Adenovirus Improves the Efﬁcacy of Adoptive T …...Research Article Adenovirus Improves the Efﬁcacy of Adoptive T-cell Therapy by Recruiting Immune Cells to and Promoting Their

Research Article

Adenovirus Improves the Efficacy of AdoptiveT-cell Therapy by Recruiting Immune Cells to andPromoting Their Activity at the TumorSiri T€ahtinen1, Susanna Gr€onberg-V€ah€a-Koskela1, Dave Lumen2, Maiju Merisalo-Soikkeli1,Mikko Siurala1,3, Anu J. Airaksinen2,4, Markus V€ah€a-Koskela1, and Akseli Hemminki1,3,5

Abstract

Despite the rapid progress in the development of novel adop-tive T-cell therapies, the clinical benefits in treatment of estab-lished tumors have remained modest. Several immune evasionmechanisms hinder T-cell entry into tumors and their activitywithin the tumor.Of note, oncolytic adenoviruses are intrinsicallyimmunogenic due to inherent pathogen-associated molecularpatterns. Here, we studied the capacity of adenovirus to overcomeresistance of chicken ovalbumin-expressing B16.OVA murinemelanoma tumors to adoptive ovalbumin-specific CD8þ T-cell(OT-I) therapy. Following intraperitoneal transfer of polyclonallyactivatedOT-I lymphocytes, control of tumor growthwas superiorin mice given intratumoral adenovirus compared with controlmice, even in the absence of oncolytic virus replication. Preexist-ing antiviral immunity against serotype 5 did not hinder thetherapeutic efficacy of the combination treatment. Intratumoral

adenovirus injection was associated with an increase in proin-flammatory cytokines, CD45þ leukocytes, CD8þ lymphocytes,and F4/80þ macrophages, suggesting enhanced tumor immuno-genicity. The proinflammatory effects of adenovirus on the tumormicroenvironment led to expression of costimulatory signals onCD11cþ antigen-presenting cells and subsequent activation of Tcells, thus breaking the tumor-induced peripheral tolerance. Anincreased number of CD8þ T cells specific for endogenous tumorantigens TRP-2 and gp100 was detected in combination-treatedmice, indicating epitope spreading. Moreover, the majority ofvirus/T-cell–treated mice rejected the challenge of parental B16.F10 tumors, suggesting that systemic antitumor immunity wasinduced. In summary, we provide proof-of-mechanism data oncombining adoptive T-cell therapy and adenovirotherapy for thetreatment of cancer. Cancer Immunol Res; 3(8); 915–25. �2015 AACR.

IntroductionImmunotherapy using ex vivo–expanded tumor-infiltrating

lymphocytes (TIL) was pioneered by Steven Rosenberg in the1980s (1), and adoptive T-cell therapy (ACT) is currentlygaining ground in the form of receptor-engineered immunecell therapy. Although CD19-expressing hematologic malig-nancies in particular seem amenable to adoptive chimericantigen receptor (CAR) T-cell therapy (2), efficacy in solidtumors is lacking possibly due to T-cell hypofunction (3–5).Recently, several approaches have been developed to improve

the activity of genetically redirected antitumor T cells in solidtumors, with the main focus being on modification of T-cellcostimulatory genes, receptor affinities, and optimal targetmolecules (3, 4). Nevertheless, these advances alone may notbe sufficient to reverse the effects of the immune-suppressivenature of the tumor microenvironment (TME). Thus, sensiti-zation of tumor milieu for T-cell therapy may prove to becrucial to achieve optimal clinical responses.

Tumor immunogenicity is the sum of several phenotypichallmarks, including the degree and type of tumor-infiltratingimmune cells, stromal cells, and expression of MHC moleculeson tumor cells. During cancer development, several immuneevasion tactics are employed by tumor cells. Immunoeditingleading to escape variants can result in failure of the hostimmune system to mount a sufficient immune responseagainst the tumor (6). Peripheral tolerance of tumor-specificT cells due to insufficient costimulation by professional anti-gen-presenting cells (APC) may result in T-cell deletion oranergy (7). Furthermore, the highly immunosuppressive TMEtypically renders infiltrating T cells incapable of killing theirtarget (tumor) cells (8). For successful cancer immunotherapy,the TME has to be immunogenic enough in order to accom-plish T-cell–mediated cytolysis and subsequent antitumorresponses.

Oncolytic virotherapy is the use of cancer cell–specific, condi-tionally replicating viruses in the treatment of cancer. Adenovirus-based oncolytic viruses have been shown to trigger potent innateand adaptive antiviral and antitumor immune responses (9),

1Cancer Gene Therapy Group, Department of Pathology and Trans-plantation Laboratory, Haartman Institute, University of Helsinki, Hel-sinki, Finland. 2Laboratory of Radiochemistry, Department of Chem-istry, University of Helsinki, Helsinki, Finland. 3TILT BiotherapeuticsLtd, Helsinki, Finland. 4Centre for Drug Research, Division of Pharma-ceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Hel-sinki, Finland. 5Department of Oncology, Helsinki University CentralHospital, Helsinki, Finland.

