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Research Article

Induction of T-cell Immunity OvercomesComplete Resistance to PD-1 and CTLA-4Blockade and Improves Survival in PancreaticCarcinomaRafael Winograd1, Katelyn T. Byrne1, Rebecca A. Evans1, Pamela M. Odorizzi2,Anders R.L. Meyer3, David L. Bajor1,4,5, Cynthia Clendenin1, Ben Z. Stanger1,4,5,Emma E. Furth3, E. John Wherry2,4, and Robert H. Vonderheide1,4,5

Abstract

Disabling the function of immune checkpoint molecules canunlock T-cell immunity against cancer, yet despite remarkableclinical success with monoclonal antibodies (mAb) that blockPD-1 or CTLA-4, resistance remains common and essentially unex-plained. To date, pancreatic carcinoma is fully refractory to theseantibodies. Here, using a genetically engineered mouse model ofpancreaticductaladenocarcinomainwhichspontaneousimmunityisminimal, we found that PD-L1 is prominent in the tumormicro-environment, a phenotype confirmed in patients; however, tumorPD-L1 was found to be independent of IFNg in this model. TumorT cells expressed PD-1 as prominently as T cells from chronicallyinfected mice, but treatment with aPD-1 mAbs, with or withoutaCTLA-4 mAbs, failed in well-established tumors, recapitulating

clinical results. Agonist aCD40mAbs with chemotherapy inducedT-cell immunity and reversed the complete resistance of pancreatictumors to aPD-1 and aCTLA-4. The combination of aCD40/che-motherapy plus aPD-1 and/or aCTLA-4 induced regression ofsubcutaneous tumors, improved overall survival, and conferredcurative protection from multiple tumor rechallenges, consistentwith immune memory not otherwise achievable. Combinatorialtreatment nearly doubled survival of mice with spontaneouspancreatic cancers, although no cures were observed. Our findingssuggest that in pancreatic carcinoma, a nonimmunogenic tumor,baseline refractoriness to checkpoint inhibitors can be rescued bythe priming of a T-cell responsewithaCD40/chemotherapy.CancerImmunol Res; 3(4); 399–411.�2015 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDA) is a lethal and aggres-

sive disease with the lowest 5-year patient survival rate of anytumor type routinely tracked (6%). The incidence of PDA is rising,and it is projected to become the second leading cause of cancer-related death in the United States by 2025 (1). PDA is distin-guished by a dense desmoplastic stroma, rich in fibroblasts,

extracellular matrix, and inflammatory leukocytes (but few infil-trating effector T cells). Although new combination chemothera-pies are increasingly effective for PDA (2, 3), tumor response ratesremain low and durability is short.

T cells are key mediators of antitumor immunity and regulatethe outcome of tumor immune surveillance (4). Critical to thisregulation are lymphocyte inhibitory receptors, such as pro-grammed cell death protein 1 (PD-1) and cytotoxic T-lympho-cyte–associated antigen 4 (CTLA-4), which restrain T-cell antitu-mor immunity (5–8). Monoclonal antibodies (mAb) that blockPD-1 or CTLA-4 induce T cell–dependent tumor regression inmany experimental systems (7, 8). Unprecedented rates of tumorregressions have been observed in patients with melanoma andmultiple carcinomas following treatment with mAbs againstCTLA-4, PD-1, or programmed death-ligand 1 (PD-L1), a ligandfor PD-1 (9–14). PD-1 engagement inhibits T-cell function pri-marily during the T-cell effector phase (15–17). Its main ligandsare PD-L1 and programmed death-ligand 2 (18). PD-L1 isexpressed on tumor cells as well as on tumor-associated leuko-cytes, and antibody blockade of either PD-1 or PD-L1 can restoreantitumor immunity inmurine cancermodels (19–21). CTLA-4 isa crucial immune checkpoint regulator expressed onT cells; loss ofCTLA-4 leads to rampant lymphoproliferation, autoimmunity,and death inmice (22). Expression of CTLA-4 is found on effectorT cells but especially regulatory T cells (Treg); CTLA-4 blockade inmurine tumor models both inhibits negative signaling in effectorcells and depletes Tregs (23–25).

1Abramson Family Cancer Research Institute, Perelman School ofMedicine, University of Pennsylvania, Philadelphia, Pennsylvania.2Department of Microbiology, Perelman School of Medicine, Univer-sity of Pennsylvania, Philadelphia, Pennsylvania. 3Department ofPathology and Laboratory Medicine, Perelman School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania. 4AbramsonCancer Center, Perelman School of Medicine, University of Pennsyl-vania, Philadelphia, Pennsylvania. 5Department ofMedicine, PerelmanSchool of Medicine, University of Pennsylvania, Philadelphia,Pennsylvania.

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

K.T. Byrne and R.A. Evans contributed equally to this article.

Corresponding Author: Robert H. Vonderheide, Abramson Family CancerResearch Institute, Perelman School of Medicine, University of Pennsylvania,8-121 TRC, Building 421, 3400 Civic Center Boulevard, Philadelphia, PA 19104.Phone: 215-573-4265; Fax: 215-573-2652; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0215

�2015 American Association for Cancer Research.

CancerImmunologyResearch

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Mechanisms of PD-1 or CTLA-4 resistance are poorly under-stood. Preexisting T-cell antitumor immunity has been hypoth-esized as a prerequisite (26–29). The majority of cancer patientstreated with these agents alone do not respond clinically, andsome tumor types, such as PDA, are fully refractory (12, 30, 31).Although combinations of aPD-1 and aCTLA-4 may improvetumor response rates inmelanoma, a large fraction of patients stillfail to respond (32). Tumor PD-L1 expression in somemalignan-cies correlates spatially with the presence of infiltrating CD8þ Tcells, suggesting that tumor cells upregulate PD-L1 in response toimmune pressure, a hypothesis termed adaptive immune resis-tance (33–35). CD8þ T cell–derived IFNg may drive PD-L1expression in malignant cells (18, 36). These data suggest thatthe efficacy of checkpoint inhibitors may require the presence ofan endogenous antitumor T-cell response. In fact, the augmen-tation of antitumor T-cell responses with vaccines, peritumoralpoly(I:C), or intratumoral oncolytic virus has been shown toimprove baseline responses to checkpoint inhibitors in murinemodels (21, 27, 37, 38).

