Cancer Research Cancer Epigenetics Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Singh, R.
Right arrow Articles by Paterson, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Singh, R.
Right arrow Articles by Paterson, Y.
[Cancer Research 66, 7748-7757, August 1, 2006]
© 2006 American Association for Cancer Research

Immunology

Vaccination Strategy Determines the Emergence and Dominance of CD8+ T-Cell Epitopes in a FVB/N Rat HER-2/neu Mouse Model of Breast Cancer

Reshma Singh and Yvonne Paterson

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Requests for reprints: Yvonne Paterson, Department of Microbiology, University of Pennsylvania School of Medicine, 323 Johnson Pavilion, 36th Street and Hamilton Walk, Philadelphia, PA 19104-6076. Phone: 215-898-3461; Fax: 215-573-4666; E-mail: yvonne{at}mail.med.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The HER-2/neu oncogene has >25 HLA epitopes, yet only one FVB/N mouse CD8+ T-cell epitope has been mapped to date. This epitope has been termed the immunodominant epitope for the FVB/N mouse, but we propose that the vaccination strategy determines the dominance of epitopes. Using a series of overlapping peptides, we have mapped another CD8+ T-cell epitope that emerges in the FVB/N mouse following vaccination with Listeria monocytogenes–based vaccines that express fragments of HER-2/neu. Following the identification of this novel H-2Kq-restricted epitope, we sought to compare the T-cell response to this epitope with the previously identified PDSLRDLSVF epitope. This newly identified epitope and the previously identified epitope lie within fragments contained in different vaccines, the PDSLRDLSVF epitope in Lm-LLO-EC2 and the newly identified PYNYLSTEV epitope in Lm-LLO-EC1; thus, it has been possible to compare the responses of these epitopes independent of any competing response between the epitopes. CTL analysis of individual peptide-pulsed target cells and intracellular cytokine stain for IFN-{gamma} produced by splenocytes from Lm-LLO-EC1 compared with Lm-LLO-EC2 vaccinated FVB/N mice shows that there is no difference between the responses generated to either of these epitopes. We also show that the avidity of the CD8+ T cells for either of these epitopes is similar based on the concentration of peptide necessary to mediate similar levels of lysis of target cells. In addition, HER-2/neu DNA vaccination followed by CTL analysis further showed that both of these peptides can emerge as epitopes. (Cancer Res 2006; 66(15): 7748-57)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD8+ T-cell population generated against a specific tumor antigen is a vital component of a potent antitumor immune response (1–5). Several tumor-associated antigens have been identified, and the specific immune responses that can be generated by targeting these antigens is being studied in both humans and in mouse models of cancer (3, 6–8). Determining the epitopes against which CTL responses can be generated is an important factor in characterizing antitumor immune responses because it allows for the direct measurement of antitumor immunity against a single specificity.

A frequently targeted tumor-associated antigen is HER-2/neu, which is a 185-kDa transmembrane glycoprotein and a member of the epidermal growth factor family of receptors (9–11). It is overexpressed in 15% to 40% of all breast cancers and contributes to the tumor phenotype, which makes it a good target for immunotherapy (12, 13). Specific anti-HER-2/neu CD8+ T-cell (14) responses have been detected in patients with HER-2/neu overexpressing breast cancers, and several epitopes against which these responses are directed have been mapped. The epitopes identified in the human HER-2/neu span the entire protein (15–20). However, although antitumor CD8+ T cells are generated by some patients, along with any antibodies or CD4+ T cells produced, they are not enough to overcome the effects of tolerance and eradicate the tumors (14, 21, 22).

Several strategies have been developed to target HER-2/neu as a method of treating breast cancers that overexpress this oncogene. These include whole-cell vaccines, vaccinia vaccines, and DNA vaccines among others (23–26). Our lab has previously described a series of Listeria monocytogenes vaccines expressing fragments of HER-2/neu, all of which are capable of inducing stasis in the growth of established implanted tumors in wild-type (wt) FVB mice (5). Each of these five vaccines can also generate anti-HER-2/neu–specific CD8+ T-cell responses, which are a key component of their antitumor efficacy. Due to its ability to exist within the host cell, primarily antigen-presenting cells (APC), L. monocytogenes can generate both a specific CD4+ and CD8+ T-cell response to secreted antigens (27–29). Through fusion of the tumor antigen to a truncated, nonhemolytic form of listeriolysin O (LLO), secretion of the tumor antigen is ensured (30). Fusion to LLO is also thought to target the fusion protein for proteasomal degradation, which allows for the foreign protein to be efficiently targeted to the MHC class I processing pathway (31, 32).

Previously, in the FVB/N mouse, only one CD8+ T-cell epitope had been mapped, the H2-Dq-restricted PDSLRDLSVF epitope, which has been called the immunodominant HER-2/neu MHC class I epitope for this mouse (33, 34). Based on our previous antitumor and CTL data, we concluded that there must be more than one FVB/N T-cell epitope present for the rat HER-2/neu protein (5). The previously mapped HER-2/neu epitope was determined to be the immunodominant epitope in the FVB/N mouse because this was the only epitope identified upon the generation of specific T-cell clones following vaccination with whole-cell tumor vaccines transduced with granulocyte macrophage colony-stimulating factor (GM-CSF) and vaccinia vaccines expressing HER-2/neu (34). The demonstration that other regions of HER-2/neu contain CTL epitopes challenges this conclusion (5).

Dominance may be determined by the type of vaccination, the dose of antigen given, and the time that the response is studied (35–38). It is also greatly determined by the potential T-cell repertoire that is available for generation of antigen-specific CD8+ T cells (39–42). Depending on the route and type of vaccine, different T-cell pools may be primed and expanded. Out of all these factors, this study looks primarily at the effect of the type of vaccination on the generation of the antigen-specific CD8+ T-cell response and the strength of this response against two different epitopes.

