Microenvironment and Immunology

STING Activation Reverses Lymphoma-MediatedResistance to Antibody ImmunotherapyLekh N. Dahal1, Lang Dou1, Khiyam Hussain1, Rena Liu1, Alexander Earley1,Kerry L. Cox1, Salome Murinello2, Ian Tracy3, Francesco Forconi3,Andrew J. Steele3, Patrick J. Duriez3, Diego Gomez-Nicola2, Jessica L. Teeling2,Martin J. Glennie1, Mark S. Cragg1, and Stephen A. Beers1

Abstract

Tumors routinely attract and co-opt macrophages to promotetheir growth, angiogenesis, and metastasis. Macrophages are alsothe key effector cell for mAb therapies. Here we report that thetumor microenvironment creates an immunosuppressive signa-ture on tumor-associated macrophages (TAM), which favorsexpression of inhibitory rather than activating Fcg receptors(FcgR), thereby limiting the efficacy of mAb immunotherapy. Weassessed a panel of TLR and STING agonists (a) for their ability toreprogram macrophages to a state optimal for mAb immuno-

therapy. Both STINGa and TLRa induced cytokine release, mod-ulated FcgRexpression, andaugmentedmAb-mediated tumor cellphagocytosis in vitro. However, only STINGa reversed the sup-pressive FcgR profile in vivo, providing strong adjuvant effects toanti-CD20 mAb in murine models of lymphoma. Potent adju-vants like STINGa, which can improve FcgR activatory:inhibitory(A:I) ratios on TAM, are appealing candidates to reprogram TAMand curb tumor-mediated immunosuppression, thereby empow-ering mAb efficacy. Cancer Res; 77(13); 3619–31. �2017 AACR.

IntroductionTumors can suppress the innate and adaptive arms of the

immune system through regulation of myeloid cells (1, 2).Central to this suppressive capacity is the regulation of macro-phages. Macrophages that have differentiated through interactionwith tumor cells play a key role in exerting local immunosup-pression and promoting tumor metastasis, neoplastic invasion ofectopic tissue, and angiogenesis (3). Although the description ofmacrophage activation is currently contentious, these tumor-promoting macrophages have been proposed to be akin to IL4/13–stimulated, anti-inflammatory "M2" macrophages generatedduring wound healing that orchestrate Th2 responses and pro-mote tissue repair and remodeling (4). Polarization of TAMtoward an LPS/IFNg–activated, inflammatory "M1" state providesthe potential to arrest these tumor-promoting activities andalleviate immunosuppression (5). Reagents capable of achieving

this have the capacity to provide antitumor effects, particularly asan adjuvant to immunotherapy, and are keenly sought in cancertherapy (6).

Toll-like receptor agonists (TLRa) are potent stimulators ofinnate immunity, acting as "danger" signals that elicit phenotypic,secretory, and transcriptomic changes in macrophages consistentwith immune activation (7, 8). Several studies have shown thatTLRa can provide adjuvants effects in human cancers; TLR9a CpGwas shown to be feasible, safe, and powerful to induce objectiveclinical response in lymphoma patients (9), and TLR7a imiqui-mod was effective in the treatment of vulvar intraepithelialneoplasia (10). Efficacy of TLRa in combination with mAb ther-apy in animal models has also been explored. Agonistic anti-CD40 mAb combined with imiquimod induced a systemic anti-tumor CD8þ T-cell and type I IFN response, significantly delayingthe growth of implanted tumors and prolonging animal survivalinmodels ofmesothelioma (11) andmelanoma (12). In our ownstudies (13), we have shown that TLR3a polyinosinic:polycy-tidylic acid (Poly I:C) augmented the agonistic activity of anti-CD40 mAb dependent upon the upregulation of activating Fc-gamma receptors (FcgR). Although TLRa-based reagents havebeen investigated in combinationwith antibody immunotherapyin humans (14), and such studies have motivated ongoing trials,they are yet to be successfully translated into the clinic (15).

Cyclic dinucleotides are a new class of immune adjuvantsbeing evaluated in preclinical studies and in an early-phase clinicaltrial in patients with advanced/metastatic solid tumors(NCT02675439).They signal via stimulator of interferon genes(STING), which is crucial for sensing DNA viruses (16, 17).Cytosolic DNA activates STING, leading to phosphorylation ofIRF3 via tank-binding kinase 1 (TBK1), and subsequent transcrip-tion of type I IFN genes (17–19). In vivo studies have shown thatSTING�/� or IRF3�/� mice fail to prime T cells against tumorantigens anddonot reject immunogenic tumors (20), emphasizing

1Antibody & Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, Universityof Southampton, SouthamptonGeneral Hospital, Southampton, United Kingdom.2Centre for Biological Sciences, University of Southampton, Southampton Gen-eral Hospital, Southampton, United Kingdom. 3Cancer Sciences Unit, CancerResearch UK and NIHR Experimental Cancer Medicine Centres, University ofSouthampton, Southampton General Hospital, Southampton, United Kingdom.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M.S Cragg and S.A Beers are co-senior authors of this article.

Corresponding Authors: Stephen A. Beers, Antibody & Vaccine Group, CancerSciences Unit, Faculty of Medicine, University of Southampton, SouthamptonGeneral Hospital, Southampton, SO16 6YD, United Kingdom. Phone: 44-2381-206593; Fax: 44-2380-704061; E-mail: sab@soton.ac.uk; and Mark S. Cragg,msc@soton.ac.uk

doi: 10.1158/0008-5472.CAN-16-2784

�2017 American Association for Cancer Research.

