Episomal Expression of Truncated Listeriolysin O in LmddA-LLO-E7 Vaccine Enhances Antitumor Efficacy...

Research Article

Episomal Expression of Truncated Listeriolysin O inLmddA-LLO–E7 Vaccine Enhances Antitumor Efficacy byPreferentially InducingExpansionsofCD4þFoxP3�andCD8þ

T Cells

Zhisong Chen1, Laurent Ozbun1, Namju Chong1, Anu Wallecha2, Jay A. Berzofsky1, and Samir N. Khleif3

AbstractStudies have shown that Listeria monocytogenes (Lm)–based vaccine expressing a fusion protein comprising

truncated listeriolysin O (LLO) and human papilloma virus (HPV) E7 protein (Lm-LLO–E7) induces a decrease inregulatoryT cells (Treg) and complete regressionof established, transplantedHPV-TC-1 tumors inmice.However,how the Lm-based vaccine causes a decrease in Tregs remains unclear. Using a highly attenuated Lm dal datDactAstrain (LmddA)–based vaccine, we report here that the vector LmddA was sufficient to induce a decrease in theproportion of Tregs by preferentially expandingCD4þFoxP3�T cells andCD8þT cells by amechanismdependenton and directly mediated by LLO. Episomal expression of a nonhemolytic truncated LLO in Lm (LmddA-LLO)significantly augmented the expansion, thus further decreasing Treg frequency. Although adoptive transfer ofTregs compromised the antitumor efficacy of the LmddA-LLO–E7 vaccine, a combination of LmddA-LLO and anLm-based vaccine expressing E7 protein (Lm–E7) induced complete regression against established TC-1 tumors.An engineered LLO-minus Lm expressing perfringolysin O (PFO) that enables the recombinant bacteria to exitfrom the phagolysosome without LLO confirmed that the adjuvant effect was dependent on LLO. These resultssuggest that LLO may serve as a promising adjuvant by preferentially inducing the expansions of CD4þFoxP3� Tcells and CD8þ T cells, thus reducing the ratio of Tregs to CD4þFoxP3� T cells and to CD8þ T cells favoringimmune responses to eradicate tumor. Cancer Immunol Res; 2(9); 911–22. �2014 AACR.

IntroductionListeria monocytogenes (Lm) is a gram-positive facultative

intracellular pathogen that causes listeriosis (1). Upon invad-ing a host cell, Lm can escape from the phagolysosome byproducing a pore-forming protein, listeriolysin O (LLO), whichlyses the vesicular membrane, allowing Lm to enter the cyto-plasm, in which it replicates and spreads to adjacent cellsmediated by the mobility of actin-polymerizing protein (ActA;ref. 2). In the cytoplasm, Lm-secreted proteins are degraded bythe proteasome and processed into peptides that associatewith MHC class I molecules in the endoplasmic reticulum (3).This unique characteristic makes it an attractive cancer vac-

cine vector in that Lm-expressed tumor antigens can bepresented with MHC class I molecules to activate tumor-specific cytotoxic T lymphocytes (CTL). Attenuated Lm strainshave been generated and developed as cancer vaccine vectorsdelivering tumor antigens or tumor-associated antigens asimmunogens to treat various types of cancer (4–10).

Human papillomavirus (HPV) infection is associated withmost cervical cancer, andHPV strain 16 (HPV-16) is detected inabout half of cervical cancer cases worldwide (11). Constitutiveexpression of HPV-16 E6 and E7 viral proteins in infected cellsdisrupts the cell cycle and inducesmalignant proliferation (12).Although prophylactic HPV vaccines are effective against HPVinfection and development of cervical intraepithelial neoplasia(13), a therapeutic vaccine for advanced-stage cervical canceris still being developed. Progress has been made in the con-struction of an Lm-LLO–E7 vaccine, a live-attenuated Lm-based vector producing and secreting a fusion protein com-prising a truncated LLO and full-length E7 antigen. The Lm-LLO–E7 vaccine induced complete regression of establishedHPV-immortalized TC-1 tumors in mice (14). CD8þ T cellshave a critical role in the antitumor activity induced by Lm-LLO–E7, as depletion of CD8þ T cells before vaccinationabrogated the inhibition of tumor growth (14). Lm-LLO–E7vaccine has been shown to decrease regulatory T cells (Treg) inmouse spleens and tumors (15). Tregs, identified as CD4þ

FoxP3þ (or CD4þCD25þ when it was first discovered), are asmall population of T cells that suppress immunity. An

1Vaccine Branch, Center for Cancer Research, National Cancer Institute,NIH, Bethesda, Maryland; 2Advaxis Inc., Princeton, New Jersey; and 3GRUCancer Center, Georgia Regents University, Augusta, Georgia

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

CorrespondingAuthors:Samir N. Khleif, Cancer Center, GeorgiaRegentsUniversity, 1410 Laney Walker Boulevard, CN-2133, Augusta, GA 30912.Phone: 706-721-0570; Fax: 706-721-8787; E-mail: [email protected]; andJay A. Berzofsky, Vaccine Branch, Center for Cancer Research, NationalCancer Institute, NIH, Building 41, Room D702D, 41 Medlars Drive,Bethesda, MD 20892, Phone: 301-496-6874; Fax: 301-480-0681,[email protected]

doi: 10.1158/2326-6066.CIR-13-0197

�2014 American Association for Cancer Research.

CancerImmunology

Research

www.aacrjournals.org 911

on April 2, 2016. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst May 28, 2014; DOI: 10.1158/2326-6066.CIR-13-0197

http://cancerimmunolres.aacrjournals.org/

effective immunotherapy must overcome the Treg obstacle totrigger helpful immune responses. It is conceivable that theLm-LLO–E7–induced reduction of Tregs contributes to its anti-tumor effect, but how the Lm-LLO–E7 vaccine induces Tregdecrease remains unclear. Studies toward identifying the mech-anism by which Lm-LLO–E7 causes Treg reduction may leadto further improvement of its antitumor efficacy, such as thedevelopment of novel therapeutic strategies tomanipulate Tregs.

