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Research Article

AMD3100 Augments the Efficacy of Mesothelin-Targeted, Immune-Activating VIC-008 inMesothelioma by Modulating IntratumoralImmunosuppressionBinghao Li1,2, Yang Zeng1, Patrick M. Reeves1, Chongzhao Ran3, Qiuyan Liu1, Xiying Qu1,YingyingLiang1,ZhaoLiu1, JianpingYuan1,PierreR.Leblanc1, ZhaomingYe2,AnnE.Sluder1,Jeffrey A. Gelfand1, Timothy A. Brauns1, Huabiao Chen1, and Mark C. Poznansky1

Abstract

AMD3100 (plerixafor), a CXCR4 antagonist, has been dem-onstrated to suppress tumor growth and modulate intratumoralT-cell trafficking. However, the effect of AMD3100 on immuno-modulation remains elusive. Here, we explored immunomodu-lation and antitumor efficacy of AMD3100 in combinationwith apreviously developed mesothelin-targeted, immune-activatingfusion protein, VIC-008, in two syngeneic, orthotopic models ofmalignant mesothelioma in immunocompetent mice. Weshowed that combination therapy significantly suppressed tumorgrowth and prolonged animal survival in two mouse models.Tumor control and survival benefit were associated withenhanced antitumor immunity. VIC-008 augmented mesothe-lin-specific CD8þ T-cell responses in the spleen and lymph nodesand facilitated intratumoral lymphocytic infiltration. However,VIC-008 treatment was associated with increased programmedcell death protein-1 (PD-1) expression on intratumoral CD8þ Tcells, likely due to high CXCL12 in the tumor microenvironment.

AMD3100 alone and in combination with VIC-008 modulatedimmunosuppression in tumors and the immune system throughsuppression of PD-1 expression on CD8þ T cells and conversionof regulatory T cells (Tregs) into CD4þCD25–Foxp3þIL2þCD40Lþ

helper-like cells. In mechanistic studies, we demonstratedthat AMD3100-driven Treg reprogramming required T cell recep-tor (TCR) activation and was associated with loss of PTEN due tooxidative inactivation. The combination of VIC-008 augmen-tation of tumor-specific CD8þ T-cell responses with AMD3100abrogation of immunosuppression conferred significant bene-fits for tumor control and animal survival. These data providenew mechanistic insight into AMD3100-mediated immuno-modulation and highlight the enhanced antitumor effect ofAMD3100 in combination with a tumor antigen–targeted ther-apy in mouse malignant mesothelioma, which could be clin-ically relevant to patients with this difficult-to-treat disease.Cancer Immunol Res; 6(5); 539–51. �2018 AACR.

IntroductionMalignant mesothelioma is an aggressive tumor that arises

from the pleural and peritoneal mesothelium. Malignant meso-thelioma is largely refractory to conventional therapies, and themedian survival after symptomonset is often less than 12months(1–4). Surgery, radiotherapy, and chemotherapy have improvedquality of life but have made little impact on survival with thistumor. Studies over the last two decades suggest that immuno-

therapy may be a very promising avenue for the treatment ofmalignantmesothelioma, given that the tumor expresses antigensthat can be targeted by the immune system (5, 6). Althoughclinical trials of various immunotherapeutic modalities haveyielded significant benefit in certain cancers, includingmelanomaand non–small cell lung cancer (7), limitations in the efficacy ofthese therapeutic approaches have been observed and can beunderstood in light of the complexities of intratumoral immunedysregulation (8), which require the development of more effi-cacious combination immunotherapies that address theseimmune evasion mechanisms.

