Immunotherapy Converts Nonimmunogenic Pancreatic Tumors into ...

Research Article

Immunotherapy Converts Nonimmunogenic PancreaticTumors into Immunogenic Foci of Immune Regulation

Eric R. Lutz1,4,5,6, Annie A. Wu1,3,4, Elaine Bigelow1,4, Rajni Sharma2, Guanglan Mo1,4,5, Kevin Soares1,3,4,5,6,Sara Solt1,4,5, Alvin Dorman1,4,5, Anthony Wamwea1,4,5, Allison Yager1,4, Daniel Laheru1,4,5,Christopher L. Wolfgang1,3,4,6, Jiang Wang7, Ralph H. Hruban1,2,4,6, Robert A. Anders1,2,4,6,Elizabeth M. Jaffee1,2,4,5,6, and Lei Zheng1,3,4,5,6

AbstractPancreatic ductal adenocarcinoma (PDAC) is considered a "nonimmunogenic" neoplasm. Single-agent immu-

notherapies have failed to demonstrate significant clinical activity in PDAC and other "nonimmunogenic" tumors,in part due to a complex tumormicroenvironment (TME) that provides a formidable barrier to immune infiltrationand function. We designed a neoadjuvant and adjuvant clinical trial comparing an irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting, allogeneic PDAC vaccine (GVAX) given as a singleagent or in combination with low-dose cyclophosphamide to deplete regulatory T cells (Treg) as a means to studyhow the TME is altered by immunotherapy. Examination of resected PDACs revealed the formation of vaccine-induced intratumoral tertiary lymphoid aggregates in 33 of 39 patients 2 weeks after vaccine treatment. Immuno-histochemical analysis showed these aggregates to be regulatory structures of adaptive immunity. Microarrayanalysis of microdissected aggregates identified gene-expression signatures in five signaling pathways involvedin regulating immune-cell activation and trafficking that were associated with improved postvaccinationresponses. A suppressed Treg pathway and an enhanced Th17 pathway within these aggregates were associatedwith improved survival, enhanced postvaccination mesothelin-specific T-cell responses, and increased intratu-moral Teff:Treg ratios. This study provides the first example of immune-based therapy converting a "nonimmu-nogenic" neoplasm into an "immunogenic" neoplasm by inducing infiltration of T cells and development of tertiarylymphoid structures in the TME. Post-GVAX T-cell infiltration and aggregate formation resulted in theupregulation of immunosuppressive regulatory mechanisms, including the PD-1–PD-L1 pathway, suggesting thatpatients with vaccine-primed PDAC may be better candidates than vaccine-na€�ve patients for immune checkpointand other immunomodulatory therapies. Cancer Immunol Res; 2(7); 616–31. �2014 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) remains a lethal

malignancy with less than 5% of patients alive at 5 years (1).Standard therapies provide only short-term benefit beforechemoresistance develops. Immunotherapy, vaccines, andimmune-modulating agents have shown progress against che-motherapy-sensitive and chemoresistant "immunogenic" can-

cers such as renal cell carcinoma (RCC) and melanoma thatnaturally attract tumor-infiltrating effector T cells (2–4). How-ever, PDAC and other malignancies that are considered "non-immunogenic" neoplasms typically lack tumor-infiltratingeffector lymphocytes (5–8) and are less responsive to immu-notherapy (9). Thus, single-agent inhibitors of regulatory T-cell(Treg) signals, such as cytotoxic T-lymphocyte antigen-4(CTLA-4) and programmed death-1 (PD-1) receptor, whichdemonstrate significant clinical activity against melanoma,RCC, and non–small cell lung cancer (NSCLC), do not haveactivity in PDAC (2, 10, 11). However, we recently reportedtumor regressions and improved survival in patients withadvanced metastatic PDAC, who were treated with PDACGVAX combinedwith ipilimumab, which targets the inhibitorymolecule CTLA-4 on T cells (12), as compared with patientstreated with ipilimumab alone. These data suggest that T cellsfirst need to be induced to provide available cells for theactivation by T-cell–modulating agents like ipilimumab andnivolumab.

Antigen-specific T-cell responses have been observed insome patients with PDAC treated with vaccines (13). Wereported the induction of systemic mesothelin-specific T-cellresponses following treatment with PDAC GVAX in patients

Authors' Affiliations: Departments of 1Oncology, 2Pathology, and 3Sur-gery; 4The Sidney Kimmel Cancer Center; 5The Skip Viragh Center forPancreatic Cancer Research and Clinical Care; 6The Sol Goldman Pan-creatic Cancer Center, Johns Hopkins University School of Medicine,Baltimore, Maryland; and 7Department of Pathology and Laboratory Med-icine, University of Cincinnati College of Medicine, Cincinnati, Ohio

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

A.A. Wu and E. Bigelow contributed equally to this work.

Corresponding Authors: Lei Zheng, Johns Hopkins University School ofMedicine, 1650 Orleans Street, CRB1, Room 488, Baltimore, MD 21231.Phone: 410-502-6241; Fax: 410-614-8216; E-mail: [email protected]; andElizabeth M. Jaffee, [email protected]

doi: 10.1158/2326-6066.CIR-14-0027

�2014 American Association for Cancer Research.

CancerImmunology

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with resected and metastatic PDAC (12, 14–18). Mesothelin isan antigen expressed by virtually all PDACs, and posttreatmentdetection of enhanced mesothelin-specific T-cell responses inperipheral blood lymphocytes (PBL) is associated withimproved disease-free survival (DFS) and overall survival (OS)in GVAX-treated patients (12, 16–18). Despite evidence ofperipheral immune activation and antitumor activity in somepatients, immune tolerance mechanisms within the tumormicroenvironment (TME) likely inhibit the full potential ofvaccines alone (13). Thus, measures of peripheral immuneactivation following treatment with immunotherapy may notrepresent the immune activation status within the TME.Tumors evolve numerous mechanisms to escape immune

recognition (19). For PDAC, suppressive monocytes, includingdendritic cells (DC), neutrophils, and myeloid-derived sup-pressor cells (MDSC), immune checkpoints (CTLA-4 and PD-1), and CD4þCD25þFoxP3þ Tregs have been reported inpreclinical and clinical studies (13). Tregs have been foundinfiltrating the TME of many human tumors, including PDAC,and elevated Treg numbers are generally associated withshorter patient survival (6, 20–23). Previous studies havesuggested that Tregs can be depleted with immune-modulat-ing doses of cyclophosphamide to enhance immunotherapies(24–28).We previously reported the induction of higher aviditymesothelin-specific T-cell responses in the periphery ofpatients with metastatic PDAC when low-dose cyclophospha-mide is given 1 day before vaccination (16). Furthermore, ourpreclinical studies suggest that cyclophosphamide primarilyaffects subsets of Tregs found infiltrating tumors, and thatstudying peripheral Tregs does not provide insight into themechanisms by which Tregs regulate immune responses with-in the TME (29). However, the effect of cyclophosphamide onintratumoral Tregs and other immune-cell populations withinhuman cancers has not been well studied.In this study, we tested the hypothesis that vaccine-based