Here, we describe the development of LmddA-LLO–E7, animproved attenuated Lm-based vaccine that decreased Tregfrequency but not its absolute cell number. Specifically,LmddA-LLO–E7 preferentially induced the expansions ofCD4þFoxP3�T cells and CD8þT cells, thus effectively decreas-ing the proportion of CD4þFoxP3þ T cells by dilution. Wefound that the LmddA vector was able to induce CD4þFoxP3�

T-cell and CD8þ T-cell expansions, but the addition of episom-al expression of a truncated LLO dramatically enhanced suchan expansion, thus further decreasing the percentage of CD4þ

FoxP3þ T cells. Lm-induced CD4þFoxP3� T-cell and CD8þ T-cell expansions were dependent on and directly mediated byLLO. Although enhancement of the expansions of CD4þ

FoxP3� T cells and CD8þ T cells by the combination ofLmddA-LLO andLm–E7 induced complete regression of estab-lished TC-1 tumors, adoptive transfer of CD4þCD25þ Tregscompromised LmddA-LLO–E7 antitumor efficacy, suggestingthat different T-cell subsets and their balance are critical andcan affect the outcome of immunotherapy.

Materials and MethodsMice

C57BL/6 mice, female, 6- to 8-weeks-old (unless statedotherwise), were purchased from the Frederick National Lab-oratory for Cancer Research (Frederick, MD). Mice werehoused in the Animal Facility of the National Cancer Institute(Bethesda, MD). Protocols for use of experimental mice wereapproved by the Animal Care and Use Committee at the NIH.

Cell lineThe TC-1 cell line, a generous gift from Professor T.C. Wu at

Johns Hopkins University (Baltimore, MD), was generated bythe transformation of primary lung epithelial cells fromC57BL/6 mice with HPV-16 E6 and E7 and activated rasoncogene (16). TC-1 cells were tested to be mycoplasma free;no other authentication was performed. The cells were grownin RPMI-1640, supplemented with 10% FBS, 100 U/mL peni-cillin, 100 mg/mL streptomycin, 2 mmol/L L-glutamine, 1mmol/L sodium pyruvate, 100 mmol/L nonessential aminoacids, and 0.4 mg/mL G418 at 37�C with 5% CO2.

L. monocytogenes strainsLmddA-LLO–E7 and the corresponding controls LmddA-

LLO and LmddA were generated in Advaxis Inc. The dal datDactA strain (LmddA) was constructed from the dal datstrain, which is based on Lm wild-type strain 10403S (17).With dal, dat, and actA mutated, LmddA is highly attenu-ated. The LmddA-LLO–E7 strain was constructed by thetransformation of LmddA with the pTV3 plasmid (18) afterdeletion of prfA and the chloramphenicol-resistant gene in

the plasmid (17). Expression and secretion of the LLO–E7fusion protein were confirmed in culture supernatants of theLmddA-LLO–E7 strain by Western blot analysis as previ-ously described (14). Construction of the LmddA-LLO con-trol strain was similar to that of the LmddA-LLO–E7 strain,but both prfA and E7 were deleted in the pTV3 plasmid. Lmwild-type strain 10403S and mutant strains Dhly, Dhly::pfo,and hly::Tn917–lac (pAM401-hly) were kindly provided byDr. D. Portnoy (University of California, Berkeley, CA). Lm–E7 strain, in which the full-length E7 gene was integratedinto the Lm chromosome, was kindly provided by Dr. Y.Paterson (University of Pennsylvania, Philadelphia, PA). Thestrain hly::Tn917–lac is a nonhemolytic mutant of wild-typeLm, in which the Tn917–lac fusion gene is inserted into thehly gene (the gene encoding LLO) to disrupt LLO hemolyticactivity. When this mutant hly::Tn917–lac strain is trans-fected with plasmid pAM401-hly expressing LLO, it regainshemolytic activity. Bacteria were cultured in brain–heartinfusion medium plus streptomycin (100 mg/mL) with orwithout D-alanine (100 mg/mL).

ReagentsFluorescence-conjugated anti-mouse antibodies CD4-

PerCP-Cy5.5 (GK1.5) and CD8-Brilliant Violet 421 (53-6.7) werefrom BioLegend. FoxP3-FITC (FJK-16s) was from eBioscience.H-2Db tetramers loaded with the E7 peptide (RAHYNIVTF)were kindly provided by the National Institute of Allergy andInfectious Diseases Tetramer Core Facility and the NIH AIDSResearch and Reference Reagent Program. CountBright abso-lute counting beads were from Life Technologies.

Tumor inoculation and mouse vaccinationTC-1 cells (105 cells/mouse) were implanted subcutane-

ously in the right flank of mice on day 0. On day 10, whentumors were 5 to 6 mm in diameter, mice were injected i.p.with LmddA-LLO–E7 vaccine or corresponding controls ata dose of 0.1 LD50. Vaccination was boosted on day 17.Tumors were measured twice a week using an electroniccaliper, and tumor size was calculated by the formula:length�width�width=2. Mice were euthanized whentumors reached 2.0 cm in diameter.

Flow cytometryMouse splenocytes or cells harvested from tumors were

stainedwithCD4-PerCP-Cy5.5, CD8-Brilliant Violet 421, andH-2Db E7 tetramer–allophycocyanin (APC) for 30 minutes. Cellswere fixed, permeabilized, and stained with FoxP3–FITC over-night. Cells were analyzed by flow cytometry. A lymphocytegate was set in which Tregs were identified as CD4þFoxP3þ.CountBright absolute counting beads were added for countingabsolute cell numbers.

Adoptive transfer of CD4þCD25þ TregsCD4þCD25þT cells were isolated frommouse spleens by the

Dynal CD4þCD25þ Treg Kit (Life Technologies). Cells wereinjected i.v. into TC-1 tumor–bearingmice at day 9 after tumorcell inoculation. One day after Treg transfer, mice were immu-nized i.p. with LmddA-LLO–E7 (0.1 LD50) twice at 1-weekinterval. Tumor growth was monitored.

Chen et al.

Cancer Immunol Res; 2(9) September 2014 Cancer Immunology Research912




Statistical analysisThe data were analyzed using the nonparametric Mann–

Whitney test. Statistical significance was determined atP < 0.05.