CancerResearch

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a critical role for host STING in immune sensing of tumors throughdendritic cell (DC) activation and T-cell priming (21, 22).

Here we document a comprehensive analysis of the efficacyof STING and TLR agonists in promoting macrophage proin-flammatory activation, and tumoricidal function in combina-tion with mAb immunotherapy. Certain TLRa were highlypotent at activating both human and murine macrophages andaugmenting antibody-dependent cellular phagocytosis (ADCP)in vitro, but did not elicit similar potency in murine in vivomodels of normal and malignant B-cell depletion. However,STINGa were potent both in vitro and in vivo, crucially reversinglymphoma-mediated immunosuppression and providing pro-tection in tumor-bearing mice where immunotherapy alonefailed. STINGa, but not TLRa, were subsequently shown toeffectively reverse the suppressive effects induced by the lym-phoma on macrophage FcgR expression, the principal immuneeffector cells in vivo.

Materials and MethodsClinical samples and ethics

Ethical approval was obtained by Southampton UniversityHospitals NHS Trust from Southampton and South West Hamp-shire Research Ethics Committee. Informed consent was providedin accordance with the Declaration of Helsinki. Chronic lympho-cytic leukemia (CLL) samples were fromHuman Tissue Authoritylicensed University of Southampton, Cancer Sciences Unit TissueBank and leukocyte cones from Southampton General HospitalNational Blood Service.

AnimalsMice were bred and maintained in local facilities and experi-

ments approved by the local ethical committee under HomeOffice license PPL30/2964. Experiments conformed to theAnimalScientific Procedure Act (UK). hCD20Tg, g-chain�/� and FcgR-null mice (23, 24) have been described with genotypes confirmedby PCR and/or flow cytometry.

In vivo B-cell depletion (adoptive transfer) assaysSplenocytes from target hCD20Tg (T) and nontarget (NT)wild-

type (unless otherwise specified)micewere labeledwith 5mmol/Land 0.5 mmol/L CFSE (Invitrogen), respectively, mixed (1:1), andintravenously injected into recipients (5–8 � 106 cells/mouse).Two doses of adjuvant [TLR1/2a Pam3CSK4: 10–100 mg, TLR2/4aLPS: 10 mg, TLR3a Poly I:C: 100 mg, TR7/8a R848: 2.5 mg, DMXAA:400 mg, type I IFN: 10,000 IU] were administered at 24 and 48hours intraperitoneally, followed by Ritm2a (25–50 mg) or iso-type control intravenously. For BALB/cmice, DMXAAwas admin-istered only once (300 mg at 24 hours). Spleen was harvested 16–20 hours after Ritm2a administration, splenocytes stained withanti-mouse CD19 APC (eBioscience), and assessed for T:NT ratio.IFNAR-blockingmAb (cloneMAR1-5A3; Leinco)was given at 500mg i.p. as indicated in the figure legend.

Generation and polarization of human monocyte-derivedmacrophages and murine bone marrow–derived macrophages

Humanmonocyte-derived macrophages (hMDM) andmurinebone marrow–derived macrophages (mBMDM) were generatedas described in refs. 23, 25. For hMDM polarization, cells werestimulatedwith TLRaor STINGa (Invivogen) for 48hours [LPS: 50ng/mL, recombinant human (rh) IFNg : 2 ng/mL (PeproTech),

rhIL4: 10 ng/mL (PeproTech), rhIL13: 10 ng/mL (PeproTech),TLR1/2: Pam3CSK4 0.1 mg/mL, TLR3: Poly I:C 40 mg/mL, TLR4:Monophosphoryl Lipid A (MPLA): 5 mg/mL, TLR5: Flagellin:125 ng/mL, TLR7/8: R848 1 mmol/L, 202-, 2030-, 3030-cGAMPs:10–50 mg/mL; type I IFN: 5–100 ng/mL]. For polarizationof mBMDMs, cells were stimulated with recombinantmurine (rm) IFNg [2 ng/mL (Peprotech)], rmIL4 [10 ng/mL(Peprotech)], rmIL13 [10 ng/mL (Peprotech)], and TLRa orSTINGa overnight (similar concentration as in hMDM).

Phenotypic analysis of hMDM/mBMDM and calculation ofFcgR activatory:inhibitory ratio

Human and murine FcgR staining is described elsewhere (26).Fluorescently conjugated mAbs were from BD Biosciences, AbD-Serotec, eBioscience, ormade in-house. hMDMswere stainedwithanti-human CD40 Alexafluor (AF)488 (Clone ChiLob 7/6),CD38 AF488 (Clone AT 13/5), both in-house, and CD11b PE(eBioscience). mBMDMs/Splenocytes were stained with anti-mouse CD11b PE, Ly6C PerCpCy5.5, Ly6G PeCy7 (eBioscience),and F4/80 APC (AbD Serotec). Samples were acquired on FACS-calibur/canto II (BD Biosciences) and data analyzed with FCSexpress (DeNovo Software). FcgR activatory:inhibitory (A:I) ratiofor hMDMs was calculated as: MFI for FcgRI� FcgRIIA� FcgRIII/FcgRIIB (FcgRI� FcgRIII� FcgRIV/FcgRII for mBMDMs) giving avalue of x for NT macrophage. The results for each test conditionwere thendividedby x so that unstimulatedmacrophages receiveda ratio of 1.

hMDM and mBMDM phagocytosis assay and phagocyticindex

Phagocytosis assay was performed as described in refs. 23and 25. hCD20 transgenic murine B cells and human CLL cellswere used as targets for mBMDM and hMDM, respectively.Phagocytic index was calculated by dividing the percentageof phagocytic macrophages under the test condition by thepercentage of phagocytic macrophages seen in the unstimu-lated condition.

