Most tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and become targets of antitumor immune responses. Antibodies and T cells specific for some TAAs have been found in healthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer has also been associated with a history of febrile viral diseases. We hypothesized that virus infections could lead to transient expression of abnormal forms of self-molecules, some of which are TAAs; facilitated by the adjuvant effects of infection and inflammation, these molecules could elicit specific antibodies, T cells, and lasting immune memory simultaneously with immunity against viral antigens. Such infection-induced immune memory for TAA would be expected to provide life-long immune surveillance of cancer. Using influenza virus infection in mice as a model system, we tested this hypothesis and demonstrated that influenza-experienced mice control 3LL mouse lung tumor challenge better than infection-naive control mice. Using 2D-difference gel electrophoresis and mass spectrometry, we identified numerous molecules, some of which are known TAAs, on the 3LL tumor cells recognized by antibodies elicited by two successive influenza infections. We studied in detail immune responses against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H4, HSP90, malate dehydrogenase 2, and annexin A2, all of which were overexpressed in influenza-infected lungs and in tumor cells. Finally, we show that immune responses generated through vaccination against peptides derived from these antigens correlated with improved tumor control. Cancer Immunol Res; 2(3); 263–73. ©2013 AACR.

Genetic mutations and epigenetic modifications can lead to cellular transformation and cancer (1). Tumor immunosurveillance is the mechanism by which the immune system recognizes and protects against abnormal cells (2). Successful tumor immunosurveillance leads to tumor elimination, which involves the recognition of tumor-specific or tumor-associated antigens (TAA) by antibodies and immune cells. T cells can recognize tumor antigens presented by the MHC class I and II molecules and kill the tumor cells via lytic granule release (CD8+ T cells) or promote cellular and humoral responses through the production of cytokines (CD4+ T cells). TAA-specific antibodies can bind to and lyse tumor cells with the help of complement or facilitate the killing of tumor cells by T and natural killer (NK) cells through antibody-dependent cell-mediated cytotoxicity (ADCC; refs. 3, 4). Molecular and biochemical characterization of tumor antigens have yielded targets for immunosurveillance and for the development of immunotherapeutic strategies.

Patients with cancer have circulating tumor-specific antibodies and T cells that have been used as reagents to characterize the individual's tumor antigens (5–8). Immune responses to several TAAs have been correlated with favorable prognoses. Even when target antigens are not known, infiltration of tumors by activated T cells has been correlated with better prognosis and longer disease-free and overall survival (9). The promising new approaches in cancer treatment include immunotherapies that are directed toward regaining immune control by targeting both the cancer and the immune system (10).

DNA sequencing has shown that tumors have, on average, a dozen or more mutations that could generate new epitopes known as tumor-specific antigens (11). Although tumors could express these epitopes as targets and adoptive transfer of T cells or antibodies could lead to their recognition and elimination, spontaneous immune responses to such epitopes (e.g., mutated Kras, EGFR, or p53) have not been found in patients with cancer as often as could be expected from the frequency of these mutations (12). Instead, the majority of the spontaneous antitumor immune responses are directed against the nonmutated self-antigens, which are expressed on tumor cells and are named TAAs. They include molecules that are overexpressed on tumor cells [e.g., Her-2neu (13), MUC1 (14), CEA (15), Cyclin B1 (5)], molecules with dysregulated stage- or tissue-specific expression [e.g., oncofetal antigens α-fetoprotein (16), cancer-testis antigens NY-ESO-1 (17), Mage 1-7 (18)], or molecules with altered posttranslational modifications [glycosylation or phosphorylation; e.g., hypoglycosylation of MUC1 (14) or aberrant phosphorylation of β-catenin (19)]. The aberrant expression of many of these antigens can be detected on premalignant precursors of various cancers early in tumor development (6, 20).

Results from several large epidemiologic studies have indicated that individuals with a history of febrile childhood infections had a reduced lifetime risk of various cancers (21–23). The mechanisms underlying this protective function are unknown. Healthy individuals with no previous history of cancer have been shown to have antibodies and/or T cells specific for several TAAs (24, 25). For example, we found that the TAA MUC1 was expressed in the tumor form (overexpressed and hypoglycosylated) on salivary gland ducts during mumps parotitis infection (26), on breast ducts during lactation and in lactational mastitis (27), and in inflammatory bowel disease (20). Furthermore, we showed that the presence of anti-MUC1 immunoglobulin G (IgG) in women, who experienced early in life one or more of these events, correlated with a significantly lower risk for ovarian cancer (28).

In this study, we present the first attempt to recapitulate these observations in an animal model. This experimental mouse model of influenza infection allows us to test the hypothesis that immunity and immune memory against abnormal self-antigens, known as TAAs, is not elicited in response to their de novo expression on tumor cells or premalignant lesions, but rather it is elicited earlier in life in response to their expression during acute inflammations accompanying viral and other infections. When some of the same self-antigens are aberrantly expressed on premalignant lesions or tumor cells, they can be recognized by the infection-primed immune memory responses, leading to tumor elimination or enhanced tumor control. We show that mice, which experienced two infections with two different influenza viruses, and which develop immunity to self-antigens abnormally expressed on infected lungs, have improved ability to control the growth of transplantable lung tumors expressing those same self-antigens. We analyzed in detail the infection-elicited immune responses to five such antigens: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H4, malate dehydrogenase 2 (MDH2), annexin A2, and HSP90. These antigens were all recognized in tumor cell lysates by postinfection sera. We show that they were overexpressed in tumor cells, as well as in influenza virus–infected lungs compared with healthy lungs, and that influenza virus infection induced antibody and CD8+ T cells specific for these antigens. We demonstrate that immunization of mice with peptides derived from these antigens effectively protects them against tumor challenge.