ResultsLmddA-LLO–E7 induces regression of established TC-1tumors accompanied by a decrease in Treg frequencyIt was reported previously that an Lm-based vaccine, Lm-

LLO–E7, which comprises a fusion protein of LLO–E7 and PrfAexpressed episomally in a prfA-negative strain of ListeriaXFL-7,induced complete regression of established TC-1 tumors (14).Here, we investigated the antitumor activity of another highlyattenuated Lm-based vaccine, LmddA-LLO–E7, which pro-duces the fusion protein LLO–E7 by a plasmid in a dal, dat,and actA mutated Lm strain (17). LmddA-LLO–E7 is safer andevenmore attenuated comparedwith Lm-LLO–E7, because thechloramphenicol resistance gene and PrfA have been removedfrom the plasmid. We found that similar to Lm-LLO–E7 (14),LmddA-LLO–E7 significantly inhibited the growth of estab-lished TC-1 tumors (Fig. 1A and B and Supplementary Fig. S1).Tumors completely regressed in approximately 40% of TC-1tumor–bearing mice after two vaccinations with LmddA-LLO–E7 (Fig. 1B and Supplementary Fig. S1). Except for one mousethat relapsed and died at 3 months, mice that showed tumorregression (33% of total animals) survived at least 6 monthswithout relapse (Fig. 1C).AlthoughLm–E7 slowed thegrowthofTC-1 tumors, it failed to induce complete tumor regression (Fig.1A andB and Supplementary Fig. S1). LmddA-LLO (without E7)was unable to significantly inhibit TC-1 tumor growth (Fig. 1Aand B and Supplementary Fig. S1), suggesting that innateimmune response is not sufficient to eradicate TC-1 tumorcells. LmddA-LLO–E7 and Lm–E7 induced similar H-2Db E7-tetramerþCD8þ T-cell response in the spleen (SupplementaryFig. S2A top, S2B, and S2D), which was consistentwith previousfinding (14). We then analyzed CD4þFoxP3þ Tregs. Unexpect-edly, we found that LmddA-LLO–E7, Lm–E7, and LmddA-LLOall significantly decreased Treg frequency in spleens and moredramatically in tumors compared with PBS control, althoughLmddA-LLO–E7 and LmddA-LLO decreased the frequencymore than did Lm–E7 (Fig. 1D–H). We note that a previousreport found that Lm–E7 was unable to decrease Tregs (15);whether the differences in these models account for the dif-ferent findings are not clear.

Lm is sufficient to induce decrease of Treg frequencyInitially, we suspected that the decrease of Treg frequency

was mediated by the truncated LLO. However, Lm–E7 did notexpress truncated LLObutwas able to decrease Treg frequency(Fig. 1D–H). This observation suggests that Lm might be ableto decrease Treg frequency. Indeed, both LmddA, the vectorcontrol for LmddA-LLO–E7, and 10403S, a wild-type Lm strainand the vector control for Lm–E7, significantly decreased Tregfrequency in spleens and more so in tumors (Fig. 2).

Lm decreases Treg frequency by preferentially inducingCD4þFoxP3� T-cell and CD8þ T-cell expansionsA relative Treg frequency (proportion of total T cells) is

determined by the numbers of Tregs, CD4þFoxP3� T cells, and

CD8þT cells. To investigate how Lmdecreases Treg frequency,we quantified the numbers of CD4þFoxP3þ Tregs, CD4þ

FoxP3� T cells, and CD8þ T cells in TC-1 tumor–bearing micetreated with either LmddA-LLO–E7, LmddA-LLO, LmddA,Lm–E7, or Lm (10403S). As shown in Fig. 3, surprisingly, wefound that LmddA did not markedly change the number ofCD4þFoxP3þ T cells in the tumors. It actually increased CD4þ

FoxP3� T cells and CD8þ T cells, thus decreasing Treg fre-quency proportionately. Episomal expression of a truncatedLLO in LmddA-LLO and LmddA-LLO–E7 further increasedthe numbers of CD4þFoxP3� T cells and the LmddA-LLO–E7also increased the numbers of CD8þ T cells, thus furtherdecreasing the frequency of CD4þFoxP3þ T cells. Wild-typeLm 10403S also induced an increase in CD4þFoxP3� T cellsand CD8þ T cells while not significantly changing CD4þ

FoxP3þ T-cell number. Lm-LLO–E7 significantly increasedthe density of CD4þFoxP3� T cells and CD8þ T cells intumors. These results demonstrate that Lm preferentiallyinduces CD4þFoxP3� T-cell expansion and to a lesser extentCD8þ T-cell expansion resulting in a decrease of CD4þ

FoxP3þ T-cell frequency.

Lm-induced expansion of CD4þFoxP3� T cells and CD8þ

T cells is dependent on and mediated by LLOLLO, encoded by the hly gene, is a pore-forming cytolysin by

which Lm can escape from a host cell phagosomal vacuoleinto the cytoplasm (19). Because LmddA-LLO–E7, Lm–E7,and their respective controls produce LLO, we used an LLO-deficient Lm-mutant derived from 10403S, in which the hlygene is deleted using a shuttle vector followed by homologousrecombination (20), to study whether LLO plays a role ininducing the expansion of CD4þFoxP3� T cells and CD8þ Tcells. We found that Dhly Lm was unable to increase CD4þ