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

M. V€ah€a-Koskela and A. Hemminki contributed equally to this article.

Corresponding Author: Akseli Hemminki, University of Helsinki, P.O. Box 21,00014, Helsinki, Finland. Phone: 358-5044-82766; Fax: 358-9191-25465; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-14-0220-T

�2015 American Association for Cancer Research.

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while still maintaining good safety profile in patients (10).Adenovirus infection of tumors results in chemokine secretion,release of tumor-associated antigens (TAA) from infected dyingtumor cells, and activation of APCs, which recognize themultiplepathogen-associated molecular patterns (PAMP) of the virus (9).Importantly, infection also results in changes in immune cellcomposition of the tumor, supporting the argument that localimmunosuppression can be overcome (11). As the efficacy ofadoptive antitumor immunotherapy may depend on support byinnate immune responses (12), combination with oncolytic virustherapy appears an attractive approach that warrants testing.

We set out to studywhether intratumoral adenovirus injectionswould benefit adoptive T-cell therapy. We used the syngeneicC57BL/6 mouse B16.OVA melanoma tumor model, which ispoorly immunogenic (13), and thus representative of advancedhuman melanomas (which are often initially immunogenic butthen become severely immunosuppressive and immunoedited atthe advanced metastatic stage). We observed superior antitumorefficacy and increased levels of tumor-infiltrating immune cells,enhancedmaturation of APCs, and higher numbers of activated Tcells in the combination-treated group compared with the grouptreated with T-cell therapy alone. In addition, induction ofendogenous antitumor T cells was seen in mice treated withadenovirus and adoptive T-cell therapy, suggesting that the com-bination approach leads to epitope spreading and systemic anti-tumor immunity.

Materials and MethodsCells

Mouse melanoma B16 cells expressing ovalbumin were a kindgift of Professor Richard Vile (Mayo Clinic, Rochester, MN;September 30, 2010) and have been tested to be pathogen-free(by Surrey Diagnostics Ltd).Mousemelanoma B16.F10 cells wereobtained from the American Type Culture Collection (November25, 2014). B16.OVA and parental B16.F10 were maintained inRPMI, 10% FBS, 1% L-Glutamine, 1% penicillin/streptomycinsolution and propagated at 37�C and 5% CO2. G418 (5 mg/mL;Roche) was added to the culture medium for B16.OVA cells.

VirusesThe treatment viruses Ad5/3-D24 and Ad5/3-D24-hGMCSF

have been described (10, 14). Briefly, the virus consists of ahuman adenovirus serotype 5 (Ad5) nucleic acid backbone,a 5/3 chimeric fiber knob, and a 24-bp deletion (D24) in theRb binding constant region 2 of adenoviral E1. Although theAd5/3-D24-hGMCSF contains a transgene, it actually resem-bles an unarmed, replication-incompetent virus, becausehuman GM-CSF is not biologically active in mouse cells(15), nor does the human adenovirus replicate productivelyin mice (16). Replication-incompetent Ad5-Luc1 is an E1-deleted serotype 5 vector expressing firefly luciferase (17), andit was used in the preimmunization experiment to model Ad5-based immunity.

Animal experimentsAll animal protocols were approved by the experimental ani-

mal committee of the University of Helsinki (Helsinki, Finland)and the Provincial Government of Southern Finland. Four- to7-week-old C57BL/6 immunocompetent female mice (HarlanLaboratories) were implanted subcutaneously with 2.5 � 105

B16.OVA cells in 50 mL RPMI, 0% FBS, in the right flank, onetumor per mouse. Roughly 10 days after tumor implantation(when tumors became injectable, �3 mm minimum diameter),mice were divided into groups and treated on 6 consecutive dayswith intratumoral injections of either 50 mL PBS or 1 � 109 viralparticles (VP)of oncolytic adenovirus in50mLPBS. Tumor growthof mice was monitored every 2 to 3 days by using electroniccalipers, and volume was calculated as 0.52 � length � width2.For Ad5 preimmunization, mice were injected twice intramuscu-larly with replication-incompetent 1� 107 VPs of Ad5-Luc1 in 20mL of PBS 3 weeks before intratumoral virus treatments. For B16.F10 challenge, B16.OVA-bearing mice (treated with oncolyticadenovirus and adoptive transfer of 2 � 106 OT-I T cells) wereimplantedwith 2.5� 105 B16-F10 cells in 50mL RPMI, 0%FBS, inthe left flank on day 13 after transfer. B16.F10-challenged micewere followed for 14 additional days for tumor emergence ortumor growth.

