Enhanced Tumor Control with Combination mTOR and PD-L1 ...MOC1 tumor cells, an effect abrogated by depleting inﬁltrating CD8 T cells from the tumor microenvironment. These data conﬂict

Research Article

Enhanced Tumor Control with CombinationmTOR and PD-L1 Inhibition in Syngeneic OralCavity CancersEllen C. Moore1, Harrison A. Cash1, Andria M. Caruso1, Ravindra Uppaluri2,James W. Hodge3, Carter Van Waes1, and Clint T. Allen1,4

Abstract

Significant subsets of patientswith oral cancer fail to respond tosingle-agent programmed death (PD) blockade. Syngeneic mod-els of oral cancer were used to determine if blocking oncogenicsignaling improved in vivo responses to PD-L1 monoclonal anti-body (mAb). Anti–PD-L1 enhanced durable primary tumor con-trol and survival when combined with mTOR (rapamycin), butnot in combination with MEK inhibition (PD901) in immuno-genic MOC1 tumors. Conversely, PD-L1 mAb did not enhancetumor control in poorly immunogenic MOC2 tumors. Rapamy-cin enhanced expansion of peripheral antigen-specific CD8 T cellsand IFNg production following ex vivo antigen stimulation. MoreCD8 T cells infiltrated and were activated after PD-L1 mAbtreatment inmice with immunogenic MOC1 tumors, which werestable or increased by the addition of rapamycin, but suppressedwhen PD901 was added. Rapamycin increased IFNg production

capacity in peripheral and tumor-infiltrating CD8 T cells. In vivoantibody depletion revealed a CD8 T-cell–dependent, and notNK cell–dependent mechanism of tumor growth inhibition aftertreatment with rapamycin and PD-L1 mAb, ruling out significanteffects from NK cell–mediated antibody-dependent cellular cyto-toxicity. Rapamycin also enhanced IFNg or PD-L1 mAb treat-ment–associated induction of MHC class I expression onMOC1 tumor cells, an effect abrogated by depleting infiltratingCD8 T cells from the tumor microenvironment. These dataconflict with traditional views of rapamycin as a universalimmunosuppressant, and when combined with evidenceof enhanced antitumor activity with the combination of rapa-mycin and PD-L1 mAb, suggest that this treatment combina-tion deserves careful evaluation in the clinical setting.Cancer Immunol Res; 4(7); 611–20. �2016 AACR.

IntroductionCarcinogen-associated head and neck squamous cell carci-

noma (HNSCC) portends poor disease-specific survival, andtreatment often leaves patients functionally disabled (1, 2). Asignificant subset of patients with HNSCC appear to haveimmunogenic tumors (3), and preliminary results of responserates to single-agent checkpoint inhibitors, such as monoclonalantibodies (mAb) that block the programmed death (PD)pathway, have been promising (4). To enhance the proportionof patients that respond to checkpoint inhibitor therapy, com-binations of checkpoint inhibitors with standard cytotoxic andtargeted therapies are being considered (5). However, many

standard cytotoxic and targeted therapies can also suppress thefunction of effector immune cells, making these combinationapproaches challenging (6).

Patients with HNSCC frequently have coactivation of the phos-phoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) and mitogen-activated protein kinase kinase/extracellularrelated signal kinases 1 and2 (MEK/ERK1/2)pathways (7),makingthese attractive targets forHNSCC treatment. Rapamycin is anFDA-approvedmTOR inhibitor (8)with preclinical and clinical promisein the treatment of HNSCC (9, 10). The MEK1/2 inhibitorPD0325901 (PD901) is an investigational small molecule withclinical activity (11) that potentiates the antitumor effects of single-agent PI3K/mTOR inhibition in HNSCC xenografts (12). PI3K/mTOR and MEK/ERK signaling promotes the development of amyeloid-rich immunosuppressive tumor microenvironmentthrough myeloid chemokine expression (13).

We previously showed in an immunogenicmodel of oral cavitycancer that tumor growth inhibition seen after rapamycin therapyis CD8 dependent (14). MEK inhibitors and immune therapieshave combinatorial and synergistic effects in other solid tumormodels (15). We hypothesized that mTOR inhibition with rapa-mycin and MEK1/2 inhibition with PD901 alone or in combina-tion may enhance the antitumor effects of PD-L1 mAb treatment.The Mouse Oral Cancer (MOC) model is carcinogen-induced,fully syngeneic on a C57BL/6 genetic background, and consists ofcell lines with genetic alterations that mirror that of human oralcancer (16, 17). MOC1 cells display a high somatic alteration rateand generate tumors with slow primary tumor growth that do notmetastasize, have high PD-L1 and MHC class I expression, and

1Tumor Biology Section, Head and Neck Surgery Branch, NationalInstitutes of Deafness and Other Communication Disorders, NationalInstitutes of Health, Bethesda, Maryland. 2Department of Otolaryn-gology-Head and Neck Surgery, Washington University in St. LouisSchool of Medicine, St. Louis, Missouri. 3Laboratory of Tumor Immu-nology and Biology, Center for Cancer Research, National CancerInstitute, Bethesda, Maryland. 4Department of Otolaryngology-Headand Neck Surgery, Johns Hopkins School of Medicine, Baltimore,Maryland.