FoxP3�T cells and CD8þT cells in the spleen ofmice on day 7after a single administration (Fig. 4A), indicating that induc-tion of expansion of CD4þFoxP3� T cells and CD8þ T cells isdependent on LLO. This dependence could be either a directeffect of LLO on the immune cells or a requirement for thebacteria to escape the phagolysosome. To address this ques-tion, we studied an Lm strain with LLO replaced by perfrin-golysin O (PFO). PFO, produced by Clostridium perfringens, is43% identical in amino acids with LLO and can also lyse thevacuolar membrane (21). The pfo gene, encoding PFO underthe control of hly promoter, was recombined into the chro-mosome of the Dhly strain to form Dhly::pfo strain (20).Although Dhly::pfo was able to escape from phagocytosis intothe cytoplasm (20), it was unable to increase the numbers ofCD4þFoxP3� T cells and CD8þ T cells in the mouse spleen(Fig. 4A). A limitation of Dhly::pfo control is that PFO is toxicto the infected host cell when secreted by Lm and does notallow productive intracellular replication of Lm within theinfected cells (22). Thus, a mutant of PFO that allows for aneffective intracellular replication in the context of an Lminfection might be a more appropriate control in the exper-iment (22). Different from the Dhly and Dhly::pfo strains, hly::Tn917–lac (pAM401-hly), a nonhemolytic Tn917–lac mutantof wild-type Lm (in which the Tn917–lac fusion gene isinserted into the hly gene to disrupt LLO hemolytic activity)transformed with an LLO-expressing plasmid pAM401-hly,

LLO Enhances Vaccine Efficacy by Expanding Non-Tregs

www.aacrjournals.org Cancer Immunol Res; 2(9) September 2014 913




induced expansions of mouse splenic CD4þFoxP3� T cellsand CD8þ T cells (Fig. 4A). These results suggest that expan-sions of CD4þFoxP3� T cells and CD8þ T cells are directlymediated by LLO independently of the hemolytic activity.

Because Lm did not induce CD4þFoxP3þ T-cell expansionsignificantly, Lm-induced Treg decrease in frequencyresulted from the increase of CD4þFoxP3� T cells andCD8þ T cells (Fig. 4A–D).

Figure 1. LmddA-LLO–E7 induces regression of established TC-1 tumors accompanied by Treg frequency decrease. C57BL/6 mice were inoculated s.c. with1� 105 TC-1 tumor cells each, and immunized i.p. with 0.1 LD50 LmddA-LLO–E7 (1� 108 CFU), Lm–E7 (1� 106 CFU), or LmddA-LLO (1� 108 CFU) in PBS(100 mL) on days 10 and 17 after tumor challenge. Tumors were measured twice a week using an electronic caliper. Tumor volumes were calculatedby the formula, length�width�width=2.Micewere sacrificedwhen tumor diameter reached approximately 2.0 cmor onday 24 for flowcytometric analysis.A, average tumor volume fromdays 10 to 24. B, tumor volume on day 24. C, survival percentage over time. D, flow cytometric profile of CD4þFoxP3þ T cells intotal CD4þ T cells. E, the percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells in the spleen. F, ratio of CD4þFoxP3þ T cells to CD8þ T cells in thespleen. G, percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells in the tumor. H, ratio of CD4þFoxP3þ T cells to CD8þ T cells in the tumor. Data,mean � SEM; �, P < 0.05; ��, P < 0.01; ��, P < 0.001 (Mann–Whitney test). Data are from three independent experiments (A and B) and are representativeof three independent experiments (C–H).

Chen et al.





Episomal expression of a truncated LLO in LmddAinduces expansion of CD4þFoxP3� T cells and CD8þ

T cells to a higher levelWe compared LmddA and LmddA-LLO to assess the effect

of episomal expression of a truncated LLO on T-cell prolifer-ation in healthy, tumor-free mice. We found that LmddAinduced a slight increase in the numbers of CD4þFoxP3� Tcells and CD8þ T cells in the spleens of mice at day 7 after asingle administration, andLmddA-LLO induced the increase ofthese T cells to a higher level (Fig. 5A). In contrast, the numberof CD4þFoxP3þ T cells was not changed significantly aftereither LmddA or LmddA-LLO infection (Fig. 5A). The admin-istration of LmddA-LLO resulted in a significant decrease ofTreg proportion compared with that in PBS control (Fig. 5B–D). We also examined the cell proliferation marker Ki67 inthese cells. LmddA increased the frequency and absolutenumber of Ki67þCD4þFoxP3� T cells and Ki67þCD8þ T cells,and LmddA-LLO increased the numbers of these cells to a

greater extent (Fig. 5E–G). The level of Ki67 expression inCD4þ

FoxP3�T cells andCD8þT cells was also increased accordingly(Fig. 5H). In contrast, the frequency and absolute number ofKi67þCD4þFoxP3þ T cells and Ki67 expression in CD4þ

FoxP3þ T cells were not markedly changed, indicating thatLmddA and LmddA-LLO did not induce their proliferation.

The combination of Lm–E7 and LmddA-LLO inducesregression of established TC-1 tumors

The Lm–E7 vaccine alone did not induce much expan-sion of CD4þFoxP3� T cells and CD8þ T cells (Fig. 3). Thismay account for its failure in the induction of TC-1 tumorregression. Because LmddA-LLO induced CD4þFoxP3� T-cell and CD8þ T-cell expansions (Figs. 3 and 5A), it isconceivable that the antitumor effect of Lm–E7 may beimproved in the presence of LmddA-LLO. Indeed, thecombination of Lm–E7 and LmddA-LLO induced nearlycomplete regression of established TC-1 tumors (Fig. 6A–C)

Figure2. Lm is sufficient to induceadecrease inTreg frequency.C57BL/6micewere inoculated s.c.with 1�105TC-1 tumor cells each, and immunized i.p.with0.1 LD50 LmddA (1� 108CFU) or 0.5 LD50wild-type Lm 10403S (1� 104 CFU) in PBS (100 mL) on days 10 and 17 after tumor challenge. Mice were sacrificedat day 24, and lymphocytes isolated from the spleens and tumors were analyzed by flow cytometry. A, flow cytometric profile of CD4þFoxP3þ T cellsin total CD4þ T cells. B, percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells in the spleen. C, ratio of CD4þFoxP3þ T cells to CD8þ

T cells in the spleen. D, percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells in the tumor. E, ratio of CD4þFoxP3þ T cells to CD8þ

T cells in the tumor. Data, mean � SEM; �, P < 0.05; ��, P < 0.01; and ��, P < 0.001 (Mann–Whitney test). Data are representative of three independentexperiments.






and prolonged survival of about 20% of the mice (Fig. 6C).In contrast, the addition of LmddA failed to augment Lm–E7-induced antitumor activity (Supplementary Fig. S3),indicating that endogenous LLO produced by the LmddAvector is not sufficient to enhance antitumor efficacy of theLm–E7 vaccine. As expected, CD4þFoxP3� T-cell and CD8þ

T-cell numbers were significantly increased in the spleensof mice that received combination treatment comparedwith those in mice treated with Lm–E7 or PBS (Fig. 6D).Again, because the number of CD4þFoxP3þ T cells wasrelatively unchanged, the increase of CD4þFoxP3� T-celland CD8þ T-cell numbers to a higher level induced bythe combined Lm–E7 and LmddA-LLO treatment resultedin a greater decrease in the CD4þFoxP3þ T-cell proportion(Fig. 6E–G).