Adoptive transfer of OT-I cellsOn the first day of the virus treatment, the mice also received 5

� 105 to 2� 106 CD8a-enriched and expanded splenocytes from4- to 8-week-old C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice(ref. 18; The Jackson Laboratories), genetically engineered to haveonly ovalbumin-specific CD8 T-cell receptors (TCR), in 100 mLRPMI and 0%FBS. CD8a enrichmentwas performed by depletionof nontarget cells with mouse CD8a (Ly-2) MicroBeads as per themanufacturer's instructions (Miltenyi Biotech). Enriched cellswere expanded in numbers for 5 days in lymphocyte medium(RPMI, 10% FBS, 20 mmol/L L-glutamine, 1� penicillin/strepto-mycin solution, 15 mmol/L HEPES, 50 mmol/L 2-mercaptoetha-nol, 1 mmol/L Na pyruvate) in the presence of 160 ng/mLrecombinant murine IL2 (R&D Systems) and 0.3 mg/mL solubleanti-mouse CD3e antibody clone 145-2C11 (Abcam). Adminis-tration into the intraperitoneal cavity was based on the notionthat total number and accumulation kinetics of adoptively trans-ferred OT-I cells into B16.OVA tumors are independent of injec-tion route (19).

Statistical analysisStatistical analysis was performed with GraphPad Prism 6

(GraphPad Software Inc.) using unpaired, two-tailed Student ttest. Tumor volume data were analyzed by repeated measuresANOVA on log-transformed values with SPSS version 21 (SPSSIBM). Differences were considered statistically significant when Pvalues were less than 0.05.

ResultsInfection with adenovirus results in low-level antitumorimmune responses

To study whether adenovirus infection results in antitumorimmune responses in mice with melanoma, subcutaneous B16.OVA tumors were injected intratumorally with either PBS or5/3 fiber-chimeric adenovirus for 6 consecutive days. As murinecells are poorly permissive to human adenovirus (16), multipleintratumoral virus injections were used to mimic virus repli-cation–induced inflammation. Relative tumor volumes in Fig.1A show that, compared with the PBS-treated mock group, Ad-treated mice showed minor tumor growth control (5,495% �1,679% vs. 2,067% � 329%, respectively). A declining copynumber of virus genomes in the tumors over time suggests

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induction of antiviral immune responses and confirms previ-ous reports regarding the absence of productive amplificationof human adenovirus in mice (Fig. 1B). These results are in linewith previous reports showing that intratumoral administra-tion of adenovirus can induce antitumor immunity (20, 21).Correspondingly, we saw virus-induced increase in secretion ofintratumoral IFNg (Fig. 1C), which was associated with a trendtoward upregulation of IFNg-inducible chemokines RANTES,MIP-1a, and MCP-1 on day 10 (Supplementary Fig. S1A–S1C).Flow cytometric analysis of the tumors revealed that adenovirusinjections increased the number of TILs (Fig. 1D) and induced alow-level endogenous antitumor response in the form of CD8þ

T cells specific for melanoma antigen glycoprotein 100 (gp100)but not for tyrosinase-related protein 2 (TRP-2; Fig. 1E and F).We did not detect significant differences in CD8þ T cells specificfor the xenoantigen chicken ovalbumin expressed by the B16.OVA cells (data not shown).

Adenovirus treatment enhances the efficacy of adoptive T-celltherapy despite inducing antiviral immunity

To assess the impact of adenovirus treatment on adoptive T-cell therapy, mice bearing subcutaneous B16.OVA tumors were

given intraperitoneal injections of polyclonally activated (withanti-CD3e and IL2) 5 � 105 CD8þ-enriched splenocytes fromOT-I mice (Supplementary Fig. S2A), in which all CD8þ T cellscarry MHC-I–restricted ovalbumin peptide SIINFEKL-specificTCRs. Beginning on the same day, tumors were either leftnoninjected or injected with PBS or 5/3 fiber-chimeric adeno-virus for 6 consecutive days (Supplementary Fig. S2B). Inter-estingly, superior tumor growth control was observed in thegroup treated with the combination of virus þ T cells com-pared with control groups receiving PBS injections and OT-Icells or OT-I cells alone (631%� 200% vs. 4,646%� 827% vs.5,565% � 1,221%, respectively; Fig. 2A). Increasing OT-I doseto 2 � 106 cells (roughly equivalent to 1 � 108 cells/kg)resulted in somewhat higher efficacy in the virus/T-cell groupversus the PBS/T-cell group (198% � 36% vs. 3,451% �1,620%, respectively) but did not alter the shape of the tumorgrowth curves (Fig. 2B). According to earlier reports (22, 23),using even higher cell numbers in adoptive transfer does nottranslate into long-term survival of B16.OVA-bearing mice,highlighting the immunosuppressive nature of the model,which is in accord with clinical observations suggesting a lackof efficacy in T-cell therapies used as single agents for the

