samples were collected by tail bleeding, and the serum was tested for the presence of MUC1-specific Abs with a MUC1-specific ELISA (10) with a few modifications. Briefly, 96-well Immulon 4 plates (Dynatech, Chantilly, VA) were coated at room temperature overnight with 10 µg/ml of 100-aa MUC1 peptide in PBS. The plates were washed three times with PBS and incubated with serial dilutions of the immune serum for 1 h at room temperature. After three washes with PBS/0.1% Tween 20, the plates were incubated with goat anti-mouse peroxidase-conjugated secondary Abs for 1 h at room temperature. The goat anti-mouse-IgM and -IgG secondary Abs were obtained from Sigma (St. Louis, MO). The goat anti-mouse-IgG1,-IgG2b, and -IgG3 Abs were obtained from Southern Biotechnology Associates (Birmingham, AL). The plates were washed three times with PBS/0.1% Tween 20 and then incubated with the substrate O-phenylenediamine dihydrochloride tablets (Sigma) for 1 h. The reaction was stopped with 2.5 M sulfuric acid, and the absorbance was measured at 490 nm.

IFN-{gamma} enzyme-linked immunospot (ELISPOT) assay

Lymph node (LN) cells were mixed with peptide-pulsed bone marrow-derived DC (at a ratio of 10:1) in MultiScreen 96-well filtration plates (Millipore, Bedford, MA) precoated with the anti-IFN-{gamma} capture Ab (BD PharMingen, San Jose, CA). The plates were incubated for 40 h at 37°C. After three washes with PBS/0.1% Tween 20, the plates were incubated with 2 µg/well of biotin-labeled anti-IFN-{gamma} Ab (BD PharMingen) at 37°C. The plates were washed, and spots developed with the Elite Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). For blocking studies, anti-CD4, anti-CD8, or isotype control Abs (BD PharMingen) were added to the wells at a final concentration of 2.5 µg/ml.

Intracellular cytokine detection assay

To analyze the T cells, bone marrow-derived DC were prepulsed with MUC1 synthetic peptide and used to stimulate LN cells at a 1:10 stimulator:responder cell ratio. Responder cells stimulated with DC alone served as the negative controls. After 30 h, PMA (20 ng/ml), ionomycin (1 µM), and monensin (3 µM) were added to the cultures for 6 h. The cells were washed twice in FACS buffer (5% FBS and 0.01% sodium azide in 1x PBS) and then stained for surface markers with anti-CD3 PERCP and anti-CD8 FITC. To analyze NK cells, LN cells and splenocytes were harvested from mice injected 24 h earlier with SB-AS2 adjuvant. The cells were stimulated in vitro with PMA (20 ng/ml), ionomycin (1 µM), and monensin (3 µM) for 4 h and then stained for surface markers with anti-NK, anti-CD16, or anti-NK1.1-FITC-conjugated Abs.

Intracellular staining for IFN-{gamma} was conducted according to a previously described protocol (59) with a few modifications. After surface staining, the cells were washed three times with FACS buffer and then fixed in a final concentration of 2% paraformaldehyde for 30 min at 4°C. The cells were permeabilized with 0.1% saponin in FACS buffer and stained with labeled anti-IFN-PE Ab. All of the Abs used in FACS assays were obtained from BD PharMingen. After a 1-h incubation at 4°C, the cells were washed and fixed in 1% paraformaldehyde solution. The samples were read on a FACSCalibur (Becton Dickinson, San Jose, CA) and the data analyzed with the CellQuest data analysis program (Becton Dickinson, San Jose, CA). A fold increase >2 over the control is considered to be significant.

Human MUC1 expression on the mouse tumor and tumor challenge

The T cell lymphoma line RMA on a C57BL6 background was transfected by electroporation with the pR/CMV-MUC1 plasmid containing full-length MUC1 cDNA with 42 tandem repeats (42). Stable transfectants were selected by using G418 (Life Technologies) and further cloned by limiting dilution. Anti-MUC1 Abs 3C6 and 4H5 (generous gifts from Jo Hilgers, Free University, Amsterdam, The Netherlands), were used to confirm cell surface expression of MUC1. Transfected RMA clones were incubated for 1 h at 4°C with anti-MUC1 Abs, washed, and then incubated with goat anti-mouse PE-conjugated secondary Ab (Biosource, Camarillo, CA) for 1 h at 4°C. The cells were washed and then fixed with a final concentration of 1% paraformaldehyde. The samples were read on aFACSCalibur (Becton Dickinson) and the data analyzed using the CellQuest data analysis program (Becton Dickinson). The RMA-MUC1 cell line that was derived maintains long-term stable expression of MUC1 in vitro and in vivo and has been used in tumor challenge experiments.

