ﬂuenza Virus Infection Elicits Protective Antibodies and T ...cancerimmunolres.aacrjournals.org/content/canimm/2/3/263.full.pdf · also been associated with a history of febrile

Research Article

Influenza Virus Infection Elicits Protective Antibodies andT Cells Specific for Host Cell Antigens Also Expressed asTumor-Associated Antigens: A New View of CancerImmunosurveillance

UzomaK. Iheagwara1, Pamela L. Beatty1, Phu T. Van3, TedM.Ross2, JonathanS.Minden3, andOlivera J. Finn1

AbstractMost tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and

become targets of antitumor immune responses. Antibodies andT cells specific for someTAAs have been found inhealthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer hasalso been associated with a history of febrile viral diseases. We hypothesized that virus infections could lead totransient expression of abnormal forms of self-molecules, some of which are TAAs; facilitated by the adjuvanteffects of infection and inflammation, thesemolecules could elicit specific antibodies, T cells, and lasting immunememory simultaneously with immunity against viral antigens. Such infection-induced immune memory for TAAwould be expected to provide life-long immune surveillance of cancer. Using influenza virus infection inmice as amodel system, we tested this hypothesis and demonstrated that influenza-experienced mice control 3LL mouselung tumor challenge better than infection-naive control mice. Using 2D-difference gel electrophoresis and massspectrometry, we identified numerous molecules, some of which are known TAAs, on the 3LL tumor cellsrecognized by antibodies elicited by two successive influenza infections. We studied in detail immune responsesagainst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H4, HSP90, malate dehydrogenase 2, andannexin A2, all of which were overexpressed in influenza-infected lungs and in tumor cells. Finally, we show thatimmune responses generated through vaccination against peptides derived from these antigens correlated withimproved tumor control. Cancer Immunol Res; 2(3); 263–73. �2013 AACR.

IntroductionGenetic mutations and epigenetic modifications can lead to

cellular transformation and cancer (1). Tumor immunosur-veillance is the mechanism by which the immune systemrecognizes and protects against abnormal cells (2). Successfultumor immunosurveillance leads to tumor elimination, whichinvolves the recognition of tumor-specific or tumor-associatedantigens (TAA) by antibodies and immune cells. T cells canrecognize tumor antigens presented by the MHC class I and IImolecules and kill the tumor cells via lytic granule release(CD8þ T cells) or promote cellular and humoral responsesthrough the production of cytokines (CD4þ T cells). TAA-

specific antibodies can bind to and lyse tumor cells with thehelp of complement or facilitate the killing of tumor cells by Tand natural killer (NK) cells through antibody-dependent cell-mediated cytotoxicity (ADCC; refs. 3, 4). Molecular and bio-chemical characterization of tumor antigens have yieldedtargets for immunosurveillance and for the development ofimmunotherapeutic strategies.

Patients with cancer have circulating tumor-specific anti-bodies and T cells that have been used as reagents to charac-terize the individual's tumor antigens (5–8). Immuneresponses to several TAAs have been correlated with favorableprognoses. Even when target antigens are not known, infiltra-tion of tumors by activated T cells has been correlated withbetter prognosis and longer disease-free and overall survival(9). The promising new approaches in cancer treatmentinclude immunotherapies that are directed toward regainingimmune control by targeting both the cancer and the immunesystem (10).

DNA sequencing has shown that tumors have, on average, adozen or more mutations that could generate new epitopesknown as tumor-specific antigens (11). Although tumors couldexpress these epitopes as targets and adoptive transfer of Tcells or antibodies could lead to their recognition and elimi-nation, spontaneous immune responses to such epitopes (e.g.,mutated Kras, EGFR, or p53) have not been found in patients

Authors' Affiliations: Departments of 1Immunology and 2Microbiologyand Molecular Genetics, University of Pittsburgh School of Medicine; and3Department of Biological Sciences, Carnegie Mellon University, Pitts-burgh, Pennsylvania

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author:Olivera J. Finn, University of Pittsburgh School ofMedicine, Department of Immunology, E1040 Biomedical Science Tower,Pittsburgh, PA 15261.Phone: 412-648-9816; Fax: 412-383-8098; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-13-0125

�2013 American Association for Cancer Research.