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

Corresponding Author: Clint Tanner Allen, National Institutes of Health, CRC4-2732, Bethesda, MD 20892. Phone: 301-827-2036; Fax: 301-402-1140; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-15-0252

�2016 American Association for Cancer Research.

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show robust effector immune cell infiltration (18, 19). Converse-ly, MOC2 cells have fewer genetic alterations, generate aggressivetumors that metastasize early to draining lymph nodes, have verylow PD-L1 and MHC class I expression, and demonstrate limitedeffector immune cell infiltration. Use of these cell lines allows themodeling of both highly and poorly immunogenic human oralcancer, and the presence of PD-L1 in the tumormicroenvironmentof both models provides a rationale for using PD-L1–targetingcheckpoint inhibition.

We report the results of our investigation into the combinationof rapamycin and/or PD901 with PD-L1mAb treatment in highly(MOC1) and poorly (MOC2) immunogenic oral cavity cancers.The addition of mTOR inhibition with rapamycin to PD-L1 mAbtreatment enhanced durable tumor responses and survival inimmunogenic MOC1 but not in poorly immunogenic MOC2tumors. Survival, tumor growth, and immune correlative datasuggested thatMEK inhibition suppresses antitumor immunity inimmunogenic MOC1 tumors. No treatment combinationinduced detectable CD8 T-cell or NK cell–mediated antitumorimmunity in poorly immunogenic MOC2 tumors. Both periph-eral and tumor-infiltrating CD8 T cells in rapamycin-treatedtumor-bearing mice had a more robust IFNg response whenactivated. Tumor-infiltrating CD8 T cells, but not NK cells, weremechanistically required for the tumor growth alteration andenhancement of tumor cell MHC class I expression observedfollowing rapamycin and PD-L1 mAb treatment. These findingschallenge traditional views of rapamycin being immunosuppres-sive in all clinical contexts and have important implications forthe rational combination of targeted and immune-activatingtherapies in the clinical trial setting.

Materials and MethodsMice and in vivo experiments

TheNational Institute onDeafness andOther CommunicationDisorders Animal Care and Use Committee (ASP1364-14)approved all in vivo studies. MOC cell lines were generated fromDMBA-induced oral cavity tumors and have been validated andpathogen tested as described (16). Experiments were carried outusing 8- to 10-week-old female C57BL/6 mice (Charles River)kept in a pathogen-free environment. MOC1 and MOC2 cellswere maintained in media as previously described (14). MOC1(1.5 � 106) or MOC2 (1 � 105) cells were transplanted subcu-taneously and allowed to engraft to a volume of 0.1 cm3 beforetreatment. Different concentrations of MOC1 and MOC2 cellswere used for tumor engraftment given the dramatic differences inprimary tumor growth rate (14). In vivo treatments and cellulardepletions were performed as detailed in the SupplementaryMethods.

Tissue flow cytometrySpleens were mechanically dissociated into single-cell suspen-

sions with frosted histologic slides and a 70-mm strainer. Freshlyresected tumor tissue was digested into a single-cell suspensionusing the mouse tumor dissociation kit from Miltenyi per pro-tocol. Cell surface, intracellular, and tetramer staining was per-formed as detailed in the Supplementary Methods.

Ex vivo antigen-specific lymphocyte stimulationFor analysis of peripheral lymphocytes, spleen single-cell sus-

pensions were plated in the presence of H2-Kb–restricted

p15E604–611 (KSPWFTTL) peptide (1 mg/mL) for 7 days. Lympho-cytes were then enriched via a histopaque gradient and stimulatedwith irradiated splenocytes (20 Gy) pulsed with 1 mg/mL ofp15E604–611 or control OVA257–264 (SIINFEKL) peptide at a10:1 ratio of antigen-presenting cell (APC) to T cell for 24 hours.A flow cytometry–based assay (IFNg secretion assay, Miltenyi)was used per protocol to detect IFNg-secreting CD8 T cells. Foranalysis of tumor-infiltrating lymphocytes (TIL), CD8þ TILswere sorted from tumor single-cell suspensions using a FAC-SAria to >99% purity and immediately stimulated for 3 hourswith PMA/ionomycin (eBioscience, 10 ng/mL, 500 ng/mL,respectively) in the presence of brefeldin-A, followed by intra-cellular staining with an antibody to mouse IFNg (eBioscience).Dead cells were excluded via LIVE/DEAD fixable viability dye.Data were collected and analyzed as described in the Supple-mentary Methods.