immunotherapy can convert PDACs from "nonimmuno-genic" into "immunogenic" tumors with infiltrating effectorlymphocytes. We evaluated the effects of GVAX, given aloneor in combination with Treg-modulating doses of cyclophos-phamide, on lymphocytes infiltrating PDAC tumors. Neoad-juvant therapy was initiated 2 weeks before surgical resec-tion to enable the direct assessment of the TME followingtreatment. Here, we show for the first time that an immune-based therapy induces the development of tertiary lymphoidaggregates within this "nonimmunogenic" neoplasm thatresemble ectopic lymph node–like structures observed insubsets of immunotherapy-na€�ve patients with more "immu-nogenic" cancers such as melanoma and NSCLC (30–33). Thedevelopment of intratumoral tertiary lymphoid aggregatesin PDAC tumors was dependent on vaccination, supportingGVAX as a trigger of antigen-specific immune responsesat the tumor site. Microdissection and gene array analysesdemonstrated that decreased Treg and increased Th17immune effector signatures within the vaccine-inducedintratumoral lymphoid aggregates were associated withenhanced systemic postvaccination mesothelin-specific T-cell responses, higher intratumoral Teff:Treg ratios, andlonger patient survival. However, as has been observed for

melanomas (34, 35) that are naturally infiltrated with T cells,the T-cell infiltration following treatment with GVAX wasalso associated with the upregulation of immunosuppressiveregulatory mechanisms within the PDAC TME that can betargeted by immune modulation. This process has beentermed adaptive resistance. Thus, these data support a newmodel for developing immunotherapy approaches in tumorslacking natural T-cell infiltration.

Materials and MethodsStudy subjects and tissue specimens

Between July 2008 and September 2012, 59 patients wereenrolled into an ongoing study (NCT00727441) of an irra-diated, allogeneic granulocyte-macrophage colony-stimulat-ing factor (GM-CSF)–secreting pancreatic tumor vaccine(GVAX) administered intradermally either alone or in com-bination with immunomodulatory doses of cyclophopha-mide as neoadjuvant and adjuvant treatment for patientswith resectable PDAC. Patients were randomized 1:1:1 intothree treatment arms (Supplementary Fig. S1, study sche-ma). In arm A, patients received GVAX alone; in arm B,patients received GVAX plus a single intravenous dose ofcyclophosphamide at 200 mg/m2 1 day before each vacci-nation; in arm C, patients received GVAX plus oral cyclo-phosphamide at 100 mg once daily for 1 week on and 1 weekoff (36). Up to six GVAX treatments were administered andall the patients remained in their initial treatment armsthroughout the duration of the study. All 59 patientsreceived the first GVAX treatment 2 weeks �4 days beforesurgery. Fifty-four patients successfully underwent pancrea-ticoduodenectomy (the Whipple surgery) and received thesecond GVAX treatment. Five patients were found to haveliver metastases during surgery, which were not radiograph-ically identified before the surgery, and instead these pati-ents underwent bypass surgery. Among the 54 patients whohad pancreaticoduodenectomy, 1 patient was found to haveampullary cancer, 1 to have neuroendocrine tumor, 2 to haveundifferentiated carcinoma, and 1 to have autoimmunepancreatitis. These patients' preoperative CT scans did notdistinguish their disease process from PDAC. In addition, 1patient had grossly residual tumors and another 9 patientshad recurrence immediately following surgery. All of thesepatients were taken off the study postoperatively. The 39patients remaining on the study received standard adjuvantchemo- and radiotherapy. Patients remaining disease freefollowing chemoradiotherapy received up to four additionalPDAC GVAX treatments every 4 weeks. A full clinical report,including survival analyses, will be submitted at the com-pletion of the study.

Tumor specimens from the 39 patients who remained inthe study and continued with adjuvant chemotherapy wereanalyzed in this study. Formalin-fixed paraffin-embedded(FFPE) tissue blocks were obtained from our pathologyarchive. For most tumors, a piece was also stored frozen at�80�C in optimal-cutting-temperature (OCT) freezing medi-um. PDAC tumor specimens collected before vaccine expo-sure from the 54 subjects treated in a previous adjuvant

Vaccine-Induced Intratumoral Lymphoid Aggregates

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vaccine study (17) and from 4 unvaccinated patients whounderwent surgery concurrently at our institution wereobtained from an Institutional Review Board (IRB)–approvedretrospective database and used as controls in this study.

Isolation of PDAC tumor-infiltrating lymphocytesAfter keeping adequate tissue for surgical pathology diag-

nosis, the majority of the remaining tissue was processed forisolating tumor-infiltrating lymphocytes (TIL). The sparedportion of each tumor was digested with enzymes into sin-gle-cell suspensions as described previously (15). Lymphocyteswere isolated from these tumor-digested single-cell suspen-sions by Percoll density gradient centrifugation and storedfrozen at �140�C. To isolate TIL subpopulations, TIL werethawed and labeled with anti-human CD3-PE, anti-humanCD4-PECy5, anti-human CD8-APC, and anti-human CD19-FITC (all monoclonal antibodies were from BD Pharmingen).CD3þCD4þ cells (CD4þ T cells), CD3þCD8þ cells (CD8þ Tcells), CD3�CD19þ cells (B cells), and CD3�CD4�CD8�CD19�

cells (non-T or -B cells) were isolated by fluorescence-activatedcell sorting (FACS) using a Beckman Coulter MoFlo FACSsorter. Dead cells were excluded using LIVE/DEAD FixableBlue Dead Cell Stain (Invitrogen).

ImmunohistochemistryImmunohistochemistry (IHC)was performed on FFPE 5-mm

sections of resected PDAC. Immunostaining for CD1a, CD3,CD4, CD8, CD20, CD56, CD68, and CD163 was performed onVentana autostainer using Ultra-view detection (VentanaMedical Systems, Inc.). The protocols and sources of antibodiesare summarized in Supplementary Table S1. Staining for IL17Aand RORgt was performed on Leica auto stainers using BondRefine detection (Leica Microsystems). For double staining ofIL17A and RORgt, Chromplex detection was used as per themanufacturer's guidelines (Bond Leica, Leica Microsystems).For some protocols, antigen retrieval was performed manuallyas indicated. PD-L1 staining was performed as previouslydescribed (37). Following standard protocol, slides were depar-affinized, hydrated, and heat-induced antigen retrieval wasperformed. Incubation with the primary antibody using opti-mal conditions was followed by development of immunostain-ing. Counterstaining with hematoxylin and eosin (H&E) wasapplied, and slides were dehydrated and coverslipped.

Microarray analysisApproximately 20 3-mm sections of FFPE tissue from each

subject were anonymized and stained with H&E immedi-ately before the lymphoid aggregates were microdissected bya pathologist (J. Wang) using a dissection microscope. Themicrodissected FFPE tissue was stored in RNALater solution(Ambion) until RNA was purified using the RecoverAll TotalNucleic Acid Isolation Kit for FFPE (Ambion) and amplifiedby the WT-Ovation FFPE System (NuGEN Technologies) asper the manufacturers' protocols. Gene expression micro-array analysis was performed using the Affymetrix HumanU133 Plus 2.0 array chips. The microarray data have beensubmitted to the Gene Expression Omnibus (Accessionnumber: GSE52171).