Here, we identify another rat HER-2/neu CD8+ T-cell epitope for the FVB/N mouse that is in a different region of the protein than the previously mapped epitope. Furthermore, we go on to characterize the immune response that can be generated targeting either of these epitopes to determine how the CD8+ T-cell response differs. Our findings show that the "dominance" of epitopes is determined by many factors, including the method of generating the antigen-specific CD8+ T cells. Given the interest in using HER-2/neu for human cancer immunotherapies, including peptide-based therapies, a complete knowledge of the epitopes in this animal model for HER-2/neu overexpressing cancers will allow for the better design and preclinical testing of therapies directed to this antigen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
L. monocytogenes vaccines. The L. monocytogenes–based vaccines for HER-2/neu used in this study were described previously (5). A similarly constructed L. monocytogenes–based vaccine for NY-ESO-1 was kindly provided by Dr. Paulo Maciag and was used as a control for antigen specificity in some experiments. Bacteria were grown in brain heart infusion medium (BD, Sparks, MD) with 50 µg/mL chloramphenicol and frozen in 1 mL aliquots at –80°C. To inject mice, the vaccines were thawed and washed twice with sterile PBS before being resuspended in PBS.

Mice. Six- to 8-week-old female FVB/N mice were purchased from Charles River Laboratories (Wilmington, MA). Animals were cared for and used in accordance with protocols approved by the Animal Care and Use Committee of the University of Pennsylvania (Philadelphia, PA).

Cell lines. The NT-2 tumor cell line was developed from a spontaneously occurring mammary tumor in an FVB/N HER-2/neu transgenic mouse (43). These cells constitutively express low levels of rat HER-2/neu and were used as a feeder cell line as a source of antigen for the restimulation of splenocytes in CTL assays. NT-2 cells were grown in RPMI 1640 supplemented with 20% FCS, 10.2 mL HEPES, 2 mmol/L L-glutamine, 100 µmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 50 units/mL penicillin G, 50 µg/mL streptomycin, 20 µg/mL insulin, and 2 µg/mL gentamicin. Several different target cell lines were used for the CTL assays. NIH 3T3 cells are a mouse fibroblast line and 3T3-neu cells were made by transducing these cells with the full-length rat HER-2/neu as previously described (33). The NIH 3T3 cells (3T3-wt) were cultured in DMEM supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 µmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 50 units/mL penicillin G, and 50 µg/mL streptomycin. Additionally, the 3T3-neu cell line was supplemented with 1 mg/mL G418. The 16-3-1N and 3-83P hybridomas (American Type Culture Collection, Manassas, VA) were used to produce antibodies used as blocking antibodies in CTL analyses. Both of these cell lines produce antibodies that are IgG2a. Cells were cultured in DMEM supplemented with 10 mmol/L HEPES, 2 mmol/L L-glutamine, solution I (1 mmol/L oxalacetic acid, 20 units/mL insulin, 0.5 mmol/L sodium pyruvate), 100 µmol/L nonessential amino acids, 10% NCTC 135, and 12% FCS. All cells were grown at 37°C with 5% CO2.

DNA vaccines. Escherichia coli cells containing the DNA vaccine plasmids of pcDNA3.1-neu and pcDNA3.1-LLO-neu were generated as described previously (5). pcDNA3.1-LLO-E7 was used as a negative control for the HER-2/neu DNA vaccines. Cultures were grown in Luria-Bertani supplemented with ampicillan at 37°C. Plasmids containing the antigens were isolated using a DNA plasmid purification maxi prep kit (Qiagen Sciences, Germantown, MD).

Peptides. Nine- and 10-mers were synthesized as lyophilized peptides by EZBiolab (Westfield, IN). All peptides were reconstituted in water at a 1 mg/mL concentration.

Flow cytometry. The 16-3-1N and 3-83P antibodies were analyzed for their ability to block the binding of a H-2Kq (KH114)–specific FITC-labeled antibody or a H-2Lq/Dq-biotinylated (KH117) antibody and a SA-FITC secondary antibody (BD Biosciences PharMingen, San Diego, CA). 3T3 cells were incubated with the blocking antibody for 1 hour and then stained for 30 minutes with an H-2Kq antibody conjugated to FITC. Samples were run on a Beckman Coulter FACSCalibur, and the data were analyzed using FlowJo (Treestar, Ashland, OR).

51Cr release assays. Six- to 8-week-old mice were vaccinated i.p. with 0.1 LD50 of Lm-LLO-EC1, Lm-LLO-EC2, or 200 µL PBS. Nine days later, splenocytes were harvested and set up to be restimulated. Briefly, whole splenocytes were plated in 24-well plates at a ratio of 10:1 with irradiated NT-2 (20,000 rad) tumor cells and 20 units/mL interleukin-2 (IL-2; Roche, Indianapolis, IN). Four days later, splenocytes were harvested and used in a standard 51Cr release assay. Target cells were labeled with chromium and then cultured with splenocytes at the effector-to-target (E:T) ratios of 200:1, 100:1, 50:1, and 25:1, in triplicate for 4 hours. Before labeling the target cells with chromium in peptide-loaded assays, cells were pulsed for 1 hour at 37°C with 1 µg/mL of the appropriate peptide. When blocking antibodies were used to block binding of the peptide to the MHC, target cells were treated with antibodies before loading with peptide. The percent specific lysis was determined as [(experimental counts per minute – spontaneous counts per minutes) / (total counts per minute – spontaneous counts per minute)] x 100.

Intracellular cytokine stain. Mice were vaccinated with either PBS, Lm-LLO-NYESO1, Lm-LLO-EC1, and Lm-LLO-EC2 to test the Listeria-based vaccines, or PBS, DNA-LLO-E7, DNA-LLO-neu, and DNA-LLO-EC1 to test the ability of the DNA vaccines to stimulate the production of IFN-{gamma}. Spleens were harvested, and splenocytes were isolated 9 days after vaccination and cultured with the appropriate stimulus, including GolgiPlug (BD, Franklin Lakes, NJ) to prevent secretion of IFN-{gamma}, in 96-well plates. Anti-CD3/CD28 stimulation was used as a positive control, and three stimulation groups consisting of the EC1, EC2, and IC1 peptides at a 1 µg/mL concentration were used during an 8-hour in vitro stimulation of the splenocytes. Cells were then surface stained with CD8 (53-6.7, FITC conjugated), CD11b PerCP, and CD62L (Mel-14, APC conjugated; BD, Franklin Lakes, NJ). Cells were then fixed and permeabilized using a cytofix/cytoperm solution (BD, Franklin Lakes, NJ) and then stained with IFN-{gamma} PE before fluorescence-activated cell sorting (FACS) analysis on a FACSCalibur. FACS data were then analyzed using Flowjo software (Treestar).

