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Research Article

Stereotactic Radiation Therapy AugmentsAntigen-Specific PD-1–Mediated AntitumorImmune Responses via Cross-Presentation ofTumor AntigenAndrew B. Sharabi1,2, Christopher J. Nirschl2, Christina M. Kochel2, Thomas R. Nirschl2,Brian J. Francica2, Esteban Velarde1, Theodore L. Deweese1,2,3, and Charles G. Drake2,3,4

Abstract

The immune-modulating effects of radiotherapy (XRT) havegained considerable interest recently, and there have beenmultiple reports of synergy between XRT and immunotherapy.However, additional preclinical studies are needed to demon-strate the antigen-specific nature of radiation-induced immuneresponses and elucidate potential mechanisms of synergy withimmunotherapy. Here, we demonstrate the ability of stereo-tactic XRT to induce endogenous antigen-specific immuneresponses when it is combined with anti–PD-1 checkpointblockade immunotherapy. Using the small animal radiationresearch platform (SARRP), image-guided stereotactic XRTdelivered to B16-OVA melanoma or 4T1-HA breast carcinomatumors resulted in the development of antigen-specific T cell–and B cell–mediated immune responses. These immune-stim-

ulating effects of XRT were significantly increased when XRTwas combined with either anti–PD-1 therapy or regulatoryT cell (Treg) depletion, resulting in improved local tumorcontrol. Phenotypic analyses of antigen-specific CD8 T cellsrevealed that XRT increased the percentage of antigen-experi-enced T cells and effector memory T cells. Mechanistically, wefound that XRT upregulates tumor-associated antigen–MHCcomplexes, enhances antigen cross-presentation in the drain-ing lymph node, and increases T-cell infiltration into tumors.These findings demonstrate the ability of XRT to prime anendogenous antigen-specific immune response and provide anadditional mechanistic rationale for combining radiation withPD-1 blockade in the clinic. Cancer Immunol Res; 3(4); 345–55.�2014 AACR.

IntroductionIonizing radiation is a locally directed therapy that induces

lethal chromosomal aberrations and activates the DNA damageresponse pathways, including ATMandp53, resulting in cell-cyclearrest and apoptosis or mitotic catastrophe (1, 2). However,radiotherapy (XRT) also activates other signal transduction path-ways and transcription factors, including protein kinase C (PKC)and mitogen-activated protein kinases (MAPK; refs. 3, 4), as wellas nuclear factor-kB (NF-kB; ref. 5), which is a master regulator ofimmune responses. Activation of these signaling pathways andtranscription factors can result in significant changes to thephenotype of cancer cells before cell death. Supporting this is agrowingbodyof literature demonstrating howXRT can change the

immunophenotype of cancer cells and alter how the immunesystem interacts with cancer cells (6–12). For example, in a studyof 23 human carcinoma cell lines treated in vitro with radiation,91% of the cell lines upregulated one or more of the surfacemolecules, including Fas, intercellular adhesion molecule-1(ICAM-1), mucin-1, carcinoembryonic antigen (CEA), and/ormajor histocompatibility (MHC) class I (7). Furthermore, theirradiated CEA/A2 colon tumor cells were more susceptible tokilling by CEA-specific CD8 cytotoxic T lymphocytes (CTL) ascompared with nonirradiated tumor cells (7). Similar directeffects of radiation on the immunophenotype of tumor cells andresponding immune cells have been corroborated by severalgroups (8–12).

There is evidence supporting the hypothesis that the immunesystem itself may play a critical role in the therapeutic efficacy ofXRT (13–17). Early data showed that the radiation dose requiredto control a fibrosarcoma tumor in 50% of mice (TCD50) wassignificantly increased in immunocompromised mice as com-pared with control mice (13). Conversely, when the immunesystem was activated with bacterial pathogens, the radiation doserequired to control the tumor was significantly reduced (13).More recent data show that CD8 T cells play a key role in theantitumor effect of standard XRT applied to B16 melanomatumors. Specifically, depleting CD8 T cells reduced the antitumoreffect of XRT and decreased survival of mice with melanomatumors (14, 15). These findings run counter to the conventionalparadigm that XRT induces tumor cell kill primarily throughDNAdamage alone and instead suggest that the immune system mayplay an underappreciated role in the therapeutic effects of XRT.

1Department of Radiation Oncology and Molecular RadiationSciences, Johns Hopkins University School of Medicine, Baltimore,Maryland. 2DepartmentofOncology, JohnsHopkinsUniversity Schoolof Medicine, Baltimore, Maryland. 3The Brady Urological Institute,Johns Hopkins University School of Medicine, Baltimore, Maryland.4Department of Molecular Biology and Genetics, Johns Hopkins Uni-versity School of Medicine, Baltimore, Maryland.

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

Corresponding Author: Charles G. Drake, Johns Hopkins Sidney Kimmel Com-prehensive Cancer Center, 1650 Orleans Street, CRB I #410, Baltimore, MD 21231.Phone: 410-614-3616; Fax: 410-614-0549; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0196

�2014 American Association for Cancer Research.