In the studies reported here, we tested the hypothesis that failedimmune recognition or poor T-cell priming underlies weak clin-ical responses to checkpoint therapy in pancreatic cancer, that is,induction of T-cell immunity is required to potentiate tumorregressions not otherwise achievable with checkpoint blockadealone. We studied the KPC mouse model of spontaneous PDA inwhich expression of oncogenic KrasG12D and mutant p53 is tar-geted to the pancreas by Cre recombinase under the control of thepancreas-specific promoter Pdx-1 (39). This model recapitulatesthe molecular, histologic, and immune parameters of the humandisease (39–43). Analysis of human PDA was performed toconfirm the clinical relevance of ourfindings in themurinemodel.We induced T-cell immunity using an agonistic aCD40 in com-bination with chemotherapy (44, 45), and studied the impact ofaPD-1/aCTLA4 mAbs.

Materials and MethodsMice

All animal protocols were reviewed and approved by theInstitutional Animal Care and Use Committee of the Universityof Pennsylvania. KrasLSL-G12D/þ, Trp53LSL-R172H/þ, Pdx1-Cre(KPC)mice (39), and KrasLSL-G12D/þ, Trp53LSL-R172H/þ, Pdx1-Cre,LSL-Rosa-YFP (KPC-Y) mice (46) were backcrossed 10 genera-tions on the C57BL/6 background. Six- to 8-week-old femaleC57BL/6 and B6.129S7-Ifngtm1Ts/J (IFNg ko) mice used forimplantable tumor studies were from The Jackson Laboratory.

Cell linesPDA cell lines from KPC or KPC-Y mice were derived from

single-cell suspensions of PDA tissue as previously described (42).Dissociated cells were plated in a 6-well dish with serum-freeDMEM. After 2 weeks, media werre changed to DMEM þ 10%FCS. After 4 to 10passages, cells were used in experiments. The celllines were tested and confirmed to be Mycoplasma free. No otherauthentication assays were performed.

In vivo mouse studiesFor implantable tumor experiments, PDA tumor cells (5� 105)

were injected subcutaneously in PBS into the flanks of mice andallowed to grow 9 to 11 days until tumor volumes averaged 30 to100 mm3. Mice were then assigned to treatment groups such that

cohorts were balanced for baseline tumor size. Mice were treatedintraperitoneally (i.p.) with aPD-1 (RMP1-14; BioXcell; 200 mg/dose) on days 0, 3, 6, 9, 12, 15, 18, and 21 (after enrollment) and/oraCTLA-4 (9H10; BioXcell; 200 mg/dose) on days 0, 3, and 6. Allantibodies were endotoxin free. Clinical grade gemcitabine (EliLilly and Company) was purchased through the Hospital of theUniversity of Pennsylvania Pharmacy; clinical grade nab-pacli-taxel was either purchased or a kind gift from Celgene. Chemo-therapy vials were resuspended and diluted in sterile PBS, andinjected i.p. at 120 mg/kg (for each chemotherapeutic) on day 1.As a control for the human albumin component of nab-paclitaxel,control cohorts were treated with human albumin at the samedose as the albumin component of nab-paclitaxel (108mg/kg) onday 1 (Sigma Life Science). All antibodieswere given i.p. AgonisticaCD40 (FGK45; BioXcell; 100 mg) was given on day 3. For T-celldepletion studies,aCD8 (2.43; BioXcell; 200 mg/dose) andaCD4mAbs (GK1.5; BioXcell; 200 mg/dose) were injected twice weeklyfor the duration of the experiment, starting on day 0 (day ofenrollment). For isotype controls, rat IgG2a (2A3; BioXcell; 100mg) and rat IgG2b (LTF-2; BioXcell; 200 mg/dose) were used. Thisapproach achieved >98% depletion of CD8þ and CD4þ T cells inperipheral blood and tumor tissue compared with that of controlmice, as monitored by flow cytometry. Formacrophage depletionstudies, clodronate-encapsulated liposomes (CEL) or PBS-encap-sulated liposomes (PEL; both at 12 mL/g; purchased fromDr.Nicovan Rooijen, Vrije Universiteit, Amsterdam, the Netherlands)were used i.p. starting on day �1 and repeated every 4 days forthe duration of the experiment; in these experiments, 2.5 � 105

PDA cells were implanted. For tumor rechallenge studies, aCD8or isotype control antibodies were injected i.p. the day before thesecond rechallenge and continued twiceweekly until day 60or themouse was sacrificed for tumor burden. To monitor growth ofsubcutaneous tumors, tumor diameters were measured by cali-pers and volume calculated by 0.5 � L � W2 in which L is thelongest diameter andW is the perpendicular diameter. Endpointcriteria for the survival studies included tumor volume exceeding1,000 mm3 or tumor ulceration. Mice that died suddenly ordeveloped vestibular signs, as described in Supplementary Fig.S8,withminimal tumor burdenwere censoredon thedayof deathor euthanasia.

For studies using the KPC model, young KPC mice weremonitored by abdominal palpation and/or ultrasonography(Vevo 2100 Imaging System with 55MHz MicroScan transduc-er, Visual Sonics) for the development of pancreatic tumors.Mice with ultrasound diagnosed tumors of volume 30 to 150mm3 were enrolled and block randomized into treatmentgroups. Tumors were visualized and reconstructed for quanti-fying tumor volume using the integrated Vevo Workstationsoftware package. Baseline tumor volume was not significantlydifferent across cohorts. KPC mice were treated with the samedose and schedule of antibodies and chemotherapeutics asnoted above in the subcutaneous model. Mice were censoredfrom study if they developed a secondary malignancy (n ¼ 1).Endpoint criteria included tumor volume exceeding 1,000mm3

(by ultrasonography), severe cachexia, or extreme weaknessand inactivity.

For viral studies, C57BL/6 mice were infected intravenouslywith 4 � 106 PFU of lymphocytic choriomeningitis viral (LCMV)clone 13, which was propagated, titrated, and used as previouslydescribed (15). Mice were sacrificed on day 30 after infection andtissues harvested for analyses.