IHC image analysisAll slides were anonymized and scanned, and whole-slide

images were analyzed using the Image Analysis Software(Aperio Technologies). The area of tumor to be analyzed wascircled by a pathologist (R.A. Anders) on the anonymized H&E-stained slides, with attention to the inclusion of the largest areapossible of continuous neoplastic tissue while excluding, to theextent possible, normal pancreatic, intestinal, and smoothmuscle tissue. The Positive Pixel Count algorithm was usedto quantify IL17 expression. The Cell Surface Count algorithmand the Nuclear Count algorithm were used to quantify thenumber of cells positive for other immune-cell surface ornuclear markers, respectively. The automatic quantificationresults were anonymized and validated first by manual quan-tification by a pathologist (R.A. Anders).

Detection of mesothelin-specific T-cell responses byIFNg ELISPOT

The mesothelin peptide sequences and enzyme-linkedImmunoSpot (ELISPOT) assay used in this study have beendescribed previously (17, 18). PBLs were isolated by densitygradient centrifugation using Ficoll-Hypaque from peripheralblood collected at baseline, before, and following each PancGVAX treatment. The postvaccination blood sample after thefirst vaccination was obtained either the day before surgery oron the day of surgery before anesthesia. Postvaccination bloodsamples for all subsequent vaccinations were obtained 1month after each vaccination (Supplementary Fig. S1). OnlyPBLs from 27 of 39 patients expressed the HLA-A�0101 and/orHLA-A�0201 alleles and were analyzed by ELISPOT. T-cellresponses to mesothelin peptides were adjusted for back-groundmeasured against irrelevant melanoma or RCC controlpeptides. Responses were measured to eight HLA-A�0101and six HLAA�0201 mesothelin peptides. Postvaccination res-ponses were considered enhanced when the frequency ofmesothelin-specific T cells increased by at least 2-fold whencompared with pretreatment baseline frequencies. Mesothe-lin-specific T-cell levels measured in 16 patients (8 respondersand 8 nonresponders) whose microdissected lymphoid aggre-gates and/or TIL were used for gene-expression or FACSanalysis are shown in Supplementary Fig. S2A and S2B.

Quantitative real-time PCR analysisQuantitative real-time PCR was performed with the SYBR

Green system (SABiosciences) as per the manufacturer's pro-tocol. Prevalidated PCR primers for each gene listed in Sup-plementary Table S2 were obtained from Real Time Primer,LLC, or SABiosciences. The expression levels of each gene werenormalized to the same scale (1–4) as previously described(Supplementary Table S3; ref. 38). The average score for the setof genes representing each pathway was used to compare Thpathway gene expression between grouped patients' TILspecimens.

Intracellular cytokine and FoxP3 staining analysis of TILCryopreserved TIL isolated from 10 vaccinated patients

treated in this study and 4 unvaccinated control subjects wereanalyzed. TIL were thawed and incubated at 37�C overnight

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either alone ("unstimulated") or at a 1:1 ratio with humanT-cell Activator CD3/CD28 Dynabeads (Invitrogen; "stimulat-ed") in Iscove's Modified Dulbecco's Medium (IMDM) supple-mented with 5% FBS. After 16 hours, Protein TransportInhibitor Cocktail (eBioscience) was added and the TIL werecultured for an additional 4 hours. Unstimulated and stimu-lated TIL were then harvested and surface stained with anti-CD3-PECy7 (BD Pharmingen), anti-CD4-PECF594 (BD Hori-zon), and anti-CD8-APCEF780 (eBioscience), and intracellularstaining was performed following permeabilization and fixa-tion using eBioscience FoxP3/transcription Factor StainingBuffers with anti-IFNg-FITC (BD Pharmingen), anti-IL17A-PE(eBioscience), and anti-FoxP3-V450 (BD Horizon). Deadcells were excluded using LIVE/DEAD Fixable Aqua DeadCell Stain (Invitrogen). Stained samples were analyzed usinga Beckman Coulter Galios flow cytometer and FlowJo soft-ware (TreeStar, Inc.).

Statistical analysisOS is defined as the time from surgery to death from any

cause. Comparisons between groups were made using two-tailed unpaired Student t tests or Wilcoxon signed-rank tests.All graphing and statistical analyses were performed usingeither GraphPad Prism software (GraphPad Software) or SASversion 9.3 statistical software (SAS Inc.). Gene set enrichmentanalysis (GSEA) was performed according to the algorithmdescribed by Subramanian and colleagues (39), using theanalysis software provided by the Broad Institute (Cambridge,MA).

ResultsVaccine-based immunotherapy induces the neogenesisof intratumoral tertiary lymphoid aggregates thatresemble ectopic lymph node–like structuresThirty-nine patientswith resectable PDACwere randomized

to receive either GVAX alone, GVAX with one dose of intra-venous cyclophosphamide 1 day before vaccine, or GVAX withdaily oral cyclophosphamide for 1 week on and 1 week off(Supplementary Fig. S1). Patients underwent pancreaticoduo-denectomy 2 weeks following GVAX treatment and theresected PDAC tumor specimens were analyzed histologically.The majority (33/39 or 85%) of tumors contained intratumorallymphoid aggregates (Fig. 1A and B). In contrast, even thoughlymphocytes were sometimes present, organized intratumorallymphoid aggregates like the ones observed in tumors fromGVAX-treated patients were not detected in 54 PDAC speci-mens collected before vaccine exposure from patients treatedin a previous study (Fig. 1A and B; ref. 17). Thus, the formationof intratumoral aggregates was dependent on GVAX treat-ment (P < 0.001).Immunohistochemical analysis demonstrated that these

vaccine-induced aggregates were organized tertiary lymphoidstructures (Fig. 1A and C) and not random clusters of lym-phocytes (Supplementary Fig. S3). They were composed oforganized T- and B-cell zones, and contained germinal center–like structuresmarked by CD21þ follicular DCs (Fig. 1C; ref. 40)and Ki67þ actively proliferating cells (Fig. 1D). In most, the

B-cell zoneswere in the center whereas the T-cell zoneswere inthe periphery of the aggregate. There was also evidence oflymphoid neogenesis including the presence of lymphaticvessels marked by D2-40 (Fig. 1E) and infiltration of CCL21-expressing cells, a chemokine known to be involved in lym-phoid neogenesis (Fig. 1F; ref. 41).

Tertiary lymphoid structures develop in response to antigenstimulation. Thus, it is not surprising that innate immune cellscapable of presenting antigens were also present in theseaggregates. Although CD56þ [natural killer (NK) cell marker]and CD1aþ cells (immature DCmarker) were not present (Fig.1G), CD83þ and DC-LAMPþ mature DCs were present withinmost aggregates (Fig. 1H). CD68þ and CD163þ cells withmorphologies consistent with monocyte/macrophages werealso commonly present in these aggregates (Fig. 1H). Thus,these vaccine-induced lymphoid aggregates are organizedstructures of adaptive immunity.

The lymphoid aggregates that formed in GVAX-treatedPDAC patients resemble naturally induced ectopic lymphnode–like structures observed in tumors from immunothera-py-na€�ve patients with other cancers that are associated withlonger survival (30–33). Consistent with these prior studies,there was an association between the formation of postvacci-nation lymphoid aggregates and longer survival (Supplemen-tary Fig. S4). Because lymphoid aggregates formed in most(85%) vaccinated patients' tumors (Fig. 1B), not all PDACtumors from patients who survived <1.5 years lacked intratu-moral lymphoid aggregates (Supplementary Fig. S4). Thus, theinduction of lymphoid aggregates alone did not accuratelypredict the postvaccination induction of a clinically effectiveantitumor response.