In vitro MOC cell treatments and flow cytometryCells (5�104)were plated into 6-well plates, allowed to adhere

overnight, and treated for 48 hours with rapamycin or IFNg (10ng/mL) alone or in combination. Subconfluent cells were har-vested with 1� TrypLE Select (Fisher Scientific) and immediatelystained with antibodies as indicated and used for flow cytometricanalysis as detailed in the Supplementary Methods. Dead cellsexcluded via 7AAD negativity.

RT-PCRFor details, please refer to Supplementary Methods.

Statistical analysisTests of statistical significance between pairs of data are

reported as P values, derived using a Student t test with a two-taileddistribution and calculated at 95%confidence.Comparisonof multiple sets of data was achieved with one- or two-wayanalysis of variance (ANOVA). Survival curves were comparedusing the log-rank (Mantel–Cox) test. When present, error barsreflect standard error of measurement (SEM). Significance was setin each case to P < 0.05. All analyses were performed usingGraphPad Prism v6.

ResultsmTOR inhibition and PD-L1 blockade in MOCtumor–bearing mice

Micewith immunogenicMOC1 tumors had durable antitumoreffects and prolonged survival when mTOR, but not MEK, wasinhibited, and the inhibition of primary tumor growthwas CD8 Tcell–dependent (14). We hypothesized that combining mTOR orMEK inhibition with PD-L1 mAb treatment could result inenhanced tumor control in immunogenic MOC1, but not poorlyimmunogenicMOC2, tumors.We combined themTOR inhibitorrapamycin and the MEK inhibitor PD901 alone, or in combina-tion with PD-L1 mAb, and assessed primary tumor growth andsurvival in both MOC1 and MOC2 tumor–bearing mice (Fig. 1).As shown in Fig. 1A, for mice bearing immunogenic MOC1tumors, treatment with PD-L1 mAb alone afforded no survivaladvantage, although primary tumor growth had a statisticallysignificant short-term delay, followed by tumor growth rebound(Supplementary Fig. S1A). MEK inhibition with PD901 did notenhance survival either alone or in combinationwith PD-L1mAb,despite the presence of activating codon 61 Ras mutations (17).

Moore et al.

Cancer Immunol Res; 4(7) July 2016 Cancer Immunology Research612




Conversely, mTOR inhibition with rapamycin alone prolongedsurvival of MOC1 tumor–bearing mice (P ¼ 0.008), and thissurvival benefit was significantly enhanced with the addition ofPD-L1 mAb (P ¼ 0.04). The combination of rapamycin andPD901 alone enhanced survival, but the addition of PD901partially negated the significantly improved survival and prima-ry tumor growth delay achieved with rapamycin plus PD-L1mAb. In poorly immunogenic MOC2 tumor–bearing mice, PD-L1 mAb monotherapy did not delay primary tumor growth orenhance survival (Fig. 1B and C; Supplementary Fig. S1B).Rapamycin and PD901 alone or in combination modestlyimproved survival in MOC2 tumor–bearing mice, but theaddition of PD-L1 mAb did not further enhance survival ordelay primary tumor growth with any combination. Thus,combination of mTOR inhibition and PD-L1 mAb enhancedtumor control over either treatment alone in immunogenicMOC1, but not in poorly immunogenic MOC2 tumor–bearingmice. However, any combination involving MEK inhibition didnot demonstrate such responses in either model.

Enhancement of peripheral antigen-specific CD8 T cells byblocking PD-L1 and mTOR

Because a durable treatment effect was observed in immuno-genic MOC1 but not in poorly immunogenic MOC2 mice, weinvestigated if correlative studies could support an immune-mediated mechanism of enhanced tumor control with combina-tion of rapamycin and PD-L1 mAb treatment. We used theendogenous retroviral envelope protein p15E as amodel antigen.It is significantly expressed in MOC1 and, to a lesser degree, inMOC2 cells (Supplementary Fig. S2). We first characterized theeffect of these treatments on peripheral CD8 T cells by flowcytometry. Total peripheral CD8 T-cell counts were diminishedfollowing treatment with PD901, but not with rapamycin(Fig. 2A). Using a tetramer specific for T-cell receptors thatrecognizes H2-Kb

–restricted p15E604–611 (KSPWFTTL), weobserved that peripheral antigen-specific CD8 T cells expandedsignificantly with combination of rapamycin and PD-L1 mAbtreatment compared with control or PD-L1mAb alone. However,using CD107a cell-surface staining as a marker of T-cell

Figure 1.Combination of rapamycin and PD-L1 mAb treatment improved survival in MOC1, but not in MOC2, tumor–bearing mice. A, treatment schema for MOC1 and MOC2tumor–bearing mice treated with PD-L1 mAb alone or in combination with rapamycin and PD901. B, Kaplan–Meier survival analysis for treated MOC1 andMOC2 tumor–bearingmice (n¼ 7–10mice/group). � , P <0.05; �� , P <0.01; �� , P < 0.001; Mantel–Cox log-rank test. C, individual growth curves for treatedMOC1 andMOC2 tumor–bearing mice (n ¼ 7–10 mice/group). Primary tumor growth was monitored following withdrawal of all treatment up to 100 days after tumortransplantation. The timing of treatments is indicated along the x-axis of each graph; solid bar represents targeted therapy treatment and arrows indicate PD-L1mAbtreatment. Results are representative of two independent experiments with similar results. aPD-L1, anti–PD-L1.