We previously described a fusion protein consisting of thebroadly immune-activating Mycobacterium tuberculosis–derivedheat shock protein 70 (MtbHsp70) and the tumor antigen–targeting activity of a single-chain variable fragment (scFv)-binding mesothelin (MSLN; ref. 9), a validated immunotherapytarget (10–12).We evaluated the antitumor efficacy of thisMSLN-targeted fusion protein as an in vivo vaccination strategy insyngeneic, immunocompetent mouse models of ovarian cancerand mesothelioma and demonstrated that this bifunctionalfusion protein significantly enhances survival and slows tumorgrowth through the augmentation of tumor-specific, cell-medi-ated immune responses (9). In vivo CD8þ T-cell depletion studiesdemonstrated that this protective antitumor effect is mediated by

1Vaccine and Immunotherapy Center, Infectious Diseases Division, Departmentof Medicine, Massachusetts General Hospital and Harvard Medical School,Charlestown, Massachusetts. 2Department of Orthopaedics, Institute ofOrthopaedic Research, Second Affiliated Hospital of Zhejiang UniversitySchool of Medicine, Hangzhou, China. 3Martinos Center for Biomedical Imaging,Department of Radiology, Massachusetts General Hospital and Harvard MedicalSchool, Charlestown, Massachusetts.

B. Li and Y. Zeng contributed equally to this article.

Corresponding Author: Huabiao Chen, Massachusetts General Hospitaland Harvard Medical School, Boston, MA 02114. Phone: 617-643-2561;Fax: 617-726-5411; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0530

�2018 American Association for Cancer Research.

CancerImmunologyResearch

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tumor-specific CD8þ T cells (9). The current version of this fusionprotein, VIC-008, was derived from the original by modifica-tions that remove redundant amino acids and introduce asingle amino acid mutation, phenylalanine to valine, at posi-tion 381 of MtbHsp70 to prevent nonspecific peptide bindingand presentation while retaining the immune-stimulatorycapacity of the original protein. This fusion protein showedsignificantly improved efficacy in tumor control and animalsurvival in a mouse model of MSLN-expressing ovarian cancerover the original protein (13). However, the intratumoralimmunosuppressive microenvironment, including, most nota-bly, the presence of regulatory T cells (Tregs), may limit theeffectiveness of VIC-008. Removal of Tregs has been shown toresult in tumor growth inhibition and the release of antitumoreffector T cells from immunosuppression (14).

The critical role of chemokine receptor 4 (CXCR4) and itsligand (CXCL12) in the pathogenesis of many tumors has beenwell-recognized (15, 16). AMD3100, a specific antagonist forCXCR4, was originally developed as an anti-HIV drug (17) andlater applied as a reagent to mobilize hematopoietic stem cellsfrom bone marrow (18). A number of studies have shown thatAMD3100 can affect tumor growth, metastasis, and angiogenesisby blockade of the CXCL12/CXCR4 axis (19–21) and subsequentinhibition of PI3K-Akt or Ras/Raf-Erk1/2 signaling (22). Wepreviously reported that the blockade of the CXCL12/CXCR4 axiswith AMD3100 as a monotherapy in ovarian tumor–bearingmice conferred a survival advantage and elicited multimodaleffects on tumor pathogenesis and intratumoral T-cell traffick-ing, including selective reduction of Tregs in the tumor (23).However, the precise mechanism of AMD3100-mediatedimmunomodulation remains unknown.

Immunomodulators have been widely used in combinationwith tumor vaccines or immunotherapies for improving antitu-mor immune responses, which include removing or inhibitingsuppressive cells such as Tregs, regulatory type II NKT cells, ormyeloid-derived suppressor cells (24–29). In this study, weevaluated the antitumor efficacy of AMD3100 and VIC-008 intwo syngeneic orthotopic models of malignant mesothelioma inimmunocompetent mice and found that AMD3100 treatmentenhanced the effects of VIC-008 in tumor control and animalsurvival. This response was associated with AMD3100-mediatedneutralization of intratumoral immunosuppression. We alsoexplored and revealed a mechanism by which AMD3100 mod-ulates immunity alone and in combination with VIC-008 inmurine models of malignant mesothelioma.

Materials and MethodsReagents

The fusion protein VIC-008 was constructed with VH and VL

from anti-MSLN p4 scFv fused to full-length MtbHsp70 with a(G4S)3 linker in between and a single amino acid mutation,phenylalanine in place of valine, at position 381 of MtbHsp70,which has been shown in our previous study (13). Proteins wereexpressed by WuXi Biologics in CHO cells and provided at apurity of above 95% by HPLC and an endotoxin level less than1.0 EU/mg. AMD3100was purchased fromAbcam (#ab120718).