Postvaccination effector T-cell trafficking into the TMEto form intratumoral lymphoid aggregates is associatedwith evidence of early T-cell activation, increasedrecruitment of Tregs, and upregulation of IFNg and thePD-1–PD-L1 T-cell suppressive pathway

The postvaccination formation of intratumoral lymphoidaggregates suggests that treatment with GVAX triggers anadaptive T-cell response at the tumor site. To assess theactivation status and functional polarity of T cells within theseaggregates, we evaluated the expression of a number of T-cellmarkers by IHC. The lymphoid aggregates contained bothCD45RAþ na€�ve and CD45ROþ antigen-experienced T cells(Fig. 2A). CD45RAþ T cells were clustered in B-cell zones,whereas CD45ROþ T cells were more enriched in T-cell zoneslocated in the periphery of the aggregates. These aggregates didnot contain many granzyme B–expressing T cells (Fig. 2B),which were found frequently in lymphocytes outside of theaggregates (Supplementary Fig. S5). In contrast, the early T-cellactivation marker CD69 and the activated T-cell traffickingchemokine receptor CXCR3 were abundantly expressed in thelymphoid aggregates (Fig. 2B). The aggregates also containedT-betþ cells consistent with the induction of IFNg-expressingeffector T-cell responses (Fig. 2B). These data suggest thatvaccine-induced aggregates are notmajor sites for cytotoxic T-cell lysis of target cells, but more likely are sites of initial T-cellactivation within the TME.






Along with signals of effector T-cell trafficking and earlyactivation, negative regulatory signals were also present inthese aggregates. FoxP3-expressing Tregswere present inmost

aggregates (Fig. 2B). In addition, PD-L1, an inhibitory ligandthat signals through the negative regulatory receptor PD-1 onactivated T cells (42), was commonly expressed by cells in

Figure 1. Intratumoral tertiarylymphoid aggregates observedin PDACs from Panc GVAX–treated patients. A, lymphoidaggregates (indicated by arrows)observed in PDACs from PancGVAX–treated (vaccinated)patients are not seen in PancGVAX–naïve (unvaccinated)patients. Lymphoid aggregatesare composed of T cells (anti-CD3 staining) and B cells (anti-CD20 staining). B, numbers ofPDACs with (TLAþ) or without(TLA�) at least one lymphoidaggregate present in theintratumoral area in 54unvaccinated patients and 39vaccinated patients. Intratumorallymphoid aggregates areobserved specifically in PDACsfrom vaccinated patients (Fisherexact test, P < 0.001). C, IHCanalysis with T-cell markers(CD3, CD4, and CD8), B-cellmarker (CD20), and follicular DCmarker (CD21). D, IHC analysis ofthe cell-proliferation markerKi67. E, IHC analysis of thelymphatic-vessel (arrows)marker D2-40. F, IHC analysis ofthe chemokine ligand CCL21(arrows). G, IHC analysis ofCD20þ B cells, CD3þ T cells,CD56þ NK cells, and CD1aþ

immature DCs. Note that anti-CD56 staining also marksneural tissues. H, IHC analysis ofCD68þ and CD163þ

macrophages and CD83þ andDC-LAMPþ mature DCs(arrows). All positive IHC signalsare in brown.

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aggregates with morphologies consistent with monocytes/macrophages (Fig. 2C). These PD-L1þ cells were surroundedby lymphocytes that expressed PD-1. Clusters of PD-L1þ cellswere also seen at the edges of intratumoral lymphoid aggre-gates where T cells tended to be located (Fig. 2D). In contrast,PD-L1–expressing cells were rarely detected in PDAC tumorsfrom vaccine-na€�ve patients (Fig. 2E), supporting our hypoth-esis that PD-L1 expression is induced by vaccine treatment.Some functional markers are difficult to assess by immuno-

histochemical analysis. To explore the functional activity of theseaggregates, we next studied postvaccination TIL isolated fromportions of the resected tumors. We compared IFNg productionby unstimulated TIL from vaccinated and unvaccinated patientsby FACS. Analysis was limited to specimens from 10 vaccinatedpatients and 4 unvaccinated patients who had enough TILrecovered for the assays. Elevated numbers of IFNg-producingT cells were detected in TIL from vaccinated patients (Fig. 3A).To evaluate whether Treg infiltration into tumors was affectedby GVAX, we compared the levels of PDAC tumor-infiltrating

Tregs between the same 10 vaccinated and 4 unvaccinatedpatients. As shown in Fig. 3B, the levels of CD3þCD4þFoxP3þ-

Tregs in TIL, quantified by FACS, were higher in vaccinatedpatients compared with that in unvaccinated patients. Weobserved a similar GVAX-induced increase in tumor-residingTregs in a murine PDAC model (Soares and colleagues; sub-mitted for publication). Thus, the activities of GVAX includedboth the recruitment of effector T cells into the TME and theupregulation of immunosuppressive regulatory mechanisms,specifically the expression of PD-L1 and Treg infiltration.

To estimate the net impact of the simultaneous infiltrationof both effector T cells and Tregs in the TME, we compared theratios between the number of IFNg-producing effector T cellsto the number of CD4þFoxP3þ Tregs, both measured by FACSdirectly in TIL without stimulation. Higher Teff:Treg ratiosmeasured by CD8þ/Foxp3þ T-cell ratio in TIL in untreatedpatients have been reported to correlate with better prognosisin multiple human cancers, including PDAC (3, 43–45). Wefound that the ratios of IFNg-producing Teff:Tregs were

Figure 2. Intratumoral lymphoid aggregates are sites of postvaccination T-cell activation and regulation. A, IHC analysis of CD3-, CD20-, CD45RO-, andCD45RA-expressing cells. B, IHC analysis of CD4-, T-bet-, Foxp3-, CXCR3-, CD69-, and granzyme B–expressing cells. C, IHC analysis of B7-H1/PD-L1- and PD-1–expressing cells. D, PD-L1 expression in a representative intratumoral lymphoid aggregate from a vaccinated patient. The squareindicates the edge of the intratumoral lymphoid aggregate that is shown in the bottom at a higher magnification. Brown staining indicates expressionof the indicated markers. E, absence of PD-L1 expression in a representative PDAC specimen from an unvaccinated patient.






higher in 7 of the 10 vaccinated patients with over a log-foldincrease in 5 of these 7 vaccinated patients when comparedwith unvaccinated patients (Fig. 3C). These data suggest thatGVAX can alter the balance of Teff to Tregs in favor of anantitumor response.