Adoptive transfer of Tregs compromises the antitumorefficacy of LmddA-LLO–E7 against established TC-1tumors

LmddA-LLO–E7 did not significantly change Treg numbers,although it decreased Treg frequency (Fig. 1D–H). The ratio ofTregs toCD4þFoxP3�Tcells or toCD8þTcells has been awell-accepted parameter to determine Treg-suppressive ability. Todetermine whether the Treg proportion has any impact on theantitumor efficacy of LmddA-LLO–E7, we isolated CD4þ

CD25þ Tregs from na€�ve C57BL/6 mice and injected themi.v. into TC-1 tumor–bearingmice followed by LmddA-LLO–E7vaccination. LmddA-LLO–E7 significantly inhibited TC-1tumor growth in the mice without adoptive transfer of Tregs(Fig. 7A and B). However, in mice given Tregs, LmddA-LLO–E7was unable to significantly inhibit TC-1 tumor growth (Fig. 7Aand B). Mice receiving Tregs showed a slight increase of Tregnumber in the spleens but higher increase in tumors (Fig. 7Fand G). On the other hand, mice that received Tregs had fewerCD4þFoxP3� T cells and CD8þ T cells after being vaccinatedwith LmddA-LLO–E7 compared with those in the LmddA-LLO–E7 control, indicating that adoptive transfer of Tregsinhibits CD4þFoxP3� T-cell and CD8þ T-cell expansions

(Fig. 7F and G). These together resulted in the increase ofTreg frequency in the Treg-recipient mice (Fig. 7C–E).

DiscussionIt is well known that tumor antigen–specific CTLs play

dominant roles in killing tumor cells, and Lm, an intracellularbacteria, can deliver antigens associated with MHC class Imolecules to activate CTLs. However, it is unclear why two Lm-based vaccines, Lm-LLO–E7 and Lm–E7, induced similar levelsof HPV E7–specific CTLs in the spleens but exhibited distinctantitumor activity, with the former inducing a much strongerantitumor effect (Fig. 1A–C and Supplementary Figs. S1 and S2;ref. 14). CD8þ T cells are known to participate in killing tumorcells, as their depletion abrogated Lm-LLO–E7-induced tumorregression (14). It is also clear that a certain level of tumorantigen–specific CTLs is necessary for killing tumor cells, asLmddA-LLO, which lacks E7 expression, was unable to signif-icantly inhibit TC-1 tumor growth (Fig. 1A–C and Supplemen-tary Fig. S1). It has been proposed that Lm–E7 induced anincrease of Tregs to suppress the host immune response, thuscompromising host antitumor immunity (15). However, wefound that both Lm–E7 and LmddA-LLO–E7 decreased Tregfrequency in a TC-1 tumor model compared with that intumor-bearing mice treated with PBS control (Fig. 1D–H).Furthermore, we found that neither Lm–E7 nor LmddA-LLO–E7 significantly increased the total number of Tregs inTC-1 tumors after vaccination (Fig. 3).

In fact, we found that a major difference in vaccine efficacybetween LmddA-LLO–E7 and Lm–E7 is that the former wasable to induce a marked increase in CD4þFoxP3� T cells andCD8þ T cells, whereas the latter induced a much smallerincrease (Fig. 3). This explains how LmddA-LLO–E7 decreasedthe Treg percentage to a greater degree than did Lm–E7 (Fig.1D–H). We observed that the Lm vector alone was sufficient toincrease CD4þFoxP3� T-cell and CD8þ T-cell numbers. How-ever, with episomal expression of a truncated LLO, Lmincreased the numbers of CD4þFoxP3� T cells and CD8þ

Figure 3. Lm decreases Tregfrequency by preferentiallyinducing CD4þFoxP3� T-cell andCD8þ T-cell expansions. C57BL/6mice were inoculated s.c. with1� 105 TC-1 tumor cells each, andimmunized i.p. with 0.1 LD50LmddA-LLO–E7 (1 � 108 CFU),LmddA-LLO (1� 108CFU), LmddA(1 � 108 CFU), Lm–E7 (1 � 106

CFU), or 0.5 LD50 wild-type Lm10403S (1 � 104 CFU) in PBS (100mL) on days 10 and 17 after tumorchallenge. Mice were sacrificed atday 24, and lymphocytes isolatedfrom the tumors were analyzed byflow cytometry. Data (mean �SEM); n ¼ 3–10; �, P < 0.05;��, P < 0.01 (Mann–Whitney test).Data are representative of threeindependent experiments.

Chen et al.





T cells to a higher level, thus decreasing Treg frequency evenfurther by dilution, but there was no change in the absolutenumber of Tregs (Fig. 5). Thus, it is conceivable that LLO mayplay a critical role in inducing increases of CD4þFoxP3� T cellsand CD8þ T cells. Indeed, not only is LLO necessary for Lm toescape from the phagolysosome, but it also directly induces theexpansions of CD4þFoxP3�T cells and CD8þT cells, as neitheran LLO-minus (Dhly) Lm strain nor Dhly::pfo, an LLO-minusstrain expressing PFO that enables Lm to enter the cytoplasm,was able to induce the proliferation of CD4þFoxP3�T cells andCD8þ T cells. Transformation of a nonhemolytic LLO-mutantLm strain with an LLO-expressing plasmid restored CD4þ

FoxP3� T-cell and CD8þ T-cell expansions (Fig. 4). LLO-mediated induction of CD4þFoxP3� T-cell and CD8þ T-cellexpansions is not related to its hemolytic activity, as episomalexpression of a nonhemolytic-truncated LLO (hly::Tn917–lac)in LmddAgreatly augmented the expansions of CD4þFoxP3�Tcells and CD8þ T cells (Fig. 5). Although the expansion of both

CD4þ T-cell and CD8þ T-cell responses by LLO seems to be anantigen-nonspecific adjuvant effect, LLO may also containimmunodominant epitopes of these two cell types. Indeed,early studies have identified that LLO bears two CD4þ T-cellepitopes (residues 189–201 and residues 215–226) and oneCD8þ T-cell epitope (residues 91–99; refs. 23–25).