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degranulation, these antigen-specific peripheral CD8 T cellsshowed no evidence of activation.

To investigate whether peripheral antigen-specific CD8 T cellsin MOC1 tumor–bearing mice could be activated in the presenceof specific peptide, we measured IFNg production followingstimulation with OVA peptide p15E604–611 (KSPWFTTL)-pulsedAPCs. Compared with mice stimulated with a control OVApeptide, baseline production of IFNg by IFNg-producing anti-gen-specific CD8 T cells in MOC1 tumor–bearing control micewas elevated and modestly, but significantly, enhanced withrapamycin treatment alone. Treatment with PD-L1 mAb signifi-cantly elevated both the percentage of IFNg-producingCD8T cellsand their IFNg mean fluorescence, which was then further

enhanced by adding rapamycin (Fig. 2B and C, quantifiedin Fig. 2D). Taken together, these data suggest that expanded,antigen-specific CD8 T cells from the periphery could be activatedin an antigen-specific fashion, and that the significantly elevatedIFNg responsiveness in mice treated with PD-L1 mAb was furtherenhanced with the addition of rapamycin.

mTOR preserves recruitment and enhances activation ofinfiltrating immune cells

To evaluate the effects of treatment on tumor-infiltratingimmune cells, we performed flow cytometry on freshly isolatedMOC1 and MOC2 tumor single-cell suspensions following treat-ment. In MOC1 tumors, total CD3þ TILs increased following

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Figure 2.Rapamycin, but not PD901, induced expansion of and enhanced IFNg production from peripheral antigen-specific CD8 T cells in MOC1 tumor–bearing mice. A, flowcytometric characterization of the presence and activation status (CD107a) of total and H2-Kb-restricted p15E604–611 specific CD8 splenocytes fromMOC1 tumor–bearingmice following treatment using p15E tetramer staining. Cells shown in A are gated 7AAD�CD45.2þCD3þCD8þ. Absolute numbers of indicated cells per 104 cellscollected are shown. Eight spleens from two independent in vivo experiments were analyzed. B, splenocytes from treated MOC1 tumor–bearing mice were incubatedfor 7 days in the presence of p15E604–611 (KSPWFTTL) peptide, then stimulated with KSPWFTTL peptide–pulsed APCs for 24 hours to determine antigen-specificIFNg production. Representative dot plots demonstrating IFNg-producing CD8 splenocytes following stimulation with specific peptide or control OVA peptide.C, IFNg MFI on stimulated CD8 splenocytes. D, quantification of B and C. Splenocyte stimulation results shown are cumulative from two independent experiments, eachinvolving 2 to 3 mice for each condition, performed in technical triplicate. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ANOVA. aPD-L1, anti–PD-L1.

Moore et al.





PD-L1 mAb treatment (P ¼ 0.009). This increase was largelypreserved following the addition of rapamycin, but reduced tolevels below baseline after adding PD901 (Fig. 3A). This changewas not due to alterations in the number of CD4 TILs. After PD-L1 mAb treatment, MOC1 tumors had enhanced infiltration oftotal (P¼ 0.01) and p15E antigen-specific (P¼ 0.007) CD8 TILsthat was again largely preserved with the addition of rapamycin,but reversed to equal to or less than control numbers after theaddition of PD901 (P < 0.01). The absolute number of activatedantigen-specific CD8 TILs positive for CD107a followed thesame pattern of suppression following the addition of PD901,indicating that PD901 inhibits, but rapamycin preserves, PD-L1mAb–induced activation of antigen-specific CD8 TILs in MOC1tumors. Figure 3B demonstrates representative cytometry dotplots of total and antigen-specific, p15E tetramer-positive CD8TILs from MOC1 tumors following treatment. Activation mar-

kers CD44 and PD-1 showed variable preservation of PD-L1mAb–induced expression as indicated (Fig. 3C). Analysis oftreated MOC2 tumors revealed that although the same trend ofdecreased total TILs following PD901 treatment was observed,the majority of TILs were CD4, and the low baseline numbers ofCD8 TILs were not enhanced by any treatment (SupplementaryFig. S3A).