Specific gene expression signatures in intratumorallymphoid aggregates define regulatory structures thatcorrelate with immune responses and OS

To evaluate possible mechanisms regulating vaccine-induced lymphoid aggregates, and to assess whether particularfunctional immune signatures within these aggregates wereassociatedwith response to treatment, wefirst performed genemicroarray analysis on RNA isolated from microdissectedlymphoid aggregates (Supplementary Fig. S6). Gene expressionwas compared among samples grouped according to threedifferent correlates of vaccine response: patient OS, postvac-cination mesothelin-specific T-cell responses in PBL (17), andthe intratumoral CD8þ Teff to FoxP3þ Treg ratio. Mesothelin-specific CD8þT cells specific for six HLA-A2–binding and eightHLA-A1–binding mesothelin epitopes were measured usingIFNg ELISPOT assays as described previously (17). Mesothelin-specific T-cell levels measured before and 12 to 14 daysfollowing the first vaccination just before surgery are shownfor 16 patients whowere either HLA-A1þ or -A2þ: 8 responderswho developed enhanced (at least 2-fold higher than baseline)postvaccination mesothelin-specific T-cell responses and 8nonresponders who did not (see Supplementary Fig. S2A andS2B, respectively). The postvaccination levels of mesothelin-specific T cells were 2.6- to 54-fold higher than baseline inresponders compared with 0.97- to 57-fold lower than baselinein nonresponders.

Gene ontology analysis of the microarray data revealed thatgenes involved in regulating immune-cell trafficking and func-tion were differentially expressed, including genes encodingmultiple integrins, chemokines and chemokine receptors,

members of the NF-kB pathway, and the ubiquitin–proteasomesystem (UPS; Supplementary Fig. S7A–S7D, respectively). Thesepathways are involved in regulating both adaptive and innateimmune cells. A few themes among the signatureswere present.Specifically, chemokine and integrin signals involved in recruit-ing immunosuppressive populations were downregulated inpatientswho had longer survival. These include the chemokinesCXCL9, CCL18, CCL19, and CCL21, and the chemokine receptorCCR7 involved in recruiting Tregs (34, 46–48), chemokine CCL2involved in recruiting immunosuppressive populations ofmonocytes/macrophages (49, 50), and the integrins ITGA4 andITGB1 that are expressed by the suppressive MDSCs (Fig. 4A;ref. 51). A specific NF-kB signature defined by the downregula-tion of canonical NF-kB genes (NFKB1, RELA, and REL) com-bined with the upregulation of noncanonical NF-kB genes(NFKB2 and RELB) was associated with improved postvacci-nation responses (Fig. 4B). Although NF-kB is a known regu-lator of all immune subsets, this signature is consistent withpolarization toward Th17 and away from Tregs (52, 53). TheUPS signature may also represent both adaptive and innateimmune signals but the underlying mechanisms of this signa-ture are less clear. However, it is interesting to note that thedownregulation of the UBE2N gene that encodes an E2 ubiqui-tin-conjugating enzyme important for maintaining Treg func-tion and preventing Tregs from acquiring effector T cell–likefunction (54) was specifically associated with longer survivaland higher intratumoral Teff:Treg ratios (Fig. 4B). In addition,multiple genes involved in the Treg pathway were downregu-lated, whereas multiple genes in the Th17 pathway wereupregulated in lymphoid aggregates frompatientswho survivedlonger and developed enhanced immune responses (Fig. 4C andSupplementary Fig. S8). In contrast, the expression of Th1 andTh2 effector pathway genes was not uniformly upregulated ordownregulated (Fig. 4C). Gene set enrichment analysis (GSEA;ref. 39) of themicroarray data further supported the hypothesisthat the differences in gene expression in the Treg and Th17

Figure 3. Postvaccination effector T-cell (Teff) activation in intratumoral lymphoid aggregates is associated with Treg infiltration into the TME. Percentagesof IFNg-producing CD4þCD8þ effector T cells (A) and CD3þCD4þFoxP3þ Tregs (B) quantified by FACS in unstimulated TIL isolated from 10 vaccinatedpatients compared with those isolated from 4 unvaccinated patients. C, IFNg-producing Teff:Treg ratios measured in unstimulated TIL from the same sets ofvaccinated and unvaccinated patients. Comparisons between vaccinated and unvaccinated TIL were made using Wilcoxon signed-rank tests, and thecalculated P values are shown.

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pathways, but not in the Th1 or Th2 pathways, in these aggre-gates may predict clinical and immune responses (Fig. 5).Because PD-L1 protein expression was shown by IHC to be

induced by vaccination, we next compared PD-L1 (CD274) andPD-1 (PDCD1) gene expression with respect to OS, enhancedT-cell responses, and Teff:Treg ratios (Fig. 4D). CD274/PD-L1expression was downregulated in patients who survived >3

years, whereas there was no correlation with enhanced T-cellresponses and Teff:Treg ratios, In contrast, PDCD1/PD-1expression was increased in lymphoid aggregates frompatients who survived >3 years and who demonstratedenhanced mesothelin-specific T-cell responses. Because PD-1 upregulation was associated with improved survival andimmune responses, it may be a marker of postvaccination

Figure 4. Gene expression signatures in lymphoid aggregates associated with immune responses and OS. Gene expression measured by microarray wascompared between microdissected lymphoid aggregates from 14 tumors grouped according to overall patient survival (OS > 3 years vs. <1.5 years),postvaccination induction of enhancedmesothelin-specific T-cell responses in PBL (Enh vs. Unenh), and the intratumoral CD8þ T effector (Teff):FoxP3þ Tregratio (high vs. low CD8:Foxp3 ratio). Gene ontology analysis revealed the enrichment of differentially expressed genes encoding numerous chemokineand chemokine receptor, integrin and adhesion molecule, NF-kB pathway, and UPS components (Supplementary Fig. S6). Differences in the Treg andTh17pathway genes, but not theTh1 or Th2 pathway genes,were also associatedwith improved postvaccination responses and survival (Supplementary Fig.S7). The linear fold-change in expression for each comparison is shown for representative genes in chemokine, chemokine receptor, and integrin genes (A),NF-kB pathway genes (B), Th pathway genes (C), and PD-1 and PD-L1 (D).






T-cell activation in these aggregates, independent of its role indownregulating antitumor T-cell responses when engaged byits ligand, PD-L1.

Decreased Tregs are associatedwith enhanced Th1/Th17signals in lymphoid aggregates and prolonged survivalfollowing vaccine therapy

Microarray analysis of microdissected intratumoral lym-phoid aggregates was performed in a subset of vaccinated

patients and IHC was used to further evaluate the in situexpression of Th pathway genes in a larger cohort of patients.We examined the expression of representative transcriptionfactors known to drive Th1 (T-bet), Th2 (GATA-3), Treg(Foxp3), and Th17 (RORgt) differentiation. As shown in Fig.6A–D, T-bet and RORgt protein expression was increased,whereas Foxp3 expression was decreased in the immuneaggregates from patients who survived longer. No differencein GATA3 expression was observed. Costaining of RORgt and

Figure 5. GSEA analyses of Th1, Th2, Th17, and Treg pathways. GSEA analyses were performed on the microarray data. A, comparison between specimensfrompatientswithOS>3years (subject numbers in gray) andpatientswithOS<1.5 years (subject numbers in yellow). B, comparisonbetween specimens frompatients who developed enhanced T-cell responses (in gray) and patients who did not (in yellow). C, comparison between specimens from patients whosetumors were infiltrated with a high CD8:FoxP3 ratio (in gray) and whose tumors were infiltrated with a low CD8:FoxP3 ratio (in yellow). Heatmaps of themicroarraygeneexpressiondata are shown.Blue shading represents downregulation and red represents upregulationof gene expression.P values are shownfor each GSEA comparison.