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Immunotherapy has recently gained mainstream recognitionas a viable anticancer therapy (18, 19). Much of the excitementabout immunotherapy revolves around checkpoint blockadeusing antibodies blocking the negative regulatory moleculescytotoxic T-lymphocyte antigen-4 (CTLA-4) and/or pro-grammed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1; refs. 20, 21). These blocking antibodies haveshown activity in multiple different tumor types, and whencombined have shown synergistic effects in metastatic mela-noma (22–24). Given that immunotherapy is now a likelyfourth pillar in the armamentarium against cancer, additionalefforts are required to understand how immunotherapy can bebest incorporated with surgery, chemotherapy, and XRT (25).Along these lines, XRT may be uniquely suited to synergizewith immunotherapy because it can be delivered preciselyto the tumor and may enhance expression of targets for theimmune system (8, 26–28). Moreover, several clinical casereports provide evidence of synergy between combined XRTand immune checkpoint blockade (29, 30).

A number of preclinical studies have combined XRT andimmunotherapy with intriguing results, including effects out-side of the radiation field—termed the abscopal effect. Initialpioneering work by Demaria and colleagues (31) combinedXRT with Flt3-L and documented an abscopal effect in contra-lateral shielded tumors that was immune mediated (31, 32). Asubsequent study combined XRT with anti–CTLA-4 antibody inTSA breast carcinoma and MC38 colorectal carcinoma andreported abscopal effects that correlated with the frequency ofIFNgþ CD8 T cells (33). Our group previously used the smallanimal radiation research platform (SARRP; ref. 34) to combineXRT with a cell-based vaccine in an autochthonous model ofprostate cancer, and showed an additive treatment effect (35).In addition, we were the first to use the SARRP to deliverstereotactic XRT combined with anti–PD-1 antibody in a gli-oma model and reported long-term survival of mice receivingcombination therapy (36). Recently, a report combining XRTwith anti–PD-L1, an antibody against the ligand of PD-1,demonstrated enhanced efficacy through a cytotoxic T cell–dependent mechanism with a synergistic reduction in tumor-infiltrating myeloid-derived suppressor cells (MDSC; ref. 37).Furthermore, a recent study combining radiation with blockadeof PD-L1 demonstrated improved local control, survival, andprotection against tumor rechallenge in colorectal and breastcancer mouse models (38). Although these preclinical studieshave shown additive or synergistic effects on tumor control, thequestion of whether XRT-induced priming augments an endog-enous antigen-specific immune response, specifically in com-bination with antibody-mediated blockade of the PD-1 recep-tor, requires further investigation.

Here, we report that combining XRT with anti–PD-1 anti-body results in the development of endogenous antigen-spe-cific antitumor immune responses in models of melanoma andbreast cancer in conjunction with enhanced tumor control.Importantly, we identify that the immune responses inducedby combined XRT and checkpoint blockade are not limited to Tcells and include development of specific B cell–mediatedantitumor antibodies. Mechanistically, we found that XRT iscapable of increasing immune-cell tumor infiltrates and directpresentation of tumor antigens, although in vivo the increase inantigen recognition mediated by XRT likely requires cross-presentation.

Materials and MethodsMouse strains and cell lines

C57BL/6, BALB/cJ, and MHC class I knockout female 6- to8-week-old mice were purchased from The Jackson Laboratory.OT1-Rag knockoutmice were bred in-house. Animal experimentswere performed in specific pathogen-free facilities accredited bythe American Association for the Accreditation of LaboratoryAnimal Care (AAALAC) with protocols approved by the AnimalCare and Use Committee of the Johns Hopkins University Schoolof Medicine (Baltimore, MD). MC38-OVA cells were a kind giftfrom Dr. Mark Smyth (QIMR Berghofer Medical Research Insti-tute, Melbourne, Australia) on April 2013. B16-OVA melanomacells were obtained from the laboratory of Dr. Hyam Levitsky(Johns Hopkins University) and cultured in RPMI completemedia plus G418 selection. 4T1-HA breast carcinoma cells wereobtained from Dr. Katharine Whartenby (Johns Hopkins Univer-sity). B3Z T-cell hybridomawas a kind gift fromDr.Nilabh Shastri(University of California, Berkeley, CA) on September 2013. Alltumor cell lines were tested before in vivo use and found to be freeof Mycoplasma. In addition, model antigen expression for oval-bumin (OVA) and hemagglutinin (HA) was confirmed by flowcytometry and Western blotting.

Flow cytometry, intracellular cytokine staining, and cytokineanalysis

Single-cell suspensions were prepared from spleens, inguinallymph nodes, and tumors. Cells were stained with fluorescent-labeled antibodies (BioLegend, BD-Bioscience Pharmingen, oreBiosciences) and analyzed by either FACSCalibur or LSR II flowcytometer (BD). The following clones were used: CD4 (RM4-5),CD8 (5H1), CD11c (HL3), Cd11b (M170), CD25 (PC61.5),SIINFEKL/H-2Kb (25-D1.16), CD44 (IM7), CD62L (MEL-14),IFNg (XMG1.2), TNFa (MP6-XT22), FAS (2495), CD40 (3/23),and Foxp3 (FJK-16s). For pentamer staining, H-2Kb (SIINFEKL)or H-2Kd (IYSTVASSL) pentamers were used followed by Pro5 R-PE Fluorotag (ProImmune). For intracellular cytokine staining,cells were activated withOVA peptide–pulsed splenocytes or withPMA (100 ng/mL) plus ionomycin (500 ng/mL) for 4 hours in thepresence of GolgiPlug (32), processed with a Cytofix/Cytopermkit (32), and stained as indicated. Gates and quadrants were setbased on isotype control staining.