ELISASupernatant from macrophage cultures was harvested and

levels of cytokines (IFNg , TNFa, IL12p70 and IL6) assessed byMSD V-Plex assay (Meso Scale Discovery) according to the man-ufacturer's instructions. Type I IFNs, IFNa and IFNb, were mea-sured by ELISA kit (PBL Assay Science).

BCL1 lymphoma model and therapyOn day 0, 8- to 12-week-old female WT or FcgR null BALB/c

mice were injected in tail vein with 1 � 104 BCL1 tumor cells.DMXAA (300 mg) and anti-CD20 18B12 (200 mg, produced in-house from patented published sequences) were administered asindicated in Fig. 7A. Anti-CD8 antibody YTS169 (500 mg, in-house) was injected intraperitoneally on day 0, 5, 10, and 14.Tumor-bearing mice were culled humanely before reaching ter-minal endpoint.

Chemokine and cytokine gene expressionRNA was purified from mice spleen using Qiagen RNAeasy

Mini Kit. RNA (500 ng) was used to synthesize cDNA using RT2

First Strand Kit and gene expression was assessed using RT2

Profiler PCR mouse cytokine and chemokine array kit (Qiagen).

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Statistical analysisStatistical analysis was performed using GraphPad Prism. To

compare differences between the experimental groups, Student ttest, Wilcoxon, paired or unpaired t test analyses were performed.Kaplan–Meier curves were produced and analyzed by Log rank(Mantel–Cox) test. A P value <0.05 was considered significant atthe 95% confidence interval. Asterisks denote statistical signifi-cance (�, P < 0.05; ��, P < 0.01; ���, P < 0.001; and ����, P < 0.0001).

ResultsTumors can generate a suppressive microenvironment with alow FcgR A:I ratio on macrophages, leading to mAb therapyresistance

To explore how tumors can regulate their microenvironment invivo, we performed adoptive transfer experiments (24) in miceharboring the syngeneicmouse lymphoma, BCL1 (27). The abilityof the anti-human CD20 mAb, Ritm2a to deplete adoptivelytransferred target human CD20 transgenic (hCD20 Tg) B cellswas examined (24).Whereas depletion of target cells was efficientinmice lacking tumor, inmice implanted with even low numbers(1 � 104) of BCL1 cells 9 days prior to mAb administration,depletion was completely abrogated (Fig. 1A). As the lymphomacells themselves lack the hCD20 target and are not deleted in thissystem, this directly demonstrates the ability of the tumor cells toelicit an immunosuppressive microenvironment. Analysis ofcytokine and chemokine gene expression showed that inocula-tion of tumor upregulated expression of IL10 and IL21 (Supple-mentary Fig. S1A). IL10 has previously been directly implicated asamajor immunosuppressive component of BCL1 lymphoma (28,29) and IL21 has recently been identified to induce B cells toproduce IL10 (30). Previously, we have shown that mice depletedof macrophages are unable to eliminate target B cells (24),implicating macrophages as the most probable cells affected bythis suppressive tumor microenvironment. We and others haveshown that FcgR expression profiles on these cells are importantfor determining mAb efficacy (24, 25, 31) Therefore, next weinvestigated the differences in FcgR expression and A:I ratio (32)induced on splenic macrophages following BCL1 inoculation.Tumor became detectable in the spleen only after day 14 post-inoculation (Fig. 1B) with numbers significantly correlating withspleen weight (Fig. 1C). Increasing tumor presence correspondedto a significant decrease in expression of the activatory FcgRs I andIII on splenic macrophages. Over the same time-course, FcgRIVwas marginally elevated, while the inhibitory FcgRII first showedan initial (day 7), small reduction and was then rapidly andsubstantially upregulated, approximately 4-fold (Fig. 1D). Thisresulted in an overall, statistically significant suppression ofmacrophage FcgR A:I ratio by day 14 (Fig. 1E), which persistedto day 21. Further evidence of the ability of the tumor to manip-ulate macrophage activation status was observed by measuringF4/80, CD40, and CD11b (Supplementary Fig. S1B). These dataillustrate the profound effects that the tumor can elicit on mac-rophage FcgR expression profiles and phenotypic markers culmi-nating in a substantially lowered A:I ratio and resistance toantibody-mediated target cell depletion (Fig. 1A); even when veryfew tumor cells were detectable (e.g., at day 7 and 14). Notably,and in accordance with their redundancy in mediating target cellclearance in the adoptive transfer model (Supplementary Fig.S1C–S1E), monocytes and neutrophils did not show such pro-found changes in their FcgR A:I ratios (Fig. 1E).

The inhibitory FcgRII has been previously shown to suppressantibody-mediated depletion (33). We therefore assessed thecontribution of elevated FcgRII levels to the defective mAb deple-tion inour system.We found that FcgRII knockoutmice harboringBCL1 lymphoma treated with anti-CD20 mAb demonstratedenhanced (�50%) deletion of target cells compared with WTmice (Fig. 1F). These results show that a significant proportion,but not all of the effects of the tumor immune suppression, is dueto elevations in FcgRII, and strategies to enhance FcgR A:I wouldlikely augment mAb activity.