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Collection of tissue samples from miceThe entire pancreas (KPC) or subcutaneous tumor was washed

in PBS, minced into small fragments, and incubated in collage-nase solution (1 mg/mL collagenase V in DMEM) at 37�C for 45minutes. Dissociated cells were passed through a 70-mm cellstrainer twice and washed three times in DMEM. Spleens andlymphnodeswere homogenized andpassed through a 70-mmcellstrainer to achieve single-cell suspensions. For spleens, red bloodcells were lysed using ACK Lysis Buffer (BioWhittaker).

Antibodies, flow cytometry, in vitro IFNg stimulation of tumorcells, and toxicology are described in Supplementary Methods.

Patient samples and analysisFormalin-fixed, paraffin-embedded tissue samples were pre-

pared after surgical resection of patients with resectable pancreaticcarcinoma according to an Institutional Review Board approvedprotocol, as noted in Supplementary Methods.

Statistical analysisDifferences between two groups were analyzed by a two-tailed

Student t test. Differences between three or more groups wereanalyzed by one-way ANOVA with the Bonferonni multiplecomparison test used as a post hoc test to assess differences betweenany two groups. Tumor growth curves were analyzed by two-wayANOVA,with Tukeymultiple comparisons ofmeans used as a posthoc test to assess differences between any two groups. Survivalcurves were assessed by the log rank (Mantel–Cox). The correla-tion between two groups was assessed by the Spearman rankcorrelation coefficient. All statistical analyses were performed onGraphPad Prism 6 (GraphPad) except two-way ANOVA andrelated post hoc testing that were performed on R StatisticalSoftware (R Core Team). P � 0.05 denotes differences that arestatistically significant.

ResultsPD-1–PD-L1 axis is highly expressed inmurine andhumanPDA

We interrogated the expression of PD-1 and PD-L1 using theKPC spontaneous genetic model of PDA. Within the microenvi-ronment of KPC tumors, few infiltrating T cells were observed, aspreviously reported (41, 42), but these T cells prominentlyexpressed PD-1 in all subsets including CD8þ, CD4þ, and regu-latory (Foxp3þ) T cells. For each subset, PD-1 expression wassignificantly higher in the tumor than in the correspondingpopulations in the spleens of the same tumor-bearing mice (Fig.1A). In the absence of a distinct marker for pancreatic epithelialcells in the KPC model, we identified tumor cells with negativegating, excluding leukocytes (CD45), endothelial cells (CD31),and mesenchymal populations (CD90) by flow cytometry ofsingle-cell suspensions of KPC tumors. KPC pancreatic tumorcells exhibited moderate expression of PD-L1 on more than 40%of the identified tumor cells (Fig. 1B). PD-L1was also expressed by10% to 50% of normal pancreatic epithelial cells identified intumor-freewild-typemice. Dendritic cells (DC) andmacrophagesin the KPC tumormicroenvironment expressed very high levels ofPD-L1, statistically significantly higher compared with PD-L1expression of these same antigen-presenting cell (APC) popula-tions in the spleens of KPC mice (Fig. 1B).

To assess whether these findings in the KPC model wereconsistent with human PDA, we examined human PDA samplesfor PD-L1 expression. In primary tumors from patients with

resected PDA, we observed moderate to intense expression ofPD-L1 on tumor andmononuclear cells in 4 of 8 (50%) resectionspecimens (Fig. 1C and Supplementary Fig. S1).We also observedthat T cells in human PDA were relatively rare within malignantfoci (mean ratio of CD8þ T cells per mm2 of tumor vs. nontumorareas was 0.065 � 0.052; range, 0.000–0.170)—again consistentwith the KPC model (42). PD-L1 expression on tumor cells inhuman samples did not correspond spatially with the presence ofCD8þ T cells; therewas no statistical correlation between intensityor extent of tumor PD-L1 expression and intratumoral CD8þ T-cell infiltration (P¼0.69; Fig. 1C). For example, of the two tumorswith themost intratumoral CD8þ T cells, one had intense and theother had minimal PD-L1 expression (Fig. 1C). These data inhumanPDAare in contrast to the correspondence of tumor PD-L1expression and T-cell infiltration previously reported for tumorsfrom patients with melanoma or kidney or head and neck carci-noma (HNSCC; refs. 33–35).

PD-1 is as highly expressed in murine PDA as it is in chronicLCMV infection

To evaluate the potential role of the PD-1–PD-L1 axis inmediating immune suppression in PDA, we first generated aPDA cell line from a backcrossed KPC mouse and establishedsubcutaneous PDA tumors in immunocompetent C57BL/6mice. Histopathologic examination of established tumors fromthis model showed recapitulation of both the cellular andextracellular components of spontaneous KPC tumors, withprominent deposition of a dense desmoplastic stroma andcomparable populations of infiltrating immunosuppressiveleukocytes, including F4/80þ macrophages (data not shown).We then examined expression of PD-1 on T cells from subcu-taneous tumor-bearing mice but did so by simultaneouslyexamining PD-1 expression on T cells from a parallel cohortof mice in which chronic LCMV infection had been establishedwith LCMV clone 13 (Fig. 2A). In many ways, this model ofchronic LCMV infection has served as a gold standard forunderstanding the transcriptional basis and phenotype ofexhausted CD4þ and CD8þ T cells (15, 16, 47–50). Two ofthe most highly upregulated genes mechanistically linked to T-cell exhaustion in response to chronic infection in this modelare PD-1 and Lag-3 (15). We therefore compared coexpressionof these markers on intratumoral and splenic T cells in micebearing established subcutaneous PDA tumors with splenic Tcells from mice with chronic LCMV (Fig. 2A). IntratumoralCD8þ, CD4þ, and Tregs coexpressed PD-1 and Lag-3 at levelscomparable with the same T-cell populations in LCMV-infectedmice (Fig. 2B). This phenotype was restricted to the tumor, assplenic T cells from tumor-bearing mice did not coexpress PD-1or Lag-3. Thus, T-cell expression of PD-1 is as prominent in thePDA tumor microenvironment as it is in chronic LCMVinfection.