Statistics. The Student's t test was used for all statistical analyses. Significance is noted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a novel HER-2/neu epitope. The immunodominant CD8+ T-cell epitope for rat HER-2/neu in the FVB/N mouse was been identified as PDSLRDLSVF (33). Based on our previous tumor regression and CTL data, we had identified regions of HER-2/neu, which contain unidentified CD8+ T-cell epitopes (5) and further narrowed these regions down to 20 mers. We found more than one CD8+ T-cell epitope exists in the EC1 region from amino acids 20 to 326, including an epitope in the 20-mer spanning amino acids 291 to 310 (data not shown). At this point, further identification of the epitope was frustrated by the fact that there are no predictive algorithms for the FVB/N mouse, which is on the H-2q background. Hansen et al. has shown that the H-2Dq and H-2Lq genes are very similar to the H-2d genes (44, 45). Using the "d" haplotype as a guide, we analyzed the sequence of the 20-mer with the Rankpep algorithm (http://mif.dfci.harvard.edu/Tools/rankpep.html) and identified 9- and 10-mers that were likely to bind to d and thus to q. The peptides that were predicted by the algorithm: VTTCPYNYL, TFGASCVTT, TFGASCVTTC, and PYNYLSTEV, were used for CTL analyses. By pulsing these peptides individually onto 3T3-wt target cells, we identified one 9-mer that would target cells for lysis by CD8+ T cells (Fig. 1D ). The response is specific to the PYNYLSTEV peptide as seen by the complete lack of response to any of the other three peptides (Fig. 1A-C).


Figure 1
View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Identification of a novel CD8+ T-cell epitope that falls into the region of HER-2/neu covered by the Lm-LLO-EC1 vaccine. CTL analysis of 3T3-wt cells pulsed with 9- and 10-mers from the identified 20-mer (291-310). Cells were labeled with 1 µg/mL of peptide. Each ratio was done in triplicate. A, VTTCPYNYL. B, TFGASCVTT. C, TFGASCVTTC. D, PYNYLSTEV. *, P < 0.05, specific lysis above background.

 
CTL responses to both the old and the new epitopes are similar. Upon identification of this novel HER-2/neu epitope, we sought to determine whether this new EC1 epitope is as potent in stimulating a CTL response as the previously identified immunodominant epitope. We did a side-by-side CTL analysis by pulsing the target cells with either the previously identified EC2 or the new EC1 epitope and using splenocytes from mice vaccinated with either Lm-LLO-EC1 or Lm-LLO-EC2 as effector cells (Fig. 2 ). There is no lysis when the peptide used as a target does not fall into the region of HER-2/neu that the vaccine spans; thus, the lysis seen is specific for the particular peptide used. At an E:T ratio of 200:1, we observed about 50% lysis of the EC1 peptide-loaded target cells with the Lm-LLO-EC1 immune splenocytes (Fig. 2A), whereas with the EC2 peptide and the Lm-LLO-EC2 splenocytes we observed about 40% lysis (Fig. 2B). There is no significant difference between the lysis seen for the two peptides at all E:T ratios. Based on this analysis, at a peptide concentration of 1 µg/mL, the known EC2 epitope is not immunodominant to the new EC1 epitope, PYNYLSTEV.


Figure 2
View larger version (8K):
[in this window]
[in a new window]
  		Figure 2. Similar levels of specific lysis are seen for the both the known and new epitopes, and the lysis is specific for the vaccine that corresponds to the region containing the epitope. A, PDSLRDLSVF. B, PYNYLSTEV. *, P < 0.05, significant levels of lysis.

 
DNA vaccines show the ability to target both epitopes at one time. One main difference between the L. monocytogenes–based vaccines and vaccination approaches where full-length HER-2/neu has been used is the fact that the Listeria-based vaccines use fragments of HER-2/neu. Although we have shown that this factor does not seem to significantly increase the antitumor effect of these vaccines, it is possible that the use of fragments increases the variety of epitopes that can be targeted through vaccination (5). Using DNA vaccines that we have previously described, we attempted to determine if we could generate a CTL response against both of these epitopes through vaccination with full-length HER-2/neu. CD8+ T-cell responses can be generated targeting both of these epitopes using an LLO-fused full-length HER-2/neu DNA vaccine, although the response to the EC2 epitope is greater than the response to the EC1 epitope (Table 1 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Levels of specific lysis targeting the PDSLRDLSVF and PYNYLSTEV epitopes from DNA-vaccinated wt FVB mice