Mice, tumor cell lines, and influenza virus

Six- to 8-week-old female C57BL/6 wild-type (WT) mice were purchased from The Jackson Laboratory and maintained in the University of Pittsburgh animal facility. All animal protocols were in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines at the University of Pittsburgh. Lewis Lung Carcinoma cell line (3LL) derived from a murine lung epithelial tumor, was maintained in complete Dulbecco's Modified Eagle Medium (c-DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 1% nonessential amino acid, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% l-glutamine, and 0.1% 2-mercaptoethanol. IG10, an epithelial tumor cell line derived from mouse ovarian epithelium, was cultured as described in ref. (29).

Influenza virus infection and tumor challenge

All mice were anesthetized with ketamine (100 mg/mL)/xylazine (20 mg/mL) solution. Mice were infected intranasally with 1.25 × 103 pfu of H1N1 influenza A/Puerto Rico/8/34 (PR8) virus and re-infected 35 days later with 1.25 × 103 pfu of H3N2 influenza A/Aichi/2/68 (Aichi) X-31 virus. Percentage weight loss was used as a measure of successful infection, and mice were weighed at 2-day intervals. On day 60 following the first infection, mice were injected subcutaneously in the right hind flank with 1 × 105 3LL tumor cells. Tumor length and width were measured every 2 days using calipers. Mice were sacrificed when the tumor diameter reached 20 mm, or the tumors became severely ulcerated, or otherwise advised by the University of Pittsburgh animal facility.

Staining of tumor cells with pre- and postinfection sera

Four days before primary influenza infection, mice were bled to obtain their preinfection sera antibody repertoire. Ten days following the second infection, mice were bled to obtain postinfection sera antibodies. Before staining, both sets of sera were diluted 1:62.5 in PBS. Then 2 × 105 3LL and IG10 tumor cells were plated in a 96-well plate and stained on ice for 1 hour with 100 μL of the pre- or postinfection sera. Cells were then stained on ice for 30 minutes with fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse IgG2a (BD Bioscience) as the secondary antibody. Cells were fixed in 1.6% paraformaldehyde and samples were run on a LSR II flow cytometer.

Affinity purification of 3LL antigens

Total cell lysates were generated from 50 × 106 3LL cells in 300 μL NP-40 lysis buffer [0.5% NP40, 0.5% Mega 9 (octylglucoside), 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L Tris pH 7.5, 2 mmol/L PMSF, 5 mmol/L iodoacetamide, and protease inhibitor (Roche)]. Lysates were precleared with the addition of Protein G Sepharose beads (Sigma-Aldrich, Inc) and the mixture incubated for 1 hour at 4°C on an orbital shaker. Protein G beads were removed by centrifugation at 1,200 rpm before affinity purification. Protein G HP SpinTrap Columns and Buffer Kits (GE Healthcare) were used according to the manufacturer's protocol with the following modifications. Preinfection and postinfection sera were pooled separately from mice (n = 6) and each set of sera was poured over multiple protein G columns (100 μL/column). Columns were washed with the wash buffer and 50 mmol/L dimethyl pimelimidate dihydrochloride (DMP) was added to covalently cross-link the bound antibodies from each set of sera to the protein G columns, as described in the manufacturer's protocol. This was done to ensure that only the bound protein fractions were eluted from the columns and not the antibodies. Then 400 μL of 3LL tumor lysate was added to both the pre- and postinfection sera columns and incubated overnight at 4°C on an orbital shaker. The following day, the columns were washed with TBS (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.5) and the bound proteins were eluted off the columns with 0.1 mol/L glycine provided from the kit with 2 mol/L urea, pH 2.9. Pooled proteins from the preinfection and postinfection antibody columns were concentrated and the elution buffers were changed to 2D-gel buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 10 mmol/L dithiothreitol (DTT), 10 mmol/L HEPES, pH 8.0) using 5,000 Molecular Weight Cut Off Vivaspin columns (Sartorius Stedim Biotech).

2D-difference gel electrophoresis and liquid chromatography/mass spectrometry analysis