Ten days after the last boost, the mice were anesthetized with Metofane (Schering-Plough Animal Health, Omaha, NE) and 5 x 104 RMA-MUC1 cells injected s.c. in the shaved right hind flank. Tumor growth was monitored every 2–3 days and tumor size determined with calipers. Mice were sacrificed when the tumor size reached 2 cm in diameter.

Statistical analysis

The statistical significance of our results was calculated in a Student unpaired t test. The two-tailed p values were determined with the statistical program INSTAT version 2.03 GraphPad Software (San Diego, California). Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential induction of MUC1-specific Ig isotypes in WT and MUC1-Tg mice

Cytokines produced by Th cells have been implicated in inducing switching from IgM to other Ab isotypes (60, 61, 62). Hence, we were interested in the quantitative as well as qualitative differences in the humoral responses elicited by different MUC1 peptide-based vaccines, not only as a direct reflection of the B cell response, but also as an indirect reflection of the Th cell response in WT vs MUC1-Tg mice. Table IGo is a summary of anti-MUC1 Abs detected in the sera of immunized mice from various groups 10 days after the last boost. Sera from unimmunized mice were used as controls. When MUC1 peptide was administered with GM-CSF as an adjuvant, the vaccine induced IgM and moderate IgG1 and IgG2b Ab responses in conventional mice but only IgM Ab in MUC1-Tg mice. Ab responses elicited by vaccination with MUC1 peptide with SB-AS2 adjuvant were very different. Strong IgG1, IgG2b, and IgG3 responses were induced in both WT and MUC1-Tg mice. In C57BL/6 mice, the IgG2a gene is deleted (63), and thus this isotype was not measured here. Even though of very high titer in both strains, IgG responses in MUC1-Tg mice were nevertheless 10-fold lower in end-point titer compared with WT mice. Low- to moderate-titer IgM and no IgG Ab responses were detected in WT and MUC1-Tg mice immunized with peptide-pulsed DC (Table IGo). This was expected, as the peptide-pulsed DC were washed before injection, and, hence, there was little soluble peptide available to activate the B cells. Addition of soluble peptide to the peptide-pulsed DC vaccine induced high-titer IgG1 and moderate IgG2b (Table IGo), an isotype profile similar to that elicited by MUC1 peptide admixed with GM-CSF.


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  		Table I. MUC1-specific Abs elicited in WT and MUC1-Tg mice