B3Z assayB3Z cellswere cultured in completemedia and split 1:20 every 2

days. Semi-confluent B16-OVA cells were trypsinized andwashedin completemedia and irradiated using a Fixed Cs source GammaIrradiator with 0, 10, or 20 Gy. B16-OVA cells were washed andthen immediately seeded into 24-well plates at 1 � 105 cells perwell and cultured in complete media þ G418 for 36 hours.Wells were washed twice in serum-free media and 5 � 105 B3Zcells and/or whole splenocytes were then added to the wells andcocultured for 16 hours. For control experiments, OVA peptide–pulsed splenocytes alone were added to wells 4 hours before B3Zcells. Supernatant was aspirated and IL2 concentrations weremeasured by IL2 ELISA (eBioscience) according to the manufac-turer's specifications.

Adoptive transfer experiments and CFSE labelingT cells from spleens and lymph nodes of OT1-Rag knockout

mice were washed twice in PBS and labeled in 5 nmol/L CFSE

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(25 � 106 cells/mL) for 8 minutes at 37�C on a shaker followedby quenching with ice-cold media. Cells were washed twice inPBS and 3 � 106 cells were injected per mouse via retro-orbitalinjection. Three days later mice were sacrificed and the spleen andlymph node cell suspensions were analyzed via flow cytometry.

Serum antibody analysisMouse serum was isolated by allowing whole blood collected

via cardiac puncture to coagulate at room temperature for 45minutes. Samples were centrifuged at 10,000 rpm for 10 minutesand serum supernatant was aspirated and analyzed via Anti-Ovalbumin IgG1 Mouse EIA Kit (Cayman Chemical).

Tumor growth experiments and TIL preparationB16-OVA melanoma and 4T1-HA breast carcinoma models

were performed as previously described with somemodifications(33, 34). Briefly, on day 0, mice were injected with 1.25 to 5.0 �105 B16 cells intradermally (i.d.) in the back or 2.0 to 5.0 � 106

4T1-HA cells subcutaneously (s.c.) in the right flank. Tumordiameter wasmeasured every 2 to 3 dayswith an electronic caliperand reported as volume using the formula (m1 � m2

2)/2. Micewere treated with 200 mg anti–PD-1 antibody via i.p. injectionevery 3 days for a total of three injections per mouse. To isolatetumor-infiltrating lymphocytes (TIL), solid tumors were excisedafter 12 to 14 days, single-cell suspensions prepared by mechan-ical dissociation, followed by density gradient centrifugation onan 80%/40% Percoll (GE Healthcare) gradient.

SARRP irradiationFor in vivo experiments, mice with established palpable tumors

were treated using the SARRP previously described by Wong andcolleagues (34). The anesthesized mice underwent CT imagingon the SARRP for image-guided localization of the tumor andplacement of isocenter before irradiation. The dose rate was 1.9Gy/min.

Statistical analysisStatistical analysis was performed using Prism 5 (GraphPad).

Unpaired two-tailed t tests were conducted and results wereconsidered statistically significant at P � 0.05 (�), 0.01 (��), and0.001 (��).

ResultsXRT enhances presentation of tumor-associated antigens

Tumor cell lines expressingmodel antigens, such asOVAorHA,are useful models with which to evaluate the effects of XRT on thedevelopment of antigen-specific immune responses. To betterunderstand how XRT affects antigen presentation, we used B16melanoma and MC38 colorectal carcinoma cell lines that wereengineered to express OVA. The gross morphologic effects of XRTon these cell lines were as expected; light microscopy of cellcultures 48 hours after XRT showed increased cell size, anincreased nuclear-to-cytoplasmic ratio, and prominent nucleoli(Fig. 1A, top). These effectsweremirrored in the increased forwardscatter and side scatter of the viable cell population as assayed byflowcytometry (Fig. 1A, bottom). To testwhether XRT could resultin increased direct (i.e., tumor-cell intrinsic) antigen presentation,we used a commercially available antibody that binds specificallyto the immunodominant OVA peptide SIINFEKL complexedwithin the MHC class I molecule Kb (39–41). Using this reagent,we observed a significant increase in antigen presentation on

irradiated cells (Fig. 1B). Although B16-OVA had a lower baselineexpression ofMHCclass I as comparedwithMC38-OVA, both celllines upregulated MHC and OVA antigen presentation uponirradiation in a dose-dependent manner (Fig. 1C). As a positivecontrol for the proimmunogenic effects of XRT, we confirmedthat XRT upregulated expression of FAS, as previously described(Fig. 1D; ref. 7). As a negative control, we evaluated CD40expression and found no significant upregulation (Fig. 1D),suggesting that XRT does not lead to a general, nonspecificexpression of cell-surface antigens on these tumor cell lines.

Radiation increases T-cell recognition of tumor cells in vitroTo study whether increased MHC–OVA expression secondary

to XRT leads to specific T-cell recognition in vitro, we used the B3Zhybridoma (42, 43). These immortalized T cells have a TCRspecific for theOVApeptide SIINFEKL, and secrete IL2 in responseto antigen recognition in a dose-dependent manner (42). We firstverified B3Z function by coculturing these cells with peptide-pulsed, syngeneic splenocytes as antigen-presenting cells (APC),and confirmed their dose-dependent IL2 secretion as assayed byELISA (Fig. 2A). Surprisingly, XRT of B16-OVA cells in the absenceof APC resulted in increased IL2 secretion by cocultured B3Z Tcells, showing that XRT enhanced direct antigen presentationfrom tumor cells (Fig. 2B). In addition to this direct presentation,it is also possible that XRT-induced cell death could result in therelease of antigen, which could then be taken up by APCs andcross-presented. To investigate this possibility, we irradiated B16-OVA cells, harvested the cell-free supernatant from those cultures24 hours after radiation, and pulsed APCs with the cell-freeconditioned media. As shown in Fig. 2C, the addition of condi-tioned media from irradiated B16-OVA to APCs did not result indetectable IL2 secretion (i.e., activation) fromB3Z. Alongwith thedata from Fig. 1, these data suggest that increased direct antigenpresentation could be one of the effects of XRT on tumor cells.