 
Production of IFN-{gamma} from splenocytes from Listeria-based vaccine-treated mice is similar for cells reactive to the PDSLRDLSVF epitope and to the PYNYLSTEV epitope, but differences in responsiveness emerge with splenocytes from DNA-vaccinated mice. Mice were vaccinated with Lm-LLO-EC1, Lm-LLO-EC2, or PBS, and splenocytes were harvested and analyzed for the production of IFN-{gamma} after stimulation with the EC1 and EC2 peptides. This stimulation, as seen through the production of IFN-{gamma}, is specific for the vaccine given and the corresponding peptide (Fig. 3A ). Regardless of the stimulus used during the 8-hour in vitro culture, the splenocytes from the PBS-treated (data not shown) or Lm-LLO-NY-ESO-1–vaccinated mice did not respond with any significant production of IFN-{gamma} (Fig. 3A). Stimulation with an irrelevant HER-2/neu peptide resulted in a negligible level of IFN-{gamma} production for all the vaccination groups (data not shown). Following stimulation with the EC2 peptide, there was an increased amount of production of IFN-{gamma} from Lm-LLO-EC2–vaccinated mice but not from Lm-LLO-EC1–vaccinated mice (Fig. 3A, bottom), and conversely, the EC1 peptide stimulation led to increased IFN-{gamma} production from Lm-LLO-EC1–vaccinated mice but not from Lm-LLO-EC2–vaccinated mice (Fig. 3A, top). Furthermore, the level of IFN-{gamma} production observed by the Lm-LLO-EC2 mice in response to the EC2 peptide and the Lm-LLO-EC1 mice in response to the EC1 peptide were very similar. Mice were also vaccinated with PBS, DNA-LLO-E7, DNA-LLO-neu, and DNA-LLO-EC1 to observe the extent of IFN-{gamma} production stimulated by the DNA vaccines. Similar to the Listeria-based vaccines, the PBS-treated (data not shown) and the DNA-LLO-E7–vaccinated mice had a negligible level of IFN-{gamma} production by the immune splenocytes regardless of which peptide was used as a stimulus (Fig. 3B). Again, stimulation with an irrelevant HER-2/neu peptide resulted in no IFN-{gamma} production by any of the DNA vaccine groups (data not shown). Stimulation with the EC2 peptide resulted in the production of IFN-{gamma} by splenocytes from DNA-LLO-neu–treated mice but not from the DNA-LLO-EC1–treated mice (Fig. 3B, bottom), and stimulation with the EC1 peptide resulted in the production of IFN-{gamma} from the DNA-LLO-EC1 and DNA-LLO-neu mice (Fig. 3B, top). Unlike the Listeria-based vaccines, where a similar level of IFN-{gamma} production was seen from the Lm-LLO-EC1 splenocytes stimulated with the EC1 peptide and the Lm-LLO-EC2 splenocytes stimulated with the EC2 peptide, a greater level of IFN-{gamma} production was seen upon stimulation with the EC2 peptide than with the EC1 peptide with the splenocytes from DNA-LLO-neu–treated mice; 5.91% of activated CD8+ T cells from DNA-LLO-EC1–vaccinated mice produced IFN-{gamma} when stimulated with the EC1 peptide. When mice are treated with DNA-LLO-neu, only 2.25% produced IFN-{gamma} in response to the EC1 peptide, but this level rose to 7.4% in response to the EC2 peptide (Fig. 3B). This shows that where similar levels of effector activity, as measured by IFN-{gamma} production, can be stimulated by Listeria-based vaccines to fragments of HER-2/neu, when the vaccine contains both of these epitopes, a greater level of the effector response seems to be directed to the EC2 epitope.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Activation and production of IFN-{gamma} is specific for cells stimulated by a vaccine containing a particular epitope, but the levels of stimulation differ based on the vaccine strategy employed. A, mice were vaccinated with PBS, Lm-LLO-NY-ESO-1, Lm-LLO-EC1, and Lm-LLO-EC2. Top, splenocytes were stimulated with 1 µg/mL of the EC1 PYNYLSTEV peptide. Bottom, splenocytes were stimulated with 1 µg/mL of the EC2 PDSLRDLSVF peptide. B, mice were vaccinated with PBS, DNA LLO-E7, DNA LLO-EC1, and DNA LLO-neu. Top, splenocytes were stimulated with 1 µg/mL of the EC1 peptide. Bottom, splenocytes were stimulated with 1 µg/mL of the EC2 peptide.

 
Avidity of the CD8+ T cells induced by the Lm-LLO-EC1 and Lm-LLO-EC2 vaccines are similar in response to the two peptides with both Listeria-based and DNA-based vaccines. At a very high peptide concentration of 1 µg/mL (Fig. 2), the lytic responses targeting the two peptides are very similar. However, it was possible that differences in the responses of the different peptide-specific CD8+ T cells could become apparent when the concentration of peptide pulsed onto the target cells is decreased. This will serve to show differences in the avidities of the T cells (46–48). A high concentration of peptide will be necessary to stimulate low-avidity T cells, whereas high-avidity T cells will be stimulated at high and low concentrations (48). Dominant epitopes are likely to be able to stimulate low avidity cells, whereas less dominant epitopes may not be capable of activating cytotoxic cells that have a low avidity for the peptide. Concentrations ranging from 1 µg/mL down to 100 pg/mL were used to target cells to elicit a lytic response from effector splenocytes to determine whether the previously identified immunodominant epitope can stimulate lower avidity cells than the new EC1 epitope. At both a high E:T ratio (Fig. 4A and B ) and a lower E:T ratio (data not shown), both of the peptides stimulate similar levels of lysis when the CD8+ T cells are primed by the Listeria-based vaccines. Very high levels of lysis can be seen at the low nanogram concentration of either peptides. Lower levels of lysis of target cells is seen with splenocytes from DNA-LLO-EC1–vaccinated mice versus the DNA-LLO-neu–vaccinated mice (Fig. 4C and D). The response towards the EC2 peptide primed by the DNA-LLO-neu vaccine is higher even at a nanogram concentration of peptide than that for the EC1 peptide, although a response can be seen against the EC1 peptide. At both high and low E:T ratios, T cells induced by Lm-LLO-EC1, Lm-LLO-EC2, DNA-LLO-neu, and DNA-LLO-EC1 are antigen specific as evidenced by the lack of lysis to 3T3-wt cells (data not shown).


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. The affinity of stimulated CD8+ T cells for the PDSLRDLSVF and the PYNYLSTEV peptides are the same at both high and low E:T ratios but differ when stimulated by DNA vaccination. A and B, splenocytes from PBS-, Lm-LLO-EC1–, and Lm-LLO-EC2–vaccinated mice at a 200:1 E:T ratio. C and D, splenocytes from PBS-, DNA-LLO-neu–, and DNA-LLO-EC1–vaccinated mice at a 200:1 E:T ratio. A and C, the target peptide is the EC1 PYNYLSTEV peptide. B and D, the target peptide is the EC2 PDSLRDLSVF peptide. *, P < 0.05, significant levels of lysis above background lysis.

 
PYNYLSTEV epitope is restricted to H-2Kq. The previously identified PDSLRDLSVF epitope has been shown to be H-2Dq restricted (33). We next sought to identify the MHC class I restriction of the new PYNYLSTEV peptide, which had been identified by Rankpep as a Kd binder. To do this, we pulsed both the peptides individually onto L cells that had been transfected with either the H-2Dq or the H-2Lq genes (45). As expected, when the PDSLRDLSVF peptide is pulsed onto L-Dq cells, there is killing of target cells, but this is not seen with peptide-pulsed L-Lq cells (data not shown). When the EC1 peptide is pulsed onto either of these cell lines, there is no killing of the target cells (data not shown). Based on this, we believe that the EC1 PYNYLSTEV peptide is actually restricted to H-2Kq, as this MHC class I molecule is present in the FVB/N mouse and the 3T3 target cell line but is absent on the L-Dq or -Lq cells.