CancerImmunology

Research

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Published OnlineFirst December 5, 2013; DOI: 10.1158/2326-6066.CIR-13-0125

http://cancerimmunolres.aacrjournals.org/

with cancer as often as could be expected from the frequency ofthese mutations (12). Instead, the majority of the spontaneousantitumor immune responses are directed against the non-mutated self-antigens, which are expressed on tumor cells andare named TAAs. They include molecules that are overex-pressed on tumor cells [e.g., Her-2neu (13), MUC1 (14), CEA(15), Cyclin B1 (5)], molecules with dysregulated stage- ortissue-specific expression [e.g., oncofetal antigens a-fetopro-tein (16), cancer-testis antigens NY-ESO-1 (17), Mage 1-7 (18)],or molecules with altered posttranslational modifications[glycosylation or phosphorylation; e.g., hypoglycosylation ofMUC1 (14) or aberrant phosphorylation of b-catenin (19)]. Theaberrant expression of many of these antigens can be detectedon premalignant precursors of various cancers early in tumordevelopment (6, 20).

Results from several large epidemiologic studies have indi-cated that individuals with a history of febrile childhoodinfections had a reduced lifetime risk of various cancers(21–23). The mechanisms underlying this protective functionare unknown. Healthy individuals with no previous history ofcancer have been shown to have antibodies and/or T cellsspecific for several TAAs (24, 25). For example, we found thatthe TAA MUC1 was expressed in the tumor form (overex-pressed and hypoglycosylated) on salivary gland ducts duringmumps parotitis infection (26), on breast ducts during lacta-tion and in lactationalmastitis (27), and in inflammatory boweldisease (20). Furthermore, we showed that the presence of anti-MUC1 immunoglobulin G (IgG) in women, who experiencedearly in life one or more of these events, correlated with asignificantly lower risk for ovarian cancer (28).

In this study, we present the first attempt to recapitulatethese observations in an animal model. This experimentalmouse model of influenza infection allows us to test thehypothesis that immunity and immunememory against abnor-mal self-antigens, known as TAAs, is not elicited in response totheir de novo expression on tumor cells or premalignantlesions, but rather it is elicited earlier in life in response totheir expression during acute inflammations accompanyingviral and other infections.When some of the same self-antigensare aberrantly expressed on premalignant lesions or tumorcells, they can be recognized by the infection-primed immunememory responses, leading to tumor elimination or enhancedtumor control. We show that mice, which experienced twoinfections with two different influenza viruses, and whichdevelop immunity to self-antigens abnormally expressed oninfected lungs, have improved ability to control the growth oftransplantable lung tumors expressing those same self-anti-gens. We analyzed in detail the infection-elicited immuneresponses to five such antigens: glyceraldehyde-3-phosphatedehydrogenase (GAPDH), histone H4, malate dehydrogenase 2(MDH2), annexin A2, and HSP90. These antigens were allrecognized in tumor cell lysates by postinfection sera. Weshow that they were overexpressed in tumor cells, as well as ininfluenza virus–infected lungs compared with healthy lungs,and that influenza virus infection induced antibody and CD8þ

T cells specific for these antigens. We demonstrate thatimmunization of mice with peptides derived from these anti-gens effectively protects them against tumor challenge.

Materials and MethodsMice, tumor cell lines, and influenza virus

Six- to 8-week-old female C57BL/6 wild-type (WT) micewere purchased from The Jackson Laboratory and maintainedin the University of Pittsburgh animal facility. All animalprotocols were in accordance with Institutional Animal Careand Use Committee (IACUC) guidelines at the University ofPittsburgh. Lewis Lung Carcinoma cell line (3LL) derived froma murine lung epithelial tumor, was maintained in completeDulbecco's Modified EagleMedium (c-DMEM) containing 10%heat-inactivated fetal calf serum (FCS), 1% nonessential aminoacid, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% L-glutamine, and 0.1% 2-mercaptoethanol. IG10, an epithelialtumor cell line derived from mouse ovarian epithelium, wascultured as described in ref. (29).

Influenza virus infection and tumor challengeAll mice were anesthetized with ketamine (100 mg/mL)/

xylazine (20 mg/mL) solution. Mice were infected intranasallywith 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 ofH3N2 influenza A/Aichi/2/68 (Aichi) X-31 virus. Percentageweight loss was used as a measure of successful infection, andmice were weighed at 2-day intervals. On day 60 following thefirst infection, mice were injected subcutaneously in the righthind flank with 1 � 105 3LL tumor cells. Tumor length andwidth were measured every 2 days using calipers. Mice weresacrificed when the tumor diameter reached 20 mm, or thetumors became severely ulcerated, or otherwise advised by theUniversity of Pittsburgh animal facility.