As peripheral CD8 T-cell activation (as investigated in Fig. 2)may not be representative of TIL functional status, we sorted CD8TILs from treated MOC1 tumors and assessed their ability tosecrete IFNg after PMA/ionomycin stimulation (Fig. 3D). Rapa-mycin treatment alone enhanced CD8 TIL activation potentialcompared with control (P < 0.001). Treatment with PD-L1 mAbdramatically increased CD8 TIL IFNg secretion potential, and thiswas further enhanced with the addition of rapamycin (P¼ 0.03).Thus, modulation of the tumor microenvironment after

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Figure 3.Rapamycin, but not PD901, preserved the infiltration and activation of PD-L1mAb–induced antigen-specific CD8TIL and enhancedCD8TIL IFNg production potentialin MOC1 tumor–bearing mice. A, flow cytometric characterization of MOC1 tumor–infiltrating TILs following treatment. Cells shown are gated 7AAD�CD45.2þCD3þ.Absolute numbers of indicated cells per 104 cells collected are shown. Eight individual tumors from two independent in vivo experiments were analyzed. B,representative dot plots demonstrating total and OVA peptide KSPWFTTL (p15E604–611) antigen-specific CD8 TILs from MOC1 tumors following treatment. C,representative histograms andquantification of cell-surface CD44 andPD-1 staining on total CD8TILs fromMOC1 tumors (n¼ 5 tumors/group). D, representative dotplots demonstrating CD8 TIL IFNg production along with quantification. Single-cell suspensions from three individual treated MOC1 tumors per condition werepooled, CD8 TILs were sorted and stimulated with PMA/ionomycin followed by IFNg intracellular staining. Staining was done in technical quadruplicate. Dead cellswere excluded via a LIVE/DEAD viability dye. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ANOVA. aPD-L1, anti–PD-L1.






rapamycin treatment alone enhanced the activation potential ofCD8 TILs. Also, whereas expression of surface activation markersCD107a, CD44, and PD-1 on CD8 TILs was unaffected by theaddition of rapamycin to PD-L1mAb treatment inMOC1 tumor–bearing mice, IFNg secretion potential was modestly but signif-icantly enhanced.

Effects of mTOR, MEK, and PD-L1 inhibition on tumor-infiltrating NK cells, MDSCs, and Tregs

Given the established role of NK cells in controlling malignantprogression, and the potential for PD-L1 mAb–mediated, NKcell–dependent, antibody-dependent cell-mediated cytotoxicity(ADCC), we measured infiltration and activation of tumor infil-trating NKs (Fig. 4A). In MOC1 tumors, NK infiltration did notincrease with PD-L1 mAb treatment alone, and this baseline wassignificantly reduced with PD901, but not rapamycin, treatment.Similar to the case with CD8 TILs, the number of CD107aþ NKcells was stable with the addition of rapamycin to PD-L1 mAbtreatment, but decreased with PD901 alone or in combination.LowbaselineNK infiltration inMOC2was not enhancedwith anytreatment (Supplementary Fig. S3B).

We alsomeasured infiltration of immunosuppressivemyeloid-derived suppressor cells (MDSC) and Tregs into the tumormicro-environment following treatment. Changes in MOC1 tumor–infiltrating MDSCs and Tregs were heterogeneous and did notreach statistical significance for any treatment group (all MDSCandTreg changes betweengroupsP>0.05), thougha trend towardincreased Tregs in the rapamycin plus PD-L1 mAb treatment

group was present (Fig. 4B and C). Largely, similar results wereobserved in MOC2 tumors with several modest but statisticallysignificant observed changes in either MDSC or Treg infiltrationfollowing treatment (Supplementary Fig. S3C and S3D).

Robust TIL and NK infiltration into MOC1 but not MOC2tumors, a PD-L1 mAb monotherapy response in MOC1 but notMOC2 tumor–bearingmice, and enhancedMOC1peripheral andTIL CD8 IFNg production capacity following rapamycin and PD-L1mAb treatment alone or in combinationprovide support for animmune mechanism of tumor control in responsive MOC1tumors. The lack of tumor growth suppression or statisticallysignificant changes in immune correlates in MOC2 is consistentwith the known poorly immunogenic status of these tumors.Consistent with previous experiments (14), MEK inhibition sig-nificantly reduced the presence of total and antigen-specific CD8TILs and NKs in MOC1 tumors. These data, along with preservedNK-cell tumor infiltration andCD107a staining inMOC1, suggestthat CD8TILs, NK cells, or both could participate in the enhanced,durable MOC1 tumor control following combination of rapa-mycin and PD-L1 mAb treatment.

Depletion of CD8 but not NK cells in vivo abrogates antitumorresponses

To differentiate between a CD8 TIL and NK cell–mediatedADCC mechanism for the observed treatment response, wedepleted in vivo CD8 T cells or NK cells after treatment withrapamycin and PD-L1mAb alone or in combination (Fig. 5). CD8but not NK-cell depletion completely abrogated the antitumor

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Figure 4.Rapamycin, but not PD901,preserves baseline infiltration andactivation of tumor-infiltrating NKcells in MOC1 tumor–bearing mice,whereas MDSC and Treg levels wereunaffected. A, flow cytometricanalysis of MOC1 tumor–infiltrating7AAD�CD45þCD3�NK1.1þ NK cellsand cell surface CD107a. B, analysisof 7AAD�CD45þGr1þCD11bþMDSCs,and C, live CD45þCD4þFoxP3þ

Tregs. Five individual tumors fromeach condition were analyzed.Absolute numbers of indicated cellsper 104 cells collected on the flowcytometer are shown. � , P < 0.05;�� , P < 0.01; ANOVA. aPD-L1,anti–PD-L1; n/s, not statisticallysignificant.