FcgR changes induced in mBMDMs by TLRa and STINGaHaving established that FcgR profiles and A:I ratios on TAM

were the key determinants of mAb-mediated target cell dele-tion, we sought to identify suitable TLR and STING ligandreagents capable of modifying these properties. The majorityof TLRa tested did not show any activity with mBMDMs, eitherin terms of modulating FcgR expression (Fig. 2A), increasingthe FcgR A:I ratio (Fig. 2B), or augmenting phagocytosis (Fig.2C). The sole exception was TLR3a (Poly I:C), which typicallyshowed an increase in FcgR A:I ratio coincident with anincrease in phagocytosis (Fig. 2B and C). In contrast to TLRa,both human (2020-, 2030- and 3030- cGAMP) and murine(DMXAA) STINGa showed increases in expression of activatingFcgRs I, III, and IV with no changes in FcgRII (Fig. 2A),culminating in an increased FcgR A:I ratio (Fig. 2B), andsignificant increases in phagocytosis (Fig. 2C; SupplementaryFig. S2). Notably, we observed a correlation between phago-cytic activity of macrophages with their FcgR A:I ratio (Fig. 2D,R2 ¼ 0.48, P ¼ 0.0041).

Stimulation of mBMDMs with STINGa also induced potenttype I IFN-a and -b responses, with TLR3a again being the onlyTLRa to induce the secretion of these cytokines (Fig. 2E).

Phenotypic and cytokine changes induced in hMDMsstimulated with TLRa or STINGa

We next examined whether our observations pertaining tomouse macrophages could be translated to human. We firstestablished that hMDMs stimulated with TLRa displayedchanges in a range of phenotypic markers; CD40, CD80, CD14,MHCII, CD206, and CD11b that were akin to changes inducedby LPS/IFNg treatment (Supplementary Fig. S3). We theninvestigated responses to a broader panel of both TLR andSTINGa for their ability to induce phenotypic changes inhMDMs in relation to the representative markers CD40, CD38,and CD11b. CD40 and CD38 are considered markers ofimmune cell activation and are generally upregulated onmacrophages following stimulation with LPS (34, 35). Accord-ingly, CD40 and CD38 were significantly upregulated inhMDMs stimulated with LPS/IFNg and decreased followingIL4/13 treatment. Although the integrin amb2 (CD11b) isgenerally considered a pan-macrophage marker, its expressionwas significantly decreased with LPS/IFNg and increased withIL4/13 (Fig. 3A and B).

hMDMs stimulated with both TLRa and STINGa showed aprofile resembling LPS/IFNg–stimulated macrophages withsignificant increases in CD40 seen with TLRa-1/2, -4, -5, and-7/8 and STINGa 2020- and 2030-cGAMP. Likewise, TLRa-1/2 andSTINGa 2020- and 2030-cGAMP resulted in increased CD38. MostTLRa resulted in decreased CD11b akin to LPS/IFNg–treatedmacrophages, with significant changes seen with TLRa-1/2, -4,

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and -7/8. The other TLRa or STINGa induced subtle changes inthose markers, which were reflective of steady-state, nonpolar-ized macrophages, demonstrating that within the spectrum of

polarization, macrophages stimulated with TLRa and STINGamostly display an inflammatory LPS/IFNg–activated profile(Fig. 3A and B).

Figure 1.

BCL1 lymphoma generates a profoundly immunosuppressive tumor microenvironment. A, WT BALB/c mice were implanted with BCL1 lymphoma cells 1 weekprior to transfer of target (T) hCD20Tg B cells and non-Tg nontarget (NT) B-cells. Mice were then treated with Ritm2a 2 days later and spleens harvestedthe following day and assessed for T:NT ratio (n¼ 5–6,Wilcoxon test). n.s., nonsignificant. B,WTBALB/cmice were implanted with BCL1 lymphoma and the numberof tumor cells in the spleen quantitated by flow cytometry. C, Correlation between spleen weight and tumor percentage in the spleen. D and E, Fold change inmacrophage FcgR expression (D) and A:I ratio on splenic macrophages, monocytes, and neutrophils (n¼ 12, Student t test; E). F, Adoptive transfer, as in A, in BCL1lymphoma bearing WT versus FcgRII KO BALB/c recipient mice with hCD20TgxFcgRII KO B cells as target (T) and FcgRIIKO B cells as nontargets (NT). �� , P < 0.01;���� , P < 0.0001.

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Consistent with an activated proinflammatory profile, super-natant from hMDMs stimulated with TLRa contained elevatedIFNg , IL6, IL12p70, and TNFa, comparable with supernatant

from LPS/IFNg–stimulated macrophages (Fig. 3C). However,such cytokine responses were not seen following stimulationwith STINGa; instead supernatant from STINGa 2020- and

Figure 2.

Phenotypic and functional changes induced by TLRa, or STINGa on mBMDMs. FcgR expression (A), A:I ratio (B), and phagocytic index (C) of mBMDMsfollowing stimulation with LPS/IFNg , (L/g), IL4/13 (4/13), TLRa, or STINGa (n ¼ 3–6; error bar � SEM; Student t test). D, Correlation between A:I ratio andphagocytic index ($, STINGa mBMDM; +, TLRa mBMDM;*, NT, 4/13, and L/g mBMDM, analyzed by linear regression). E,mBMDM supernatant assessed for IFNaand IFNb (n ¼ 3; error bars � SEM; Student t test). �� , P < 0.01; ��� , P < 0.001.