The immunoprecipitated proteins were subjected to difference gel electrophoresis (DIGE; ref. 30) to identify proteins largely or uniquely precipitated by postinfection sera. Protein labeling, isoelectric point focusing, and second dimension SDS-PAGE were conducted as described by Minden (31) with the following modifications. Of note, 2.5 μg of preinfection proteins and postinfection proteins were reduced in 10 mmol/L Tris(2-carboxyethyl)phosphine (TCEP; Sigma) for 60 minutes in the dark at 37°C. Briefly, 10 mmol/L CyDye DIGE Fluor Cy3 or Cy5-maleimide saturation dyes (GE Healthcare) diluted in dimethylformamide (Sigma), which label all available TCEP reduced cysteines on all proteins, were added to each sample for 30 minutes at 37°C. Labeling was quenched with 7 mol/L DTT. Samples were then combined and immobilized pH gradient (IPG) buffer (GE Healthcare) was added at 1 μL/40 μL of sample. Labeling of the two samples was reversed (reciprocal labeling) and run concurrently on a second gel to eliminate dye-dependent differences. Proteins were separated in the first dimension on 13-cm pH3-10NL IPG strips on an IPGphor apparatus (GE Healthcare) for 35,000 Volt-hours. The samples were then separated on the second dimension SDS-PAGE in precast 10% to 20% gradient polyacrylamide gels encased in low fluorescent glass (www.precastgels.com) in standard Tris–glycine–SDS running buffer. Fluorescent images of reciprocal gels were taken as described in ref. (31). The Bioinformatics Analysis Core of the University of Pittsburgh Genomics and Proteomics core laboratories analyzed the resultant fluorescent images and selected spots that were then cut from the gels and identified via nano-liquid-chromatography-electrospray ion/tandem mass spectrometry (LC-ESI-MS/MS), as described in ref. (32).

Western blot and densitometry analyses

Lung tissues were homogenized with a 2-mL Dounce homogenizer and total lysates were obtained in NP-40 lysis buffer. The same procedure was applied to generate total cell lysates from 3LL and IG-10 tumor cells. Before Western blotting, protein concentrations were determined via Bradford assay; 50 μg of protein from various groups was separated on 10% TGX precast gels (Bio-Rad) and immunoblotted onto polyvinylidene difluoride (PVDF) membranes. The following antibodies were used to probe for their respective proteins on separate blots: anti-HSP 90α/β (1:100; Santa Cruz Biotechnology), anti-annexin II (1:100; Santa Cruz Biotechnology), anti-histone H4 (1:1,000; Abcam), anti-GAPDH (1:1,000; Abcam), anti-MDH2 (1:100; Abcam), anti-actin (1:15,000; Sigma-Aldrich, Inc), goat anti-mouse horseradish peroxidase (HRP; 1:5,000; Santa Cruz Biotechnology), and goat anti-rabbit HRP (1:5,000; Santa Cruz Biotechnology). All Western blots were scanned on Kodak Image Station 4000 MM and band densitometry analysis was performed on all blots using ImageJ (NIH). All bands were normalized according to their actin control. Once normalized, all experimental bands and lanes were compared with a normal uninfected mouse lung.

ELISA

To examine the differences in antibody recognition between pre- and postinfection sera, 15 μg/mL of one of the following proteins was coated on Immulon 4HBX ELISA plates (Thermo Scientific) in duplicate wells: MDH2 (Novus Biologicals), GAPDH (Abcam), histone H4 (New England Biolabs), HSP90α (Abcam), annexin A2 (Novoprotein). Human proteins were used because of their high conservation between mouse and human. Duplicate wells that were not coated with antigen served as controls for nonspecific binding. Plates were then placed on an orbital shaker overnight at 4°C. The next day pre- and postinfection sera were diluted (1:62.5) in PBS, added to ELISA plates, and placed on an orbital shaker for 2 hours at room temperature. Plates were washed and 0.3% hydrogen peroxide was added to the wells to block background peroxidase activity. Plates were washed and rat anti-mouse IgG-HRP (1:500) was added. Plates were again washed and TMB substrate was added for 15 minutes and 2N sulfuric acid was added to stop the developing signal. ELISA plates were then read at 450 nm on a Gen 5 plate reader. Data were represented using the average of duplicate antigen-coated wells after subtracting the value from the no-antigen control wells.

Peptide identification and MHC-I binding assays

Candidate peptide sequences were identified using the Immune Epitope Database (IEDB) MHC-I binding predictor program with a percentile rank of 5 or less (33). MDH251–260 (MAYAGARFVF), GAPDH300–310 (ALNDNFVKLIS), annexin A2184–191 (SVIDYELI), H487–95 (VVYALKRQG), histone H4 with an amino acid substitution (H4-sub VVYAFKRQG) peptides were synthesized by the Peptide Synthesis Core of the University of Pittsburgh Genomics and Proteomics core laboratories as described in ref. (34). MHC-I binding was verified performing RMA-S, a TAP-deficient cell line, MHC-Class I stabilization assays. In short, 2 × 105 RMA-S cells were plated in a 96-well plate. The cells were cultured overnight at 29°C. Unloaded RMA-S cells served as controls. Each peptide candidate was added in triplicates to the plate from 10−4 mol/L to 10−9 mol/L for 2 hours and 30 minutes in a 29°C incubator. Cells were placed in a 37°C incubator for 1 hour and 30 minutes. RMA-S cells were fixed in 1.6% paraformaldehyde and then stained with anti-H2-Kb or anti-H2-Db (BD Bioscience). Samples were run on the LSR II flow cytometer (BD Bioscience) and analyzed using FACSDiva software (Supplementary Fig. S1).