 
DC vaccine activates CD4+ T cells in WT mice but not in MUC1-Tg mice

The ability of the two adjuvant-based vaccines to elicit multiple IgG isotypes suggested that MUC1-specific CD4+ T cells were being stimulated. The differences that were seen between the WT and MUC1-Tg mice suggested that the CD4+, MUC1-specific T cells were impaired in Tg mice but could be efficiently activated depending on the immunization protocol, MUC1 plus SB-AS2 in this case appearing to be a better vaccine than MUC1 plus GM-CSF. Because IFN-{gamma} plays an important role in isotype switching and in particular in switching to IgG3, we expected to find Ag-specific, IFN-{gamma}-producing Th cells, especially in mice where we detected MUC1-specific IgG3 Ab. Contrary to our expectations, we were not able to detect any such cells in mice immunized with adjuvant-based vaccines. The peptide-pulsed DC vaccine was the only one capable of inducing IFN-{gamma}-producing T cells in WT (Fig. 1GoA) as well as in MUC1-Tg mice (Fig. 1GoB). These cells were MUC1-specific, as stimulation with DC in the absence of MUC1 peptide resulted in a significantly lower number of background spots in the ELISPOT assay. The number of IFN-{gamma}-producing T cells was ~5-fold higher in WT mice compared with MUC1-Tg mice. More importantly, in WT mice, blocking with either anti-CD4 or anti-CD8 Abs resulted in a significant decrease in the total number of IFN-{gamma} spots, indicating that both CD4+ and CD8+ cells contributed to MUC1-specific IFN-{gamma} production (Fig. 1GoA). However, in MUC1-Tg mice, blocking with anti-CD8 Ab eliminated almost all IFN-{gamma} production, whereas blocking with anti-CD4 Ab had no significant effect (Fig. 1GoB). The ELISPOT data were confirmed with the intracellular cytokine assay in which we could directly evaluate the phenotype of IFN-{gamma}-producing cells. As seen in Table IIGo, only CD8+ T cells from MUC1-Tg mice immunized with peptide-pulsed DC were capable of producing IFN-{gamma} in response to MUC1 peptide. Moreover, when cultured for 2 days in the presence of MUC1 peptide, the percentage CD8+ T cells increased from 20% in the starting population to 66%, showing Ag-specific proliferation of CD8+ cells. In vitro, these cells remain cytokine producing, without any measurable CTL activity. After in vitro stimulation, we were able to detect on occasion very low MUC1-specific CTL activity in bulk LN cells from WT and MUC1-Tg mice immunized with the peptide-pulsed DC vaccine (data not shown), but these results were not always reproducible. CTL activity was never detected in bulk LN or spleen cells from mice immunized with adjuvant-based vaccines (data not shown).



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  		FIGURE 1. Cytokine production by MUC1-specific T cells from immunized WT (A) and MUC1-Tg (B) mice. ELISPOT assays were conducted with LN cells from immunized and control mice stimulated for 40 h with MUC1 peptide-pulsed (+Ag) or no peptide (-Ag) control DC. The LN cells were pooled from four mice per group. Anti-CD4 or anti-CD8 Abs were added to the T cells before the addition of the DC to the cultures for the duration of the assay. *, The only statistically significant differences: A, anti-CD4, p = 0.02; anti-CD8 p = 0.01; B, anti-CD8, p = 0.01.

 

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  		Table II. Detection of intracellular IFN-{gamma} in LN T cells from immunized MUC1-Tg mice following 2 days in vitro culture1

 
Efforts to detect IL-4-producing Th cells were unsuccessful because of the difficulty of detecting Ag-specific IL-4 production in either the ELISPOT or the intracellular cytokine assay in the murine system.

Adjuvant-activated non-T Cells are a source of IFN-{gamma} required for MUC1-specific IgG3 induction

To identify the source of the IFN-{gamma} responsible for isotype switching and in particular to IFN-dependent IgG3 in mice immunized with the MUC1 peptide admixed with the SB-AS2 adjuvant, we injected WT mice with SB-AS2 alone and 24 h later analyzed their spleen and draining LN for production of IFN-{gamma} by non-T cells. SB-AS2 induced a 2- to 3-fold increase in the percentage of NK+ or NK1.1+ cells in the spleen, compared with untreated mice (Fig. 2GoA). This increase in the percentage of NK cells was unique to the spleen because no such difference was observed in LN from SB-AS2-treated and untreated control mice. In addition to the overall increase in cell number, we also saw an increase in the percentage of IFN-{gamma}-producing NK+ and NK1.1+ cells in the spleen and not in the LN (Fig. 2GoB). There was no difference in the percentage of CD16+ cells in either the spleen or LN in response to SB-AS2, as would be expected because CD16 is a marker found on a variety of cells other than NK cells, such as macrophages, B cells, and DC (Fig. 2GoA). However, the percentage of CD16+ cells that produced IFN-{gamma} in response to SB-AS2, was greatly increased in the spleen and not in the LN (Fig. 2GoB). Addition of MUC1 peptide to the adjuvant does not change the above results, as indicated by the induction of an IgG3 Ab response by the MUC1 peptide admixed with SB-AS2 vaccine in the absence of a T cell source of IFN-{gamma} (Table IGo).



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  		FIGURE 2. Non-T cells (A) and IFN-{gamma} production by non-T cells (B) in spleens of SB-AS2-injected mice. Splenocytes and LN cells from uninjected mice were used as controls.