In the subcutaneous PDA model, about 60% to 70% of tumorcells isolated from established tumors expressed PD-L1 (Fig. 2C),similar to the expression of PD-L1 on this cell line grown in vitro(Fig. 2D). These findings were confirmed using a YFPþ tumor cellline established from a pancreatic tumor isolated from a KPC-Ygenetically engineered mouse; in this model, YFP serves as avalidated lineage tracer for pancreatic epithelium (46). Aftersubcutaneous tumor implantation and growth in syngeneic hosts,we found that on average 66.7% of YFPþ tumors cells expressedPD-L1, as measured by flow cytometry (Supplementary Fig. S2).

Extending Reach of Checkpoint Blockade in Pancreatic Cancer

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Tumor CD8s

IsotypePD-1

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Moreover, high levels of PD-L1 on both DCs and macrophageswere observed in the tumor microenvironment of the KPC sub-cutaneous tumors (Fig. 2C), mirroring PD-L1 expression on theseAPC subsets in spontaneous tumors of KPC mice. Both a higherpercentage of PD-L1þ APCs and a higher (�3–4-fold) meanfluorescence intensity (MFI) of PD-L1 were observed comparedwith that of the corresponding APC populations in the spleens ofthe samemice (Fig. 2C). These data indicate that PD-L1 expressionis prevalent in the PDA microenvironment.

Tumor PD-L1 expression in PDA is not IFNg dependentPD-L1 expression in human melanoma and HNSCC correlates

spatially with T-cell infiltration (33, 35), and inmelanoma, tumorexpression of PD-L1 is dynamically upregulated in response toIFNg secreted by infiltrating CD8þ T cells (36). To determinewhether this samemechanism is responsible for PD-L1 expressionin PDA, we assessed the ability of our PDA cell line to upregulatePD-L1 in response to IFNg ; in vitro, IFNg stimulation resulted inincreased expression of PD-L1 by PDA cells (Fig. 2D). In vivo, weevaluated tumor and APC PD-L1 expression in the presence orabsence of T cells and IFNg . Subcutaneous PDA tumors wereestablished inmice thatwere genetically lacking IFNg , systemicallydepleted of CD4þ and CD8þ T cells, or both. Tumor growth ratein vivo was the same for each condition compared with that ofcontrol (data not shown). Analysis of these tumors showed nosignificant change in tumorPD-L1 expression (either percentage orMFI) with regard to IFNg or T-cell status (Fig. 2E). Analysis of theAPCpopulations in these tumors indicated that IFNg plays aminorrole in the regulation of PD-L1 expression on intratumoral DCsand macrophages. Small but statistically significant differenceswere observed in the percentage and MFI of PD-L1 expression onintratumoral APCs between IFNg-sufficient and IFNg-deficienthosts (Fig. 2F). In contrast, the presence or absence of T cells didnot affect PD-L1 expression by APCs regardless of host IFNg status(Fig. 2F), recapitulating the lack of correspondence betweenCD8þ T cells and PD-L1 expression in human PDA (Fig. 1C).

T-cell stimulation with CD40/gemcitabine/nab-paclitaxelconverts PDA from being fully refractory to highly sensitive tocheckpoint blockade

We then tested the antitumor in vivo efficacy of PD-1-blockingmAbs either with or without CTLA-4 blocking mAbs (Fig. 3A).CTLA-4 is expressed at higher levels on intratumoral CD8þ andCD4þ T-cell populations (including Treg) when compared withthat of splenic populations, suggesting that blockade of thisnegative checkpoint may improve antitumor immunity (Supple-mentary Fig. S3). Even with aCTLA-4, aPD-1 did not affect tumorgrowth or survival (Fig. 3B), even though a comparable aPD-1dosing schedule reproducibly improves clinical outcomes inmice

chronically infected with LCMV clone 13 (50, 51). This lack ofantitumor efficacy is similar to the lack of responses observed todate in patients with advanced PDA treated with aPD-L1 oraCTLA-4 (12, 30, 31).

These same reagents have shown efficacy in patients with othermalignancies; one possible distinction may be the presence of anantitumor immune response at baseline in subsets of thesepatients (28, 52). We therefore hypothesized that the inductionof a T-cell response would be required to overcome refractorinessto aPD-1 and aCTLA-4 in PDA and achieve clinical benefit.Agonist aCD40 antibody facilitates cancer vaccines (53) and cansynergizewith chemotherapy-induced immunogenic cell death toinitiate a T cell–dependent antitumor regression, providing avaccine effect in model systems for which a tumor-rejectionantigen is not characterized (45, 54). APCs in KPC-derived sub-cutaneous tumors express CD40 (Supplementary Fig. S4), as wehave previously shown (42). Here, we studied gemcitabine andnab-paclitaxel because this combination was recently approvedby the FDA for the treatment of metastatic PDA (3), and gemci-tabine has been shown to cooperate immunologically withaCD40 (45). Treatment of mice with established subcutaneousPDA tumorswithaCD40/chemotherapy altered the phenotype oftumor-infiltrating T cells although the percentage of T cells infil-trating the tumors did not change. There were statistically signif-icantly fewer CD8þ T cells that coexpressed the inhibitory PD-1and Lag-3 markers in treated tumors, and more proliferatingCD4þ and CD8þ T cells were found in the tumors of treated micecompared with controls (Supplementary Fig. S5).

The combination of gemcitabine and nab-paclitaxel at themaximum-tolerated dose did not induce regression of establishedsubcutaneous PDA tumors; however, the addition of aCD40 tothis chemotherapy regimen inhibited tumor growth andimproved survival compared with control-treated tumor-bearingmice (Fig. 3C). These effects were macrophage independent asCEL failed to abrogate the effect (Supplementary Fig. S6). Giventhat we have previously observed thataCD40 plus chemotherapyin the subcutaneous KPC model manifests an antitumor effectthat is T-cell dependent (55), we added aPD-1, aCTLA-4, or bothto treatment with aCD40/chemotherapy in an effort to enhanceT-cell immunity. The addition of aPD-1, aCTLA-4, or both toaCD40/chemotherapy enhanced tumor growth inhibition andled to an increase in survival in mice bearing subcutaneous PDAtumors (Fig. 3C). Moreover, these combinations led to completerejection of established tumors and long-term tumor-free survivalin significant proportions of mice treated with the combinedregimen (Fig. 3D). Treatment with aPD-1 and aCTLA-4 did notalter CD40 expression on APCs in the tumor microenvironment(Supplementary Fig. S4). The highest rates of tumor regressionwere observed in mice treated with both aPD-1 and aCTLA-4,