Tumor cells40L and AE17 mouse mesothelioma cell lines were kind gifts

from Dr. Agnes Kane (Department of Pathology and Laboratory

Medicine, Brown University, Providence, RI). The cell lines wereauthenticated bymorphology check undermicroscope. Cellswerecultured at 37�C in DMEM (HyClone) supplemented with 1%L-glutamine, 1% penicillin–streptomycin, and 10% FBS (all fromGibco). Mycoplasma detection was performed before use by aMycoplasma detection kit (Thermo Fisher; #4460626). Cells inthe logarithmic phase were used for further assays.

Animal modelsFive-week-old female C57BL/6 mice were obtained from the

Jackson Laboratory and maintained in the gnotobiotic animalfacility of Massachusetts General Hospital (MGH) in compliancewith institutional guidelines and policies. After 1-week acclima-tization, tumors were initiated with 4 � 106 40L cells or 2 � 106

AE17 cells per mouse administered i.p. A subset of the mice fromeach group were euthanized with i.p. administration of ketamine(9 mg/mL in saline) and xylazine (0.9 mg/mL in saline; Sigma) 7days after the last treatment, and samples were harvested forimmune profiling of tumors, inguinal and axillary lymph nodes,and spleens (processing described below).

The remaining animals in each group were monitored forsurvival. For survival studies, we observed the mice daily afterinoculation of tumor cells. Tumor generation was consistentlyfirst evident via the appearance of abdominal distension, sec-ondary to malignant ascites, and tumor-bearing mice wereeuthanized using carbon dioxide at the endpoint when signsof distress, including fur ruffling, rapid respiratory rate,hunched posture, reduced activity, and progressive ascites for-mation, were observed.

Splenocytes from T-Red/FoxP3-GFP transgenic mice, whichwere generated by Dr. Thorsten Mempel and were a kind giftfrom Dr. Gilles Benichou at MGH, were used as a source offluorescently tagged Tregs by cell sorting, as described below.T-Red/FoxP3-GFP transgenic mice were generated by inductionof uniform and selective expression of ds-RedII in all T cells andFoxP3-GFP. The method consists of inserting the red fluorescentprotein ds-RedII under the control of the CD4 promoter andproximal enhancer. This construct lacks the intronic silencer,which prevents CD4 promoter activity in CD8þ cells. The micewere then fully backcrossed with FoxP3-GFP mice from theJackson Laboratory. The FoxP3 construct expresses GFP undercontrol of the forehead box p3 (FoxP3) promoter. The construct isdesigned such that no FoxP3 protein is produced, preventingoverexpression. All animal studies were approved by the Institu-tional Animal Care and Use Committee of MGH.

TreatmentBeginning 7 days after tumor inoculation, treatments were

administrated by i.p. injection once a week for 4 successive weeks.VIC-008 was administrated i.p. at 20 mg in 100 mL of saline permouse once a week, and AMD3100 was given once a week by i.p.injection at 1 mg/kg of body weight in 100 mL of saline.

Immune profiling by flow cytometryTumors harvested at day 7 after the last treatmentweremechan-

ically disaggregated using sterile razor blades anddigested at 37�Cfor 2 hours in RPMI 1640 with collagenase type IV for 40L tumorsor type I for AE17 tumors (2mg/mL; Sigma), DNase (0.1 mg/mL;Sigma), hyaluronidase (0.1 mg/mL; Sigma), and BSA (2 mg/mL;Sigma). Cell suspensions were passed through 100 mm filters to

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Cancer Immunol Res; 6(5) May 2018 Cancer Immunology Research540




remove aggregates. Lymph nodes and spleens were mashed bytwo microslides and filtered through 40 mm strainers. Cells werewashed with staining buffer (#420201; BioLegend) and stainedwith the conjugated antibodies for surface markers. In brief, 1 �106 cells from lymph nodes or spleens or up to 1� 107 cells fromtumors were resuspended in staining buffer and distributed 100mL/tube of suspension into plastic tubes. Fc receptors wereblocked by anti-mouse CD16/32 (BioLegend; #101319). Conju-gated primary antibodies were added and incubated on ice for 20minutes in dark. Total live cells were determined by LIVE/DEADstaining (Thermo Fisher; #L23105).