 
DC-based vaccine leads to tumor-free survival in both WT and MUC1-Tg mice

The mouse tumor cell line RMA was transfected with human MUC1 cDNA and, as seen in Fig. 3Go, epitopes present on both glycosylated normal MUC1 (recognized by Ab 3C6), as well as on underglycosylated tumor MUC1 (recognized by Ab 4H5) are expressed on the transfected RMA MUC1 tumor cells. RMA MUC1 grows well as a s.c. solid tumor, and we determined in preliminary experiments that injection of 5 x 104 tumor cells resulted in 80% tumor take in 18 days (data not shown).



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  		FIGURE 3. MUC1 expression on RMA-MUC1 tumor cells. Ab 3C6 (A) recognizes MUC1 irrespective of its glycosylation state, while 4H5 (B) recognizes only the underglycosylated (tumor) form of MUC1.

 
All mice were challenged 10 days after the last immunization with RMA-MUC1 tumor cells injected s.c., and tumor growth was monitored up to 60 days. By day 30, ~60% of unimmunized control WT and MUC1-Tg mice were sacrificed because their tumors reached a size of 2 cm. WT and MUC1-Tg mice that had been immunized with MUC1 peptide admixed with either GM-CSF or SB-AS2 also had to be sacrificed because they failed to reject the tumors (Fig. 4Go). Before concluding that the humoral response alone was not being effective against this MUC1-expressing tumor, it was important to determine whether the Abs elicited by the synthetic MUC1 peptide-based vaccines that recognized the immunizing peptide in the ELISA were also capable of recognizing the native MUC1 molecule on the RMA-MUC1 tumor. We stained RMA-MUC1 cells with a 1:100 dilution of immune serum from mice immunized with MUC1 peptide admixed with SB-AS2 and could clearly show MUC1 protein-specific immune reactivity (Fig. 5Go). This confirmed that MUC1 peptide and adjuvant-based vaccines did induce Abs capable of recognizing the tumor, but that recognition did not result in tumor rejection.



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  		FIGURE 4. Potential for tumor rejection in mice immunized with the three different vaccines. A, WT mice. B, MUC1-Tg mice. Mice were injected three times, 3 wk apart. Ten days after the last boost, the mice were challenged s.c. with tumor. Unimmunized mice served as the control group. The results are from 20–35 mice per vaccine group. The data are representative of two independent experiments.

 


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  		FIGURE 5. Recognition of MUC1 on the surface of RMA MUC1 tumor cells by sera of mice immunized with MUC1 peptide admixed with SB-AS2 adjuvant. Staining of RMA-MUC1 (filled peak) and RMA (unfilled peak) with sera used at 1:100 dilution. Data from three different mice are shown.

 
Only the MUC1 peptide-pulsed DC vaccine resulted in an impressive tumor rejection in both WT and MUC1-Tg mice. Ninety percent of immunized mice were completely tumor free up to 60 days after tumor challenge (Fig. 4Go). The survival curves were comparable between the WT and MUC1-Tg mice, even though in WT mice the vaccine had elicited CD4+ as well as CD8+ MUC1-specific T cells, but only CD8+ MUC1-specific T cells in MUC1-Tg mice.

MUC1-expressing tissues, lung, pancreas, liver, and kidney, were harvested from MUC1-Tg mice after immunization and/or after tumor rejection to investigate whether the immune responses elicited by vaccination alone or also boosted through tumor rejection would show reactivity against normal tissues. As signs for autoimmunity, we looked by hematoxylin and eosin staining for mononuclear infiltrates in the tissues, especially around the MUC1+ ducts, as well as evidence of tissue destruction. We saw no difference in the appearance of these tissues between immunized and control mice (data not shown), concluding that the MUC1-specific immune response that was strong enough to cause tumor rejection did not cause autoimmune damage to normal MUC1+ tissues.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We chose the long MUC1 synthetic peptide as the immunogen because of our previous studies that showed that this peptide is efficiently processed and presented by APCs such as DC (21, 22). In addition, studies in vitro with human T cells as responders have shown that DC pulsed with this form of MUC1 can prime both CD8+ and CD4+ T cells (21, 23). Our experiments demonstrate important differences in immune responses that can be elicited by the same tumor Ag administered in different vaccine formulations. As we expected, we saw additional differences in response to these vaccines between MUC1-Tg and WT mice. Thus, we saw that MUC1 peptide vaccine with GM-CSF adjuvant induces IgG responses only in conventional mice, and primarily those that do not depend on IFN-{gamma}. The vaccine with SB-AS2 as adjuvant induces IgG responses in both conventional and MUC1-Tg mice, including the IFN-{gamma} dependent isotype IgG3. It is important to note that the titer of IgG Abs elicited in MUC1-Tg mice was 10-fold lower than in WT mice. Inasmuch as there was no Th cell activation in either the WT or the MUC1-Tg mice, these results support previous reports that MUC1-Tg mice display some level of B cell tolerance (64, 65).