Figure 1.Expression of PD-1 and PD-L1 in murine and human PDA. A, representative histograms and quantification of PD-1 expression on tumor-infiltrating CD8þ

(gated on live, CD45þ, CD3þ, CD8þ), CD4þ (gated on live, CD45þ, CD3þ, CD4þ), or regulatory (Tregs; gated on live, CD45þ, CD3þ, CD4þ, FoxP3þ) T cells in tumors (n¼ 6–11) or spleens (n ¼ 4–17) from tumor-bearing KPC mice. �� , P � 0.01; �� , P � 0.0001. B, representative histograms and quantification of PD-L1 expression ontumor cells, normal pancreatic epithelial cells (gated on live, CD45neg, CD31neg, CD90neg), DCs (gated on live, CD45þ, F4/80neg, CD19neg, CD11cþ), and macrophages(Macs; gated on live, CD45þ, F4/80þ) in tumors (n ¼ 4–11) or spleens (n ¼ 25) from tumor-bearing KPC mice; and normal pancreata (n ¼ 5) from healthyC57BL/6 mice. �� , P� 0.01; �� , P� 0.001. C, histology and quantification of PD-L1 expression and CD8þ T-cell infiltration in human pancreatic cancer sections. Lefttwo, PD-L1 expression on malignant cells of a PDA tumor (PD-L1 expression score of 4þ (intense). See Materials and Methods and Supplementary Fig. S1.�40 and�400magnification for top and bottom, respectively). Right top, CD8 expression in serial section of the tumor on left, showing few tumor-infiltrating CD8þ

T cells (�40 magnification). Right bottom, plot describing correlation between intratumoral CD8 count and tumor PD-L1 score (n ¼ 8). P ¼ 0.69.






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with 39% (17 of 44) of mice achieving long-term completeremission and survival after treatment with all three antibodiesplus chemotherapy (Fig. 3D). Tumor growth was delayed innearly all mice treated with aCD40/chemotherapy and aPD-1/aCTLA-4, even in those mice not completely rejecting theirtumors, suggesting that the tumor response rate is even higherthan the tumor rejection rate in thismodel (SupplementaryFig. S7).Although treatment was well tolerated in the vast majority ofmice,in 6.3% of mice treated with aCD40/chemotherapy and at leastone checkpoint blocking mAb we noted clinical deteriorationconsistent with an infectious syndrome (Supplementary Fig. S8).

Rejection of PDA tumors by aCD40/chemotherapy andcheckpoint blockade is T cell mediated

To determine whether the antitumor effect we observed was Tcellmediated,we repeated the studywitha cohort ofmicedepletedof CD4þ andCD8þ T cells, starting on the day before the initiationof therapy. In the absence of T cells, the treatment did not inhibittumor growth or afford survival advantage, and no T cell–depletedmice rejected the tumor or survived long term (Fig. 4A).

To understand the effect of our treatment on intratumoral T-cellpopulations, we treated cohorts of tumor-bearingmicewithaPD-1/aCTLA-4, aCD40/chemotherapy, both, or neither (control),and sacrificed mice 1 week after treatment with aCD40 (orcontrol) to analyze tumors for T-cell infiltration. Tumors frommice treated with aPD-1/aCTLA-4 plus aCD40/chemotherapyhad a significantly increased (7-fold) CD8:Treg ratio comparedwith that of control-treated mice (Fig. 4B). This phenotype wasalso seen in some of the mice treated with aCD40/chemotherapyor aPD-1/aCTLA-4, although neither one of these two groupsexhibited as consistent an increase in the CD8:Treg ratio as themice treated with aPD-1/aCTLA-4 plus aCD40/chemotherapy(Fig. 4B). The CTLA-4 mAb clone 9H10 partly mediates its anti-tumor effect by depletion of Tregs, which express CTLA-4 (24);however, we observed that mice treated with aPD-1/aCTLA-4alone did not have a significantly decreased percentage of Tregsamong CD4þ T cells (Fig. 4B) or among total CD45þ cells. Rather,the administration of aCD40/chemotherapy (either with orwithout aPD-1/aCTLA-4) was associated with a significantdecrease in Treg percentages comparedwith that of control-treatedmice; all groups treated with immunotherapy demonstrated aslight increase in the CD8þ T-cell infiltrate, although the changeswere not statistically significant (Fig. 4B). These data suggest thataCD40/chemotherapy changes the immune microenvironmentin this PDA model and leads to a decreased percentage of Tregsand increased CD8:Treg ratio, an effect that is augmented furtherwith the addition of checkpoint blockade. The greatest changes inTreg percentage and CD8:Treg ratio were associated with the

highest rates of complete remission and long-term survival acrosscohorts reported in Fig. 3D.

To test whether mice that had completely rejected establishedPDA tumors had developed immune memory, we rechallengedcohorts of mice that were in long-term complete remission withthe same number of cells of the same PDA tumor line but on theopposite flank (Fig. 4C). We observed that 67% to 86% of suchmice rejected the PDA tumor cells implanted on the oppositeflank without any additional therapy (Fig. 4C), consistent withthosemice having developed immunologic memory. Because themost likely effector memory T-cell population mediating thiseffect is a CD8þ T cell, we further studied mice that had rejectedboth the initial tumor and the first rechallenge on the oppositeflank, and either depleted these mice of CD8þ T cells or admin-istered an isotype control antibody that did not deplete CD8þ Tcells. All mice were then rechallenged with the same number ofcells of the same cell line on the originalflank. Allmice depleted ofCD8þ T cells rapidly developed progressively growing tumors atthe site of the second rechallenge, whereas 4 of 6 isotype-treatedmice rejected this second tumor rechallenge (Fig. 4D). This effecttranslated into a statistically significant difference in overallsurvival after the second rechallenge (Fig. 4D).

aCD40/chemotherapy cooperates with PD-1 blockade toimprove survival of mice with established tumors in the KPCgenetic model of PDA