Moore et al.





effect observed in MOC1 tumor–bearing mice following PD-L1mAb alone or in combination with rapamycin, and partiallyabrogated the effect observed with rapamycin monotherapy.Depletion of NK and CD8 cells from the periphery and fromwithin the tumor microenvironment was validated at multipletime points (Supplementary Fig. S4). Thus, CD8 T cells were theeffector immunecellsmediating the enhanced survival anddurabletumor growth suppression following withdrawal of treatment inMOC1 tumor–bearing mice.

Rapamycin enhances IFNgproductionorPD-L1mAb-inducibleMHC class I expression

To explore putative mechanisms of enhanced antitumorimmunity in MOC1 tumor–bearing mice treated with rapamy-cin and/or PD-L1 mAb, we measured cell-surface MHC class Iexpression on tumor cells in treated mice (Fig. 6). Althoughrapamycin alone did not enhance tumor cell-surface class Iexpression, it further increased class I expression over PD-L1mAb treatment alone (Fig. 6A). Thus, the enhanced class Iexpression on MOC1 tumor cells in vivo may have been inresponse to increased IFNg in the tumor microenvironment, sowe modeled MOC1 cell exposure to IFNg in vitro. Whereasrapamycin treatments from 10 to 100 nmol/L did not alterMOC1 cell-surface class I expression, lower dose rapamycin (10or 20 nmol/L) significantly enhanced IFNg-inducible H2-Kb

and H2-Db expression (Fig. 6B). Given that IFNg can beproduced by TILs and NK cells in the tumor microenvironment,we explored whether depletion of CD8 TILs or NK cells fromMOC1 tumors could reverse this enhanced class I expression onMOC1 tumor cells (Fig. 6C). Depletion of CD8 T cells, but notNK cells, reduced tumor-cell H2-Kb and H2-Db expression tonear baseline, suggesting that CD8 TILs are a significant sourceof tumor microenvironment IFNg that drives tumor cell class Iexpression in treated MOC1 tumors.

DiscussionConcepts of howbest to combine different cytotoxicmodalities

with immunotherapy to maximize antitumor effects are evolvingrapidly. The use of either standard anticancer treatments, such asplatinum-based agents and radiation (20), or small-moleculetargeted therapies, such as tyrosine kinase inhibitors (5), candamage tumor cells and lead to enhanced antigen release, APCactivation, and adaptive antitumor immunity. Yet, many of thesesame agents can potently suppress cells of innate and adaptiveimmunity (6). Here, we demonstrate that the addition of themTOR inhibitor rapamycin to PD-L1 mAb treatment enhancesboth immediate and durable tumor control in an immunogenicmodel of oral cavity cancer. This durable response was notobserved in the same model when PD-L1 mAb is combined withaMEK inhibitor, despite the fact that this mouse carried a DMBA-induced codon 61 Ras-mutant tumor. In support of our previouswork demonstrating suppressed effector immune cell infiltrationfollowing MEK inhibition, we showed that PD901 treatmentreverses the enhanced infiltration and activation of antigen-spe-cific CD8 TILs observed following PD-L1 mAb treatment alone.Conversely, the addition of mTOR inhibition to PD-L1 mAbtreatment preserved antigen-specific CD8 TIL and NK-cell infil-tration in immunogenic MOC1 tumors and enhanced IFNgsecretion capacity in both peripheral CD8 T cells and CD8 TILs.The different responses of rapamycin- and PD901-treated micedid not appear to be due to significant changes in the infiltrationof MDSCs or Tregs, and our previous work indicates that rapa-mycin itself had no direct cytotoxic or antiangiogenic effect onMOC tumors, despite high baseline activity of the PI3K/mTORpathway in MOC cells (14). Mechanistically, primary tumorgrowth suppression following treatment with rapamycin andPD-L1 mAb alone or in combination was dependent upon thepresence of CD8 TILs but not NK cells in the tumor microenvi-ronment. Enhanced IFNg and PD-L1 mAb-inducible MHC class I

Figure 5.CD8 but not NK cell depletion abrogated the in vivo antitumor effects of rapamycin and PD-L1 mAb treatment alone and in combination in immunogenic MOC1tumors. In independent experiments, MOC1 tumor–bearing mice were treated with rapamycin and PD-L1 mAb alone or in combination as detailed in Fig. 1with and without either CD8 (clone YTS169.4) or NK (clone PK136) antibody-based cellular depletion (200 mg each antibody twice weekly; n ¼ 5–7 mice/group).For each group, depletion was started 1 day before rapamycin and/or PD-L1 mAb treatment. Timing of rapamycin and PD-L1 mAb treatment indicated alongthe x-axis of each graph. CD8 and NK cell depletion (>95%) from both the periphery and tumor microenvironment was verified in separate in vivo experiments(Supplementary Fig. S4). aCD8, anti-CD8; aNK, anti-NK; aPD-L1, anti–PD-L1.






expression on MOC1 tumor cells was reversed again with deple-tion of CD8 but not NK cells, indicating that enhanced ability oftumor cells to present antigen is likely to play a role in the durableantitumor immunity induced with this treatment combination.