Antigen-specific T-cell detection

Animals were sacrificed 6 days following the second influenza infection. Lungs and spleens were harvested and cells were isolated as described in ref. (35). Then, 1 × 106 cells from each set of tissues were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies (BD Bioscience). Dimer-X soluble dimeric mouse H-2Kb:Ig fusion protein and H-2Db:Ig fusion protein (BD Bioscience) were used according to the manufacturer's protocol to detect MDH251–260, GAPDH300–310, annexin A2184-191, and H487–95 peptide-specific CD8+ T cells. In addition, the presence of influenza-specific T cells was evaluated using peptides PA224–233 and NP147–155 purchased from GenScript USA. Of note, 100,000 events were collected and samples were run on the LSR II flow cytometer (BD Bioscience), gated (example in Supplementary Fig. S2), and analyzed using FACSDiva software.

Vaccination and tumor challenge

D1 dendritic cells are an established growth factor–dependent immature dendritic cell line. D1 dendritic cells were grown and maintained as described and used in all vaccinations (36). A total of 1.25 × 106 D1 dendritic cells per mouse were cultured in 6-well plates, loaded separately with 100 μg of MDH251–260, GAPDH300–310, annexin A2184–191, or H487–95, and matured with 12.5 μg/mL of Poly IC:LC adjuvant. Briefly, 0.25 × 106 D1 dendritic cells loaded with individual peptides were pooled together for a total number of 1 × 106 D1 dendritic cells. An additional 50 μg of each soluble peptide per mouse was added to the mixture and injected into mice in the right hind flank. Unloaded, Poly IC:LC matured D1 dendritic cells were injected into control mice. Animals were vaccinated at weeks 0, 2, and 6, and challenged with 1 × 105 3LL cells in the right hind flank, 2 days following the week 6 vaccination. Tumor length and width were measured at 2-day intervals using calipers.

Statistical analysis

Statistical analysis was performed using GraphPad Prism v6.0 software (GraphPad Inc.). Results were represented as means ± SEM. Statistical means and significance were analyzed using unpaired two-tailed Student t test. Kaplan–Meier survival curves were analyzed with the log rank test. Significance for all experiments was defined as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Influenza virus infection induces antibodies to multiple host cell antigens, some of which are known TAAs

Mice were infected with influenza virus PR8 and then 35 days later with the second influenza strain Aichi, as described in Materials and Methods. Mice experienced the general signs of malaise and lost close to 20% of their starting body weight in the first week following each infection. Seven to 8 days after infection, mice began to recover, and by days 16 to 18, their weight returned to baseline (Fig. 1A). At day 25 after the second infection, animals were injected subcutaneously with 3LL tumor cells. Tumors became palpable 8 days after injection (Fig. 1B). At day 14, tumor growth kinetics between the influenza-experienced animals and the naïve mock-infected group began to diverge. The growth of tumors in the influenza-experienced animals was slower from days 14 to 22. On day 16, the average tumor size in the influenza-experienced mice was 20.67 mm2 versus 51.63 mm2 in the control group. The size difference was still significant at day 18, when the average tumor size in the influenza-experienced group was 31.78 mm2 compared with 73.25 mm2 in the control mice.

Figure 1.

Influenza virus infection delays tumor growth at early time points. A, animals were intranasally infected with PR8 and Aichi influenza viruses on day 0 and day 35, respectively. Mice were weighed every 2 days. B, influenza-experienced animals and naïve animals were challenged subcutaneously with 1 × 105 3LL tumor cells in the right hind flank. Tumor length and width were measured every 2 days using calipers. Data are representative of two experiments with at least 8 mice per group and are expressed as means ± SEM. C, sera from animals (n = 7) 4 days before PR8 infection (preinfection) and 10 days after Aichi infection (postinfection) were diluted 1:62.5 and used to stain 3LL and IG10 tumor cells. Cells were subsequently stained with FITC-conjugated goat anti-mouse IgG2a antibody and analyzed on the LSR II flow cytometer. Results are shown as MFI. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 1.

Influenza virus infection delays tumor growth at early time points. A, animals were intranasally infected with PR8 and Aichi influenza viruses on day 0 and day 35, respectively. Mice were weighed every 2 days. B, influenza-experienced animals and naïve animals were challenged subcutaneously with 1 × 105 3LL tumor cells in the right hind flank. Tumor length and width were measured every 2 days using calipers. Data are representative of two experiments with at least 8 mice per group and are expressed as means ± SEM. C, sera from animals (n = 7) 4 days before PR8 infection (preinfection) and 10 days after Aichi infection (postinfection) were diluted 1:62.5 and used to stain 3LL and IG10 tumor cells. Cells were subsequently stained with FITC-conjugated goat anti-mouse IgG2a antibody and analyzed on the LSR II flow cytometer. Results are shown as MFI. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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We examined the postinfection sera for the ability to stain 3LL tumor cells, which would suggest that flu infections elicited antibodies against tumor cell surface proteins. In the experiment illustrated in Fig. 1C, we obtained sera from mice (n = 7) pre- and postinfluenza infection. Tumor cell surface staining was performed with individual sera and the results were pooled into pre- and postinfection groups and represented as an average value. Average mean fluorescence intensity (MFI) of staining of 3LL tumor cells with postinfection sera was significantly higher than that with preinfection sera. The same result was obtained after staining another mouse epithelial tumor cell line IG10 with the same sera.