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2030-cGAMP–treated macrophages displayed a type I IFN cyto-kine profile with release of both IFNa and IFNb (Fig. 3D),thereby demonstrating a divergence in STING and TLR inflam-matory signaling with respect to cytokine induction in humanmyeloid cells.

FcgR changes and phagocytic activity of hMDMs stimulatedwith TLRa or STINGa

We subsequently examined the expression pattern of activating(FcgRI, -IIA, -III) and inhibitory (FcgRIIB) FcgR on hMDMsstimulated with TLRa or STINGa (Fig. 4A) and calculated the

Figure 3.

Phenotypic changes and cytokine profile of hMDMs stimulated with TLRa or STINGa. A and B, FcgR changes (open black histogram, Iso ctrl for NT macrophage;filled black histogram, surface marker on NT macrophage; open gray histogram, Iso ctrl for TLRa/STINGa–treated macrophage; filled gray histogram, surfacemarker for TLRa/STINGa–treated macrophage; A) and fold change in CD40, CD38, and CD11b following stimulation with LPS/IFNg (L/g), IL4/13 (4/13), TLRa, orSTINGa (n¼ 6–25, Wilcoxon test; B). C and D,Macrophage supernatant assessed for cytokines by ELISA (error bars� SEM; Student t test). � , P < 0.05; �� , P < 0.01;��� , P < 0.001; ���� , P < 0.0001.

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A:I ratio (Fig. 4B). TLRa-1/2, -5, and -7/8 showed an increase in theexpression of activating FcgRs and decrease in inhibitory FcgRIIB,resulting in an increased A:I ratio (Fig. 4A and B), similar to LPS/IFNg–polarized macrophages. STINGa 2020 and 2030-cGAMP alsoresulted in an increase in the expression of activatory receptorsFcgRIIA and FcgRIII, and a decrease in FcgRIIB, resulting in astatistically significant increase in A:I ratio (Fig. 4B; Supplemen-tary Fig. S4A). In contrast, we observed a decrease in A:I ratio withIL4/13 polarized macrophages, largely mediated through anupregulation in FcgRIIB.

The phenotypic changes, cytokine profile, and FcgR changes ofhMDMs treated with TLRa and STINGa were indicative of proin-flammatory tumoricidal effectors. We therefore assessed whetherthese cells also displayed augmented functional activity in termsof their ability to phagocytose antibody-opsonized CLL cells (Fig.4C; Supplementary Fig. S4B). Macrophages incubated in thepresence of IL4/13 showed a decrease in phagocytic index whileLPS/IFNg–stimulatedmacrophagesmaintained a lower or similarphagocytic profile compared with unstimulated macrophages,

perhaps due to the induction of higher levels of F-actin via theIFNg-mediated mechanism involving PI3K reported previously(36). TLRa-1/2, 5, and -7/8 and all STINGa-stimulated hMDMsshowed a significant increase in their ability to phagocytose CLLcells (Fig. 4C), correlating with their A:I ratios (Fig. 4D, R2¼ 0.34,P ¼ 0.0007). It is not clear whether the contrasting activities ofTLR3a in hMDMs andmBMDMs, both in terms of ADCP and typeI IFN production, relate to differences in TLR signaling pathwaysin human and murine cells, but cell type- and species-specificresponses to TLR3 stimulation in human andmurine effector cellshave been previously reported (37).

STINGa, but not TLRa, enhance mAb-mediated target B-celldepletion in the presence of the tumor microenvironment

Having established the relative abilities of TLRa and STINGa toaugment FcgR-mediated cell depletion with both murine andhumanmacrophages in vitro, we sought to determine their efficacyin vivo. Consistent with our findings with mBMDMs, among thepanel of TLRa assessed, only TLR3a significantly increased the

Figure 4.

FcgR expression and ADCP of tumor cells following stimulation of hMDMs with TLRa or STINGa. A–C, FcgR changes (open black histogram, Iso ctrl for NTmacrophage; filled black histogram, expression of FcgR on NT macrophage; open gray histogram, Iso ctrl for TLRa/STINGa–treated macrophage; filled grayhistogram, expression of FcgR on TLRa/STINGa–treated macrophage; A), A:I ratio (B), and phagocytic index of hMDMs following stimulation with LPS/IFNg (L/g),IL4/13 (4/13), TLRa, or STINGa (C); (n¼ 6–25; Wilcoxon test).D, Correlation between A:I ratio and phagocytic index ($, STINGa hMDM; +, TLRa hMDM;*, NT, 4/13,and L/g hMDM, analyzed by linear regression). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0001.

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mAb-mediated depletion of target B cells in adoptive transferassays in vivo. Approximately 40% of target B cells were deleted byRitm2a alone, whereas inmice primedwith TLR3a approximately60% of target B cells were depleted following Ritm2a adminis-tration (Fig. 5A). Intriguingly, the TLRa-1/2 that was potent withhMDMs in vitro failed to enhance target B-cell depletion inmice invivo and appeared to be inhibitory. We next investigated thedifferences in FcgR expression and A:I ratio induced on splenicmacrophages following TLRa-1/2 and -3 treatment (Fig. 5B). Nosignificant change in FcgR A:I ratio was observed in macrophagesfrom TLR1/2a–treated mice, while TLR3a induced a significantincrease in FcgR A:I ratio, likely explaining the elevated depletionwith this reagent.