Radiation increases T-cell recognition of tumor antigen in vivoby cross-presentation

To further explore the effects of XRT on tumor antigen presen-tation, we used an in vivo model, in which congenically marked,CFSE-labeled OVA-specific CD8 T cells (OT-1) were adoptivelytransferred into animals bearing established B16-OVA tumors intheir flanks. Using the SARRP (34), animals received 12 Gy� 1 ofimage-guided stereotactic XRT to their flank tumors 1 day beforeadoptive transfer of CFSE-labeledOT-1, and their draining lymphnodes (DLN) were harvested on day 4. As shown in Fig. 2D, XRTresulted in significantly increased antigen-specific proliferation asmeasured by CFSE dilution. We tested whether this recognitionwas proinflammatory by performing intracellular staining foreffector cytokines, and found increased expression of IFNg andTNFa (Fig. 2D and E, and data not shown). Similar levels ofspecific T-cell activation were noted with a fractionated XRTscheme of 7 Gy � 3 (Supplementary Fig. S1). We next testedwhether CD11cþ dendritic cells (DC) in the tumor DLNs werepresenting antigen by staining them with the SIINFEKL/Kb-spe-cific antibody used in Fig. 1. For these studies, we used mice thatexpress OVA ubiquitously (CAG-OVA) as a positive control. Asshown in Fig. 2F, CD11cþ DCs in the tumor DLNs indeedexpressed the specific MHC–OVA complex, suggesting that inaddition to direct presentation, in vivo XRT might also augmenttumor antigen recognition via cross-presentation. To evaluate therelative importance of direct versus cross-presentation in XRT-

Cross-Presentation of Tumor Antigens Augmented by Radiotherapy

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mediated upregulation of tumor antigen recognition, we repeatedthese studies using MHC class I knockout mice, in which onlydirect presentation would be possible. In these animals, weobserved a reduction in the proliferation of adoptively transferredOT-1 T cells (Fig. 2G). Furthermore, the absence ofMHC class I onhost cells also abrogated the ability of radiation to enhanceactivation (i.e., cytokine secretion) of tumor antigen–specific Tcells (Fig. 2G). Taken together, these results demonstrate thatlocally directed XRT can increase the activation and proliferationof an antigen-specific antitumor T-cell population in theDLN andthat this effect likely involves cross-presentation via MHC class I–expressing professional APCs. It should benoted that althoughwedid not directly measure the radiation dose to the DLNs in theseexperiments, in previous studies, we used IHC for gH2AX todemonstrate that the SARRP is capable of precisely irradiating atarget structure while sparing nearby normal tissues (35).

Priming of endogenous antigen-specific T cells and B cells byXRT

To test whether XRT could increase specific recognition oftumors by endogenous (as opposed to adoptively transferred)T cells, we performed antigen-specific pentamer staining in an invivo treatmentmodel.Wefirst established such amodel by treatingimplanted B16-OVA tumors with either XRT, anti–PD-1, or thecombination. For these studies, we chose to block the immunecheckpoint PD-1, because of clinical activity, as well as its gener-ally tolerable side effect profile. As previously demonstrated byour group (36) and others (33, 37), the combination of directedXRT and immune checkpoint blockade resulted in increasedinhibition of tumor outgrowth (Fig. 3A). Using SIINFEKL-MHCpentamer staining, we next tested whether the combined treat-ment increased antigen-recognition and drove the expansion oftumor antigen–specific T cells. As shown in Fig. 3B, XRT aloneresulted in amoderate increase inOVA-specific T cells in theDLNs

and spleens. However, when single-fraction XRT was combinedwith PD-1 blockade, we observed significant increases in OVA-specific T cells (Fig. 3B andC).Of interest, the increasedprevalenceof pentamer-positive T cells in the spleens supports thenotion thatlocal irradiation can result in the development of a systemicimmune response outside of the radiation field. To assay thefunctionality of the endogenous immune cells induced by eithersingle or combined treatment, we assayed the ability of these cellsto prevent tumor outgrowth after adoptive transfer into na€�vemice. To perform those studies, we adoptively transferred sple-nocytes from untreated or treated mice into wild-type (WT) na€�vemice, and then challenged those animals with primary B16-OVAtumor cells and followed tumor outgrowth. As shown in Fig. 3D,endogenous immune cells primed by XRTþ anti–PD-1 were ableto significantly delay tumor cell outgrowth in na€�ve hosts, sup-porting their functionality. Although previous groups havedescribed the ability of XRT to induce or expand antibodies(44), the relative specificity of those antibodies for tumor cellsis less clear. To address that issue, we assayed the ability of XRTþPD-1 blockade to induce OVA-specific antibodies inmice bearingOVA-expressing tumors. As shown in Fig. 3E, although the abilityof XRT � PD-1 blockade to induce IgG antibodies to OVA wasquite variable, we observed an increased frequency and magni-tude of IgG1 OVA-specific antibodies in the sera from animals inthe combined treatment group. Taken together, these data suggestthat combined XRT þ checkpoint blockade can induce tumorantigen–specific responses among endogenous immune cells.