Staining of tumor cells with pre- and postinfection seraFour days before primary influenza infection,micewere bled

to obtain their preinfection sera antibody repertoire. Ten daysfollowing the second infection, mice were bled to obtainpostinfection sera antibodies. Before staining, both sets ofsera were diluted 1:62.5 in PBS. Then 2 � 105 3LL and IG10tumor cells were plated in a 96-well plate and stained on ice for1 hour with 100 mL of the pre- or postinfection sera. Cells werethen stained on ice for 30 minutes with fluorescein isothiocy-anate (FITC)–conjugated rat anti-mouse IgG2a (BD Biosci-ence) as the secondary antibody. Cells were fixed in 1.6%paraformaldehyde and samples were run on a LSR II flowcytometer.

Affinity purification of 3LL antigensTotal cell lysates were generated from 50 � 106 3LL cells in

300 mL NP-40 lysis buffer [0.5% NP40, 0.5% Mega 9 (octylgluco-side), 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L Tris pH7.5, 2 mmol/L PMSF, 5 mmol/L iodoacetamide, and proteaseinhibitor (Roche)]. Lysates were precleared with the additionof Protein G Sepharose beads (Sigma-Aldrich, Inc) and themixture incubated for 1 hour at 4�C on an orbital shaker.Protein G beads were removed by centrifugation at 1,200 rpmbefore affinity purification. Protein G HP SpinTrap Columnsand Buffer Kits (GE Healthcare) were used according to themanufacturer's protocol with the following modifications.

Iheagwara et al.

Cancer Immunol Res; 2(3) March 2014 Cancer Immunology Research264




Preinfection and postinfection sera were pooled separatelyfrom mice (n ¼ 6) and each set of sera was poured overmultiple protein G columns (100 mL/column). Columnswere washed with the wash buffer and 50 mmol/L dimethylpimelimidate dihydrochloride (DMP) was added to cova-lently cross-link the bound antibodies from each set of serato the protein G columns, as described in the manufac-turer's protocol. This was done to ensure that only thebound protein fractions were eluted from the columns andnot the antibodies. Then 400 mL of 3LL tumor lysate wasadded to both the pre- and postinfection sera columnsand incubated overnight at 4�C on an orbital shaker. Thefollowing day, the columns were washed with TBS (50mmol/L Tris, 150 mmol/L NaCl, pH 7.5) and the boundproteins were eluted off the columns with 0.1 mol/L glycineprovided from the kit with 2 mol/L urea, pH 2.9. Pooledproteins from the preinfection and postinfection antibodycolumns were concentrated and the elution buffers werechanged to 2D-gel buffer (7 mol/L urea, 2 mol/L thiourea,4% CHAPS, 10 mmol/L dithiothreitol (DTT), 10 mmol/LHEPES, pH 8.0) using 5,000 Molecular Weight Cut OffVivaspin columns (Sartorius Stedim Biotech).

2D-difference gel electrophoresis and liquidchromatography/mass spectrometry analysisThe immunoprecipitated proteins were subjected to

difference gel electrophoresis (DIGE; ref. 30) to identifyproteins largely or uniquely precipitated by postinfectionsera. Protein labeling, isoelectric point focusing, and sec-ond dimension SDS-PAGE were conducted as described byMinden (31) with the following modifications. Of note, 2.5mg of preinfection proteins and postinfection proteins werereduced 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 sat-uration dyes (GE Healthcare) diluted in dimethylforma-mide (Sigma), which label all available TCEP reducedcysteines on all proteins, were added to each sample for30 minutes at 37�C. Labeling was quenched with 7 mol/LDTT. Samples were then combined and immobilized pHgradient (IPG) buffer (GE Healthcare) was added at 1 mL/40mL of sample. Labeling of the two samples was reversed(reciprocal labeling) and run concurrently on a second gelto eliminate dye-dependent differences. Proteins were sep-arated in the first dimension on 13-cm pH3-10NL IPG stripson an IPGphor apparatus (GE Healthcare) for 35,000 Volt-hours. The samples were then separated on the seconddimension SDS-PAGE in precast 10% to 20% gradientpolyacrylamide gels encased in low fluorescent glass(www.precastgels.com) in standard Tris–glycine–SDS run-ning buffer. Fluorescent images of reciprocal gels weretaken as described in ref. (31). The Bioinformatics AnalysisCore of the University of Pittsburgh Genomics and Prote-omics core laboratories analyzed the resultant fluorescentimages and selected spots that were then cut from the gelsand identified via nano-liquid-chromatography-electro-spray ion/tandem mass spectrometry (LC-ESI-MS/MS), asdescribed in ref. (32).