To identify molecules specifically recognized by postinfection sera, pre- and postinfection antibodies were bound to Protein G columns for affinity purification of proteins from 3LL tumor cell lysates. Tumor proteins bound to the antibody columns were eluted, labeled with two different cyanine-based saturation dyes, and resolved by 2D-DIGE as described in Materials and Methods. In a concurrently run reciprocal gel, labeling was reversed such that the sample previously labeled with Cy3 was labeled with Cy5 and vice versa. Gel images were false colored for analysis: green for Cy3 and red for Cy5. Overlays were then created using Delta2D software, in which proteins unique to one sample appeared either green or red, whereas proteins common to both samples appeared yellow (green and red combined). Figure 2 shows the gel used to identify antigens described below. Green dots mark proteins eluted from the preinfection antibody columns, whereas red dots mark proteins eluted from the postinfection antibody columns. Spots that were most remarkable (yellow circles) were cut out and subjected to mass spectrometry analysis and protein sequencing. Many proteins with a wide variety of functions were identified. They included voltage-dependent ion channels, proteasome subunits, mitochondrial and cytosolic enzymes, HSPs, and structural proteins (data not shown). We selected five identified proteins: histone H4, MDH2, annexin A2, GAPDH, and HSP90 for further study (Table 1). Several reports based on methods and approaches unrelated to ours had already identified these molecules as TAA in tumor-bearing mice or in patients with cancer (37–41).

Figure 2.

Influenza infection induces antibodies against specific proteins of tumor cells. 3LL tumor lysates were affinity purified on protein G columns to which postinfection and preinfection antibodies were covalently bound and the eluted proteins were compared by 2D-DIGE gels. Green spots mark proteins eluted from the preinfection antibody columns, and red spots mark proteins from the postinfection antibody columns. Yellow color marks overlapping spots. Yellow circles indicate spots picked for sequencing. Spots were chosen according to image analysis provided by the University of Pittsburgh Bioinformatics Analysis Core (BAC) of the Genomics and Proteomics core laboratories.

Figure 2.

Influenza infection induces antibodies against specific proteins of tumor cells. 3LL tumor lysates were affinity purified on protein G columns to which postinfection and preinfection antibodies were covalently bound and the eluted proteins were compared by 2D-DIGE gels. Green spots mark proteins eluted from the preinfection antibody columns, and red spots mark proteins from the postinfection antibody columns. Yellow color marks overlapping spots. Yellow circles indicate spots picked for sequencing. Spots were chosen according to image analysis provided by the University of Pittsburgh Bioinformatics Analysis Core (BAC) of the Genomics and Proteomics core laboratories.

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Table 1.

Biochemical characterization of selected tumor antigens detected by postinfection antibodies

Spot numberAccession numberaProtein namebPeptide sequencecX-corrdΔ ScoreeChargefM/Z (Da)gMH+h (Da)ΔMi (ppm)MWj (kDa)pIk% Sequence coveragelIons matchedm
IPI00885367.1 Annexin A2 TNQELQEINR 2.74 0.36 622.81384 1244.62041 −2.08 19.6 5.96 10.80 14/18 
   QDIAFAYQR 2.55 1.00 556.28143 1111.55559 2.22    14/16 
IPI00468203.3 Annexin A2 TPAQYDASELK 3.52 0.65 611.80090 1222.59453 −0.43 38.7 7.69 22.71 15/20 
   AKDGSVIDYELIDQDAR 3.46 0.68 954.94586 1908.88445 1.16    17/32 
   SLYYYIQQDTK 3.30 0.47 711.35215 1421.69704 1.62    16/20 
   TNQELQEINR 3.08 0.47 622.81384 1244.62041 −2.08    15/18 
   WISIMTER 2.67 1.00 518.26892 1035.53057 1.29    13/14 
   DIISDTSGDFR 2.53 1.00 613.28820 1225.56914 −0.36    16/20 
   QDIAFAYQR 2.41 0.60 556.28027 1111.55327 0.13    15/16 
IPI00407339.7 Histone H4 ISGLIYEETR 3.39 0.68 590.81536 1180.62346 2.19 11.4 11.36 38.83 16/18 
   TVTAMDWYALK 3.22 0.74 655.85559 1310.70391 1.06    16/22 
   TVTAmDVVYALK 2.88 0.42 663.85327 1326.69927 1.39    14/22 
   DAVTYTEHAK 2.61 0.52 567.77410 1134.54094 −1.47    15/18 
   VFLENVIR 2.56 0.36 495.29345 989.57964 1.76    12/14 
IPI00622795.2 GAPDH LENPAKYDDIK 2.36 8.03 653.33881 1305.67035 0.20 35.8 8.03 3.30 10/20 
IPI00323592.2 MDH2 IFGVTTLDIVR 2.82 0.41 617.36487 1233.72246 0.41 35.6 8.68 3.25 14/20 
              