As there is no cell line that expresses only H-2Kq, we studied the binding of the EC1 peptide to this MHC through the use of an H-2Kq-specific blocking antibody and as a control a nonspecific antibody (Fig. 5A-D ). First, we tested target cells to confirm that the antibody specifically binds to H-2Kq and not to other "q" restricted MHC molecules by FACS analysis. Cells were treated with FITC-labeled H-2Kq or biotinylated H-2Lq/Dq, in the presence or absence of 16-3-1N or 3-83P. The 16-3-1N antibody specifically blocks H-2Kq (Fig. 5A and B), whereas the 3-83P antibody does not block H-2Kq, Lq, or Dq (Fig. 5C and D) and can thus be used as a negative control for the 16-3-1N antibody. We then tested the ability of 16-3-1N to block lysis of target cells pulsed with the EC1 epitope by splenocytes from Lm-LLO-EC1–treated mice. When target cells were treated with 16-3-1N before loading with peptide, splenocytes from Lm-LLO-EC1–treated mice showed no cytolytic activity against target cells (Fig. 5E). However, target cells treated with 3-83P and pulsed with the EC1 peptide were killed by splenocytes from Lm-LLO-EC1–treated mice (Fig. 5F). Based on these data, we conclude that the PYNYLSTEV peptide is restricted to H-2Kq. These data also suggest that the peptide-binding motifs of Kd and Kq are similar.


Figure 5
View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. The PYNYLSTEV peptide is H-2Kq restricted. Two antibodies were tested for their abilities to block the H-2Kq class I molecule. 3T3 cells are treated with the 16-3-1N or the 3-83P blocking antibodies before staining with fluorochrome conjugated MHC class I antibodies for flow cytometry, to determine the blocking potential of 16-3-1N and 3-83P. A, pretreatment of 3T3 cells with 16-3-1N abrogates the binding of the Kq-specific FACS antibody. Black line, 16-3-1N-treated cells; gray line, cells that were not treated with a blocking antibody; shaded area, isotype control. B, pretreatment of 3T3 cells with 16-3-1N does not block the binding of the Lq/Dq FACS antibody. Black line, 16-3-1N-treated cells; gray line, cells that were not treated with a blocking antibody; shaded area, isotype control. C and D, pretreatment of 3T3 cells with the 3-83P antibody does not block the binding of either the Kq- or the Lq/Dq-specific antibodies. Black line, cells treated with the 3-83P antibody; gray line, cells that were not treated with a blocking antibody; shaded area, isotype control. The PYNYLSTEV peptide is pulsed onto target cells after treatment with the blocking antibodies to determine if this epitope is Kq restricted. A, 3T3-wt target cells treated with the 16-3-1N blocking antibody. B, 3T3-wt target cells treated with the 3-83P blocking antibody. *, P < 0.05, significant levels of lysis.

 
Both the PDSLRDLSVF and the PYNYLSTEV epitopes are naturally processed and presented by tumor cells. Although unlikely, it is possible that the new EC1 epitope was not previously identified due to the fact that it is an epitope that is not naturally processed and presented by tumor cells. We tested for this possibility using a CTL assay where the in vitro stimulus was changed from irradiated tumor cells to peptide, and the target cells were tumor cells (3T3-wt, 3T3-neu, and NT-2 cells). When no peptide is provided as a stimulus, as expected, there is no lysis of any of the target cells from the PBS, Lm-LLO-EC1, or the Lm-LLO-EC2 splenocytes (Fig. 6A, D, and G ). Use of the PYNYLSTEV peptide as a stimulus results in no lysis of 3T3-wt cells from any of the vaccine groups, but the Lm-LLO-EC1 splenocytes are capable of killing both the 3T3-neu cells and the NT-2 cells (Fig. 6B, E, and H). Similarly, stimulating the splenocytes using the PDSLRDLSVF peptide leads to no killing of 3T3-wt cells from any of the groups, but splenocytes from Lm-LLO-EC2 mice kill both the 3T3-neu and NT-2 cells (Fig. 6C, F, and I). These results clearly show that both of these epitopes are naturally processed and presented by the NT-2 and 3T3-neu tumor cells and further, that the HER-2/neu peptides are similar in their ability to expand splenocytes to kill tumor cells expressing HER-2/neu.


Figure 6
View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. The PYNYLSTEV and the PDSLRDLSVF epitopes are naturally processed and presented by tumor cells. Splenocytes from vaccinated mice were in vitro restimulated with no peptide (A, D, and G), the PYNYLSTEV peptide (B, E, and H), or the PDSLRDLSVF peptide (C, F, and I). A to C, there was no killing of 3T3-wt target cells regardless of the stimulus used for restimulation. D and G, there was no killing of either the 3T3-neu or the NT-2 target cells when no peptide was used for the restimulation. E and H, when the PYNYLSTEV peptide is used for restimulation, only splenocytes from Lm-LLO-EC1–vaccinated mice are capable of killing either the 3T3-neu or the NT-2 target cells. F and I, only the Lm-LLO-EC2 splenocytes are capable of killing either the 3T3-neu or NT-2 tumor cells when restimulated with the PDSLRDLSVF peptide. Percent specific lysis was calculated as % = 100 x[(experimental – spontaneous) / (total – spontaneous)]. *, P < 0.05, significance.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have identified a novel rat HER-2/neu epitope in the FVB/N mouse revealed by L. monocytogenes–based vaccination. In addition, the previously mapped immunodominant epitope is not in actual fact "dominant" to this epitope, with this vaccination strategy. Our previous tumor regression and CTL analyses have indicated that there are multiple epitopes for rat HER-2/neu in the FVB/N mouse that have not yet been identified (5). Additionally, there have been many CD8+ T-cell epitopes mapped for the human HER-2/neu, which are spread throughout the entire length of the HER-2/neu protein (8, 15–17, 19, 20, 44). The human and rat proteins are extremely similar to each other, with a >90% homology (49). This information and our data led us to conclude that there were unmapped epitopes for rat HER-2/neu in the FVB/N mouse.

To map other epitopes, we have continued with the mapping strategy that we had developed previously, which is to use CTL analysis as an indicator for the presence of a CD8+ T-cell epitope (5). Using overlapping 20-mers, we first narrowed down the areas of HER-2/neu containing an epitope and then used the Rankpep algorithm and estimates of peptide binding to H-2d molecules to break these 20-mers into smaller peptides that are the correct length for MHC class I binding. The areas containing an epitope have been further mapped. H-2d has previously been shown to have structural homology to Hq (44, 45, 50). The four peptides that were predicted for an EC1 20-mer were then used to identify a novel class I epitope for the FVB/N mouse, and a subsequent series of experiments were done to show that EC2 is not dominant with EC1 using our vaccine strategy.