Western blot and densitometry analysesLung tissues were homogenized with a 2-mL Dounce

homogenizer and total lysates were obtained in NP-40 lysisbuffer. The same procedure was applied to generate total celllysates from 3LL and IG-10 tumor cells. Before Western blot-ting, protein concentrations were determined via Bradfordassay; 50 mg of protein from various groups was separated on10% TGX precast gels (Bio-Rad) and immunoblotted ontopolyvinylidene difluoride (PVDF) membranes. The followingantibodies were used to probe for their respective proteins onseparate blots: anti-HSP 90a/b (1:100; Santa Cruz Biotechnol-ogy), 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 scannedon Kodak Image Station 4000 MM and band densitometryanalysis was performed on all blots using ImageJ (NIH). Allbands were normalized according to their actin control.Once normalized, all experimental bands and lanes werecompared with a normal uninfected mouse lung.

ELISATo examine the differences in antibody recognition between

pre- and postinfection sera, 15 mg/mL of one of the followingproteins was coated on Immulon 4HBX ELISA plates (ThermoScientific) in duplicate wells: MDH2 (Novus Biologicals),GAPDH (Abcam), histone H4 (New England Biolabs), HSP90a(Abcam), annexin A2 (Novoprotein). Human proteins wereused because of their high conservation between mouse andhuman. Duplicate wells that were not coated with antigenserved as controls for nonspecific binding. Plates were thenplaced on an orbital shaker overnight at 4�C. The next daypre- and postinfection sera were diluted (1:62.5) in PBS,added to ELISA plates, and placed on an orbital shaker for 2hours at room temperature. Plates were washed and 0.3%hydrogen peroxide was added to the wells to block back-ground peroxidase activity. Plates were washed and rat anti-mouse IgG-HRP (1:500) was added. Plates were again washedand TMB substrate was added for 15 minutes and 2N sulfuricacid was added to stop the developing signal. ELISA plateswere then read at 450 nm on a Gen 5 plate reader. Data wererepresented using the average of duplicate antigen-coatedwells after subtracting the value from the no-antigen controlwells.

Peptide identification and MHC-I binding assaysCandidate peptide sequences were identified using the

Immune Epitope Database (IEDB) MHC-I binding predictorprogram with a percentile rank of 5 or less (33). MDH251–260

(MAYAGARFVF), GAPDH300–310 (ALNDNFVKLIS), annexinA2184–191 (SVIDYELI), H487–95 (VVYALKRQG), histone H4 withan amino acid substitution (H4-sub VVYAFKRQG) peptideswere synthesized by the Peptide Synthesis Core of the Uni-versity of Pittsburgh Genomics and Proteomics core labora-tories as described in ref. (34). MHC-I binding was verifiedperforming RMA-S, a TAP-deficient cell line, MHC-Class I

Viral Infections Elicit Immunity against Tumor Antigens

www.aacrjournals.org Cancer Immunol Res; 2(3) March 2014 265




stabilization assays. In short, 2 � 105 RMA-S cells were platedin a 96-well plate. The cells were cultured overnight at 29�C.Unloaded RMA-S cells served as controls. Each peptide can-didate was added in triplicates to the plate from 10�4 mol/Lto 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 30minutes. RMA-S cells were fixed in 1.6% paraformaldehydeand then stained with anti-H2-Kb or anti-H2-Db (BD Biosci-ence). Samples were run on the LSR II flow cytometer (BDBioscience) and analyzed using FACSDiva software (Supple-mentary Fig. S1).

Antigen-specific T-cell detectionAnimals were sacrificed 6 days following the second influ-

enza infection. Lungs and spleens were harvested and cellswere isolated as described in ref. (35). Then, 1� 106 cells fromeach set of tissues were stained with anti-CD3, anti-CD4, andanti-CD8 antibodies (BD Bioscience). Dimer-X soluble dimericmouseH-2Kb:Ig fusion protein andH-2Db:Ig fusion protein (BDBioscience) were used according to the manufacturer's pro-tocol to detect MDH251–260, GAPDH300–310, annexin A2184-191,and H487–95 peptide-specific CD8þ T cells. In addition, thepresence of influenza-specific T cells was evaluated usingpeptides PA224–233 and NP147–155 purchased from GenScriptUSA. Of note, 100,000 events were collected and samples wererun on the LSR II flow cytometer (BD Bioscience), gated(example in Supplementary Fig. S2), and analyzed using FACS-Diva software.