IPI00885367.1 Annexin A2 TNQELQEINR 3.06 0.47 622.81384 1244.62041 −2.08 19.6 5.96 5.68 14/18 
Spot numberAccession numberaProtein namebPeptide sequencecX-corrdΔ ScoreeChargefM/Z (Da)gMH+h (Da)ΔMi (ppm)MWj (kDa)pIk% Sequence coveragelIons matchedm
IPI00885367.1 Annexin A2 TNQELQEINR 2.74 0.36 622.81384 1244.62041 −2.08 19.6 5.96 10.80 14/18 
   QDIAFAYQR 2.55 1.00 556.28143 1111.55559 2.22    14/16 
IPI00468203.3 Annexin A2 TPAQYDASELK 3.52 0.65 611.80090 1222.59453 −0.43 38.7 7.69 22.71 15/20 
   AKDGSVIDYELIDQDAR 3.46 0.68 954.94586 1908.88445 1.16    17/32 
   SLYYYIQQDTK 3.30 0.47 711.35215 1421.69704 1.62    16/20 
   TNQELQEINR 3.08 0.47 622.81384 1244.62041 −2.08    15/18 
   WISIMTER 2.67 1.00 518.26892 1035.53057 1.29    13/14 
   DIISDTSGDFR 2.53 1.00 613.28820 1225.56914 −0.36    16/20 
   QDIAFAYQR 2.41 0.60 556.28027 1111.55327 0.13    15/16 
IPI00407339.7 Histone H4 ISGLIYEETR 3.39 0.68 590.81536 1180.62346 2.19 11.4 11.36 38.83 16/18 
   TVTAMDWYALK 3.22 0.74 655.85559 1310.70391 1.06    16/22 
   TVTAmDVVYALK 2.88 0.42 663.85327 1326.69927 1.39    14/22 
   DAVTYTEHAK 2.61 0.52 567.77410 1134.54094 −1.47    15/18 
   VFLENVIR 2.56 0.36 495.29345 989.57964 1.76    12/14 
IPI00622795.2 GAPDH LENPAKYDDIK 2.36 8.03 653.33881 1305.67035 0.20 35.8 8.03 3.30 10/20 
IPI00323592.2 MDH2 IFGVTTLDIVR 2.82 0.41 617.36487 1233.72246 0.41 35.6 8.68 3.25 14/20 
              
IPI00885367.1 Annexin A2 TNQELQEINR 3.06 0.47 622.81384 1244.62041 −2.08 19.6 5.96 5.68 14/18 

aProtein Accessions in the IPI mouse database.

bProtein name.

cPrimary amino acid sequence for identified peptides.

dThe cross-correlation of the experimental and theoretical spectra from SEQUEST search.

eA measure of the difference between the top two scores for the peptides identified by that spectrum.

fCharge stage of precursor ion.

gObserved mass-to-charge ratio of precursor ion.

hMonoisotopic mass for protonated molecular ion MH calculated on the basis of observed m/z.

iDeviation from expected MH.

jMolecular weight.

kIsoelectric point.

lPercentage of the database protein sequence covered by matching peptides.

mThe number of fragment ions matched out of the total number of fragment masses for the peptide.

Overexpression of GAPDH, histone H4, MDH2, annexin A2, and HSP90 in tumor cells and influenza virus–infected lungs leads to specific immunity

We hypothesized that postinfection antibodies against these proteins were elicited because of differences in their expression in infected versus normal lungs, analogous to their abnormal expression in tumors. Western blot analysis showed that these proteins were constitutively overexpressed in both epithelial tumor cell lines, 3LL and IG10, and also at various time points in the influenza-infected lungs (2–7.5-fold higher compared with healthy lungs; Fig. 3). Expression of GAPDH in influenza-infected lungs appeared to be the highest at day 3 postinfection. Histone H4 protein levels were constitutively elevated in tumor cells and at all time points after influenza infection by 5 to 7-fold higher than in normal lung. MDH2 levels were elevated 12 hours postinfection and decreased to normal levels at day 3 postinfection. Annexin A2 remained 3-fold higher than in normal lung at all time points. HSP90 protein level was the highest at day 2 postinfection.

Figure 3.

Histone H4, MDH2, GAPDH, and HSP90 are elevated in epithelial tumor cells lines and influenza-infected lungs. Whole cell lysates were generated from 3LL and IG10 cell lines and from normal and influenza-infected lungs; 50 μg of protein was loaded on each gel, resolved by electrophoresis, and transferred onto PVDF membranes. Membranes were blotted using antibodies against GAPDH, histone H4, MDH2, annexin A2, and HSP90. Actin loading controls were used to normalize each band. Densitometry analysis was performed using ImageJ. All lanes were compared with normal lung. Data are representative of two experiments.

Figure 3.

Histone H4, MDH2, GAPDH, and HSP90 are elevated in epithelial tumor cells lines and influenza-infected lungs. Whole cell lysates were generated from 3LL and IG10 cell lines and from normal and influenza-infected lungs; 50 μg of protein was loaded on each gel, resolved by electrophoresis, and transferred onto PVDF membranes. Membranes were blotted using antibodies against GAPDH, histone H4, MDH2, annexin A2, and HSP90. Actin loading controls were used to normalize each band. Densitometry analysis was performed using ImageJ. All lanes were compared with normal lung. Data are representative of two experiments.

Close modal

Even though these antigens were identified by affinity purification on postinfection sera bound to Protein G columns, we wanted to confirm in another assay the specificity of postinfection antibodies for each of these individual molecules. Figure 4 shows that in most mice there was an increase after infection in IgG specific for GAPDH, histone H4, MDH2, annexin A2, and HSP90α, as determined by ELISA.

Figure 4.