Figure 1.Injection of oncolytic adenovirusalone induces low-level antitumorimmunity in a poorly permissivemouse model of melanoma. A, micebearing syngeneic B16.OVA tumors ontheir right flank were treatedintratumorally with either 50 mL PBSor 1 � 109 VPs of 5/3 fiber chimericoncolytic adenovirus in PBS for 6consecutive days (0–5), and tumorgrowthwasmonitored (n¼ 20 on day0). A set of mice was sacrificed ondays 6, 10, and 14 after treatment wasstarted, and tumors werehomogenized for further analysis(n¼ 4–5). B, virus kineticswas studiedby qPCR specific for the adenovirusE4 region. C, expression levels ofintratumoral IFNg were measuredwith CBA Flex sets. D, proportion oftumor-infiltrating cytotoxiclymphocytes (TIL) and (E and F)endogenous antitumor CD8þ T cellswas analyzed with flow cytometry.Data, mean � SEM. � , P � 0.05; and�� , P � 0.01 by repeated measuresANOVA (A) or unpaired t test (C–F).ns, not statistically significant; OVA,ovalbumin.

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treatment of solid tumors. Keeping in mind that humanadenovirus does not productively replicate in or lyse murineB16.OVA cells, the observed synergy between adenovirus andthe transferred T cells was likely the result of favorable immuneresponses instigated by infection per se.

As most humans have preexisting memory T cells againstseveral adenovirus serotypes (especially Ad5), we also studiedthe role of preimmunization by injecting a group of mice twicewith 1 � 107 VPs of Ad5-Luc1 intramuscularly 3 weeks beforethe aforementioned treatment regimen. As shown in Fig. 2C,preexisting antiviral immunity did not hinder the efficacy ofvirus/T-cell combination therapy. Furthermore, similar levels ofantiviral T cells, characterized by IFNg production of CD8þ

CD44þ splenocytes upon HAdV-5 peptide stimulation, were

generated by both preimmunization and treatment regimens(Fig. 2D).

Trafficking of transferred T cells into tumor is not significantlyenhanced by adenovirus injection

To investigate the mechanisms of adenovirus-facilitatedboosting of adoptive T-cell therapy, the number and locali-zation of OT-I cells in tumors and periphery were studied exvivo by flow cytometry and in vivo by SPECT/CT imaging.Surprisingly, no statistically significant differences in thelevels of tumor-infiltrating OT-I cells between groups at anytime point were detected (Fig. 2E). Overall, the amount ofOT-I infiltration decreased over time in all treatment groups(on average from 3.8% to 1.1%). These results were further

Figure 2.Combining oncolytic adenovirus injections with adoptive transfer of T cells improves treatment efficacy but not through enhanced tumor trafficking of transferredcells. B16.OVA-bearing mice were adoptively transferred with (A, E) 5 � 105 or (B) 2 � 106 CD8aþ-enriched OT-I lymphocytes intraperitoneally, and tumorswere either left noninjected or injected with 50 mL PBS or 1 � 109 VPs of 5/3 fiber chimeric oncolytic adenovirus in PBS (n ¼ 13 on day 0). Tumor growth wasmonitored every 2 to 3 dayswith an electronic caliper. C andD, to study the effect of preexisting adenoviral immunity on the efficacy of combination treatment,micewere preimmunized intramuscularly with 1 � 107 VPs of serotype 5 adenovirus (Ad5) 3 weeks before intratumoral virus treatments (n ¼ 5). D, antiviral T-cellresponses were evaluated by the frequency of CD8þ CD44þ splenocytes positive for IFNg following peptide stimulation. E, levels of transferred ovalbumin-specific OT-I cells in the tumors were quantified on days 1, 7, and 14 after transfer using SIINFEKL-H-2Kb pentamer and flow cytometry (n¼ 3–6). F, CD8aþ-enrichedOT-I cells (6 � 106) were labeled with 111In-oxine and adoptively transferred into B16.OVA-bearing mice to quantify early biodistribution of transferred cellsusing nanoSPECT/CT (n ¼ 3). Percentage of total radioactivity normalized to tumor size (mm3) on days 4 and 7 after transfer. Data, mean � SEM. � , P � 0.05;�� , P � 0.01; and �� , P � 0.0001 by unpaired t test (D) or by repeated measures ANOVA (A–C). ns, not statistically significant; OVA, ovalbumin.

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corroborated by an imaging experiment, where accumulationof 111In-oxine–radiolabeled OT-I cells into tumors was quan-tified by SPECT/CT in vivo and by gamma counting ex vivo(Fig. 2F, Supplementary Fig. S3). Data on early biodistribu-tion showed presence of transferred OT-I lymphocytes in thetumor, spleen, axillary lymph nodes, and the lymphaticsystem (Supplementary Fig. S3C). Despite a slight increasein radioactivity in virus-treated tumors, signal levels detectedbetween treatment groups at different time points after trans-fer did not reach statistical significance (Fig. 2F, Supplemen-tary Fig. S3D).