One issue that arises in identifying CD8+ T-cell epitopes through CTL analyses is whether the new class I epitope arises by natural processing and presentation of HER-2/neu expressed in tumor cells. To generate CD8+ T cells for CTL analysis immune splenocytes were expanded ex vivo for 4 days before being used in the chromium release assay. In our assays, the source of antigen given to restimulate the antigen-specific T cells are whole-irradiated tumor cells. The APCs present in the culture of whole splenocytes takes up these dying tumor cells and processes and presents antigens to the T cells. This implies that the EC1 epitope is naturally processed by the APCs because CTL are generated that recognize it as well as the EC2 epitope. However, to confirm this, we expanded Lm-LLO-EC1 splenocytes ex vivo with the peptide and showed that the peptide-specific CTL could lyse tumor cells that express HER-2/neu.

Several factors are involved in determining the hierarchy of dominance of epitopes and if there is an immunodominant epitope in a system. This begins with antigen processing and then progresses to recognition by the T-cell of the peptide-MHC complex (35, 36, 38, 39, 41, 42, 51–53). Involved in this is the T-cell repertoire that is present and capable of being amplified in response to vaccination with a particular tumor antigen. In the case of the epitope described here and the previously described epitope, there should be no difference in the basic T-cell repertoire that is capable of being amplified due to the fact that the same strain of mouse is used in both studies.

In a simple attempt to determine if the EC2 epitope is immunodominant to the EC1 epitope, we conducted CTL analysis along with intracellular cytokine production analysis for IFN-{gamma}, and based on these analyses, neither epitope seems dominant to the other (Figs. 2 and 3). There are several factors that do differ in how this epitope was defined versus the previous epitope that was defined by the Jaffee lab using irradiated whole 3T3 cells transduced with GM-CSF as a vaccine in addition to a vaccinia vaccine containing full-length HER-2/neu (23, 33). They used T-cell clones derived from vaccinated mice and overlapping peptides in CTL analyses to determine the sequence of the EC2 epitope as PDSLRDLSVF. All of the T-cell clones generated, responded to this single peptide, which for this reason, was determined to be the immunodominant epitope for HER-2/neu in the FVB/N mouse (33). To map the EC1 epitope in this article, we used L. monocytogenes–based vaccines containing fragments of HER-2/neu. We did not use T-cell clones, but instead, we used a polyclonal primary T-cell pool in our CTL analyses to identify CD8+ T-cell epitopes.

It is possible that the different methods of generating an anti-HER-2/neu immune response could result in a very different pool of CD8+ T cells. Irradiated whole-cell vaccines administer a dose of antigen that is determined by the amount of neu expression by the 3T3 cells (33). The vaccinia vaccine on the other hand could lead to the production of higher levels of antigen in APCs and potentially allow for a varied pool of responder CD8+ T cells to be generated. It is interesting to note therefore that T-cell clones with similar specificities were generated using both of these vaccination strategies. However, T-cell clones do not necessarily represent the responding T-cell pool. T-cell clones are stimulated and kept alive through the continual administration of IL-2 along with a source of antigen, which generally results in the generation of a clone that is selected through its ability to respond robustly by division to the given stimulus (54). Clearly, this may not be the most highly responding, or more importantly, the only CD8+ T-cell capable of responding in vivo during an immune response. We have shown that there is a group of T cells within a polyclonal population with high avidity for the EC1 epitope. We believe that there are many other epitopes in HER-2/neu, which we are continuing to map, because all five of our Lm-HER-2/neu fragment vaccines generate CD8+ T cells that have potent antitumor activity (5).

The DNA vaccines used in this study further support the idea that the vaccination strategy determines what epitopes emerge upon vaccination and the dominance of these epitopes (Table 1; Fig. 3B; ref. 55). pcDNA-LLO-neu induces both a CTL and IFN-{gamma} response targeting the novel PYNYLSTEV epitope, but the response to this epitope is much lower than the response to the previously defined PDSLRDLSVF epitope. This suggests that with other vaccine strategies epitopes could be undetectable, as seems to have been the case for the EC1 epitope with the strategies used by Erocolini et al. (33). In the case of the Listeria-based vaccines that target these epitopes, we see no difference in the CTL response against these epitopes (Figs. 2-3). This could be because when stimulated simultaneously, the EC2 epitope is the immunodominant epitope, but taken side-by-side with individual vaccines, there is no dominance among these epitopes. Titrating the peptides down to look at the avidity of the CD8+ T cells shows that the same avidity T-cell can be activated targeting both the EC1 and the EC2 peptides. What differs though is the level of lysis that can be induced targeting either peptide, with a greater response directed towards the EC2 peptide (Fig. 4C and D). Using Listeria, no difference is seen between either the avidity or the level of lysis directed towards either peptide. Based on this, it becomes clear that the vaccination strategy does indeed determine the dominance of CD8+ T-cell epitopes and not some inherent characteristic of the epitope itself.

When discussing dominance and the generation of CD8+ T cells capable of responding to different epitopes another consideration is the possible presence of CD4+CD25+ regulatory T cells (56). In the case of the whole-cell vaccinations and the vaccinia vaccines, it is possible that regulatory T cells are being induced, which are not being generated with this Listeria-based vaccine system. We have previously shown that our episomal based Listeria vaccines do not induce a significant production of regulatory T cells in contrast to other vaccine approaches (57, 58). In the case of the DNA vaccines (Table 1), whole-cell vaccines, and vaccinia vaccines, it may be that regulatory T cells prevent the response of CD8+ T cells that are specific for epitopes other than the PDSLRDLSVF epitope (34).


   Acknowledgments
 
Grant support: Department of Defense grant W81XWH-04-1-0338 (R. Singh).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Ted Hansen (Washington University School of Medicine, St. Louis, MO) for the generous donation of the MHC class I expressing cell lines and Dr. Paulo Maciag (University of Pennsylvania School of Medicine) for allowing us to use Lm-LLO-NY-ESO-1 before publication.


   Footnotes
 
Note: Y. Paterson wishes to disclose that she has a financial interest in Advaxis, Inc., a vaccine and therapeutic company that has licensed or has an option to license all patents from the University of Pennsylvania that concern the use of Listeria or listerial products as vaccines.