Combining XRT with PD-1 blockade increases endogenousT-cell infiltration of established B16 tumors

To assay the effects of increased antigen recognition mediatedbyXRTþPD-1blockade on the endogenous T-cell population,wetested whether the combined treatment increased tumor infiltra-tion with either bulk or specific (OVA pentamer–positive) T cells.

Figure 1.XRT enhances MHC presentation oftumor-associated antigens in B16-OVA melanoma and MC38-OVAcolorectal carcinoma. A, top, lightmicroscopy images of irradiatedB16-OVA melanoma cells cultured for48 hours; bottom, flow cytometryforward scatter and side scatter (SSC)plots of unstained, irradiated B16-OVAcultures after 48 hours. B, flow plotsand percentages of irradiatedB16-OVA and MC38-OVA cell culturesat 48 hours stained with anti-mouseSIINFEKL/H-2Kb antibody [eBio25-D1.16]. C, representative histograms ofirradiated cells stained with isotype,H-2Kb, or SIINFEKL/H-2Kb anti-mouse antibodies. D, flow plots ofirradiated cells stained with anti-mouse FAS or CD40 antibodies.Experiments repeated three timeswith similar results.

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As shown in Fig. 4A, untreated B16-OVA tumors or tumors treatedwith anti–PD-1 alone had scant immune-cell infiltrates and anotable absence of CD4 or CD8 lymphocytes. Remarkably, XRTalone drove a significant intratumoral infiltrate, composed ofboth endogenous CD4 and CD8 T cells (Fig. 4A, top). Addinganti–PD-1 to XRT further enhanced the percentage of CD8 T cellswithin B16-OVA tumors, with a significant proportion of these

cells being specific for OVA as determined by pentamer staining(Fig. 4A, bottom). To further quantify these changes, we calcu-lated the absolute numbers of CD4 and CD8 T cells per 50,000gated events. As shown in Fig. 4B and C, XRT alone significantlyincreasedCD4 andCD8T-cell infiltration, but this was not furtherincreased by the addition of PD-1 blockade. As the immunologiceffects of effector cells within the tumor parenchyma likely

Figure 2.XRT enhances cell-mediated tumor antigen presentation and results in increased proliferation and activation of antigen-specific T cells in the DLN. A, IL2 ELISAconcentrations for 16 hours in supernatant of B3Z cells cocultured with splenocytes (spleen) with or without indicated concentrations of OVA peptides.B, IL2 concentrations in supernatant of untreated (0 Gy) or irradiated (10 or 20 Gy) B16-OVAmelanoma cells alone or coculturedwith B3Z cells or whole splenocytesas APCs; experiment repeated three times with similar results (� , P < 0.05). C, IL2 concentrations in supernatant of B3Z cells with or without splenocytes(spleen) cultured with indicated concentration of OVA peptides, or with cell-free conditioned media (CM) from untreated or irradiated B16-OVA cells. D, flowhistograms anddot plots of ex vivoCFSE-labeledOT1 T cells harvested fromDLNs; representative of three independent experiments (n¼4/group). E, change in IFNgpositivity in OT1 T cells fromD (Student t test; P < 0.01). F, dot plots of ex vivo CD11bþCD11cþDCs isolated from non–tumor-bearing or B16-OVA tumor–bearingmice,or CAG-OVAmice constitutively expressingOVAdriven via the b-actin promoter; representative of three independent experiments. G,flowhistograms anddot plotsof ex vivo CFSE-labeled OT1 T cells harvested from untreated or irradiated MHC Class I knockout mice bearing B16-OVA tumors; repeated twice with similar results.






represent a balance between regulatory T cell (Treg) and effector T-cell function, we also quantified Treg infiltration, as well as theratioofCD8:Treg for eachof the treatment groups. Thesedata (Fig.4D and E) show that XRT alone significantly increases the per-centage of CD4 TILs that are FoxP3þ, but that this effect issignificantly abrogated by the addition of PD-1 blockade to XRT.Indeed, the combination of XRT þ PD-1 blockade results in anincrease in the ratio of antigen-specific (OVApentamer–positive):Treg ratio; this parameter in the tumormicroenvironment appearsto correlate with the treatment effects shown in Fig. 4A. To furtherexplore the role of Tregs in this model, we performed depletionstudies using the anti-CD25 antibody PC-61. As shown in Sup-plementary Fig. S2, PC-61–mediated Treg depletion added to XRTin tumor control, confirming the notion that Tregs likely attenuatethe capacity of XRT to augment antitumor immune responses.

Combining XRT with PD-1 blockade increases endogenousT-cell infiltration of established 4T1 tumors

To investigate whether the ability of combined XRT þ PD-1blockade to enhance an endogenous antigen-specific immuneresponse was confined to the poorly immunogenic B16 cell line,we used an engineered version of the 4T1 breast carcinoma linethat expresses HA as a model antigen. Similar to B16-OVA,established 4T1-HA tumors treated with anti–PD-1 or XRT aloneshowed a significant growth delay (Fig. 5A). However, when XRTwas combinedwith anti–PD-1, we observed significant regressionof tumors and tumor control (Fig. 5A). We analyzed the devel-

opment of antigen-specific CD8 T-cell responses using HA pep-tide–MHC pentamers and found that either anti–PD-1 or radia-tion alone resulted in increased HA-specific CD8 T cells (Fig. 5BandC). However, when XRTwas combinedwith anti–PD-1, therewas a statistically significant increase in HA-specific CD8 T cellscompared with that treated with anti–PD-1 alone. Interestingly,and as was the case for B16-OVA, we found that XRT increased theproportion of intratumoral CD4 T cells with a Treg phenotype,and this increase was abrogated by the combined treatment(Fig. 5D). This XRT-mediated increase in Treg percentagesappeared to be a localized phenomenon; we did not observe anyincrease in Tregs in the DLNs (Fig. 5E) or in the spleens (data notshown) in either the 4T1-HA or B16-OVA models.