Vaccination and tumor challengeD1 dendritic cells are an established growth factor–

dependent immature dendritic cell line. D1 dendritic cellswere grown and maintained as described and used in allvaccinations (36). A total of 1.25 � 106 D1 dendritic cells permouse were cultured in 6-well plates, loaded separately with100 mg of MDH251–260, GAPDH300–310, annexin A2184–191, orH487–95, and matured with 12.5 mg/mL of Poly IC:LC

Figure1. Influenza virus infection delays tumor growth at early timepoints.A, animals were intranasally infectedwith PR8 and Aichi influenza viruseson day 0 and day 35, respectively. Mice were weighed every 2 days. B,influenza-experienced animals and naïve animals were challengedsubcutaneouslywith 1� 105 3LL tumor cells in the right hind flank. Tumorlength and width were measured every 2 days using calipers.Data are representative of two experiments with at least 8mice per groupand are expressed as means � SEM. C, sera from animals(n ¼ 7) 4 days before PR8 infection (preinfection) and 10 days after Aichiinfection (postinfection) were diluted 1:62.5 and used tostain 3LL and IG10 tumor cells. Cells were subsequently stained withFITC-conjugated goat anti-mouse IgG2a antibody and analyzedon the LSR II flow cytometer. Results are shown as MFI.�, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Figure 2. Influenza infection induces antibodies against specificproteins of tumor cells. 3LL tumor lysates were affinity purified onprotein G columns to which postinfection and preinfection antibodieswere covalently bound and the eluted proteins were compared by 2D-DIGE gels. Green spots mark proteins eluted from the preinfectionantibody columns, and red spots mark proteins from the postinfectionantibody columns. Yellow color marks overlapping spots. Yellowcircles indicate spots picked for sequencing. Spots were chosenaccording to image analysis provided by the University of PittsburghBioinformatics Analysis Core (BAC) of the Genomics and Proteomicscore laboratories.

Iheagwara et al.





Tab

le1.

Bioch

emical

charac

teriz

ationof

selected

tumor

antig

ensdetec

tedbypos

tinfectionan

tibod

ies

Spot

number

Acc

ession

number

aProtein

nameb

Pep

tidese

que

ncec

X-corr

dDSco

ree

Cha

rgef

M/Z

(Da)

gMHþh

(Da)

DMi(ppm)

MW

j

(kDa)

pIk

%Seq

uenc

eco

veragel

Ions

match

edm

2IPI008

8536

7.1

Ann

exin

A2

TNQELQ

EINR

2.74

0.36

262

2.81

384

1244

.620

41�2

.08

19.6

5.96

10.80

14/18

QDIAFA

YQR

2.55

1.00

255

6.28

143

1111

.555

592.22

14/16

3IPI004

6820

3.3

Ann

exin

A2

TPAQYDASELK

3.52

0.65

261

1.80

090

1222

.594

53�0

.43

38.7

7.69

22.71

15/20

AKDGSVIDYELIDQDAR

3.46

0.68

295

4.94

586

1908

.884

451.16

17/32

SLY

YYIQ

QDTK

3.30

0.47

271

1.35

215

1421

.697

041.62

16/20

TNQELQ

EINR

3.08

0.47

262

2.81

384

1244

.620

41�2

.08

15/18

WISIM

TER

2.67

1.00

251

8.26

892

1035

.530

571.29

13/14

DIIS

DTS

GDFR

2.53

1.00

261

3.28

820

1225

.569

14�0

.36

16/20

QDIAFA

YQR

2.41

0.60

255

6.28

027

1111

.553

270.13

15/16

4IPI004

0733

9.7

Histone

H4

ISGLIYEETR

3.39

0.68

259

0.81

536

1180

.623

462.19

11.4

11.36

38.83

16/18

TVTA

MDWYALK

3.22

0.74

265

5.85

559

1310

.703

911.06

16/22

TVTA

mDVVYALK

2.88

0.42

266

3.85

327

1326

.699

271.39

14/22

DAVTY

TEHAK

2.61

0.52

256

7.77

410

1134

.540

94�1

.47

15/18

VFL

ENVIR

2.56

0.36

249

5.29

345

989.57

964

1.76

12/14

4IPI006

2279

5.2

GAPDH

LENPAKYDDIK

2.36

8.03

265

3.33

881

1305

.670

350.20

35.8

8.03

3.30

10/20

4IPI003

2359

2.2

MDH2

IFGVTT

LDIVR

2.82

0.41

261

7.36

487

1233

.722

460.41

35.6

8.68

3.25

14/20

5IPI008

8536

7.1

Ann

exin

A2

TNQELQ

EINR

3.06

0.47

262

2.81

384

1244

.620

41�2

.08

19.6

5.96

5.68

14/18

aProtein

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adjuvant. Briefly, 0.25 � 106 D1 dendritic cells loaded withindividual peptides were pooled together for a total numberof 1 � 106 D1 dendritic cells. An additional 50 mg of eachsoluble peptide per mouse was added to the mixture andinjected into mice in the right hind flank. Unloaded, Poly IC:LC matured D1 dendritic cells were injected into controlmice. Animals were vaccinated at weeks 0, 2, and 6, andchallenged with 1 � 105 3LL cells in the right hind flank,

2 days following the week 6 vaccination. Tumor length andwidth were measured at 2-day intervals using calipers.