Having observed that the induction of T-cell immunity viaaCD40/chemotherapy potentiates the efficacy of checkpointinhibitors in the subcutaneous model of PDA, we then testedthis approach in the autochthonous KPC model of PDA. Obser-vations in the KPC model have previously been shown to predictclinical responses in PDA patients treated with the same orhomologous agents (42, 56–58). We therefore performed arandomized, controlled study of checkpoint inhibition in com-binationwithaCD40/chemotherapy in cohorts of tumor-bearingKPCmice. Given the striking expression of PD-1 and PD-L1 in theKPC tumor microenvironment, we chose to test our hypothesisusing the aPD-1 mAb. Mice diagnosed with pancreatic tumors of30 to 150 mm3 were randomized to treatment with aCD40 plusgemcitabine/nab-paclitaxel, aPD-1, aCD40/chemotheray plusaPD-1, or control (as described in Materials and Methods andsummarized in Fig. 5A), using the samedose and schedule as usedfor mice in the subcutaneous PDA studies. We observed a statis-tically significant increase in overall survival for mice receivingaCD40/chemotherapy plusaPD-1 compared with that of control(P ¼ 0.015, log-rank Mantel-Cox; Fig. 5B). The effect was large:Combination treatment nearly doubled the median overall sur-vival from 23 days in the control arm to 41.5 days in the

Figure 2.PD-1–PD-L1 axis is highly expressed and is not IFNg-dependent in a subcutaneous murine model of PDA. A, experimental design for establishment of subcutaneousPDA tumors or chronic LCMVclone 13 (Cl-13) infection simultaneously in two cohorts of C57BL/6mice. B, representative flowplots andquantification of coexpressionof PD-1 and Lag-3 on CD8þ (gated on live, lymphocytes, B220neg, NK1.1neg, CD8þ), CD4þ (gated on live, lymphocytes, B220neg, NK1.1neg, CD4þ) and regulatory(Tregs; gated on live, lymphocytes, B220neg, NK1.1neg, CD4þ, FoxP3þ) T cells from spleens of mice infected with LCMV Cl-13 (day 30) or the tumors and spleensof mice bearing PDA tumors (day 14). C, representative histograms and quantification of PD-L1 expression on tumor cells, DCs, and macrophages (Mac) insubcutaneous PDA tumors or spleens from the samemice (day 14), gated as in Fig. 1B. �� , P�0.0001. See also Supplementary Fig. S2. D, histogram of KPC-derivedPDA cell line interrogated for PD-L1 expression in vitro with or without IFNg in the culture, representative of three experiments. E, quantification and MFIof PD-L1 expression on tumor cells from subcutaneous PDA tumors established in either C57BL/6 (B6) or IFNg�/� (IFNg ko) mice with or without CD4þ and CD8þ T-cell depletion (TCD; day 16; n ¼ 6-8 mice per cohort). F, quantification and MFI of PD-L1 expression on DCs and macrophages in subcutaneous PDA tumorsgrown in either B6 or IFN-g ko mice with or without TCD (day 16; n¼ 6–8 mice per cohort). One-way ANOVA:%DCs PD-L1þ, P¼ 0.015; DC PD-L1 MFI, P¼ 0.0039;%Macs PD-L1þ, P ¼ 0.58; Macs PD-L1 MFI, P ¼ 0.0007. Post hoc test P values are indicated where statistically significant: � , P � 0.05; �� , P � 0.01.






P = 0.19

A

Treatment cohort Long-term survivors (%)

Isotype alone 0/25 (0%)

PD-1 alone 0/16 (0%)

CTLA-4 alone 0/10 (0%)

PD-1 + CTLA-4 1/19 (5%)

CD40/G/nP 3/25 (12%)

CD40/G/nP + PD-1 6/23 (26%)

CD40/G/nP + CTLA-4 7/22 (32%)

CD40/G/nP + PD-1 + CTLA-4 17/44 (39%)

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Figure 3.T-cell stimulationwithaCD40/gemcitabine/nab-paclitaxel potentiates efficacy of checkpoint blockade inmurinemodel of PDA. A, experimental design for study ofsubcutaneous PDA tumors treated with checkpoint inhibitors and aCD40/chemotherapy, as further described in Materials and Methods (G, gemcitabine; nP,nab-paclitaxel; q3d, antibody administered every 3 days). B, tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as indicated(n ¼ 9–10 per cohort; results for control and aPD-1 þ aCTLA-4 cohorts representative of three independent experiments). See also Supplementary Fig. S7. C,tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as indicated (n ¼ 9–10 per cohort; findings representative of threeindependent experiments). Two-way ANOVA: P � 0.0001. Post hoc test P values are indicated where statistically significant: � , P � 0.05; ��, P � 0.001;�� , P � 0.0001. See also Supplementary Fig. S7. D, percentage of mice bearing subcutaneous PDA tumors treated with indicated regimens that rejectedtheir tumors and survived tumor-free long-term (median follow-up of 42 days; range, 23–222 days). Data were compiled from five independent experiments.

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Treatment cohort

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Figure 4.Rejection of PDA tumors after aCD40/chemotherapy and checkpoint blockade is T cell–mediated. A, tumor growth and survival analyses of mice bearingsubcutaneous PDA tumors treated as indicated (n ¼ 9–10 per cohort; G, gemcitabine; nP, nab-paclitaxel; TCD, CD4/CD8 depletion). B, flow cytometricanalysis of subcutaneous PDA tumors treated as indicated (day 18 after tumor injection, day 7 after aCD40 treatment; P, aPD-1; C, aCTLA-4). One-wayANOVA%CD8s of live cells: P¼ 0.17; one-way ANOVA%Tregs of CD4þ T cells: P¼ 0.0004; one-way ANOVA CD8:Treg ratio: P¼ 0.0005. Post hoc test P values areindicated where statistically significant: � , P� 0.05; �� , P� 0.01; �� , P� 0.001. C, experimental design for first tumor rechallenge experiments. The table quantifiesfraction and percentage of mice that rejected tumor rechallenge in mice that had rejected the initial tumor implantation and were tumor free for at least 43 days.Data were compiled from three independent experiments. D, experimental design for second tumor rechallenge experiment. The second rechallenge occurredon days 31 to 49 after the first rechallenge. The table quantifies fraction and percentage of mice that rejected the second tumor rechallenge in mice that hadrejected a first tumor rechallenge. Host mice in this experiment were either treated with aCD8 (n ¼ 11) or isotype (Iso; n ¼ 6) antibodies. Survival analysisof mice after the second rechallenge with or without CD8 depletion is shown. Data were compiled from two independent experiments.






experimental arm with a hazard ratio of 0.334 (0.0584–0.657,95% confidence interval). Neither PD-1 alone nor aCD40/che-motherapy significantly improved overall survival. These datasuggest that, as predicted by our findings in the subcutaneousPDA model, the induction of a T-cell response is needed for theaPD-1 antitumor effect in PDA.