Combination of adoptive T-cell therapy and adenovirusincreases the level and activation of endogenousanti-melanoma T cells

As we did not see clear evidence of increased trafficking ofadoptively transferred T cells into tumors, we investigated tumorlocalization of endogenous T cells that may cooperate withadoptively transferred T cells for antitumor efficacy (24), as wellas other immune cell subsets, such as natural killer (NK) or CD4þ

cells, which may in other experimental systems control B16tumors (25, 26). On day 14 after transfer, virus-treated tumorscontained more CD45þ leukocytes, CD3þ T lymphocytes, and

Figure 3.Combination therapy leads toincreased levels of TILs and induces anendogenous antitumor T-cell responsetargeting melanoma antigens. B16.OVA-bearing mice were injectedintraperitoneally with 5 � 105

CD8aþ-enriched OT-I lymphocytesand treated intratumorally with either50 mL PBS or 1 � 109 VPs of 5/3 fiberchimeric oncolytic adenovirus in PBSor left noninjected. Levels of tumor-infiltrating (A) CD45þ leukocytes, (B)CD3þ T lymphocytes, and (C) CD8þ

cytotoxic T lymphocytes wereassessed on day 14 after transfer(n ¼ 5–6). D, representative FACSplots showing TRP-2– and gp100-specific CD3þ CD19� CD8þ T-cellpopulation. E and F, proportion ofendogenous antitumor CD8þ TILs wasanalyzed with flow cytometry.Data, mean � SEM. � , P � 0.05; and�� , P � 0.01 by unpaired t test. ns, notstatistically significant; OVA,ovalbumin.






CD8þ cytotoxic T lymphocytes than control tumors (Fig. 3A–C),whereas the levels of CD19þ B lymphocytes, CD4þ T lympho-cytes, and NK1.1þ NK cells did not differ significantly betweenthe groups (Supplementary Fig. S4A–S4C). Upon pentamer anal-

ysis, we detected a statistically significant increase in tumor-infiltrating CD8þ T cells specific for MHC-I–restricted endoge-nous TRP-2 and gp100 antigens in the virus-treated group com-pared with that of controls (Fig. 3D–F). To establish the level of

Figure 4.Combination of oncolytic adenovirusand adoptive T-cell transfer results insystemic antitumor immune response.Mice with subcutaneous B16.OVAtumors on their right flank weretreated intraperitoneally with 2 � 106

CD8aþ-enriched OT-I lymphocytesand intratumorally with either 50 mLPBS or 1 � 109 VPs of 5/3 chimericoncolytic adenovirus in PBS. Tumorswere measured every 2 to 3 days withan electronic caliper, and mice weresacrificed at specific time points. A,count of IFNg-producing CD8þ TILsafter 6-hour ex vivo stimulation withPMA/ionomycin. B, representativeELISPOT wells showing secretion ofIFNg by splenocytes from micesacrificed on day 14 after transfer(n ¼ 3). C, number of spot-formingcolonies (SFC) per 1 � 105 livesplenocytes indicating broad immuneresponse against both viral and tumorpeptides. D, tumor metastases in thedLNs (melaninþ). E, 13 days aftertransfer, Ad- and OT-I–treated miceand a set of tumor-na€�ve mice wereinjected with 2.5 � 105 parental B16.F10 cells into the left flank (n ¼ 6–10).Tumor emergence and tumor growthwere followed for 14 days afterimplantation. Data, mean � SEM.� , P� 0.05; �� , P� 0.01; �� , P�0.001;and �� , P� 0.0001 by unpaired t test(A–C) or by repeated measuresANOVA (E). OVA, ovalbumin.

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activation of these cells, we performed a flow cytometric analysisof intracellular IFNg and found that virus-treated tumors wereenriched for IFNg-expressing CD8þ T cells compared with controltumors (Fig. 4A). In addition, an IFNg ELISPOT assay withsplenocytes incubated with mouse CD8–specific peptides forovalbumin, human adenovirus, and melanoma antigens gp100and TRP-2 revealed that T-cell–mediated immune reactions to allstudied peptide epitopes were stronger in virus-treated micecompared with control mice treated with PBS (Fig. 4B and C).

Finally, as a dramatic reduction of draining lymph node tumorburden was observed in adenovirus-treated mice compared withPBS-injected animals (Fig. 4D), wewanted to studywhether thesemice could reject parental B16.F10 tumors not expressing thetargetmolecule ovalbumin. Indeed, when B16.OVA-bearingmicewere treated with the virus/T-cell combination, 67% of micerejected the challenge of B16.F10 tumors, whereas only 10% ofna€�vemicewere tumor-free onday 14 after implantation (Fig. 4E).This observation led us to conclude that combination treatmentcan result in systemic tumor control, despite local injection ofvirus. Overall, the data indicate that combination of adenovirus

and adoptive cell therapy resulted in induction of specific anti-tumor immunity to a degree that could not be achieved by eitheradenovirus or T-cell transfer alone.