Statistical analysisStatistical analysis was performed using GraphPad Prism

v6.0 software (GraphPad Inc.). Results were representedas means � SEM. Statistical means and significance wereanalyzed using unpaired two-tailed Student t test. Kaplan–

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

Iheagwara et al.





Meier survival curves were analyzed with the log ranktest. Significance for all experiments was defined as follows:�, P < 0.05; ��, P < 0.01; ��, P < 0.001.

ResultsInfluenza virus infection induces antibodies to multiplehost cell antigens, some of which are known TAAsMice were infected with influenza virus PR8 and then 35

days later with the second influenza strain Aichi, as describedin Materials and Methods. Mice experienced the general signsof malaise and lost close to 20% of their starting body weight inthe first week following each infection. Seven to 8 days afterinfection, mice began to recover, and by days 16 to 18, theirweight returned to baseline (Fig. 1A). At day 25 after the secondinfection, animals were injected subcutaneously with 3LLtumor cells. Tumors became palpable 8 days after injection(Fig. 1B). At day 14, tumor growth kinetics between theinfluenza-experienced animals and the na€�ve 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 was20.67 mm2 versus 51.63 mm2 in the control group. The sizedifference was still significant at day 18, when the averagetumor size in the influenza-experienced group was 31.78 mm2

compared with 73.25 mm2 in the control mice.We examined the postinfection sera for the ability to stain

3LL tumor cells, which would suggest that flu infectionselicited antibodies against tumor cell surface proteins. In theexperiment illustrated in Fig. 1C, we obtained sera from mice(n ¼ 7) pre- and postinfluenza infection. Tumor cell surfacestaining was performed with individual sera and the resultswere pooled into pre- and postinfection groups and repre-sented as an average value. Average mean fluorescence inten-sity (MFI) of staining of 3LL tumor cells with postinfection serawas significantly higher than that with preinfection sera. Thesame result was obtained after staining another mouse epi-thelial tumor cell line IG10 with the same sera.

Figure 4. Antibodies specific forGAPDH, histone H4, MDH2,annexin A2, and HSP90a increase,following influenza infection.Preinfection sera (4 days before thefirst infection) and postinfectionsera (10 days before the secondinfection) were assayed on ELISAplates in duplicate wells coatedwith individual proteins. Uncoatedwells served as nonspecificbinding controls and their valueswere subtracted from the values inmatching antigen-coated wells.Results are represented as meanoptical density (OD) � SEM of twoexperimental repeats with n ¼ 7.






To identify molecules specifically recognized by postinfec-tion sera, pre- and postinfection antibodies were bound toProtein G columns for affinity purification of proteins from3LLtumor cell lysates. Tumor proteins bound to the antibodycolumns were eluted, labeled with two different cyanine-basedsaturation dyes, and resolved by 2D-DIGE as described inMaterials and Methods. In a concurrently run reciprocal gel,labeling was reversed such that the sample previously labeledwith Cy3 was labeled with Cy5 and vice versa. Gel images werefalse colored for analysis: green for Cy3 and red for Cy5.Overlays were then created using Delta2D software, in whichproteins 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 toidentify antigens described below. Green dots mark proteinseluted from the preinfection antibody columns, whereas reddots mark proteins eluted from the postinfection antibodycolumns. Spots that were most remarkable (yellow circles)were cut out and subjected to mass spectrometry analysis andprotein sequencing. Many proteins with a wide variety offunctions were identified. They included voltage-dependention channels, proteasome subunits, mitochondrial and cyto-solic 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 onmethods and approaches unrelated to

ours had already identified these molecules as TAA in tumor-bearing mice or in patients with cancer (37–41).

Overexpression of GAPDH, histone H4, MDH2, annexinA2, and HSP90 in tumor cells and influenza virus–infected lungs leads to specific immunity

Wehypothesized that postinfection antibodies against theseproteinswere elicited because of differences in their expressionin infected versus normal lungs, analogous to their abnormalexpression in tumors. Western blot analysis showed that theseproteins were constitutively overexpressed in both epithelialtumor cell lines, 3LL and IG10, and also at various time pointsin the influenza-infected lungs (2–7.5-fold higher comparedwith healthy lungs; Fig. 3). Expression of GAPDH in influenza-infected lungs appeared to be the highest at day 3 postinfec-tion. Histone H4 protein levels were constitutively elevated intumor cells and at all time points after influenza infection by 5to 7-fold higher than in normal lung. MDH2 levels wereelevated 12 hours postinfection and decreased to normal levelsat day 3 postinfection. Annexin A2 remained 3-fold higher thanin normal lung at all time points. HSP90 protein level was thehighest at day 2 postinfection.