DiscussionThe clinical success of checkpoint inhibitors, including FDA

approval of ipilimumab, pembrolizumab, and nivolumab formelanoma, has prompted investigations to replicate these resultsmore broadly in oncology. Early findings, however, suggest thatmany tumors are resistant, with pancreatic carcinoma appearingcompletely refractory to monotherapy with checkpoint blockade(12, 30, 31). Here, using a genetically engineeredmouse model ofPDA, which like human PDA exhibits minimal spontaneousimmunity, we demonstrate that despite robust expression ofPD-1 and PD-L1 in the tumor microenvironment, treatment withaPD-1 with or without aCTLA-4 has minimal antitumor impact,replicating results in PDA patients treated with analogous agents(12, 30, 31).However, in the context ofaCD40/chemotherapy,wedemonstrate that the induction of T-cell immunity converts PDAfroma tumor that is completely refractory toaPD-1and/oraCTLA-4 into one in which checkpoint blockade controls tumor growthand significantly improves survival in a CD8þ T cell-dependentmanner. In particular, the combined regimen of aCD40/chemo-therapy plus aPD-1 nearly doubles the median overall survival ingenetically engineered KPCmice with preestablished spontaneouspancreatic tumors. Moreover, the capability of the treated mice toreject second and third subcutaneous tumor challenges in a CD8þ

T cell–dependent fashion thereby rendering long-term survival,suggests that the combined regimen induced the establishment ofantitumor immune memory with curative potential. Thesefindings indicate that poorly immunogenic tumors, epitomizedby theKPCpancreatic tumormodel, cannevertheless be controlledby the adaptive immune system provided a dual approachof therapeutic T-cell induction and checkpoint blockade is used.

Immunologically, the PDA tumor microenvironment is con-sidered especially suppressive, but increasingly, there is an appre-ciation from studies in KPC and other PDA models of an under-lying sensitivity of PDA tumor cells to T-cell cytotoxicity (59).Unlikemelanoma, PDAdoesnot commonly presentwith a robusttumor infiltration of CD8þ T cells (60–63). In genetically engi-neered mouse models of PDA, a prominent network of immu-nosuppression is dominant even at the earliest stages of disease(40–43, 64). We demonstrated that PD-1 is as prominent in theKPC tumor microenvironment as it is systemically in mice chron-ically infected with LCMV (50). We propose therefore, that thelack of responses to treatment with checkpoint inhibitors in KPCmice likely reflects a tumor microenvironment without an under-lying antitumor T-cell response.

The lack of PDA immunogenicity does not necessarily indicatean inherent lack of antigenicity of PDA tumors cells; indeed, PDAcells might be unexpectedly sensitive to T-cell killing because theyhave not been exposed to Darwinian-like T-cell selective pressurein vivo. Without T-cell pressure, T-cell escape and classical immu-noediting may not be necessary for pancreatic tumor growth as itis for highly immunogenic tumors (4, 59). Thus, in this study, weinterpret the antitumor effects of aCD40/chemotherapy plusaPD-1/aCTLA-4 as a strategy that overcomes acquired immuneprivilege in PDA. Other pathways and cells in the PDA tumor

A

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23 29 30.5 41.5

G, gemcitabine; nP, nab-paclitaxel.

Days after enrollment

Per

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rviv

alFigure 5.Combination of aCD40/chemotherapy and PD-1 blockadeimproves survival in KPC geneticmodel of PDA. A, experimental designfor randomized, controlled study intumor-bearing KPC mice, treated withaCD40/chemotherapy and aPD-1, asdescribed in Materials and Methods.G, gemcitabine; nP, nab-paclitaxel;q3d, antibody administered every3 days. B, overall survival analysis oftumor-bearing KPC mice treated asindicated (n ¼ 6–8 per cohort). aPD-1alone versus isotype alone P ¼ 0.39;CD40/G/nP vs. isotype aloneP¼0.76;CD40/G/nPþ aPD-1 vs. isotype aloneP¼ 0.015. C, median overall survival oftumor-bearing KPC mice treated asindicated.


Winograd et al.




microenvironment may also be "targetable" as part of novelimmunotherapeutic approaches (43, 65, 66).

Despite the 80% increased overall survival observed in KPCmice treated with aCD40/chemotherapy and aPD-1 comparedwith that of the controls, all mice succumbed to their disease. Itis worth noting that there are no published reports of cures ofKPC mice bearing established invasive tumors. A few groupshave reported improved overall survival (without cures) withtreatment in this model, including the recent demonstrationthat the vitamin D analogue calcipotriol improves survival by57% (57, 67, 68). Given the difference we observed in theresponse to treatment between the subcutaneous and KPC PDAmodels, we hypothesize that there is additional complexityin the tumor microenvironment of spontaneous KPC tumorsthat limits therapeutic responses. Potential other immunosup-pressive pathways contributing to treatment resistance includemyeloid-derived suppressor cells (MDSC), macrophages, andfibroblast-activating proteinþ (FAPþ) mesenchymal cells,among others (43, 65, 66, 69).