XRT þ anti–PD-1 increases tumor-antigen specific centralmemory cells in the DLN

Our ability to identify tumor antigen–specific endogenousCD8 T cells in two different models afforded us with the oppor-tunity to quantify the effects of either single or combined treat-ment on the phenotype of induced/expanded CD8 T cells. Forthese studies, we defined na€�ve cells as CD62Lþ and CD44� (Fig.6A). In a similar manner, the effector memory phenotype wasdefined as CD44þCD62L�, and central memory cells as CD44þ

CD62Lþ (45). As in previous experiments, we identified and gatedon tumor antigen–specific CD8 T cells in the DLNs using penta-mer staining (see Supplementary Fig. S3 for gating scheme). In thepoorly immunogenic B16-OVA model, we found that XRT

Figure 3.Stereotactic XRT combined with anti–PD-1 immunotherapy significantlyimproves tumor control and enhancesdevelopment of antigen-specificT cell– and B cell–mediated antitumorimmune responses. A, tumor volumesof mice (n¼ 8/group) inoculated with3 � 105 B16-OVA tumor cells andirradiated (12 Gy� 1) on day 12 and/ortreated with i.p. injections of 200 mganti–PD-1 Abs starting 1 day beforeirradiation and every 3 days for a totalof three injections; experimentrepeated three times with similarresults. B, percentages of CD8þ

SIINFEKL Pentamerþ T cells isolatedon day 10 after irradiation from WT(non–tumor-bearing) or B16-OVAtumor–bearing mice treated asindicated; representative of threeindependent experiments. C, scatterplot quantifying significant increase inSIINFEKL Pentamerþ T cells (� , P <0.05; �� , P < 0.01) in mice treated withXRT þ anti–PD-1 Abs. D, tumorvolumes measured at day 18 in WTmice that received 12 � 106 i.v.adoptively transferred splenocytesfrom mice treated as indicated(harvested on day 14) followed 1 daylater by tumor challenge with 2 � 105

B16-OVA cells. E, OVA Ab ELISAmeasuring concentration of anti-OVAIgG1 present in sera of mice (n ¼ 8–11mice/group) treated as indicatedharvested on day 14 after XRT.

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decreased the relative percentage of na€�ve tumor-specific CD8 Tcells and increased the proportion of tumor-specific CD8 T cellswith an effectormemory phenotype (Fig. 6A andB), but that PD-1blockade did not significantly add to this skewing. We alsoobserved significant increases in the absolute numbers oftumor-specific effectormemory CD8 T cells induced by XRT aloneor XRT combined with anti–PD-1 immunotherapy (Fig. 6C). Thephenotype of tumor antigen–specific CD8 T cells was also alteredby XRT in the 4T1-HAmodel (Fig. 6D–F), but here XRT alone wasless effective in increasing the absolute number of effector mem-ory CD8 T cells compared with that of combine therapy (Fig. 6F),possibly reflecting differences in the tumor microenvironmentand immunogenicity of 4T1 compared with B16 cells. Takentogether, these two datasets support the concept that XRT com-bined with PD-1 blockade increases tumor-specific CD8 T cellswith a memory phenotype.

DiscussionTumor cell lines expressing model antigens are useful for

studying induction of endogenous antigen-specific immune

responses because of the numerous epitope-specific reagentsavailable to detect responses to these model antigens. Here, weused these reagents to test the ability of XRT alone or in combi-nation with immune checkpoint blockade to induce immuneresponses against OVA expressed by B16 melanoma and HAexpressed by 4T1 breast carcinoma. Studies from several researchgroups have shown that XRT increases the expression of MHCclass I in a dose-dependentmanner in anumber of different tumortypes (7, 12). This is a critically important finding because manytumor types, including melanoma, may downregulate MHCexpression to protect from CD8 T cell–mediated cytotoxicity. Byupregulating MHC, XRTmay prevent tumor cells from remainingundetected by CD8 T cells. Indeed, we found that XRT increasedthe expression of specific tumor model antigenic epitopes pre-sented in MHC on the cell surface (Fig. 1). The H2kb-SIINFEKLantibody stains for the direct targets of CD8 OT1 T cells orendogenously generated CD8 T cells specific for the SIINFEKLepitope. Thus, upregulating the targets for CD8 T cells is onemechanism by which XRT directly increases the susceptibility oftumor cells to CD8 T cell–mediated cytotoxicity. One potentiallimitation of model antigen systems like these is that the relative

Figure 4.Single-dose stereotactic XRT inducesTILs and synergizes with anti–PD-1checkpoint blockade. A, dot plots ofCD4 versus CD8 (top) and SIINFEKLPentamerþ versus CD8 (bottom) TILsisolated on day 14 from tumor-bearingmice treated as indicated,representative of three independentexperiments. B andC, quantification ofsignificant increase in absolutenumber of CD8 and CD4 TILs in micetreated with XRT and anti–PD-1 Abs.D, changes in percentages of CD4þ