While TLR3a mediated a modest increase in the depletion ofadoptively transferred target B cells in these experiments, themurine STING ligand DMXAA mediated a dramatic, approxi-

mately 90%, depletion of target B cells in combination withRitm2a (Fig. 5C). We also observed a statistically significant, 4-fold increase in the FcgR A:I ratio on splenic macrophages of micethat were primed with DMXAA compared with na€�ve mice (Fig.5D) demonstrating that among all reagents assessed, DMXAAwasthe most potent in both upregulating the A:I ratio and depletingtarget B cells.

The adoptive transfer experiments discussed above provided arobustmodel to study target B-cell depletion in the absence of anycomplexity arising from a suppressive tumor microenvironment.However, when BCL1 cells were inoculated into recipient mice 1week prior to adoptive transfer, the most potent TLRa Poly I:Cfailed to induce mAb target cell deletion, while DMXAA retainedits efficacy even in the presence of the tumor (Fig. 5E). ThisDMXAA effect on Ritm2a activity was an antibody Fc–dependentprocess as FcR g chain KO mice, devoid of activatory FcgRs, were

Figure 5.

Effect of TLRa andmurine STINGa onmAb-mediated target B-cell depletion in vivo.A,WTC57BL/6mice were primed with TLRa-1/2, -2/4, -3, or -7/8 after adoptivetransfer and assessed for T:NT ratio as in Fig. 1A (n ¼ 9–20, Wilcoxon test). B, Representative histograms showing changes in FcgR expression (gray, na€�vemice; black, TLRa-treatedmice) andA:I ratio of splenicmacrophages. n.s., nonsignificant.C,Adoptive transfer as inAwith STINGaDMXAAprior to Ritm2a treatment(n ¼ 6, Wilcoxon test). D, Representative histograms showing changes in FcgR expression (gray, na€�ve mice; black, DMXAA-treated mice) and A:I ratio insplenic macrophages from WT C57BL/6 mice that were primed with DMXAA. E, Adoptive transfer in WT BALB/c or FcR g chain�/� mice conducted a weekafter implantation of BCL1 cells. Mice were treated with TLR3a or DMXAA prior to Ritm2a administration and T:NT ratio determined (n ¼ 2–5, Wilcoxon test).F, A:I ratio of splenic macrophages from E. G, Flow cytometric analysis of the spleen of BALB/c mice that were treated with DMXAA 15 days after inoculationwith BCL1 cells. � , P < 0.05; �� , P < 0.01.

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unable to delete target B cells in the same setting (Fig. 5E). Whensplenic macrophages from TLR3a- or DMXAA-treated tumor-bearing mice were assessed for FcgR changes, we found thatDMXAA, but not TLR3a, was able to completely reverse thesuppressive effect of the tumor on the macrophage FcgR A:I ratio(Fig. 5F). Hence, the observed changes in FcgR A:I ratios providean explanation as to why TLR3a loses its efficacy in tumor-bearinghosts, but DMXAA retains its ability to enhancemAb-mediated B-cell depletion even within an immunosuppressive tumor micro-environment. We were also able to exclude that this augmenteddeletion of target cells in the presence of tumor microenviron-ment was the result of any direct cytotoxic effect of DMXAA, as thepercentage of both tumor andnontumor B cells retrieved 24hoursafter DMXAA injection into BCL1-bearing mice was equivalent tothat of mice not treated with DMXAA (Fig. 5G). Notably, admin-istration of DMXAA induced upregulation of type I IFN geneexpression and also reduced expression of both IL10 and IL21 thatwere upregulated following inoculation of the tumor (Supple-mentary Fig. S5).

Type I IFN elicitsmacrophage FcgR skewing and augmentsmAbtarget cell deletion

In both human and murine cells, STINGa induced type I IFN(Figs. 2 and 3; Supplementary Fig. S5). Type I IFN is a keydownstream mediator of STING activation (18), and thereforewe interrogated whether type I IFNs were also able to modulateFcgR A:I ratio changes and target B-cell deletion. We found that inhMDMs, type I IFN induced upregulation of FcgRIIA and III,leading to an increase in A:I ratio and phagocytosis of target CLLcells, in a manner similar to that mediated by STINGa (Fig. 6A).Likewise, administration of type I IFN to mice led to a significantincrease in splenicmacrophage FcgRA:I ratio,mostlymediated byan increase in FcgRIV, as seen with DMXAA (Fig. 6B). mBMDMsincubated in the presence of type I IFN also phagocytosed targetcells more effectively, with a phagocytic index comparable withSTINGa-treated mBMDMs (Fig. 6B). Furthermore, Ritm2a-medi-ated deletion of target B cells in adoptive transfer assays were alsosignificantly enhanced in mice that were primed with type I IFNprior to mAb administration (Fig. 6C). Most importantly, thetarget B-cell–deleting ability ofDMXAAwas completely abrogatedin mice that received IFNAR-blocking antibody, further strength-ening the role of type I IFN in downstreammechanisms governingthe STING-mediated effects (Fig. 6D).