We correctly hypothesized that the lack of an Ab response in WT and MUC1-Tg mice immunized with peptide-pulsed DC was attributable to the absence of sufficient concentrations of soluble peptide that could simultaneously activate B cells. An IgG response was elicited when soluble peptide was added to the DC vaccine. Even though, to the extent that we have been able to evaluate in this particular tumor model, Abs did not play an important role, this is unlikely to be a common observation. In most cases, the most comprehensive immune response will likely be the most effective, and vaccines based on DC that have already processed the Ag will require additional soluble Ag to elicit both cellular and humoral immune responses.

IFN-{gamma} is the cytokine responsible for up-regulating IgG3 isotype Ab in mice (62, 66). It is interesting to note that the DC vaccine with added soluble peptide was unable to induce IgG3 Abs in WT mice even though it was capable of inducing CD4+ IFN-{gamma}-producing cells in these mice. This suggests to us that the quantities of IFN-{gamma} produced by these T cells were not sufficient to cause the switch to IgG3. Previous reports have suggested that Th cell-independent IgG3 can be induced by IFN-{gamma} produced by NK cells (62, 67). We were able to confirm that SB-AS2, the adjuvant in the vaccine that elicited MUC1-specific IgG3, when administered alone increased the total number of NK cells and the number of NK cells producing IFN-{gamma} in the spleen. The MUC1 peptide composed of five identical 20-aa repeats is a multivalent Ag that binds to B cell receptors and cross-links them sufficiently to activate the B cell. These activated B cells then can use IFN-{gamma} produced in their vicinity by activated NK cells to switch their Ab production from IgM to other isotypes, in particular IgG3. These results show yet again the importance of including in vaccine preparations adjuvants that are capable of stimulating the components of the innate immune system, major producers of important cytokines (68). Although the SB-AS2-based vaccine did not induce MUC1-specific T cells in our model system, recent work has shown that immunization of E7-Tg mice and control wild-type mice with recombinant HPV 16 E7 protein coadministered with SB-AS2 as an adjuvant elicits E7-specific T cell responses (69).

Based on previous publications (53, 64), we expected to find signs of tolerance of MUC1-specific CD4+ T cells in MUC1-Tg mice, but we were surprised that we could not overcome it with the DC-based vaccine. The same MUC1 peptide loaded onto human DC can prime human MUC1-specific CD4+ T cell responses in vitro (23). We have also shown that it is possible to induce CD4+ T cell responses in chimpanzees with the same MUC1 peptide admixed with Leishmania initiation factor as an adjuvant (42). We also have seen CD4+ T cell proliferation in response to MUC1 peptide in patients vaccinated with this peptide (unpublished data). CD4+ T cell tolerance in the MUC1-Tg mice appears to be more profound than in humans and may be a peculiarity of this particular Tg model.

MUC1-specific Abs of three different isotypes, IgG1, IgG2b, and IgG3, elicited by the adjuvant-based vaccines, could not provide protection against subsequent tumor challenge. It was previously reported that passive transfer of polyclonal sera containing MUC1-specific IgG1 Abs could not protect against another tumor, B16 melanoma transfected with MUC1 (65). Previous work investigating the role of Abs in tumor immunity against SV40-transformed cells suggested that Ab-dependent cellular cytotoxicity (ADCC) is an important protective mechanism against virus-induced tumors. Abs of the IgG2a isotype are thought to represent the principal element of ADCC mediated via their interaction with type II FcR (70). By working in C57BL/6 mice that lack the gene for IgG2a, we were unable to test the effect of this isotype that works primarily through ADCC. Cellular immunity clearly plays an important role in the rejection of MUC1+ tumors in C57BL/6 mice, and Abs alone are ineffective. However, Abs may complement the cellular responses by contributing to the prevention of the metastatic spread of the tumor, an issue that was not addressed to date and that we are currently exploring.