Even though these antigens were identified by affinitypurification on postinfection sera bound to Protein G columns,we wanted to confirm in another assay the specificity ofpostinfection antibodies for each of these individual

Figure 5. The amount of antigen-specific CD8þ T cells increases inthe lungs and spleens followinginfluenza infection. Mice wereinfected with PR8 and Aichiinfluenza viruses or mock PBSinfected on day 0 and day 35,respectively. Six days afterthe second infection, spleens andlungs were harvested andanalyzed by flow cytometry for thepresence of T cells staining withthe Dimer-X reagent (asdescribed in Materials andMethods). Data are representativeof two experimental repeats ofleast n ¼ 3 animals per group andare expressed as means � SEM.

Iheagwara et al.





molecules. Figure 4 shows that in most mice there was anincrease after infection in IgG specific for GAPDH, histone H4,MDH2, annexin A2, and HSP90a, as determined by ELISA.Influenza virus infection also induced an increase in CD8þ T

cells specific for all of these antigens. Spleens and lungs wereharvested frommice 6 days after the second influenza infectionand from uninfected control mice. Peptides GAPDH300–310

(ALNDNFVKLIS), annexin A2184–191 (SVIDYELI), MDH251–260

(MAYAGARFVF), histone H487-95 (VVYALKRQG), and histoneH4 with an amino acid substitution (H4-sub VVYAFKRQG;ref. 38), were selected from IEDB and confirmed to bind toMHC-I in RMA-S stabilization assays (Supplementary Fig. S1).Each peptide was loaded onto DimerX H-2Kb or H-2Db mole-cules andused to detect specificCD3þCD8þTcells. Therewereno CD8 T cells specific for these peptides in the lungs andspleens of uninfected control mice but in influenza-experi-enced animals they were present in similar numbers to theinfluenza-specific T cells (Fig. 5). The highest numbers both inthe lungs and in the spleens were H2-Kb-restricted GAPDH-specific and H2-Db-restricted MDH2-specific T-cells.

Vaccinationwith dendritic cells loadedwith the newTAApeptides delays tumor growth and promotes survivalWe loaded the D1 dendritic cells with MDH2251–260,

GAPDH300–310, annexin A2184–191, and H487–95 and vaccinatedmice as described in Materials and Methods. Control micewere vaccinated with unloaded D1 cells. Two days followingthe second boost, mice were challenged subcutaneously with3LL tumor cells. In vaccinated animals, tumor growth began toslow down significantly by day 12 (Fig. 6A), resulting in allvaccinated animals still surviving at day 40, compared withonly 2 animals surviving in the unloaded dendritic cell–vac-cinated controls (Fig. 6B). On day 12, the average tumor size inpeptide-loaded dendritic cell–vaccinated animals was 10.67mm2 compared with 30.67 mm2 for the controls.

DiscussionThe data we present here add a new dimension to our

understanding of the process of cancer immunosurveillanceand its targets. We show that immune responses againstabnormally expressed self-antigens, many of which havebeen characterized as TAAs, are generated during nonma-lignant infectious inflammatory events that occur muchearlier in life than malignancies. We propose that immunememory for these antigens is later recruited for cancerimmunosurveillance.We used amousemodel to demonstrate that two bouts with

influenza virus infection led to the ability of the host's immunesystem to slow down tumor growth. This effect was small andtransient, which may be all that could be expected from alimited exposure of themice to this one type of infection and tono other pathogens before influenza infection as the mice arekept in pathogen-free conditions. The significance of this smalldelay in tumor growth was confirmed by the induction ofantibodies against multiple molecules in the tumor cell lysate.Focusing on five antigens identified by infection-elicited anti-bodies, GAPDH, histoneH4,MDH2, annexin A2, andHSP90, we

showed that these antigens were abnormally expressed (over-expressed) in influenza-infected lungs and in mouse epithelialtumor cell lines, and that in addition to IgG, the influenzainfection induced antigen-specific CD8 T cells against thesemolecules. As predicted by our hypothesis, vaccination withpeptides derived from GAPDH, histone H4, MDH2, andannexin A2 led to a much more profound slowing down oftumor growth compared with that elicited by the virus infec-tion, 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 lympho-cytic choriomeningitis virus–infected mice were reported tohave developed antibodies against many host cell antigens,some of which are orthologs of human TAAs (42). Humanfibroblasts infected with varicella-zoster virus (VZV) or humancytomegalovirus (CMV) overexpress cyclin B1 in the cytoplasmin a similar fashion to tumor cells where cyclin B1 wasidentified as a TAA (43, 44). Cyclin B1 has been found in VZV