Received 12/20/05. Revised 3/24/06. Accepted 5/17/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pan ZK, Ikonomidis G, Lazenby A, et al. A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat Med 1995;1:471–7.[CrossRef][Medline]
  2. Pan ZK, Weiskirch L, Paterson Y. Regression of established B16F10 melanoma with a recombinant Listeria monocytogenes vaccine. Cancer Res 1999;59:5264–9.[Abstract/Free Full Text]
  3. Huang AY, Gulden PH, Woods AS, et al. The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc Natl Acad Sci U S A 1996;93:9730–5.[Abstract/Free Full Text]
  4. Peng L, Kjaergaard J, Plautz GE, et al. Helper-independent, L-selectin low CD8+ T cells with broad anti-tumor efficacy are naturally sensitized during tumor progression. J Immunol 2000;165:5738–49.[Abstract/Free Full Text]
  5. Singh R, Dominiecki ME, Jaffee EM, et al. Fusion to listeriolysin O and delivery by Listeria monocytogenes enhances the immunogenicity of HER-2/neu and reveals subdominant epitopes in the FVB/N mouse. J Immunol 2005;175:3663–73.[Abstract/Free Full Text]
  6. Tuting T, Steitz J, Bruck J, et al. Dendritic cell-based genetic immunization in mice with a recombinant adenovirus encoding murine TRP2 induces effective anti-melanoma immunity. J Gene Med 1999;1:400–6.[CrossRef][Medline]
  7. Alexander M, Salgaller ML, Celis E, et al. Generation of tumor-specific cytolytic T lymphocytes from peripheral blood of cervical cancer patients by in vitro stimulation with a synthetic human papillomavirus type 16 E7 epitope. Am J Obstet Gynecol 1996;175:1586–93.[CrossRef][Medline]
  8. Kawakami Y, Eliyahu S, Delgado CH, et al. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci U S A 1994;91:6458–62.[Abstract/Free Full Text]
  9. Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 1986;319:226–30.[CrossRef][Medline]
  10. Kim H, Muller WJ. The role of the epidermal growth factor receptor family in mammary tumorigenesis and metastasis. Exp Cell Res 1999;253:78–87.[CrossRef][Medline]
  11. King CR, Kraus MH, Aaronson SA. Amplification of a novel v-erB-related gene in human mammary carcinoma. Science 1985;229:974–6.[Abstract/Free Full Text]
  12. Disis ML, Cheever MA. HER-2/neu protein: a target for antigen-specific immunotherapy of human cancer. Adv Cancer Res 1997;71:343–71.[Medline]
  13. Knutson KL, Schiffman K, Rinn K, et al. Immunotherapeutic approaches for the treatment of breast cancer. J Mammary Gland Biol Neoplasia 1999;4:353–65.[CrossRef][Medline]
  14. Peoples GE, Smith RC, Linehan DC, et al. Shared T cell epitopes in epithelial tumors. Cell Immunol 1995;164:279–86.[CrossRef][Medline]
  15. Brossart P, Stuhler G, Flad T, et al. Her-2/neu-derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes. Cancer Res 1998;58:732–6.[Abstract/Free Full Text]
  16. Fisk B, Blevins TL, Wharton JT, et al. Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J Exp Med 1995;181:2109–17.[Abstract/Free Full Text]
  17. Kawashima I, Hudson SJ, Tsai V, et al. The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors. Hum Immunol 1998;59:1–14.[Medline]
  18. Rongcun Y, Salazar-Onfray F, Charo J, et al. Identification of new HER2/neu-derived peptide epitopes that can elicit specific CTL against autologous and allogeneic carcinomas and melanomas. J Immunol 1999;163:1037–44.[Abstract/Free Full Text]
  19. Scardino A, Alves P, Gross DA, et al. Identification of HER-2/neu immunogenic epitopes presented by renal cell carcinoma and other human epithelial tumors. Eur J Immunol 2001;31:3261–70.[CrossRef][Medline]
  20. Scardino A, Gross DA, Alves P, et al. HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy. J Immunol 2002;168:5900–6.[Abstract/Free Full Text]
  21. Coronella JA, Telleman P, Kingsbury GA, et al. Evidence for an antigen-driven humoral immune response in medullary ductal breast cancer. Cancer Res 2001;61:7889–99.[Abstract/Free Full Text]
  22. Tuttle TM, Anderson BW, Thompson WE, et al. Proliferative and cytokine responses to class II HER-2/neu-associated peptides in breast cancer patients. Clin Cancer Res 1998;4:2015–24.[Abstract]
  23. Wolope ME, Lutz ER, Ercolini AM, et al. HER-2/neu-specific monoclonal antibodies collaborate with HER-2/neu-targeted granulocyte macrophage colony-stimulating factor secreting whole cell vaccination to augment CD8+ T cell effector function and tumor-free survival in Her-2/neu-transgenic mice. J Immunol 2003;171:2161–9.[Abstract/Free Full Text]
  24. Chen Y, Hu D, Eling DJ, et al. DNA vaccines encoding full-length or truncated neu induce protective immunity against neu-expressing mammary tumors. Cancer Res 1998;58:1965–71.[Abstract/Free Full Text]
  25. Rovero S, Amici A, Di Carlo E, et al. DNA vaccination against rat Her-2/neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol 2000;165:5133–42.[Abstract/Free Full Text]
  26. Mukai K, Yasutomi Y, Watanabe M, et al. HER2 peptide-specific CD8(+) T cells are proportionally detectable long after multiple DNA vaccinations. Gene Ther 2002;9:879–88.[CrossRef][Medline]
  27. Southwick FS, Purich D. Intracellular pathogenesis of Listeriosis. N Engl J Med 1996;334:770–6.[Free Full Text]
  28. Mackaness G. Cellular resistance to infection. J Exp Med 1962;116:381–406.[Abstract]
  29. Gedde MM, Higgins D, Tilney LG, et al. Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes. Inf Immunity 2000;68:999–1003.
  30. Ikonomidis G, Paterson Y, Kos FJ, et al. Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes. J Exp Med 1994;180:2209–18.[Abstract/Free Full Text]
  31. Sewell DA, Shahabi V, Gunn GR III, et al. Recombinant Listeria vaccines containing PEST sequences are potent immune adjuvants for the tumor-associated antigen human papillomavirus-16 E7. Cancer Res 2004;64:8821–5.[Abstract/Free Full Text]
  32. Decatur AL, Portnoy DA. A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity. Science 2000;290:992–5.[Abstract/Free Full Text]