These results support emerging concepts that immune-activat-ing treatments such as checkpoint inhibitors are effective inimmunogenic tumors with high genetic alteration rates and noteffective in tumors with low baseline immunogenicity and effec-tor immune cell infiltration (21–23). Our use of both MOC1 "T-cell–inflamed" and MOC2 "non–T-cell-inflamed" tumors allowsthe modeling of both immunogenic and poorly immunogenicmalignancies, each ofwhich is represented roughly equally in oralcavity cancers (3, 24). MOC1 tumors represent immunogenictumors that are likely to respond to immune-activating therapies,whereas MOC2 tumors represent very aggressive, poorly immu-nogenic tumors likely to be resistant to immunotherapy. Studyingboth types of tumors will be critical moving forward as we aim toexpand the number of patients that have a durable response tocheckpoint inhibition.

Ourwork also suggests that in thismodel PD-L1mAb treatmentworks primarily through a CD8 T-cell and not an NK-cell mech-anism, such as ADCC. ADCC against human tumor cells in vitro

mediated by an anti-human PD-L1 mAb, demonstrated by Boy-erinas and colleagues (25), indicates that PD-L1 mAb–inducedADCCmay bemodel or target cell dependent. These data add to agrowing body of literature indicating that MEK1/2 inhibitionsuppresses effector immune cell function, despite the activatingRas mutations in MOC cells. Other preclinical studies havedemonstrated suppression of antigen-specific T-cell responses invitrowithMEK, but not upstreamBRAF inhibition (23, 26), whichis in direct contrast with work showing enhancement of on-treatment responses to PD-1mAbmonotherapywith the additionof trametinib, a MEK1/2 inhibitor similar to PD901 (15). Theseauthors demonstrate decreased tumor infiltration of total CD8 Tcells following MEK inhibition alone, but dramatically enhancedinfiltration following concurrent PD-1 mAb and trametinib treat-ment. These discrepancies may be due to differences in sensitivityto checkpoint inhibitor or MEK inhibitor monotherapy betweenmodels and highlight the remarkable heterogeneity in therapeuticresponses that can exist between different syngeneic models ofmurine carcinoma.

Our findings that rapamycin enhanced both the IFNg produc-tion capacity of peripheral and tumor-infiltrating CD8 T cells andthe induced expression of MHC class I expression onMOC tumor

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Figure 6.Rapamycin enhances IFNg or PD-L1 mAb-inducible MHC class I expression in MOC1 cells and tumors in a CD8 T-lymphocyte–dependent fashion. A, tumor cell gatingstrategy, representative histograms, and quantification of MHC class I H2-Kb and H2-Db in treated MOC1 tumors. Tumor cells are 7AAD�, CD31� (endothelial), andCD45� (hematopoietic). Previous data indicate low density (<5%) of CD140aþ fibroblasts in this CD45�CD31� population (data not shown). Quantitationperformed on 8 individual tumors harvested from two independent in vivo experiments. B, in vitro stimulation of MOC1 cells with rapamycin and IFNg (10 ng/mL)alone or in combination, then flow cytometric analysis of H2-Kb and H2-Db. Representative histograms are shown, as well as quantification from three independentexperiments each performed in technical triplicate. C, quantification of H2-Kb and H2-Db expression on MOC1 tumor cells as in A but following CD8 orNK-cell depletion (n ¼ 5 tumors/group). � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ANOVA. aPD-L1, anti–PD-L1.

Moore et al.





cells suggest that rapamycin hasmultiple anddiverse effects on theMOC tumormicroenvironment. The ability of IFNg to upregulateMHC class I expression is well established (27, 28). The use ofrapamycin to enhance an immune-activating treatment seemscounterintuitive, given its use clinically to suppress rejection oftransplanted solid organs. However, patients receiving mTORinhibitors following solid organ transplantation have a signifi-cantly reduced incidence of developing squamous cell carcinoma(reviewed in ref. 29). Mechanistically, mTOR inhibition enhancesmemory T-cell development upon viral challenge (30) and theantitumor effects of therapeutic peptide vaccines (31, 32). Com-bining agonist CD40 mAbs with AZD8055, a potent inhibitor ofboth mTORC1 and mTORC2, led to synergistic antitumor effectsin a model of metastatic renal carcinoma (33). Given thatmTORC2 inhibition is required for the development of memoryT cells (34), it is possible that dosing and duration of rapamycintreatment are factors affecting whether immune responses areenhanced or inhibited. These data, along with ours demonstratingenhanced antitumor immunity with the combination of rapamy-cin and PD-L1 mAb treatment, contrast with a report showingimpairment of an HPV-peptide–based therapeutic vaccine afterrapamycin treatment (35).Here, regressionof a subset ofHPV-E7þ

TC-1 tumors following administrationof a therapeutic vaccinewasreversed following administrationof rapamycin at doses similar tothose of our experiments. Rapamycin treatment alone induced noregression of TC-1 tumors, suggesting significant differences inintrinsic sensitivity to rapamycin in the TC-1 and MOC models.