CD25þFoxp3þ Tregs in TILs fromtreated groups. E, significant increasein the ratio of CD8þPentamerþ

Effector T cells to CD4þCD25þFoxp3þ

Tregs in TILs of mice treated with XRTplus anti–PD-1 Abs (�, P < 0.01;�� , P < 0.05; ns, not statisticallysignificant).






immunogenicity of exogenously introduced antigens such asOVAor HA could render them more susceptible to immunologicintervention. For B16, this is unlikely; implanted B16 and B16-OVA have similar growth curves in vivo (data not shown), but itdoes remain a minor concern for 4T1-HA. The interpretation ofthese studies should also be tempered by observation that bothB16 (46) and 4T1 (47) express PD-L1 in vivo, whereas expressionof PD-L1 in human tumors is variable.

In addition to upregulation of MHC class I, multiple otherpotential mechanisms exist by which XRT can increase tumor-cellsusceptibility. Upregulation of FAS (7) could increase tumor-cellsusceptibility to undergoing FAS-L–mediated apoptosis (Fig. 1D).Interestingly, FAS-L–mediated induction of apoptosis could betriggered by tumor-specific immune cells as well as bystanderimmune cells that are not necessarily specific for tumor antigens.Taken together, these observations would support the hypothesisgenerated by previous studies that the ability of XRT to controlcertain tumors depends on the general immune status of the host(13), and more specifically on CD8 T cells (14, 15).

The B3Z T-cell hybridoma is an important cell line for probingthe expression of OVA antigen presented by tumor cells or otherengineered cell lines. The B3Z T-cell hybridoma is selected tosecrete IL2 in a dose-dependent manner upon TCR ligation withMHC loaded with SIINFEKL epitope, and thus is similar in somerespects to an immortalized OT-1 cell line. Here, we adapted thiscell line to quantify the level of OVA antigen presentation intumor cells after irradiation. We confirmed that XRT increasesdirect tumor-mediated cell–cell antigen presentation asmeasuredby IL2 ELISA (Fig. 2). This raised the question whether XRT could

potentially convert tumor cells into APCs capable of primingimmune responses. Using an in vitro assay coculturing irradi-ated B16-OVA melanoma cells with na€�ve CFSE-labeled OT-1 Tcells, we found no evidence of T-cell priming or proliferation(data not shown). Furthermore, using MHC class I knockoutmice in which only the injected B16-OVA tumor cells werecapable of presenting antigen to adoptively transferred OT-1 Tcells, we confirmed that the ability of XRT to enhance T-cellproliferation in the DLN requires MHC class I–expressing APCs.This raises the intriguing question of whether stereotactic XRT,which does not generally include the DLNs, may be superior tolarger field or conventional XRT in terms of inducing antitumorimmune responses. One limitation of these results is that wedid not specifically quantify the radiation dose to the DLNs,although in previous studies, we were able to target the prostategland without irradiating either the bladder or DLNs (35).Studies in which the DLNs are specifically targeted or sparedare ongoing to more precisely address this issue.

One potential downside of increased direct tumor cell–medi-ated antigen presentation is increased T-cell anergy or conversionof na€�ve T cells to Tregs. Indeed, we did observe that XRT aloneincreased the relative percentage of Tregs in the tumor microen-vironment but not in the DLN (Figs. 4 and 5). Increased Tregswould be deleterious to antitumor immune responses, and thuswe investigated strategies to block Tregs using PC61-depletingantibody. We observed increases inMHCpentamer-specific T-cellpopulations and B16-OVA tumor control when XRT was com-bined with Treg depletion (Supplementary Fig. S2). It remains tobe determined whether this increase in Tregs is a general

Figure 5.Stereotactic XRT combined with anti–PD-1 immunotherapy significantly improves 4T1-HA tumor control and enhances development of antigen-specific T cells. A,tumor volumes in mice (n ¼ 6/group) inoculated with 1 � 106 4T1-HA cells irradiated on day 9 and/or treated with i.p. injections of 200 mg anti–PD-1 Abs starting1 day before irradiation and every 3 days for a total of three injections; representative of two independent experiments. B, percentages of CD8þ HA Pentamerþ

(H2-Kd IYSTVASSL) cells harvested at day 14 from the DLNs of WT or 4T1-HA tumor–bearing mice treated as indicated; representative of two independentexperiments. C, quantification of increase in CD8þPentamerþ cells with XRT and anti–PD-1 immunotherapy. D, changes in CD4þCD25þFoxp3þ Tregs in theTILs of 4T1-HA tumor–bearing mice treated as indicated. E, no significant changes of CD4þCD25þFoxp3þ Tregs in the DLNs; representative of two independentexperiments.

Sharabi et al.





phenomenon of XRT for multiple tumor types. However, strat-egies combining XRTwith Treg-depleting antibodies, such as anti-GITR or low-dose cyclophosphamide, may be worthy of furtherclinical investigation.