The antitumor effect of LmddA-LLO–E7may derive from itsability to induce a significant increase in CD4þFoxP3� T cellsandCD8þTcells. In contrast, the inability of Lm–E7 to induce amarked increase in CD4þFoxP3� T cells and CD8þ T cells mayaccount for its inefficiency in eradicating tumors, as thecombination of Lm–E7 and LmddA-LLO, which dramaticallyincreased CD4þFoxP3� T cells and CD8þ T cells comparedwith that by Lm–E7 alone, induced nearly complete regressionof established TC-1 tumors (Fig. 6). As a control, LmddA(without the LLO plasmid) in combination with Lm–E7 didnot show this effect (Supplementary Fig. S3), implying that thetruncated LLO was necessary. Our data indicate that the

Figure 4. Lm-induced expansions of CD4þFoxP3� T cells and CD8þ T cells are dependent on and mediated by LLO. C57BL/6 mice were injected i.p.with 1 � 104 CFU 10403S, Dhly, Dhly::pfo, or hly::Tn917–lac (pAM401-hly) in PBS (100 mL). Mice were sacrificed on day 7 after injection, and lymphocytesisolated from the spleens were analyzed by flow cytometry. A, T-cell numbers in the spleen. B, flow cytometric prolife of CD4þFoxP3þ T cells in totalCD4þ T cells. C, percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells. D, ratio of CD4þFoxP3þ T cells to CD8þ T cells; �, P < 0.05; ��, P < 0.01; and��, P < 0.001 (Mann–Whitney test). Data are representative of three independent experiments.






LmddA-LLO–E7-induced decrease in Treg frequency is theconsequence of an increase in the numbers of CD4þFoxP3�

T cells and CD8þ T cells. The ratio of Tregs to CD4þFoxP3�

T cells or to CD8þ T cells is critical, at least in an in vitro Treg-suppression assay, to suppress the function of CD4þFoxP3�

T cells and CD8þ T cells. Indeed, we found that increasingthe Treg ratio in vivo by adoptive transfer of Tregs into tumor-bearing mice followed by LmddA-LLO–E7 vaccination inhib-ited the expansion of CD4þFoxP3� T cells and CD8þ T cellsand consequently compromised the antitumor efficacy of thevaccine (Fig. 7).

It is noteworthy that besides preferentially inducing theexpansion of CD4þFoxP3� T cells and CD8þ T cells, truncatednonhemolytic LLO may make other contributions to improv-ing the antitumor efficacy of the LmddA-LLO–E7 vaccine. Wenoticed that although Lm–E7 and LmddA-LLO–E7 inducedsimilar expansion of E7-specific CD8þ T cells in the spleen, thisis not the case in the tumor. With episomal expression of thetruncated LLO (LmddA-LLO–E7), more E7-specific CD8þ Tcells were induced in the tumor (Supplementary Fig. S2E) thanby Lm–E7. We found that LmddA-LLO–E7 upregulated theexpression of chemokine receptors CCR5 and CXCR3 on CD4þ

Figure 5. Episomal expression of a truncated LLO in LmddA induces expansion of CD4þFoxP3� T cells and CD8þ T cells to a higher level. C57BL/6mice wereinjected i.p. with 1 � 108 CFU LmddA or LmddA-LLO in PBS (100 mL). Mice were sacrificed on day 7 after injection, and lymphocytes isolated from thespleens were analyzed by flow cytometry. A, T-cell numbers in the spleen. B, flow cytometric prolife of CD4þFoxP3þ T cells in total CD4þ T cells.C, percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells. D, ratio of CD4þFoxP3þ T cells to CD8þ T cells. E, flow cytometric prolife of Ki67þ T cells.F, percentage of Ki67þ T cells. G, fluorescence intensity of Ki67þ T cells. H, histograms showing fluorescence intensity of Ki67 on each subpopulation ofT cells after immunization with LmddA or LmddA-LLO or control PBS. Data, mean � SEM; �, P < 0.05; ��, P < 0.01; and ��, P < 0.001 (Mann–Whitney test).Data are representative of three independent experiments.

Chen et al.





FoxP3� T cells and CD8þ T cells, but not on CD4þFoxP3þ Tcells (unpublished data). CCR5 and CXCR3 are crucial for Th1and CD8þ T-cell trafficking (26). Our results suggest that LLOmay induce CD4þFoxP3� T-cell and CD8þ T-cell migration to

the tumor microenvironment through upregulation of CCR5and CXCR3. In addition, it is known that truncated LLO isrequired for the efficient secretion of antigens from Lm (27),and antigens that are not secreted from the Lm vector induced

Figure 6. Combination of Lm–E7 and LmddA-LLO induces regression of established TC-1 tumors. C57BL/6micewere inoculated s.c. with 1� 105 TC-1 tumorcells each, and immunized i.p. with 0.05 LD50 Lm–E7 (5 � 105 CFU), 0.05 LD50 LmddA-LLO (5 � 107 CFU), 0.05 LD50 Lm–E7 plus 0.05 LD50 LmddA-LLOin PBS (100 mL) on days 10 and 17 after tumor challenge. Tumors were measured twice a week using an electronic caliper and tumor volumes werecalculated by the formula, length�width�width=2. Mice were observed for survival or sacrificed on day 24, and lymphocytes isolated from the spleenswere analyzed by flow cytometry. A, average tumor volume from days 10 to 24. B, tumor volume on day 24. C, survival percentage over time. D, T-cellnumber in the spleen. E, flowcytometric prolife ofCD4þFoxP3þTcells in total CD4þTcells. F, percentage ofCD4þFoxP3þT cells relative to total CD4þTcells.G, ratio of CD4þFoxP3þ T cells to CD8þ T cells. Data, mean � SEM; �, P < 0.05; ��, P < 0.01; and ��, P < 0.001 (Mann–Whitney test).Data are representative of two independent experiments.






less effective antigen-specific antitumor immunity (28). Hence,the lack of potent antitumor activity of the Lm–E7 vectormightnot be only due to its inability to effectively expand CD4þ

FoxP3� T cells and CD8þ T cells but also due to its inefficientsecretion of antigens from Lm in the context of an infectedAPC, resulting in the priming of an ineffective antigen-specificT-cell response.