Figure 6. Vaccination with GAPDH, histone H4, MDH2, and annexin A2peptides loaded on dendritic cells (DC) confers early protection andprolonged survival against 3LL tumor challenge. A,micewere vaccinatedin 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 receivedthe same number of unloaded D1 dendritic cells at the same time points.Two days after the week 6 boost, animals were challengedsubcutaneouslywith 1� 105 3LL tumor cells in the right hind flank. Tumorgrowth was measured with calipers at 2-day intervals and expressed aslength � width (mm2). B, survival post tumor challenge. Data arerepresentative of two experiments with at least n¼ 8mice per group andare expressed as means � SEM.






virions (45). Many healthy individuals, presumably havingexperienced these infections, have cyclin B1–specific IgG andmemory T cells (24). It has been reported that GAPDH andannexin A2 are found in influenza virions produced by infectedepithelial cell lines Vero andA549 (46). HSP90, Annexin A2, andGAPDH were also found within human CMV particles (47). Astudy examining the measles virus effect on presentation ofself-peptides onMHC class I during infection showed that twoabundant self-peptides on HLA-A�0201 measles-infected cellscould induce auto-reactive CD8þ T cells. One of the peptidesidentified was HSP90b570–578 (ILDKKVEKV; ref. 48). TheHSP90b570–578 peptide has been found in melanoma cell lines(49). It is possible that these "auto-reactive" T cells contributeto increased tumor immune surveillance. None of these obser-vations were followed by experiments to test the potentialantitumor effects of either the viral infections or the immuneresponses against the identified molecules, with the exceptionof cyclin B1 that we showed was a target of antitumor immuneresponses (24).

The same protective effect of influenza virus–primed immu-nity specific for abnormally expressed self-antigens that weshowed here could be a collateral benefit of other viral,bacterial, and parasitic infection or various acute inflamma-tory conditions of unknown etiologies. Therefore, we proposethat the molecules abnormally expressed in these differentdisease states and also in cancer cells, which are currentlyreferred to as TAAs, should be renamed as disease-associatedantigens (DAA; ref. 50). Preexisting immune responses toseveral known tumor antigens that are candidate DAAs havebeen reported to increase the odds of successfully eliminatingspontaneously arising tumors (28). The arrival of memoryDAA-specific T cells to the site of the tumor as a secondaryimmune response could promote priming of tumor-specificresponses directed against individual mutations and epitopespreading, adding to the efficacy of immunosurveillance. IfDAA-specific immune memory is lacking or is weak due tolimited early exposures to infections, this may lead to estab-

lishment of chronic inflammation at the tumor site due tounopposed innate immune responses, which is likely to pro-mote tumor development. Therefore, a better understanding ofthe events early in life that prepare the immune system toprotect an individual against known and unknown pathogens,as well as future malignancies, will help direct vaccines andother immunemanipulation toward strengthening rather thanimpairing the establishment of life-long immunosurveillance.In addition, these findings support the use of vaccines based onDAAs/TAAs for cancer prevention.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: U.K. Iheagwara, T.M. Ross, O.J. FinnDevelopment of methodology: U.K. Iheagwara, J.S. Minden, O.J. FinnAcquisition 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. FinnAnalysis 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. FinnWriting, review, and/or revision of the manuscript: U.K. Iheagwara,P.L. Beatty, P.T. Van, T.M. Ross, J.S. Minden, O.J. FinnAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): U.K. Iheagwara, O.J. FinnStudy supervision: U.K. Iheagwara, O.J. Finn

AcknowledgmentsThe authors thank the University of Pittsburgh Genomics and Proteomics

core laboratories for their assistance. This work used the Biomedical MassSpectrometry Center and UPCI Cancer Biomarker Facility that are supported inpart by award P30CA047904. This work was supported by the NIH (CA056103 toO.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 GraduateStudent Dissertation Fellowship (U.K. Iheagwara).

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

Received August 13, 2013; revised November 18, 2013; accepted November 18,2013; published OnlineFirst December 5, 2013.

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2014;2:263-273. Published OnlineFirst December 5, 2013.Cancer Immunol Res Uzoma K. Iheagwara, Pamela L. Beatty, Phu T. Van, et al. ImmunosurveillanceTumor-Associated Antigens: A New View of CancerSpecific for Host Cell Antigens Also Expressed as Influenza Virus Infection Elicits Protective Antibodies and T Cells

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ﬂuenza Virus Infection Elicits Protective Antibodies and T ...cancerimmunolres.aacrjournals.org/content/canimm/2/3/263.full.pdf · also been associated with a history of febrile