The presence of immune-cell infiltrate has been correlated withimproved patient outcomes in multiple different solid tumors,including melanoma, colorectal, breast, and prostate carcinoma(48–51). In fact, the type and location of immune cells in human

Figure 6.XRT decreases the percentage of na€�ve antigen-specific T cells in the DLNs and increases antigen-specific effector memory T cells. A, flow plots of putative memorymarkers on CD4�CD8þSIINFEKL Pentamerþ endogenous T cells (see Supplementary Fig. 3 for gating scheme) isolated fromDLNs ofWT or B16-OVA tumor–bearingmice on day 14 treated as indicated, representative of three independent experiments. B, pie charts showing relative percentages of memory populations intreated groups. C, quantification of absolute number of na€�ve, putative central, and effector memory T cells in DLNs of WT of B16-OVA treatment groups.D–F, same respective memory charts as above except isolating CD4�CD8þIYSTVASSL Pentamerþ T cells from 4T1-HA tumor–bearing mice, representativeof two independent experiments.






colorectal cancer was previously reported to be a better predictorof survival than traditional stage groupings (48). Similarly, theabsence of immune-cell infiltrate has been associated with worsepatient outcomes, possibly as a sign of tumor immunosuppres-sion or physical barriers to immune-cell migration and tumorinfiltration. Thus, strategies that enhance immune-cell engage-ment and infiltration into tumors could potentially result insignificant clinical benefit. Here, we demonstrated that intact,untreated B16 melanoma has a relatively scant immune-cellinfiltrate consistent with the poorly immunogenic nature of thistumor line (Fig. 4A). XRT of B16 melanoma significantlyincreased both CD4 and CD8 T-cell infiltrates (Fig. 4). Interest-ingly, simply driving recruitment of T cells into a tumor is likelynot sufficient to activate an antitumor immune response given theimmunosuppressive tumor microenvironment. Indeed, analysisof the CD4 T-cell population showed that the majority of thesecells are CD25þFoxP3þ Tregs (Figs. 4D and 5D). However, thecombination of XRT and anti–PD-1 immunotherapy altered theratio of CD4 to CD8 T cells and resulted in decreased percentagesof CD4 Tregs and absolute increases in CD8 T-cell populations,likely as a result of diminished inhibitory signaling from the PD-1pathway. These findings provide a clear rationale for combiningXRT with checkpoint blockade immunotherapy and further workis certainly warranted to determine whether these findings holdtrue in human tumors.

Here, we chose to study radiation combined with anti–PD-1checkpoint blockade primarily because a drug blocking the PD-1pathway (pembrolizumab;Merck) has recently been approved bythe FDA and additional agents blocking the PD-1 pathway are inphase III clinical trial development. Given that these agents aregenerally well tolerated with favorable adverse event profiles,strategies incorporating anti–PD-1 immunotherapy with currenttreatment modalities, including chemotherapy, targeted thera-pies, and/or XRT, are of critical importance. To this end, datasuggest that XRT may be uniquely suited to combine with check-point blockade immunotherapy and specifically PD-1 blockade.Recently, there have been reports that radiation alone can result inincreased expression of PD-1 ligands on the surface of tumor cells(37, 38), including melanoma and breast cancer model cell lines.Upregulation of PD-1 ligands might serve to dampen effectorimmune responses and potentially counteract the positive immu-nogenic effects of radiation such as increased MHC expression,antigen presentation, and immune-cell infiltration. Thus, concur-rent blockade of the PD-1 pathway may synergize with radiationby specifically blocking a counterproductive effect of radiation onimmune responses.

Taken together, these data have several clinical implications.First, these findings clearly support the coadministration of XRTwith checkpoint blockade immunotherapy to improve localtumor control. The trend toward increased Tregs in the tumorafter XRT alone suggests that strategies combining Treg depletionwith XRT may have additional clinical benefit. Furthermore, theincrease in antigen-specific T cells and antitumor antibodies withconcurrent XRT and anti–PD-1 immunotherapy suggest that thecombinationmayalsopotentially aid in systemicor distant tumorcontrol via the abscopal effect. Finally, the requirement of hostAPCs and cross-presentation for XRT to enhance immuneresponses raises the intriguing hypothesis that stereotactic XRTcould be superior to conventional field XRT by sparing irradiationof DLN regions. These data provide a preclinical rationale forevaluating these questions in prospective clinical trials.

Disclosure of Potential Conflicts of InterestC.G. Drake reports receiving commercial research grants from Bristol-Myers

Squibb and Janssen; has ownership interest (including patents) in NexImmuneand Compugen; and is a consultant/advisory board member for Bristol-MyersSquibb, Compugen, Novartis, Potenza Therapeutics, Roche/Genentech, andJanssen. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.B. Sharabi, C.M. Kochel, C.G. DrakeDevelopment of methodology: A.B. Sharabi, B.J. Francica, E. VelardeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.B. Sharabi, C.J. Nirschl, E. Velarde, C.G. DrakeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.B. Sharabi, C.J. Nirschl, B.J. Francica, E. Velarde,C.G. DrakeWriting, review, and/or revisionof themanuscript:A.B. Sharabi, T.L. Deweese,C.G. DrakeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.B. Sharabi, C.M. Kochel, T.R. Nirschl,E. VelardeStudy supervision: C.G. Drake

Grant SupportThis work was supported, in part, by the American Society for Radiation

Oncology (ASTRO) Resident/Fellow in Radiation Oncology Research SeedGrant RA2013-1.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 16, 2014; revised December 2, 2014; accepted December 3,2014; published OnlineFirst December 19, 2014.

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2015;3:345-355. Published OnlineFirst December 19, 2014.Cancer Immunol Res Andrew B. Sharabi, Christopher J. Nirschl, Christina M. Kochel, et al. Tumor AntigenMediated Antitumor Immune Responses via Cross-Presentation of

−Stereotactic Radiation Therapy Augments Antigen-Specific PD-1

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