Although we have established a CD8 T-cell mechanism for anantitumor effect that includes increased IFNg production capacityand MHC class I expression, other mechanisms may also play arole. Rapamycin-induced metabolic perturbations or changes intumor vascularity could enhance oxygenation and immune cellaccess to tumor antigens (36). Inhibition of tumor cell PD-L1 candirectly suppress mTOR activity, thus enhancing tumor microen-vironment glucose levels and lifting tumor cell–induced meta-bolic restrictions on TILs (37). Rapamycin and PD-L1 mAb couldwork in an additive fashion to block tumor cellmTOR signaling toachieve similar results in MOC1 tumors. Signaling throughexpression of the PD-1 receptor on tumor cells can directlycontribute to tumor cell growth and survival (38), but MOC cellshave no detectable expression of PD-1 (data not shown). Differ-ent doses or sequencing of rapamycin with PD-L1 mAbs couldhave profound effects on the development of antitumor immuneresponses, given the known modulation of effector and memoryT-cell phenotypes by rapamycin (30, 34). Additionally, combinedrapamycin and PD-L1 mAb treatment did not cure T-cell–inflamed MOC1 tumors, suggesting that mechanisms of resis-tance to immune-mediated tumor elimination, such as the per-sistence of tumor-infiltrating immunosuppressive MDSCs andTregs, may need to be addressed to maximize the potential ofthis therapy. Because mTOR is a key node in regulating thedevelopment and suppressive function of MDSCs in tumors

(39), how rapamycinmay or may not alter the T-cell–suppressivecapacity of MDSCs needs to be explored in our model.

In summary, we demonstrate antitumor immune responses toPD-L1 mAb treatment that are enhanced following mTOR inhi-bition, but suppressed following MEK inhibition, in T-cell–inflamed MOC1 tumors. This antitumor effect was CD8 T-cell–,and not NK cell–, dependent and was associated with more IFNgproduction capacity and increased expression of MHC class I onMOC cells. Noninflamed MOC2 tumors, in contrast, did notinduce CD8 T-cell– or NK cell–mediated antitumor immunitywhen treated with combinations of targeted and checkpoint inhi-bitors. This report demonstrates enhanced antitumor immunityfollowing combination of mTOR and PD-L1 mAb checkpointinhibition in a syngeneic carcinoma model. Given that both ofthese agents are FDA approved for the treatment of solid tumorsandhave acceptable safety profiles, the combination ofmTOR andcheckpoint inhibition deserves careful investigation in the clinicalsetting.

Disclosure of Potential Conflicts of InterestR. Uppaluri reports receiving commercial research support and service as a

consultant/advisory board member for Merck. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: H.A. Cash, C. Van Waes, C.T. AllenDevelopment of methodology: E.C. Moore, H.A. Cash, C.T. AllenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.C. Moore, H.A. Cash, A.M. Caruso, C.T. AllenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.C. Moore, H.A. Cash, A.M. Caruso, J.W. Hodge,C. Van Waes, C.T. AllenWriting, review, and/or revision of the manuscript: E.C. Moore, H.A. Cash,R. Uppaluri, C. Van WaesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.C. Moore, H.A. Cash, R. Uppaluri, C.T. AllenStudy supervision: H.A. Cash, C.T. Allen

Grant SupportThis work was supported by the Intramural Research Program of the NIH,

NIDCD, project number ZIA-DC000087. H.A. Cash was supported through theNIH Medical Research Scholars Program, a public–private partnership sup-ported jointly by the NIH and generous contributions to the Foundation for theNIH from Pfizer, Inc., the Doris Duke Charitable Foundation, the NewportFoundation, the American Association forDental Research, theHowardHughesMedical Institute, and the Colgate-Palmolive Company, as well as other privatedonors. C.T. Allen received further support through the American Academy ofOtolaryngology/American Head and Neck Society Duane Sewell Young Inves-tigators Combined Award.

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 October 3, 2015; revisedMarch 11, 2016; accepted March 11, 2016;published OnlineFirst April 13, 2016.

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Moore et al.




2016;4:611-620. Published OnlineFirst April 13, 2016.Cancer Immunol Res Ellen C. Moore, Harrison A. Cash, Andria M. Caruso, et al. Inhibition in Syngeneic Oral Cavity CancersEnhanced Tumor Control with Combination mTOR and PD-L1

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Enhanced Tumor Control with Combination mTOR and PD-L1 ...MOC1 tumor cells, an effect abrogated by depleting inﬁltrating CD8 T cells from the tumor microenvironment. These data conﬂict