Our observations that aCD40/chemotherapy converts PDAfrom a tumor fully refractory to checkpoint inhibition to onethat is sensitive are important in the context of prior efforts toextend the therapeutic rangeofaPD-1 andaCTLA-4.Our goalwasto use a murine model that reproduces the lack of clinical effectobserved to date with aPD-1 and aCTLA-4 in PDA, in contrast tomany tumormodels that exhibit baseline levels of responsivenesstoaCTLA-4,aPD-1,aPD-L1, or combinations of these agents. Forexample, in models of colon carcinoma, melanoma, ovariancancer, bladder cancer, and neuroblastoma, checkpoint therapyalone inhibits tumor growth, improves survival, and occasionallymediates complete rejection (21, 70–73). In studies using thePanc02 subcutaneous PDA model, tumor rejection was observedin 50% ofmice after treatment with aCTLA-4 (74), and treatmentwith aPD-L1 (with or without gemcitabine) leads to significantdecreases in tumor growth (75), findings not representative of theclinical record of these agents in patients with PDA (12, 30, 31)and possibly related to Panc02 being a carcinogen-induced andlikely hypermutated tumor [whereas human PDA is not a hyper-mutated tumor (76)]. Previous work in these types of modelsdemonstrates that T-cell stimulatory therapies can improve base-line effects of checkpoint blockade (21, 27, 37, 38). For example,vaccination of mice bearing metastatic Panc02 tumors with agranulocyte macrophage colony-stimulating factor–secretingtumor cell vaccine was recently described to cooperate withanti–PD-1 therapy, which has single-agent activity in this model(77, 78). In PDA, the stimulation of a T-cell response usingaCD40/chemotherapy is able to transform a tumor that is refrac-tory to checkpoint inhibition into a sensitive one, rather than onlyimproving upon baseline activity of checkpoint mAbs.

In the absence of a defined tumor antigen in ourKPCmodel, wetherapeutically induced T cells with chemotherapy followed by anagonist aCD40 (45). Although other agents may also synergizewith aCD40 (79), gemcitabine, in particular, cooperates withaCD40 (45). Here, we added nab-paclitaxel given the recentregulatory clinical approval of the combination. We have previ-ously demonstrated in KPC mice with spontaneous tumors thataCD40 can reprogram macrophages to mediate (T cell–indepen-dent) stromal involution and short-term decreases in tumorvolume without improvement in overall survival (42); here, wefound that a one-time treatment with chemotherapy followed byaCD40 enabled a macrophage-independent T-cell response,

engendering immune memory and durable complete remissionsin a large fraction ofmice harboring subcutaneous PDA tumors—and extending survival in KPC mice with spontaneous tumors.Both the use of nab-paclitaxel and the subcutaneous setting maybe responsible for the differences in these observations, thesubject of current investigations. Although of interest, we couldnot evaluate in Fc receptor-null mice whether Treg changes fromaPD-1/aCTLA-4 were Fc receptor-dependent because the effectsof agonistic aCD40 are lost in such mice (80).

In certain human cancers, tumor cell PD-L1 expression colo-calizes with lymphocytic immune infiltrates, suggesting adaptiveresistance by the tumor and the potential root of sensitivity toaPD-1 (33–35, 81). In the case of melanoma, tumor cell PD-L1expression has been demonstrated to be dependent on CD8þ Tcells and the secretion of IFNg (36). In contrast, we found in ourPDA mouse models harboring minimal intratumoral T cells,tumor cells express PD-L1 at moderate levels and tumor-associ-ated DCs and macrophages express high levels of this inhibitoryligand.We further observed that PD-L1 expression inmurine PDAis not dependent on T cells or IFNg , indicating that PD-L1 tumorexpression does not appear to be an adaptive response to immunepressure. Thesefindings in theKPCmodel are corroborated byourobservations in human PDA in which there was no spatialcorrelation between tumor PD-L1 expression and the presenceof intratumoral CD8þ T cells. Tumor PD-L1 expression has beenreported to be regulated by oncogenes such as EGFR (82), sug-gesting that the regulation of PD-L1 in PDA may differ from theregulation of PD-L1 in other solid malignancies.

In summary, induction of T-cell immunity reduces resistance toPD-1 and CTLA-4 blockade and improves survival in pancreaticcarcinoma. It is notable that four out of the five reagents thatwe used in these studies have an FDA-approved human homolog(gemcitabine, nab-paclitaxel, aPD-1, and aCTLA-4), whichshould facilitate clinical translation.

Disclosure of Potential Conflicts of InterestE.J. Wherry has ownership interest (including patents) in Surface Oncology;

is a consultant/advisory boardmember for SurfaceOncology; and has providedexpert testimony for Genentech/Roche. R.H. Vonderheide reports receivingcommercial research grants from Roche and Pfizer. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: R. Winograd, E.J. Wherry, R.H. VonderheideDevelopment ofmethodology: R.Winograd, K.T. Byrne, R.A. Evans, E.E. Furth,R.H. VonderheideAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Winograd, K.T. Byrne, R.A. Evans, P.M. Odorizzi,A.R.L. Meyer, D.L. Bajor, C. Clendenin, E.E. Furth, R.H. VonderheideAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Winograd, K.T. Byrne, R.A. Evans, P.M. Odorizzi,C. Clendenin, B.Z. Stanger, E.E. Furth, R.H. VonderheideWriting, review, and/or revision of the manuscript: R. Winograd, K.T. Byrne,R.A. Evans, E.E. Furth, E.J. Wherry, R.H. VonderheideAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Winograd, A.R.L. Meyer, C. Clendenin,E.E. Furth, R.H. VonderheideStudy supervision: R.H. Vonderheide

AcknowledgmentsThe authors thank Amy Ziober, Michael Feldman, Erin Dekleva, Tim Chao,

Andrew Rech, Mark Diamond, Steve Albelda, Jim Riley, Ellen Pure, and AnilRustgi (Penn) and Dafna Bar-Sagi (New York University) for helpfuldiscussions.






Grant SupportThis study was supported by the NIH grants R01 CA169123 (to R.H. Von-

derheide and B.Z. Stanger), T32 CA009140 (to R. Winograd), T32 HL007439(to K.T. Byrne), T32 HL007775 (to D.L. Bajor), P30 CA016520 (to R.H. Von-derheide), U19 AI 082630, P01 AI112521 (to E.J. Wherry), Stand Up To Cancer(to R.H. Vonderheide), Pancreatic Action Cancer Network-AACR (to R.H. Von-derheide), and Translational Center of Excellence in Pancreatic Cancer of theAbramson Cancer Center (to R.H. Vonderheide, C. Clendenin, and E.E. Furth).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 16, 2014; revised February 6, 2015; accepted February 6,2015; published OnlineFirst February 24, 2015.

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2015;3:399-411. Published OnlineFirst February 12, 2015.Cancer Immunol Res Rafael Winograd, Katelyn T. Byrne, Rebecca A. Evans, et al. CarcinomaPD-1 and CTLA-4 Blockade and Improves Survival in Pancreatic Induction of T-cell Immunity Overcomes Complete Resistance to

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