Antibodies specific for GAPDH, histone H4, MDH2, annexin A2, and HSP90α increase, following influenza infection. Preinfection sera (4 days before the first infection) and postinfection sera (10 days before the second infection) were assayed on ELISA plates in duplicate wells coated with individual proteins. Uncoated wells served as nonspecific binding controls and their values were subtracted from the values in matching antigen-coated wells. Results are represented as mean optical density (OD) ± SEM of two experimental repeats with n = 7.

Figure 4.

Antibodies specific for GAPDH, histone H4, MDH2, annexin A2, and HSP90α increase, following influenza infection. Preinfection sera (4 days before the first infection) and postinfection sera (10 days before the second infection) were assayed on ELISA plates in duplicate wells coated with individual proteins. Uncoated wells served as nonspecific binding controls and their values were subtracted from the values in matching antigen-coated wells. Results are represented as mean optical density (OD) ± SEM of two experimental repeats with n = 7.

Close modal

Influenza virus infection also induced an increase in CD8+ T cells specific for all of these antigens. Spleens and lungs were harvested from mice 6 days after the second influenza infection and from uninfected control mice. Peptides GAPDH300–310 (ALNDNFVKLIS), annexin A2184–191 (SVIDYELI), MDH251–260 (MAYAGARFVF), histone H487-95 (VVYALKRQG), and histone H4 with an amino acid substitution (H4-sub VVYAFKRQG; ref. 38), were selected from IEDB and confirmed to bind to MHC-I in RMA-S stabilization assays (Supplementary Fig. S1). Each peptide was loaded onto DimerX H-2Kb or H-2Db molecules and used to detect specific CD3+CD8+ T cells. There were no CD8 T cells specific for these peptides in the lungs and spleens of uninfected control mice but in influenza-experienced animals they were present in similar numbers to the influenza-specific T cells (Fig. 5). The highest numbers both in the lungs and in the spleens were H2-Kb-restricted GAPDH-specific and H2-Db-restricted MDH2-specific T-cells.

Figure 5.

The amount of antigen-specific CD8+ T cells increases in the lungs and spleens following influenza infection. Mice were infected with PR8 and Aichi influenza viruses or mock PBS infected on day 0 and day 35, respectively. Six days after the second infection, spleens and lungs were harvested and analyzed by flow cytometry for the presence of T cells staining with the Dimer-X reagent (as described in Materials and Methods). Data are representative of two experimental repeats of least n = 3 animals per group and are expressed as means ± SEM.

Figure 5.

The amount of antigen-specific CD8+ T cells increases in the lungs and spleens following influenza infection. Mice were infected with PR8 and Aichi influenza viruses or mock PBS infected on day 0 and day 35, respectively. Six days after the second infection, spleens and lungs were harvested and analyzed by flow cytometry for the presence of T cells staining with the Dimer-X reagent (as described in Materials and Methods). Data are representative of two experimental repeats of least n = 3 animals per group and are expressed as means ± SEM.

Close modal

Vaccination with dendritic cells loaded with the new TAA peptides delays tumor growth and promotes survival

We loaded the D1 dendritic cells with MDH2251–260, GAPDH300–310, annexin A2184–191, and H487–95 and vaccinated mice as described in Materials and Methods. Control mice were vaccinated with unloaded D1 cells. Two days following the second boost, mice were challenged subcutaneously with 3LL tumor cells. In vaccinated animals, tumor growth began to slow down significantly by day 12 (Fig. 6A), resulting in all vaccinated animals still surviving at day 40, compared with only 2 animals surviving in the unloaded dendritic cell–vaccinated controls (Fig. 6B). On day 12, the average tumor size in peptide-loaded dendritic cell–vaccinated animals was 10.67 mm2 compared with 30.67 mm2 for the controls.

Figure 6.

Vaccination with GAPDH, histone H4, MDH2, and annexin A2 peptides loaded on dendritic cells (DC) confers early protection and prolonged survival against 3LL tumor challenge. A, mice were vaccinated in the right hind flank with 1 × 106 D1 cells per mouse (a pool of 2.5 × 105 D1 dendritic cells per peptide) at weeks 0, 2, and 6. Control mice received the same number of unloaded D1 dendritic cells at the same time points. Two days after the week 6 boost, animals were challenged subcutaneously with 1 × 105 3LL tumor cells in the right hind flank. Tumor growth was measured with calipers at 2-day intervals and expressed as length × width (mm2). B, survival post tumor challenge. Data are representative of two experiments with at least n = 8 mice per group and are expressed as means ± SEM.

Figure 6.

Vaccination with GAPDH, histone H4, MDH2, and annexin A2 peptides loaded on dendritic cells (DC) confers early protection and prolonged survival against 3LL tumor challenge. A, mice were vaccinated in the right hind flank with 1 × 106 D1 cells per mouse (a pool of 2.5 × 105 D1 dendritic cells per peptide) at weeks 0, 2, and 6. Control mice received the same number of unloaded D1 dendritic cells at the same time points. Two days after the week 6 boost, animals were challenged subcutaneously with 1 × 105 3LL tumor cells in the right hind flank. Tumor growth was measured with calipers at 2-day intervals and expressed as length × width (mm2). B, survival post tumor challenge. Data are representative of two experiments with at least n = 8 mice per group and are expressed as means ± SEM.