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IL17A (Fig. 6E) confirmed that RORgtþ cells also expressedIL17A, although many IL17Aþ T cells did not express RORgt,likely because Th17 cytokine expression is also regulated byother transcription factors (55). Depending on the cytokinemilieu, Th17 cells can also express T-bet and possess Th1-likeeffector functions (56), but it is not yet clear whether T-betþ

cells in these lymphoid aggregates were producing Th1- orTh17-like cytokines, or both. Regardless of the specific tran-scription factor(s) driving IL17A expression, we found thatIL17A protein expression increased significantly in lymphoidaggregates frompatientswho survived>3 years comparedwiththose who survived <1.5 years (Fig. 6F).

Signatures within TIL reveal reduced Tregs but nodifferences in Th1/Th2/Th17 gene expression inassociation with survivalWe next evaluated the gene expression in TIL isolated

from PDAC tumor specimens to determine whether thesame T-cell signatures in the aggregates were also presentin TIL. TIL were sorted from 20 PDAC TIL specimens for theanalysis of gene expression in individual CD4þ, CD8þ, andnon–T cell subpopulations. Consistent with signatures in theaggregates, Th1 and Th2 pathway gene expression was notassociated with any parameters of response (SupplementaryTable S3), and Treg pathway gene expression was decreased

in CD4þ TIL from patients with longer survival (Supplemen-tary Table S3 and Supplementary Fig. S9A). However, unlikesignatures in the aggregates, Th17 pathway gene expressionin CD4þ TIL and CD8þ TIL was not different betweenpatients who survived >3 years and those who survived<1.5 years (Supplementary Fig. S9B and S9C, respectively,and Supplementary Table S3). This result may not be sur-prising as some key Th17 pathway cytokines, such as IL23A,are not expressed by T cells but by other cell types (Sup-plementary Table S4) that are abundant in the aggregates.However, the majority of TIL specimens also did not havedetectable levels of IL17 expression (Supplementary TableS4). These data suggest that gene expression signatures inthe total population of T cells that infiltrate tumors aresimilar, but not identical to gene signatures in T cells withinthe lymphoid aggregates. Furthermore, these data suggestthat IL17A-producing T cells represent a relatively smallpopulation of T cells within the TME that are enriched inlymphoid aggregates of vaccine responders. In support ofthis notion, only 0.124% to 2.27% of CD4þ TIL and 0% to0.410% of CD8þ TIL from vaccinated patients producedIL17A following overnight stimulation with anti-CD3- andanti-CD28–coated beads (Supplementary Fig. S10). Takentogether, these data suggest that vaccine-induced Th17signatures reside mainly within the lymphoid aggregates.

Figure 6. In situ expression ofsignature genes in the Th1, Th2,Th17, and Treg pathways. A, anti-Foxp3 antibody staining for Tregs.B, anti-T-bet staining for Th1 cells.C, anti-RORgt staining for Th17cells. D, anti-GATA-3 staining forTh2 cells. For each target, onerepresentative lymphoid aggregatefrom a patient with OS > 3 years and1 from a patient with OS < 1.5 yearsis shown. All positive signals are inbrown. E, costaining of IL17A (inred) and RORgt (in brown) in arepresentative lymphoid aggregate.F, cumulative IL17 signals for allintratumoral lymphoid aggregatesfor each patient's tumor sectionanalyzed were quantified usingImage Analysis Software (Aperio),and the expression levels werecompared between patients withOS > 3 years (n ¼ 6) and OS < 1.5years (n ¼ 6). Medians and the Pvalue calculated using theWilcoxonsigned-rank test are shown.






Figure 7. Low-dose cyclophosphamide (Cy) reduces intratumoral Treg numbers and promotes enhanced T-cell trafficking and activation within the tertiarylymphoid aggregates. A, comparison of the densities of Foxp3þ T cells in lymphoid aggregates between patients receiving GVAX alone (GVAX) and patientsreceiving GVAX plus Treg-modulating doses of cyclophosphamide (Cy þ GVAX). B, IHC staining of Foxp3, T-bet, CD45RA, and CD45RO in representativelymphoid aggregates in PDACs from patients with high Teff:Treg ratios in the TME compared with those with low Teff:Treg ratios in the TME. C, IHCstaining of CD69 and CXCR3 in representative PDAC tumor areas from patients with high versus low Teff:Treg ratios in the TME. All positive IHC signals are inbrown. D, proposed model of vaccine-induced T-cell infiltration into the PDAC TME. At baseline, the "nonimmunogenic" PDAC TME is primarilyimmunosuppressed and infiltrated with Tregs and other immunosuppressive populations of innate immune cells, and low numbers of effector T cells.Vaccination induces antigen-specific T cells systemically that traffic to the TME and initiate an inflammatory reaction. (Continued on the following page.)

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Low Treg numbers in lymphoid aggregates enableenhanced T-cell activation and trafficking within PDACtumorsBecause decreasedTreg signatures in the aggregates andTIL

are associated with improved postvaccination responses, andthe intended purpose of low-dose cyclophosphamide is Tregdepletion, we next evaluated by IHC the effects of cyclo-phosphamide on the number of Tregs in vaccine-inducedaggregates. A trend toward increased numbers of aggregatesinfiltrating PDACswas observed in patients treatedwithGVAXplus cyclophosphamide (Supplementary Fig. S11A). Althoughthe overall numbers of CD4þ T cells were not different (Sup-plementary Fig. S12), fewer Foxp3þ Tregs were detected in theaggregates from patients treated with GVAX plus cyclophos-phamide versuswithGVAX alone (Fig. 7A). Because both formsof cyclophosphamide used to deplete Tregs showed similarresults, the two cyclophosphamide groups were combined forthe analyses. Even though we only evaluated TIL from 10treated patients (Fig. 3C), consistent with the IHC data,CD3þCD4þFoxP3þTreg levels measured in TIL by FACS werealso lower in patients who received GVAX plus cyclophospha-mide versus GVAX alone (Supplementary Fig. S11B). Thesedata suggest that cyclophosphamide can lower Treg levels intheTME, andTregsmay inhibit lymphoid aggregate formation.To assess the impact of Teff:Treg ratios on T-cell activation

within the TME, we evaluated the expression of T-cell activa-tion markers in tumors with low and high Teff:Treg ratiosdefined by IHC. Teff:Treg ratios were considered high if theywere above 10, and low if theywere below 5. Increased numbersof CD45ROþ antigen-experienced and T-betþ T cells anddecreased numbers of CD45RAþ-na€�ve T cells were detectedin lymphoid aggregates from vaccinated patients who hadhigher Teff:Treg ratios in their TME (Fig. 7B). In patients withhigher Teff:Treg ratios, greater numbers of activated lympho-cytes expressing CD69 and CXCR3 were also found traffickingoutside of lymphoid aggregates into the stroma surroundingneoplastic cells (Fig. 7C). Thus, these data suggest that treat-mentwith cyclophosphamide skews the postvaccination intra-tumoral Teff:Treg ratio and promotes effector T-cell activationand trafficking within the TME.