  33. Ercolini AM, Machiels JP, Chen YC, et al. Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/neu-transgenic mice. J Immunol 2003;170:4273–80.[Abstract/Free Full Text]
  34. Ercolini AM, Ladle BH, Manning EA, et al. Recruitment of latent pools of high-avidity CD8(+) T cells to the antitumor immune response. J Exp Med 2005;201:1591–602.[Abstract/Free Full Text]
  35. Sette A, Vitiello A, Reherman B, et al. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J Immunol 1994;153:5586–92.[Abstract]
  36. Yewdell JW, Bennink JR. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 1999;17:51–88.[CrossRef][Medline]
  37. van der Most RG, Murali-Krishna K, Lanier JG, et al. Changing immunodominance patterns in antiviral CD8 T-cell responses after loss of epitope presentation or chronic antigenic stimulation. Virology 2003;315:93–102.[CrossRef][Medline]
  38. Vitiello A, Yuan L, Chesnut RW, et al. Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kbrestricted epitopes. J Immunol 1996;157:5555–62.[Abstract]
  39. Yewdell JW, Eisenlohr LC, Cox JH, et al. Processing and presentation of antigens to class I restricted T lymphocytes. Year Immunol 1989;6:38–50.[Medline]
  40. Kedl RM, Kappler JW, Marrack P. Epitope dominance, competition and T cell affinity maturation. Curr Opin Immunol 2003;15:120–7.[CrossRef][Medline]
  41. Chen W, Bennink JR, Yewdell JW. Quantitating presentation of MHC class I-restricted antigens. Methods Mol Biol 2001;156:245–54.[Medline]
  42. Bennink JR, Anderson R, Bacik I, et al. Antigen processing: where tumor-specific T-cell responses begin. J Immunother 1993;14:202–8.[Medline]
  43. Reilly RT, Gottleib MBC, Ercolini AM, et al. HER-2/neu is a tumor rejection target in tolerized HER-2/neu transgenic mice. Cancer Res 2000;60:3569–76.[Abstract/Free Full Text]
  44. Rubocki RJ, Lee DR, Lie WR, et al. Molecular evidence that the H-2D and H-2L genes arose by duplication. Differences between the evolution of the class I genes in mice and humans. J Exp Med 1990;171:2043–61.[Abstract/Free Full Text]
  45. Lee DR, Rubocki RJ, Lie WR, et al. The murine MHC class I genes, H-2Dq and H-2Lq, are strikingly homologous to each other, H-2Ld, and two genes reported to encode tumor-specific antigens. J Exp Med 1988;168:1719–39.[Abstract/Free Full Text]
  46. Redmond WL, Sherman LA. Peripheral tolerance of CD8 T lymphocytes. Immunity 2005;22:275–84.[CrossRef][Medline]
  47. Lyman MA, Nugent CT, Marquardt KL, et al. The fate of low affinity tumor-specific CD8+ T cells in tumor-bearing mice. J Immunol 2005;174:2563–72.[Abstract/Free Full Text]
  48. Morgan DJ, Kreuwel HT, Fleck S, et al. Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity. J Immunol 1998;160:643–51.[Abstract/Free Full Text]
  49. Disis ML, Shiota FM, Cheever MA. Human HER-2/neu protein immunization circumvents tolerance to rat neu: a vaccine strategy for ‘self’ tumour antigens. Immunol 1998;93:192–9.[CrossRef][Medline]
  50. Lie WR, Myers NB, Connolly JM, et al. The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure. J Exp Med 1991;173:449–59.[Abstract/Free Full Text]
  51. Chen ZW, Li Y, Zeng X, et al. The TCR repertoire of an immunodominant CD8+ T lymphocyte population. J Immunol 2001;166:4525–33.[Abstract/Free Full Text]
  52. Davenport MP, Fazou C, McMichael AJ, et al. Clonal selection, clonal senescence, and clonal succession: the evolution of the T cell response to infection with a persistent virus. J Immunol 2002;168:3309–17.[Abstract/Free Full Text]
  53. Deng Y, Yewdell JW, Eisenlohr LC, et al. MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL. J Immunol 1997;158:1507–15.[Abstract]
  54. Gullo CA, Esser MT, Fuller CL, et al. Generation of IL-2-dependent cytolytic T lymphocytes (CTLs) with altered TCR responses derived from antigen-dependent CTL clones. J Immunol 1999;162:6466–72.[Abstract/Free Full Text]
  55. McKay PF, Barouch DH, Santra S, et al. Recruitment of different subsets of antigen-presenting cells selectively modulates DNA vaccine-elicited CD4+ and CD8+ T lymphocyte responses. Eur J Immunol 2004;34:1011–20.[CrossRef][Medline]
  56. Haeryfar SM, DiPaolo RJ, Tscharke DC, et al. Regulatory T cells suppress CD8+ T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities. J Immunol 2005;174:3344–51.[Abstract/Free Full Text]
  57. Hussain SF, Paterson Y. What is needed for effective antitumor immunotherapy? Lessons learned using Listeria monocytogenes as a live vector for HPV-associated tumors. Cancer Immunol Immunother 2005;54:577–86.[CrossRef][Medline]
  58. Hussain SF, Paterson Y. CD4+CD25+ regulatory T cells that secrete TGFbeta and IL-10 are preferentially induced by a vaccine vector. J Immunother 2004;27:339–46.



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. M. Seavey, P. C. Maciag, N. Al-Rawi, D. Sewell, and Y. Paterson
An Anti-Vascular Endothelial Growth Factor Receptor 2/Fetal Liver Kinase-1 Listeria monocytogenes Anti-Angiogenesis Cancer Vaccine for the Treatment of Primary and Metastatic Her-2/neu+ Breast Tumors in a Mouse Model
J. Immunol., May 1, 2009; 182(9): 5537 - 5546.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. M. Seavey, Z.-K. Pan, P. C. Maciag, A. Wallecha, S. Rivera, Y. Paterson, and V. Shahabi
A Novel Human Her-2/neu Chimeric Molecule Expressed by Listeria monocytogenes Can Elicit Potent HLA-A2 Restricted CD8-positive T cell Responses and Impact the Growth and Spread of Her-2/neu-positive Breast Tumors
Clin. Cancer Res., February 1, 2009; 15(3): 924 - 932.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. Singh and Y. Paterson
Immunoediting Sculpts Tumor Epitopes during Immunotherapy
Cancer Res., March 1, 2007; 67(5): 1887 - 1892.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Singh, R.
Right arrow Articles by Paterson, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Singh, R.
Right arrow Articles by Paterson, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online