Adoptive T-cell therapy and virotherapy specifically result insimultaneous upregulation of T-cell activation markers andreduction of anergy markers

Induction of endogenous antitumor CD8þ T-cell activity in thevirus/T-cell–treated tumors implied that the melanoma-inducedT-cell immunosuppressivemechanisms were perturbed. To assessthis, we compared activation markers CD25 and CD69 withknown anergy markers CTLA-4, PD-1, and TIM-3. Virus-treatedmice had a higher level of CD8þCD25þCD69þ TILs 14 days aftertransfer compared with that of the PBS control group (Fig. 5A). Atthe same time, while the expression levels of immune checkpointmolecules CTLA-4 and PD-1 were equal in all treatment groups,TIM-3 expressionwas significantly reduced inAd- andPBS-treatedtumors compared with noninjected control tumors (Fig. 5B,Supplementary Fig. S5). Thus, simultaneous T-cell activation andreduction of anergy markers occurred only in the virus/T-cellcombination group.

Adenovirus-mediated inflammation enhances antigen cross-presentation

To study how tumor immunosuppression may have beenovercome in the virus/T-cell combination group, we assessed thedegree of APC activation in relation to putative immunosuppres-sive stromal cell presence. Notably, although total levels ofCD11cþ dendritic cells did not increase postvirus in either thetumor or tumor-draining lymph node (dLN), a higher number ofCD11cþ cells expressing maturation marker CD86 was observedin the virus-treated group (Fig. 6A–D), arguing for virus-triggeredactivationofAPCs. In addition, the frequency ofCD11bþmyeloidcells and CD11bþ F4/80þ macrophages was increased in virus-treated tumors compared with the PBS group (Fig. 6E and F).However, these cells did not exhibit putative cell surface markersof immunosuppressivemyeloid-derived suppressor cells (MDSC)orM2macrophages, cell types that have been proposed to be ableto interfere with T-cell induction (refs. 27, 28; Supplementary Fig.S4D–S4G). Moreover, because antigen presentation occurs in thelymphatic system, which is subverted by melanoma cells to animmunosuppressive phenotype (29, 30), we enumerated lym-phatic endothelial cells (LEC), presented asCD45�CD31þ gp38þ

cells, in the dissociated tissues. We observed significantly fewerLECs in adenovirus-injected tumors and dLNs compared withthose in PBS-injected tumors (Fig. 7A andC). In addition, levels ofCD45� CD31� gp38þ fibroblastic reticular cells (FRC) weresignificantly higher in the adenovirus-treatedmice comparedwithboth control groups (Fig. 7B and D), possibly facilitating inter-actions between APCs and T cells (31, 32). Taken together, ourresults suggest that B16 immunosuppression, which normallyprevents APC-mediated T-cell activation and leads to T-cell tol-erance, was overcome by the combined action of transferredCD8þ T cells and adenovirus infection.

DiscussionConsidering the good safety profile of oncolytic adenovirus in

patients and the potential of tumor-specific T cells, merging thesetwo forms of immunotherapy is an appealing approach for cancertreatment. In a recent study, Nishio and colleagues reported that

Figure 5.Mice bearing subcutaneous B16.OVA tumors were treated intraperitoneallywith 5� 105CD8aþ-enrichedOT-I lymphocytes and intratumorallywith either50 mL PBS or 1 � 109 VPs of 5/3 chimeric oncolytic adenovirus or leftnoninjected (n ¼ 5–6). A, activation status of CD8þ TILs was evaluated bysurface expression of T-cell activation markers CD25 and CD69 and (B) byintratumoral expression of immune checkpointmolecule TIM-3. Data,mean�SEM. � , P � 0.05; and �� , P � 0.01 by unpaired t test. ns, not statisticallysignificant; OVA, ovalbumin.






adoptive T-cell therapy can be enhanced by oncolytic adenovirusarmed with chemokine RANTES and cytokine IL15, thus improv-ing the trafficking and survival of CAR T cells in an immunode-ficientmousemodel (33). Although the results from these authorsare valuable, the lack of an immune system overlooks many keyattributes of ACT. We set out to study the immunologic aspects ofthe combination of adenovirus and adoptive T-cell therapy in afully immunocompetent setting because tumor-infiltratingimmune cells and components of stroma can have amajor impacton the treatment outcome. For this, we used the B16 model, ahighly immunosuppressive murine model of melanoma, whichhas been referred to as the ultimate preclinical test for immu-notherapies (13). B16 cells expressing xenoantigen ovalbumin aregenerally considered immunogenic (based on tumor incidencerate after implantation and the induction of NK cells), but this

model also exerts nonimmunogenic properties (very low expres-sion of MHC class I, highly aggressive growth, and resistance toOT-I T cells). Anti–CTLA-4 monoclonal antibodies, which havebeen approved by the FDA for treatment of human melanoma,lack efficacy in this model (34).