DiscussionHere, we report the first description of tertiary lymphoid

aggregates infiltrating humanPDAC. These aggregates develop

2 weeks after a single treatment with a PDAC vaccine. Thesenovel structures express TME signatures that describe previ-ously unrecognized foci of immune regulation within PDAC,uncover PDAC signaling pathways associated with long-termsurvival, and identify potential targets for collaborativeimmune modulation in the setting of a PDAC vaccination.Taken together, these lymphoid aggregates provide the firstdemonstration that an immune-based therapy can convert a"nonimmunogenic" neoplasm into one that is similarly "immu-nogenic" to melanomas and RCCs.

Tertiary lymphoid structures play a critical role in numerousautoimmune and inflammatory diseases (57). Different fromprimary and secondary lymphoid structures, their formationdepends on antigen stimulation and represents an ongoingadaptive immune response. However, their role in cancerdevelopment and cancer therapy is not clear. In immunother-apy-na€�ve patients with colon cancer, NSCLC, and melanoma,increased numbers of lymphoid structures have correlatedwith better prognoses (31–33) and may be useful for selectingpatients who are better candidates for immunotherapy (31).However, whether these structures are formed in response totumor-antigen stimulation or to the pathogens and inflam-matory conditions towhich they are often exposed is unknown.Furthermore, the anticancer role of these structures is notestablished. In fact, under chronic inflammatory conditions,similar lymphoid structures are also associated with cancerdevelopment (58).

We have established for the first time that GVAX can inducesimilar intratumoral tertiary lymphoid structures not naturallyformed in PDAC. Our data support the notion that thesestructures are newly formed on the basis of the expression ofneogenesis markers within these structures together withantigen-experienced and activated T cells not commonlyfound in untreated PDACs. Whether these tertiary lymphoidstructures are unique toGVAXor can be elicited by other formsof immune-based therapy is not yet known. Similar to reportsin the literature (32, 33), these structures were associated withprolonged survival in some but not all GVAX-treated patients.This is not surprising as our analyses support their role asorganized structures of immune regulation. Evidence support-ing their role as regulators includes the expression of earlymarkers of T-cell activation, IFNg , as well as the recruitmentof suppressive cell populations and the upregulation of T-cell–suppressing regulatory pathways. It is possible that the

(Continued.) Chemokines, including CCL2, CCL18, CCL19, and CCL21, and other signals produced during this inflammatory reaction, recruit additionalimmune effector and regulatory cells and induce the neogenesis of tertiary lymphoid aggregates within the TME, converting the PDAC TME into an"immunogenic" environment. These lymphoid aggregates are composed of effector T cells, B cells, Tregs, and antigen-presenting cells (APC) and serve asnodal sites for immune activation and regulation within the TME. Paradoxically, higher expression of the chemokines associated with the development ofthese aggregates favors the recruitment of immunosuppressive immune cells into the lymphoid aggregates in the TME. Lymphoid aggregates dominated byeffector T cells and immune activation signals ("effector lymphoid aggregates"), including increased CD8þ effector T cells and effector T cells with Th17signatures, increased PD-1 expression on effector T cells, decreased Tregs, and lower expression of PD-L1, are associated with higher Teff:Treg ratiosin TIL and enhanced postvaccination T-cell responses in PBL and generate productive antitumor responses that can prolong survival. In contrast,lymphoid aggregates dominated by immunosuppressive immune cells and signals ("suppressor lymphoid aggregates"), including decreased CD8þ effectorT cells and effector T cells with Th17 signatures, decreased PD-1 expression on effector T cells, increased Tregs, and higher expression of PD-L1, areassociated with decreased Teff:Treg ratios in TIL and unenhanced T-cell responses in PBL, do not generate productive antitumor responses, and cannotprolong survival. Useof targeted immunemodulatorsmay tip thebalancebetween thesesignals in favor of thedevelopment of "effector lymphoid aggregates"and enhance the intratumoral immune responses induced by vaccines. However, without a vaccine, an "immunogenic" TME containing effector T cellsand lymphoid aggregates is not available for immune modulators to act on.






formation of lymphoid aggregate results from the activationand re-organization of immune cells present in the TME beforevaccination. Because GVAX is delivered intradermally and notat the tumor site, presumably additional immune cells maytraffic into the tumors following vaccination. In this study, wedid not obtain prevaccination tumor biopsies because it is notthe standard of care at our institution. For future studies,collection of pretreatment tumor biopsies will be necessary toevaluate changes in the TME following vaccination and toelucidate the mechanisms driving the development of post-vaccination lymphoid aggregates. Additional studies will berequired to determine whether T cells that are activatedwithin the lymphoid aggregates migrate directly into thetumor tissue or reenter the circulation first. We have assumedthat mesothelin-specific T cells measured in the PBL areactivated in the vaccine-draining lymph nodes. However, itis possible that some mesothelin-specific T cells are actuallyactivated within the aggregates. In fact, it is possible thatdifferent routes can be used to enter the tumor depending onwhether a T cell is activated in the periphery or within alymphoid aggregate.

In some PDACs, the lymphoid aggregates were dominatedby immunosuppressive signals, such as IL6, STAT3, and TGFb,while others had proinflammatory signals, such as IL12, IFNg ,and IL17, which are associated with activated T-cell responses.The immunosuppressive signaling proteins PD-1 and PD-L1were present in all intratumoral lymphoid aggregates. BecauseIFNg can induce PD-L1 expression (34, 35), it is likely that thePD-L1 expressed in the intratumoral lymphoid aggregates wasinduced by the IFNg produced by the lymphoid aggregate-residing CD4þ and CD8þ T cells. This could explain partiallywhy IFNg gene expression was lower in lymphoid aggregates inpatients who demonstrated improved postvaccinationresponses and longer survival. In contrast, PD-1 was upre-gulated in the lymphoid aggregates from patients whodemonstrated improved survival and enhanced postvacci-nation peripheral T-cell responses. PD-1 is a marker of T-cellactivation and likely requires the engagement with PD-L1 toconfer a downregulatory signal to the T cell. However, PD-L1was not naturally upregulated in treatment-na€�ve patients,or in untreated mice bearing implanted PDAC tumors(Soares and colleagues; submitted for publication). Our datasupport prior reports that PD-L1 is upregulated by IFNg-secreting T cells recruited into the TME (34, 35). However,patients with lower levels of PD-L1 within their lymphoidaggregates demonstrated longer OS, which is consistent withthe increased antitumor effect observed in PDAC-bearingmice treated with GVAX in combination with PD-1/PD-L1–targeted immune modulation (Soares and colleagues; sub-mitted for publication).

This study also aimed to address the role of Tregs in alteringT-cell responses within the TME. Prior reports from our groupand others have shown that Treg infiltration in PDAC andother cancers is associated with T-cell suppression within theTME in human patients and in mouse models (16, 21–29).Some reports have also correlated baseline Treg-infiltrationwith poorer prognosis in patients with PDAC (20, 43). Pre-clinical data have indicated that the Teff:Treg ratio, rather

than the absolute numbers of Tregs infiltrating the TME,correlates with antitumor immunity (59). This is the firststudy conducted in human patients confirming that it is thebalance between the infiltrating effector T cells and Tregs thatcorrelates with treatment outcomes. This is also the firststudy to show in patients that cyclophosphamide given withvaccination does indeed alter the balance in favor of aneffector T-cell response. However, not all patients who didnot receive cyclophosphamide had a lower Teff:Treg ratio,supporting differences in the natural inflammatory compo-sition of PDACs among patients, which will require furtherexploration.