In summary, we have provided evidence demonstrating thatepisomal expression of a nonhemolytic truncated LLO in anLmddA-LLO–E7 vaccine preferentially induced the expansionsof CD4þFoxP3� T cells and CD8þ T cells, thus enhancing theantitumor activity the vaccine. Our results suggest that manyfactors, including threshold levels of antigen-specific CTLs andnontumor antigen-specific CD4þFoxP3� T cells and CD8þ

Figure 7. Adoptive transfer of Tregs compromises the antitumor efficacy of LmddA-LLO–E7 against established TC-1 tumors. 11-week-old C57BL/6 micewere injected s.c.with 1�105TC-1 tumorcells each, and i.v.withCD4þCD25þTregs (1�106 cells/each) onday9after tumor challenge.Micewere immunizedi.p. with 0.1 LD50 LmddA-LLO–E7 (1�108 CFU) in PBS (100 mL) on days 10 and 17 after tumor challenge. Tumors were measured twice a weekusing an electronic caliper, and tumor volumes were calculated by the formula, length� width�width=2. Mice were sacrificed on day 24, and lymphocytesisolated from the spleens were analyzed by flow cytometry. A, average tumor volume from days 10 to 24. B, Tumor volume on day 24. C, flowcytometric prolife of CD4þFoxP3þ T cells in total CD4þ T cells. D, percentage of CD4þFoxP3þ T cells relative to total CD4þ T cells in the spleen. E, percentageof CD4þFoxP3þ T cells relative to total CD4þ T cells in the tumor. F, T-cell number in the spleen. G, T-cell number per million tumor cells. Data, mean� SEM;�, P < 0.05; ��, P < 0.01; and ��, P < 0.001 (Mann–Whitney test). Data are representative of two independent experiments.

Chen et al.





T cells, and a decreased Treg proportion, are needed to trig-ger an effective antitumor immune response. Our study indi-cates that LLO may be a promising vaccine adjuvant in thatit preferentially induces the expansion of CD4þFoxP3� T cellsand CD8þ T cells, thus decreasing Treg frequency and favoringimmune responses to kill tumor. Our future studies will aimto investigate how LLO preferentially induces the expansion ofprotective immune cells without changing the absolute num-ber of immunosuppressive CD4þFoxP3þ T cells.

Disclosure of Potential Conflicts of InterestA. Wallecha has ownership interest (including patents) in Advaxis Inc. J.A

Berzofsky reports receiving a commercial research grant from Advaxis. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Z. Chen, N. Chong, A. Wallecha, S.N. KhleifDevelopment of methodology: Z. Chen, N. Chong, A. Wallecha, J.A. Berzofsky

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Chen, L. OzbunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Chen, A. Wallecha, J.A. BerzofskyWriting, review, and/or revision of the manuscript: Z. Chen, L. Ozbun,A. Wallecha, J.A. Berzofsky, S.N. KhleifAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.N. KhleifStudy supervision: J.A. Berzofsky, S.N. Khleif

Grant SupportThis work was supported by the Intramural Research Program of the Center

for Cancer Research, National Cancer Institute, NIH. In addition, this work wasfunded in part by Advaxis through a collaborative research and developmentagreement.

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 November 8, 2013; revised April 22, 2014; accepted May 12, 2014;published OnlineFirst May 28, 2014.

References1. Eitel J, Suttorp N, Opitz B. Innate immune recognition and inflamma-

some activation in Listeria monocytogenes infection. Frontiers Micro-biol 2010;1:149.

2. de las Heras A, Cain RJ, BieleckaMK, Vazquez-Boland JA. Regulationof Listeria virulence: PrfA master and commander. Current OpinMicrobiol 2011;14:118–27.

3. Pamer EG, Sijts AJ, Villanueva MS, Busch DH, Vijh S. MHC class Iantigen processing of Listeria monocytogenes proteins: implicationsfor dominant and subdominant CTL responses. Immunol Rev 1997;158:129–36.

4. Jia Y, Yin Y, Duan F, Fu H, Hu M, Gao Y, et al. Prophylactic andtherapeutic efficacy of an attenuated Listeria monocytogenes-basedvaccine delivering HPV16 E7 in a mouse model. Int J Mol Med2012;30:1335–42.

5. Shahabi V, Seavey MM, Maciag PC, Rivera S, Wallecha A. Develop-ment of a live and highly attenuated Listeria monocytogenes-basedvaccine for the treatment of Her2/neu-overexpressing cancers inhuman. Cancer Gene Ther 2011;18:53–62.

6. Sciaranghella G, Lakhashe SK, Ayash-Rashkovsky M, Mirshahidi S,Siddappa NB, Novembre FJ, et al. A live attenuated Listeria mono-cytogenes vaccine vector expressing SIV Gag is safe and immuno-genic inmacaques andcan be administered repeatedly. Vaccine 2011;29:476–86.

7. Seavey MM, Pan ZK, Maciag PC, Wallecha A, Rivera S, Paterson Y,et al. A novel human Her-2/neu chimeric molecule expressed byListeria 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 2009;15:924–32.

8. Maciag PC, Radulovic S, Rothman J. The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a phase I safety study ofLm-LLO-E7 inpatientswith advancedcarcinomaof the cervix. Vaccine2009;27:3975–83.