Close modal

The data we present here add a new dimension to our understanding of the process of cancer immunosurveillance and its targets. We show that immune responses against abnormally expressed self-antigens, many of which have been characterized as TAAs, are generated during nonmalignant infectious inflammatory events that occur much earlier in life than malignancies. We propose that immune memory for these antigens is later recruited for cancer immunosurveillance.

We used a mouse model to demonstrate that two bouts with influenza virus infection led to the ability of the host's immune system to slow down tumor growth. This effect was small and transient, which may be all that could be expected from a limited exposure of the mice to this one type of infection and to no other pathogens before influenza infection as the mice are kept in pathogen-free conditions. The significance of this small delay in tumor growth was confirmed by the induction of antibodies against multiple molecules in the tumor cell lysate. Focusing on five antigens identified by infection-elicited antibodies, GAPDH, histone H4, MDH2, annexin A2, and HSP90, we showed that these antigens were abnormally expressed (overexpressed) in influenza-infected lungs and in mouse epithelial tumor cell lines, and that in addition to IgG, the influenza infection induced antigen-specific CD8 T cells against these molecules. As predicted by our hypothesis, vaccination with peptides derived from GAPDH, histone H4, MDH2, and annexin A2 led to a much more profound slowing down of tumor growth compared with that elicited by the virus infection, and to a prolonged survival of tumor-bearing animals.

Other viral infections may be capable of inducing TAA-specific antibodies and T cells. Vaccinia virus– and lymphocytic choriomeningitis virus–infected mice were reported to have developed antibodies against many host cell antigens, some of which are orthologs of human TAAs (42). Human fibroblasts infected with varicella-zoster virus (VZV) or human cytomegalovirus (CMV) overexpress cyclin B1 in the cytoplasm in a similar fashion to tumor cells where cyclin B1 was identified as a TAA (43, 44). Cyclin B1 has been found in VZV virions (45). Many healthy individuals, presumably having experienced these infections, have cyclin B1–specific IgG and memory T cells (24). It has been reported that GAPDH and annexin A2 are found in influenza virions produced by infected epithelial cell lines Vero and A549 (46). HSP90, Annexin A2, and GAPDH were also found within human CMV particles (47). A study examining the measles virus effect on presentation of self-peptides on MHC class I during infection showed that two abundant self-peptides on HLA-A*0201 measles-infected cells could induce auto-reactive CD8+ T cells. One of the peptides identified was HSP90β570–578 (ILDKKVEKV; ref. 48). The HSP90β570–578 peptide has been found in melanoma cell lines (49). It is possible that these “auto-reactive” T cells contribute to increased tumor immune surveillance. None of these observations were followed by experiments to test the potential antitumor effects of either the viral infections or the immune responses against the identified molecules, with the exception of cyclin B1 that we showed was a target of antitumor immune responses (24).

The same protective effect of influenza virus–primed immunity specific for abnormally expressed self-antigens that we showed here could be a collateral benefit of other viral, bacterial, and parasitic infection or various acute inflammatory conditions of unknown etiologies. Therefore, we propose that the molecules abnormally expressed in these different disease states and also in cancer cells, which are currently referred to as TAAs, should be renamed as disease-associated antigens (DAA; ref. 50). Preexisting immune responses to several known tumor antigens that are candidate DAAs have been reported to increase the odds of successfully eliminating spontaneously arising tumors (28). The arrival of memory DAA-specific T cells to the site of the tumor as a secondary immune response could promote priming of tumor-specific responses directed against individual mutations and epitope spreading, adding to the efficacy of immunosurveillance. If DAA-specific immune memory is lacking or is weak due to limited early exposures to infections, this may lead to establishment of chronic inflammation at the tumor site due to unopposed innate immune responses, which is likely to promote tumor development. Therefore, a better understanding of the events early in life that prepare the immune system to protect an individual against known and unknown pathogens, as well as future malignancies, will help direct vaccines and other immune manipulation toward strengthening rather than impairing the establishment of life-long immunosurveillance. In addition, these findings support the use of vaccines based on DAAs/TAAs for cancer prevention.

No potential conflicts of interest were disclosed.

Conception and design: U.K. Iheagwara, T.M. Ross, O.J. Finn

Development of methodology: U.K. Iheagwara, J.S. Minden, O.J. Finn

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): U.K. Iheagwara, P.L. Beatty, P.T. Van, J.S. Minden, O.J. Finn

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): U.K. Iheagwara, P.L. Beatty, P.T. Van, T.M. Ross, J.S. Minden, O.J. Finn

Writing, review, and/or revision of the manuscript: U.K. Iheagwara, P.L. Beatty, P.T. Van, T.M. Ross, J.S. Minden, O.J. Finn

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): U.K. Iheagwara, O.J. Finn

Study supervision: U.K. Iheagwara, O.J. Finn

The authors thank the University of Pittsburgh Genomics and Proteomics core laboratories for their assistance. This work used the Biomedical Mass Spectrometry Center and UPCI Cancer Biomarker Facility that are supported in part by award P30CA047904. This work was supported by the NIH (CA056103 to O.J. Finn) and (GM083602 to T.M. Ross), the National Science Foundation (NSF-IDBR 1063236 to P.T. Van and J.S. Minden) and the UNCF/Merck Graduate Student Dissertation Fellowship (U.K. Iheagwara).

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.

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