Reflecting the shortcomings of single-agent immunothera-pies in advanced and immunosuppressive tumors, the treat-ment effect of either adenovirus or tumor-specific T cells alonein the B16.OVA model remained poor, whereas prominenttumor suppression was achieved by combining these twotreatments (Figs. 1A and 2A). Unexpectedly, the number ofadoptively transferred cells present at the tumor could notexplain the superior antitumor efficacy, as tumor traffickingwas not significantly enhanced by adenovirus (Fig. 2E and F).Instead, activation of tumor-infiltrating T cells was increased

Figure 6.Maturation of dendritic cells isincreased in dLNs and tumorsfollowing combination therapy.B16.OVA-bearing mice wereadoptively transferred with 5 � 105

CD8aþ-enriched OT-I lymphocytesintraperitoneally, and tumors wereeither left non-injected or injected with50 mL PBS or 1 � 109 VPs of 5/3 fiberchimeric oncolytic adenovirus inPBS (n ¼ 5–6). A and C, proportion ofCD11cþ dendritic cells and (B, D)relative levels of dendritic cellsexpressingmaturationmarker CD86ontheir cell surface were analyzed on day14 after transfer from dLNs and tumors.E, levels of CD11bþmyeloid cells and (F)CD11bþ F4/80 macrophages weredetected from day 14 tumors. Data,mean � SEM. � , P � 0.05; �� , P � 0.01;and ��, P � 0.001 by unpaired t test.ns, not statistically significant; OVA,ovalbumin.

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following adenovirus treatment (Figs. 4A and 5A), indicatingthat the phenotypic activation status is essential in T-celleffector function and a key to successful immunotherapy.

Although resulting in significant tumor growth control, theunaltered status in tumor expression of immune checkpointmolecules CTLA-4 and PD-1 (Supplementary Fig. S5) mayexplain why combination treatment with ACT and adenoviruswas incapable of curing mice from established tumors.Although systemic antitumor immunity contributed to antitu-mor effects and reduced dLN metastasis (Figs. 2B and 4D andE), the microenvironment remained highly immunosuppres-sive even in adenovirus-treated tumors. On the other hand,adenovirus infection did not increase the intratumoral levels ofimmunosuppressive cells (Supplementary Fig. S4D–S4G). Cur-rently several immunotherapies are being combined withimmune checkpoint inhibitors (35), raising the question ofwhether checkpoint blockade in our approach would enhanceefficacy further.

Besides TCR engagement, costimulatory signals on APCs areneeded to induce T-cell activation. After encountering and ingest-ing tumor antigens, APCs such as dendritic cells andmacrophagesundergo maturation and migrate to local lymph nodes. Combi-nation of peptide:MHC complexes and costimulatory surfacemolecules (such as CD80 or CD86) on APCs activates T cells,which, in turn, triggers T-cell proliferation, differentiation, andmigration (36).WithoutAPC costimulation, TCR interactionwithspecific peptide:MHC causes antigen-specific tolerance (7).Indeed, upregulation of CD86 maturation marker on dendriticcells was observed after virus treatment in both tumor and dLN

(Fig. 6B and D). This enhanced DC activation through pathogenalarm signals may have contributed to the induction of endog-enous antitumor T cells (Figs. 3D–F and 4B and C).

Macrophages are highly plastic immune cells, and their phe-notype, ranging from protumor to antitumor, is strongly depen-dent on the signals from the TME (37).Whether themacrophagesdetected as shown in Fig. 6F acted as professional APCs (38) or iftheir accumulation was part of antiviral response (39), theirimmunosuppressiveness was not increased (Supplementary Fig.S4F–S4G). Despite growing evidence that tumor-associatedmacrophages (TAM) are linked to poor prognosis in several cancertypes (37), further experiments would be needed to confirm theactual role of these macrophages detected in the adenovirus-treated tumors.

An important aspect is the immune response against adenovi-rus (Figs. 2C andD and 4B and C), whichmight have contributedto overall efficacy through immune targeting of virus-infectedtumor cells (21). In human patients, a correlation betweenantiviral and antitumor T-cell immunity has been reported(40). As human adenoviruses do not lyse mouse cells, the onco-lytic effect is not accounted for in our B16.OVA model. It istempting to speculate whether active oncolysis would haveresulted in further efficacy, as adenoviral replication would resultin continuous viral spread, tumor debulking activity, subsequentreduction in mass-related immunosuppressive mechanisms, andmore pronounced levels of antiviral and possibly antitumorresponses. Unfortunately, current preclinical animal models donot allow us to study this possibility in the context of adoptive T-cell transfer.

Figure 7.Presence of LECs and FRCs in tumorsand dLNs may contribute to tumorimmunogenicity. Mice bearingsyngeneic B16.OVA tumors weretreated with 5 � 105 CD8aþ-enrichedOT-I lymphocytes intraperitoneally,and tumors were either not injected orinjected with 50 mL PBS or 1 � 109 VPsof 5/3 fiber chimeric oncolyticadenovirus in PBS (n ¼ 5–6).Proportion of LECs in (A) dLN and (C)tumor was analyzed with flowcytometry by gating on CD45� CD31þ

gp38þ cell population. Increased LEClevels in PBS-treated tumors suggestan active role of lymphatics in dLNmetastasis, as seen in Fig. 5. CD45�

CD31� gp38þ FRCs in dLN (B) andtumor (D) of adenovirus-treated miceindicate enhanced APC:T-cellinteraction. Data, mean � SEM.� , P� 0.05; �� , P� 0.01; �� , P� 0.001;and �� , P� 0.0001 by unpaired t test.ns, not statistically significant; OVA,ovalbumin.