STINGa DMXAA enhances mAb immunotherapy in BCL1lymphoma in an FcgR-dependent manner

Finally, having established that STING activation was capableof overcoming a suppressive lymphoma microenvironment todelete normal B cells in a transplant system, we assessed whetherthe same approach could provide effective deletion of the malig-nant B cells themselves in a therapy model. BCL1-bearing micewere treated with anti-mouse CD20 mAb (18B12; ref. 38) in thepresence or absence of DMXAA as depicted in Fig. 7A. When wemonitored FcgR modulation in the therapy model, we observedthat the tumor substantially downregulated activatory FcgRI andcaused a dramatic increase in the inhibitory FcgRII. Both of thesechanges were reversed by DMXAA, which also induced a 4-foldincrease in FcgRIII and IV (Fig. 7B); allowing DMXAA to reversethe A:I changes induced by the lymphoma microenvironment(Fig. 7C). This was reflected in the survival of experimentalanimals: anti-CD20 and DMXAAmonotherapies produced mod-

est therapeutic effects, whereas �90% of mice that were primedwith DMXAA prior to anti-CD20 administration were effectivelycured, surviving for longer than 100 days (Fig. 7D). This signif-icant enhancement in tumor protection over anti-CD20 (mediansurvival 36 days; P < 0.0001) or DMXAA alone (median survival32 days; P < 0.0001) produced by the combination therapy waslost when mice were devoid of FcgRs (FcgR-null; median survival30 days; Fig. 7E) showing that modulation of FcgRs is crucial tothe combination effect. In contrast, the induction of an adaptivecytotoxic CD8þ T-cell response is not required as 100% of micereceiving the combination therapy following CD8þ T-cell deple-tion were also cured (Fig. 7F).

DiscussionTAMs are typically immunosuppressive and have the capacity

to negatively impact on anticancer immunotherapy strategies.Although deletion of TAM could overcome this issue, it will alsoimpede tumor cell clearance with direct targeting antibodies,which rely on macrophages for their mode of action (24, 31)and are an important component of lymphoma treatment. There-fore, an alternative approach to restrict or prevent tumor growthand yet retain or provide an increased capacity of the TAM todeliver immunotherapy treatments is highly desired (39).

Here we identify that lymphoma cells can elicit a microenvi-ronment that is profoundly suppressive, leading to resistance ofmAb-mediated deletion of target cells and therapy. Lowering ofthe TAM FcgR A:I ratio was a cardinal feature of the immuno-suppressive tumor microenvironment illustrated here, achievedthrough a reduction of the activating FcgR and elevation of theinhibitory FcgRII. Almost all mAbs used in the clinic to date, withthe exception of "true blockers," such as anti-PD1, rely on theappropriate FcgR engagement for their optimal therapeutic activ-ity (40, 41). Recalibration of the FcgR expression profile and A:Iratio is proposed to be one of themost important mechanisms bywhichmAb function can bemodified. Clearly, tipping the balancein the favor of higher A:I ratios may be beneficial in cancerimmunotherapy treatments employing direct targeting mAbs.With regard to anti-CD20 mAb and lymphoma, these changesappear to be more important in the macrophage population,as both monocytes and neutrophils were relatively unaffected bythe tumor.

We demonstrate that the majority of TLRa tested polarizedhMDMs toward an activated phenotype, including an enhancedFcgR A:I ratio, induced a broad, proinflammatory cytokineresponse, and augmented their mAb-mediated phagocytic activ-ity; all parameters desirable for antitumor immunity and yet hadmore modest effects on mBMDMs. The reasons for this arecurrently unclear but may reflect established species-specific dif-ferences (37). Alternatively, these differencesmay be attributed tothe alternative progenitors from which the macrophages weregenerated (i.e., peripheral blood monocytes versus bone marrowprecursors). Conversely, STINGa elicited more reproduciblechanges in FcgR, and type I IFN production, in both hMDMs andmBMDMs, effectively augmenting ADCP and performed consis-tently inmurine assays both in vitro and in vivo. FcgRmodulation islikely to be an important mechanism behind the activity ofSTINGa in vivo as its activity was completely abrogated in gchain�/�mice lacking activating FcgR. Furthermore, the enhancedB-cell–depleting activity induced by STINGa was directly

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Figure 6.

STINGa-mediated effects can be recapitulated by type I IFN and prevented by IFNAR blocking. A, FcgR expression (left), A:I ratio (middle), and phagocyticindex (right) of hMDMs stimulated with STINGa 2030-cGAMP or IFNa (n ¼ 3; error bar � SEM). B, FcgR expression (left), A:I ratio (middle) of splenicmacrophages from WT C57BL/6 mice injected with murine STINGa DMXAA or IFNa, and mBMDM phagocytic index (right). C, WT C57BL/6 mice were primedwith IFNa after adoptive transfer as in Fig. 1A and T:NT ratio determined. D, Adoptive transfer in WT C57BL/6 mice primed with DMXAA and receiving anti-IFNAR(day �1, 0, and 1, adoptive transfer on day 0; analyzed by Student t test). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; n.s., nonsignificant.

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correlated with an enhanced A:I ratio, both in nontumor andtumor-bearing adoptive transfer assays.

Following its engagement, STING stimulates the transcriptionof numerous innate immune genes, including type I IFN with thecapacity to augment macrophage activation (42). Our observa-tions here indicate that type I IFN, togetherwith FcgRmodulation,rather than classical myeloid proinflammatory cytokine produc-tion, may play an important role in augmenting STINGa-basedmAb immunotherapy. This point is of interest, as it is known thatafter engagement of certain cyclic and double-stranded DNAmolecules, STING elicits its effects on macrophages in a manner

different to, and apparently independently of, other DNA recog-nizing receptors, such as TLR9 (17). Generation of similarresponses in terms of FcgR modulation, A:I ratio changes,enhanced phagocytosis, in vivo deletion, and the inability ofSTINGa to delete target cells in the presence of IFNAR-blockingantibodies indicate that type I IFN largely govern the downstreameffects of STING agonism observed here, which are conservedbetween mice and humans.