The complex nature of the TME is demonstrated by thefunctional and signaling differences between the lymphoidaggregates and the TIL that were evaluated in a smaller subsetof patients. The lymphoid aggregates expressed signals of T-cell activation and regulation; TIL expressed signals of activa-tion predominantly. Notably, the aggregates expressed mixedTh1 and Th17 signals, whereas TIL mostly expressed Th1-likesignals. In fact, differentially expressed genes in the five sig-naling pathways (Th17/Treg, NF-kB, ubiquitin–proteasome,chemokines/chemokine receptors, and integrins/adhesionmolecules) within the intratumoral lymphoid aggregates wereassociated with improved postvaccination responses. Thesepathways have known effects on immune responses and thedevelopment of lymphoid structures. In particular, the activa-tion of the noncanonical NF-kB pathway following lympho-toxin/lymphotoxin receptor engagement is critical for lym-phoid structure development (60). Our results are consistentwith prior studies showing that Tregs can suppress the devel-opment of tertiary lymphoid structures (61, 62), whereas cellswith Th17 subsignatures can promote their development (63,64). Procancer inflammatory signals, including IL6 and STAT3(65) that are also positive regulators of Th17 responses (55, 66),were not upregulated in the vaccine responders. Our datasuggest that the upregulation of the Th17 pathway is associ-ated with improved responses to GVAX, but the exact role ofIL17-producing cells in the postvaccination TME is not yetknown. Given that Th17 cells can possess both Treg and Th1-like effector functions (56, 67–70), it is possible that particularTh17 subsignatures are correlated more closely with improvedpostvaccination immune responses (65, 71–73). Although it isnot clear how the vaccine-induced lymphoid aggregates iden-tified in this study compare with naturally occurring tertiarylymphoid structures, several of the 12 chemokines (includingCCL2, CCL18, CCL19, CCL21, and CXCL9) that are associatedwith melanoma (31) were expressed but downregulated inpostvaccination aggregates in patients who demonstratedprolonged survival and elevated intratumoral Teff:Treg ratios.These chemokines may affect the formation of vaccine-induced lymphoid aggregates by attracting both effector Tcells and Tregs; our data suggest that at higher levels, thesechemokines may favor Tregs and a more immunosuppressiveTME. Additional studies are required to refine these signaturesand determine which members of these pathways are criticalfor regulating the trafficking of lymphocytes into the tumor, theformation of lymphoid aggregates, and the evolution of thePDAC TME following vaccination.

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On the basis of our findings, we propose a new model inwhich traditionally "nonimmunogenic" tumors can be con-verted into "immunogenic" tumors responsive to combinationimmune-based therapies. In thismodel (Fig. 7D), a systemicallyadministered vaccine is required to initiate the induction ofimmune responses that can traffic into tumors and create aninflammatory environment. This environment has the poten-tial to support an anticancer immune response, but it containsnumerous immunosuppressive components, such as infiltrat-ing Tregs and other suppressive cell populations, and theupregulation of PD-L1, which is a natural response to theinitial inflammatory "invasion" of the TME. At this stage, theorganized inflammatory infiltrates are sites of immune regu-lation. In some patients, this initial inflammation is enough toshift the balance from immune suppression to immune acti-vation, particularly when a vaccine is givenwith Treg-depletingtherapy. However, in most patients, additional immune mod-ulation is needed, which may include targeting MDSC, tumor-associated macrophages (TAM), and tumor-associated neu-trophils (TAN), to more effectively shift the balance frompredominantly cumulative T-cell downregulatory signalstoward a majority of T-cell–activating signals. Additionalstudies are required to determine the role of these immuno-suppressive cell subsets in modulating the postvaccinationTME.On the basis of the evidence available, this model explains

why vaccination and immune modulation as single-agenttherapies have failed in patients with PDAC. This model isfurther supported by our recent study demonstrating objectiveresponses in patients with metastatic PDAC treated with thecombination of GVAXþ ipilimumab but not with ipilimumabalone (12). Ourmodel provides the strongest rationale so far forcombining immune-modulating agents with vaccination inpatients whose tumors do not naturally contain abundanteffector T cells.

Disclosure of Potential Conflicts of InterestR.A. Anders has received commercial research support from Bristol-Myers

Squibb. No potential conflicts of interest were disclosed by the other authors.

DisclaimerUnder a licensing agreement between Aduro BioTech, Inc. and the Johns

Hopkins University and E.M. Jaffee, the University is entitled to milestonepayments and royalty on sales of the vaccine product described in this article.

Authors' ContributionsConception and design: E.R. Lutz, D. Laheru, R.A. Anders, E.M. Jaffee, L. ZhengDevelopment of methodology: E.R. Lutz, R. Sharma, A. Yager, D. Laheru,J. Wang, L. ZhengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.R. Lutz, A.A.Wu, E. Bigelow, G.Mo, K. Soares, S. Solt,C.L. Wolfgang, J. Wang, R.A. Anders, L. ZhengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.R. Lutz, E. Bigelow, R. Sharma, S. Solt, A. Wamwea,A. Yager, D. Laheru, C.L. Wolfgang, R.H. Hruban, E.M. Jaffee, L. ZhengWriting, review, and/or revision of the manuscript: E.R. Lutz, R. Sharma,S. Solt, A. Yager, D. Laheru, C.L. Wolfgang, J. Wang, R.H. Hruban, E.M. Jaffee,L. ZhengAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.A. Wu, E. Bigelow, S. Solt, A. DormanStudy supervision: L. Zheng

AcknowledgmentsThe authors thank Dr. Katie Bever, who provided technical assistance in

the beginning of this study. The authors also thank Lee Shu-Fune Wu, ConnieTalbot, Soonweng Cho, and Donger Zhou for their advice on microarray dataanalyses.

Grant SupportThis work was supported by the NIH K23 CA148964-01 (to L. Zheng), Johns

Hopkins School of Medicine Clinical Scientist Award (to L. Zheng), an AmericanSociety of Clinical Oncology Young Investigator Award (to L. Zheng), ViraghFoundation and the Skip Viragh Pancreatic Cancer Center at Johns Hopkins (toD. Laheru, E.M. Jaffee, E.R. Lutz, and L. Zheng), The National Pancreas Foun-dation (to L. Zheng), Lefkofsky Family Foundation (to L. Zheng), the NCI SPOREin Gastrointestinal Cancers P50 CA062924 (to E.M. Jaffee, L. Zheng, and E.R.Lutz), Lustgarten Foundation (to E.M. Jaffee and L. Zheng), and the Sol GoldmanPancreatic Cancer Center (to E.R. Lutz and L. Zheng), AACR-FNABFellowsGrantfor Translational Pancreatic Cancer Research Award 09-30-14-LUTZ (to E.R.Lutz). E.M. Jaffee is the first recipient of the Dana and Albert "Cubby" BroccoliEndowed Professorship.

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 indicate thisfact.

Received February 17, 2014; revised March 20, 2014; accepted April 3, 2014;published OnlineFirst June 18, 2014.

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2014;2:616-631. Published OnlineFirst June 18, 2014.Cancer Immunol Res Eric R. Lutz, Annie A. Wu, Elaine Bigelow, et al. into Immunogenic Foci of Immune RegulationImmunotherapy Converts Nonimmunogenic Pancreatic Tumors

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