Many previous studies combining virotherapy and ACT havefocused on oncolytic viruses encoding TAAs, thus relying mostlyon oncolysis and vaccination effect against a particular tumorepitope (8, 41–43). In our hands, the mere adenoviral backboneacted as an adjuvant and boosted the endogenous antitumorimmunity elicited by ACT. The increase in frequency of TRP-2 andgp100-specific T cells in the adenovirus-treated mice (Figs. 3D–Fand 4B and C) indicated that intratumoral adenovirus treatmentcombined with ACT can induce a potent antitumor response andlead to epitope spreading even in the absence of active oncolysis.Our results with adoptive transfer of ovalbumin-targeted T cellsare in accord with previous immunotherapy reports, in whichepitope spreading and broadened antitumor responses have beendetected after targeting a single defined TAA (44–47). Further-more, T-cell transfer has been shown to enable repertoire expan-sion through induction of endogenous antitumor T cells (48).From the clinical perspective, the importance of epitope spreadingcannot be overstated. Tumors have tremendous capacity foradaptation under selective pressure, such as immune responseagainst a single epitope, or blocking of a single pathway, explain-ing the failure of many cancer vaccines and targeted therapies. Inthis aspect, epitope spreading and systemic antitumor immunitymay be a prerequisite of successful immunotherapy of advancedtumors.

In conclusion, the results described here focus on the immu-nologic synergy between adoptive T-cell transfer and adenovi-rus treatment. Importantly, these two therapies are not merelyan attractive combination, but could represent an effectivemultimodal approach to treat solid tumors. Conversely,because it has been seen that single-agent oncolytic adenoviruslacks curative power in most patients with advanced andmetastatic tumors (10, 49), the best use of the technologycould well be related to the tremendous capacity for immu-nostimulation and breaking of tumor-associated tolerance(9, 40). We propose that adding an adenoviral danger signalcan enhance the efficacy of T-cell–based approaches and pro-vide the means to immunologically target and destroy other-wise nonimmunogenic solid tumors. Given the encouragingsafety of oncolytic adenovirus in patients, this combinationrepresents a highly feasible translation into clinical trials.

Disclosure of Potential Conflicts of InterestM. Siurala is a staff scientist at TILT Biotherapeutics Ltd. A. Hemminki is CEO

at TILT Biotherapeutics Ltd., reports receiving a commercial research grant fromOncos Therapeutics, and has ownership interest (including patents) in TILTBiotherapeutics and Oncos Therapeutics. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: S. T€ahtinen, A.J. Airaksinen, M. V€ah€a-Koskela,A. HemminkiDevelopment of methodology: S. T€ahtinen, S. Gr€onberg-V€ah€a-Koskela,D. Lumen, M. V€ah€a-KoskelaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. T€ahtinen, D. Lumen, M. Siurala, A.J. Airaksinen,M. V€ah€a-KoskelaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. T€ahtinen, D. Lumen, A.J. Airaksinen, M. V€ah€a-Koskela, A. HemminkiWriting, review, and/or revision of the manuscript: S. T€ahtinen, M. Siurala,A.J. Airaksinen, M. V€ah€a-Koskela, A. HemminkiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Gr€onberg-V€ah€a-Koskela, D. Lumen,M. Merisalo-Soikkeli, M. V€ah€a-Koskela, A. HemminkiStudy supervision: M. V€ah€a-Koskela, A. Hemminki

AcknowledgmentsThe authors thank Marjo Vaha, Saila Pesonen, Simona Bramante, Mari

Hirvinen, Vincenzo Cerullo, Saija Kaikkonen, andMinna Oksanen for excellentexpert assistance.

Grant SupportThis study has been supported by TILT Biotherapy Ltd, BiocentrumHelsinki,

Biocenter Finland, the European Research Council, the ASCO Foundation,HUCH Research Funds (EVO), the Sigrid Juselius Foundation, the Academyof Finland, the Emil Aaltonen Foundation, the Finnish Konkordia Fund, theUniversity of Helsinki Funds, the Finnish Cultural Foundation, and the FinnishCancer Organizations. A. Hemminki is the Jane and Aatos Erkko Professor ofOncology at the University of Helsinki.

The costs of publication of this articlewere defrayed inpart 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 22, 2014; revised April 16, 2015; accepted May 7, 2015;published OnlineFirst May 14, 2015.

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2015;3:915-925. Published OnlineFirst May 14, 2015.Cancer Immunol Res Siri Tähtinen, Susanna Grönberg-Vähä-Koskela, Dave Lumen, et al. TumorRecruiting Immune Cells to and Promoting Their Activity at the Adenovirus Improves the Efficacy of Adoptive T-cell Therapy by

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