The ability of STINGa, but not TLRa, to eliminate malignant Bcells in vivomirrors how well they reverse the lower FcgR A:I ratioin the macrophage population. Previously, we have shown that

Figure 7.

STINGa enhances mAb immunotherapy in BCL1 lymphoma in an FcgR-dependent manner. A, Experimental plan to assess the effect of DMXAA on mAbimmunotherapy.B andC,FcgRexpression (B) andA:I ratio (C) on splenicmacrophages onday21 after BCL1 inoculation (error bar�SEM).D–F,Kaplan–Meier survivalcurve forWT BALB/c mice (D), FcgR-null BALB/c (E), orWT BALB/c (F) in the presence or absence of CD8þ T-cell–depleting antibody (n¼ 8–10, Mantel–Cox test).

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the inhibitory FcgRIIB helps tumor cells to escape deletion byrituximab (43, 44). Here we observed a significant improvementin deletion of target B cells in FcgRII knockout mice harboringlymphoma, which suggests that some but not all of the immunesuppression mediated by the tumor is due to the upregulation ofFcgRII onmacrophages, and strengthens the hypothesis that targetcell deletion is a composite function of FcgR A:I ratio. Therefore,adjuvants, such as STINGa, capable of eliciting such FcgR A:I ratiochanges can be exploited in mAb immunotherapy. Importantly,and in marked contrast to TLRa, we have also demonstrated acommonality of response to STING agonism betweenmouse andhumanmacrophages strongly supporting the translational natureof our observations. Some studies have suggested that STINGa candirectly eradicate malignant B cells and mediate tumor regression(45) or disrupt tumor vasculature (46) leading to tumor necrosis,butwedidnot observe anydirect cytotoxic killing of tumor cells inour study. Although disruption of tumor vasculature has beenobserved in certain subcutaneously grown tumor models andendothelial cells may be directly affected by DMXAA, we andothers (47) have established thatDMXAAprimarily acts to inducemacrophage activation and modulation of intratumoral macro-phage phenotype to augment immunotherapy. Presumably, thediscrepancies are attributed to the type of STINGa, differences indosing strategy, and the models used in the studies.

A principal finding of our study was the impressive potency ofSTINGa in augmenting macrophage activation and mAb-medi-ated effector mechanisms both in vitro and in vivo. Although theSTINGa DMXAA showed very impressive effects in the murinelymphoma model studied here, early human cancer trials usingDMXAA failed (48). Subsequent studies revealed that thiswas dueto an inability of DMXAA to activate human STING (49). Sincethen, novel STINGa capable of engaging human STINGhave beengenerated and demonstrated potent adjuvant effects in the radio-therapeutic management of pancreatic cancer (50) and we have,for the first time, demonstrated its efficacy in a lymphoma modelin conjunction with immunotherapy, in a manner dependent onFcgR A:I ratio changes, and in a context where TLRa failed. Studiesshould now be directed to the development, selection, andformulation of cyclic dinucleotide analogues specific to humanSTING, which deliver enhanced pharmacokinetics and definedpotency.

Disclosure of Potential Conflicts of InterestA.J. Steele has received speakers bureau honoraria from Portola Pharmaceu-

ticals. M.S. Cragg is a consultant at Bioinvent International, reports receiving acommercial research grant from Bioinvent International, and has receivedspeakers bureau honoraria from Baxalta. S.A. Beers reports receiving othercommercial research support from Bioinvent International and is a consul-tant/advisory board member for Astex Pharmaceuticals.

Authors' ContributionsConception and design: L.N. Dahal, M.J. Glennie, M.S. Cragg, S.A. BeersDevelopment of methodology: L.N. Dahal, L. Dou, A. Earley, A.J. Steele,S.A. BeersAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.N. Dahal, L. Dou, K. Hussain, R. Liu, A. Earley,S. Murinello, F. ForconiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.N. Dahal, L. Dou, K. Hussain, A. Earley, A.J. Steele,M.J. Glennie, M.S. Cragg, S.A. BeersWriting, review, and/or revision of the manuscript: L.N. Dahal, F. Forconi,A.J. Steele, J.L. Teeling, M.J. Glennie, M.S. Cragg, S.A. BeersAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.L. Cox, I. Tracy, F. Forconi, P. Duriez,D. Gomez-NicolaStudy supervision: M.S. Cragg, S.A. Beers

AcknowledgmentsWe thank patients and volunteers who donated specimens, Mark J.

Shlomchik for hCD20Tg mice, Sjef Verbeek for FcgR-null mice, colleaguesfrom the Cancer Sciences Unit and antibody production team, BiomedicalResearch Facility for animal husbandry, I. Henderson and K.N. Potter forcollection and characterization of CLL samples.

Grant SupportThis work was supported by grants from Bloodwise (12050 to M.S. Cragg),

and CRUK (A12343 to S.A. Beers). This work was also supported by theSouthampton ECMC grant C24563/A15581 and a CRUK Southampton Centregrant C34999/A18087.

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

Received October 12, 2016; revised February 24, 2017; accepted April 19,2017; published OnlineFirst May 16, 2017.

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