Approximately 90% of the mice immunized with the DC vaccine rejected the RMA-MUC1 tumor, and in the case of MUC1-Tg mice, apparently through MUC1-specific CD8+ T cells as the only effector mechanism. These T cells, as far as we were able to determine, were primarily IFN-{gamma} producers, rather than CTL. It is possible that the function we measured in vitro does not correspond to their more comprehensive in vivo function, which might also include CTL activity. Alternatively, these cells may have destroyed the tumor cells through noncytolytic mechanisms that were first reported to operate in viral systems (71, 72, 73) and more recently in tumor rejection as well (74). It has been reported that bacterial CpG-DNA up-regulates costimulatory molecules and stimulates DC to produce IL-12, thereby "licensing" them to activate CD8+ T cell responses in a Th cell-independent fashion (75). Thus, it is possible that in our study, the MUC1-peptide-pulsed DC that were generated in vitro were sufficiently activated to prime MUC1-specific CD8+ T cells in a Th cell-independent fashion. The MUC1-specific T cells that caused tumor rejection did not infiltrate into or show destruction of normal MUC1-expressing tissues such as pancreas, kidney, and lung. However, we know that binding of MUC1 peptides from the tandem repeat region to H-2Kb and Db, C57BL6 MHC-class I alleles, is of such low affinity that we have never been able to detect it with the standard class I stabilization assay (unpublished data). In agreement with those observations, we also have never been able to generate class I-restricted CTL to the MUC1 tandem repeat region in C57BL6 mice. Thus, low-level expression of MUC1 in normal cells and low-affinity binding of MUC1 tandem repeat-derived peptides to H-2b alleles may account for the lack of recognition of normal tissues by MUC1-specific T cells. For that reason, we are now repeating immunizations and tumor rejections studies in double-Tg mice that not only express human MUC1, but also human HLA-A11 allele, known to be a restricting element for MUC1 specific CTL (17).


   Acknowledgments
 
We thank Dr. Simon Barratt-Boyes for transfecting and cloning the RMA-MUC1 cell line. We also thank Dr. Joseph Ahearn and Jeanine Navratil for the use of the FACSCalibur.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant 5RO1 CA56103 (to O.J.F.) and Department of Defense Training Grant DAMD 17-99-1-9352 (to M.M.S.). Animal purchase and maintenance were supported in part by a grant to O.J.F. from an anonymous donor. Back

2 Address correspondence and reprint requests to Dr. Olivera J. Finn, Department of Molecular Genetics and Biochemistry, W1142 Biomedical Science Tower, University of Pittsburgh, Pittsburgh PA 15261. E-mail address: ojfinn{at}pitt.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; Tg, transgenic, ELISPOT, enzyme-linked immunospot; LN, lymph node, ADCC, Ab-dependent cellular cytotoxicity. Back

Received for publication January 18, 2001. Accepted for publication March 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. Charalambous, M. Oks, G. Nchinda, S. Yamazaki, and R. M. Steinman
Dendritic Cell Targeting of Survivin Protein in a Xenogeneic Form Elicits Strong CD4+ T Cell Immunity to Mouse Survivin
J. Immunol., December 15, 2006; 177(12): 8410 - 8421.
[Abstract] [Full Text] [PDF]

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 GlycobiologyHome page
A. L. Sorensen, C. A. Reis, M. A. Tarp, U. Mandel, K. Ramachandran, V. Sankaranarayanan, T. Schwientek, R. Graham, J. Taylor-Papadimitriou, M. A. Hollingsworth, et al.
Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance
Glycobiology, February 1, 2006; 16(2): 96 - 107.
[Abstract] [Full Text] [PDF]

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M. Gerloni, P. Castiglioni, and M. Zanetti