9. Sewell DA, Pan ZK, Paterson Y. Listeria-based HPV-16 E7 vaccineslimit autochthonous tumor growth in a transgenic mouse model forHPV-16 transformed tumors. Vaccine 2008;26:5315–20.

10. Souders NC, Sewell DA, Pan ZK, Hussain SF, Rodriguez A, Wal-lecha A, et al. Listeria-based vaccines can overcome tolerance byexpanding low avidity CD8þ T cells capable of eradicating a solidtumor in a transgenic mouse model of cancer. Cancer Immunity2007;7:2.

11. Liaw KL, Hildesheim A, Burk RD, Gravitt P, Wacholder S, Manos MM,et al. A prospective study of human papillomavirus (HPV) type 16 DNAdetection by polymerase chain reaction and its association withacquisition and persistence of other HPV types. J Infectious Dis2001;183:8–15.

12. AssmannG, Sotlar K. [HPV-associated squamous cell carcinogenesis].Pathologe 2011;32:391–8.

13. Szarewski A. Prophylactic HPV vaccines. Eur J Gynaecol Oncol2007;28:165–9.

14. GunnGR, Zubair A, Peters C, Pan ZK,Wu TC, Paterson Y. Two Listeriamonocytogenes vaccine vectors that express different molecularforms of human papilloma virus-16 (HPV-16) E7 induce qualitativelydifferent T-cell immunity that correlates with their ability to induceregression of established tumors immortalized by HPV-16. J Immunol2001;167:6471–9.

15. Hussain SF, Paterson Y. CD4þCD25þ regulatory T cells that secreteTGFbeta and IL-10 are preferentially induced by a vaccine vector.J Immunother 2004;27:339–46.

16. Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT,Pardoll DM, et al. Treatment of established tumorswith a novel vaccinethat enhances major histocompatibility class II presentation of tumorantigen. Cancer Res 1996;56:21–6.

17. Wallecha A,MaciagPC, Rivera S, PatersonY, Shahabi V. Constructionand characterization of an attenuated Listeria monocytogenes strainfor clinical use in cancer immunotherapy. Clin Vaccine Immunol2009;16:96–103.

18. Verch T,PanZK, PatersonY. Listeriamonocytogenes-based antibioticresistance gene-free antigen delivery system applicable to otherbacterial vectors and DNA vaccines. Infect Immun 2004;72:6418–25.

19. Geoffroy C, Gaillard JL, Alouf JE, Berche P. Purification, characteri-zation, and toxicity of the sulfhydryl-activated hemolysin listeriolysin Ofrom Listeria monocytogenes. Infect Immun 1987;55:1641–6.

20. Jones S, Portnoy DA. Characterization of Listeria monocytogenespathogenesis in a strain expressing perfringolysin O in place of lister-iolysin O. Infect Immun 1994;62:5608–13.

21. Tweten RK. Nucleotide sequence of the gene for perfringolysin O(theta-toxin) from Clostridium perfringens: significant homology withthe genes for streptolysin O and pneumolysin. Infect Immun 1988;56:3235–40.

22. JonesS, Preiter K, PortnoyDA.Conversion of an extracellular cytolysininto a phagosome-specific lysin which supports the growth of anintracellular pathogen. Mol Microbiol 1996;21:1219–25.

23. Verma NK, Ziegler HK, Wilson M, Khan M, Safley S, Stocker BA, et al.Delivery of class I and class II MHC-restricted T-cell epitopes oflisteriolysin of Listeria monocytogenes by attenuated Salmonella.Vaccine 1995;13:142–50.

24. Rodriguez-Del Rio E, Frande-Cabanes E, Tobes R, Pareja E, Lecea-Cuello MJ, Ruiz-Saez M, et al. The intact structural form of LLO inendosomes cannot protect against listeriosis. Int J Biochem Mol Biol2011;2:207–18.






25. PamerEG,Harty JT,BevanMJ.Precise prediction of adominant class IMHC-restricted epitope of Listeria monocytogenes. Nature 1991;353:852–5.

26. Franciszkiewicz K, Boissonnas A, Boutet M, Combadiere C, Mami-Chouaib F. Role of chemokines and chemokine receptors in shapingthe effector phase of the antitumor immune response. Cancer Res2012;72:6325–32.

27. Huang JM, La Ragione RM, Cooley WA, Todryk S, Cutting SM.Cytoplasmic delivery of antigens, by Bacillus subtilis enhances Th1responses. Vaccine 2008;26:6043–52.

28. Shen H, Miller JF, Fan X, Kolwyck D, Ahmed R, Harty JT.Compartmentalization of bacterial antigens: differential effects onpriming of CD8 T cells and protective immunity. Cell 1998;92:535–45.


Chen et al.




2014;2:911-922. Published OnlineFirst May 28, 2014.Cancer Immunol Res Zhisong Chen, Laurent Ozbun, Namju Chong, et al.

T Cells+ and CD8−FoxP3+Expansions of CD4E7 Vaccine Enhances Antitumor Efficacy by Preferentially Inducing

−Episomal Expression of Truncated Listeriolysin O in LmddA-LLO

Updated version

10.1158/2326-6066.CIR-13-0197doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerimmunolres.aacrjournals.org/content/suppl/2014/06/02/2326-6066.CIR-13-0197.DC1.html

Access the most recent supplemental material at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/2/9/911.full.html#ref-list-1

This article cites 28 articles, 10 of which you can access for free at:

Citing articles

http://cancerimmunolres.aacrjournals.org/content/2/9/911.full.html#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

[email protected]

To request permission to re-use all or part of this article, contact the AACR Publications Department at



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-13-0197

http://cancerimmunolres.aacrjournals.org/content/suppl/2014/06/02/2326-6066.CIR-13-0197.DC1.html

http://cancerimmunolres.aacrjournals.org/content/2/9/911.full.html#ref-list-1

http://cancerimmunolres.aacrjournals.org/content/2/9/911.full.html#related-urls

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


Episomal Expression of Truncated Listeriolysin O in LmddA-LLO-E7 Vaccine Enhances Antitumor Efficacy...

Documents

Transcript of Episomal Expression of Truncated Listeriolysin O in LmddA-LLO-E7 Vaccine Enhances Antitumor Efficacy...