For intracellular cytokine detection, after staining of surfacemarkers, cells were fixed and permeabilized with fixation/per-meabilization reagents fromBioLegend (#424401)or eBioscience(#00-5521-00) and stained with the conjugated antibodies forintracellular markers.

Conjugated antibodies from eBioscience were as follows:PerCP-Cy5.5 CD40L (clone MR1) and APC-Cy7 PD-1 (cloneJ43). The following conjugated antibodies were purchased fromBioLegend: Brilliant Violet 421 CD3 (clone 17A2), Brilliant Violet711CD4 (cloneGK1.5), PE-Cy7CD8a (clone 53-6.7), Alexa Fluor488 Foxp3 (clone MF-14), Brilliant Violet 605 IL2 (clone JES6-5H4), and Alexa Fluor 488 IFNg (clone XMG1.2). APC CD25(clone PC61) and PE PTEN (clone A2B1) antibodies were fromBD Biosciences.

Flow cytometric analyseswere performedusing BDLSRFortessaX-20 (BD Biosciences). Gating strategies were determined by theFluorescenceMinus One. Flow data were analyzed by FlowJo V10(TreeStar).

Ex vivo culturing of splenocytes and cytokine detectionSplenocytes were treated with red blood cell lysis buffer (Bio-

Legend; #420301). Note that 2 � 106 splenocytes per well wereplated in 24-well plates in RPMI 1640 medium supplementedwith 1% L-glutamine and stimulated with recombinant mouseMSLN (2 mg/mL; BioLegend; #594006) for 72 hours. Brefeldin Aand monesin (BioLegend; #420601 and #420701, respectively)were added into the culture medium during the last 5 hours.Splenocytes were then harvested, and intracellular cytokine stain-ing was performed using anti-IFNg antibodies for cytometricanalysis.

Ex vivo treatment of intratumoral CD3þCD8þ T cells byAMD3100

In untreated AE17 tumor–bearingmice, tumors were harvestedand digested (as described above). The intratumoral CD3þCD8þ

cells were sorted out by a FACSAria (BD Biosciences) and thentreated with AMD3100 (5 mg/mL) for 24 hours. After treatment,the cells were harvested, blocked by anti-CD16/32, then stainedwith conjugated antibodies specific for CD3, CD8, and pro-grammed cell death protein-1 (PD-1; as described above), andanalyzed by flow cytometry.

In vitro reprogramming of TregsT-Red/FoxP3-GFP transgenic mice express GFP in Foxp3þ Tregs.

Spleens were collected from these mice and mashed and filteredthrough 40 mm strainers. Cells expressing GFP-Foxp3 from CD4þ

splenocytes were sorted on a FACSAria (BD Biosciences) and thenexposed to AMD3100 (5 mg/mL) in the presence or absence ofanti-CD3/CD28antibody (1mg/mL) for 24hours. BrefeldinA andmonesin (BioLegend; #420601 and #420701, respectively) were

added into the culture medium during the last 5 hours. The cellswere then harvested and stained with conjugated antibodiesspecific for CD3, CD4, CD25, Foxp3, IL2, CD40L, and PTEN andanalyzed by flow cytometry.

Enzyme-linked immunosorbent assayTumors, spleens, and inguinal and axillary lymph nodes were

collected and then mashed with the help of liquid nitrogen forprotein extraction. Tissue CXCL12 was evaluated by an ELISA kit(R&D Systems; #DY460 and #DY008), following the manufac-turer's instructions. For tissue CXCL12 quantification, 20 mg oftotal protein from each sample was added in each well of 96-wellplates.

Western blotFor analyses of protein expression, sorted Tregs were lysed and

run in nonreducing conditions or reducing conditions by addingreducing agent (Thermo Fisher; #NP004) and antioxidant(Thermo Fisher; #NP0005). The concentration of protein wastested using the BCA Protein Assay Kit (Thermo Fisher; #23227).Note that 40 mg total protein per sample was used for analysis.Before loading into the gels, lysates were mixed with loadingbuffer (Thermo Fisher; #NP0007) and heated in 70�C for 10minutes. Cell lysates were then separated by Bis-Tris protein gels(Thermo Fisher; #NP0321) in running buffer (Thermo Fisher;#NP0002), transferred to methanol-soaked PVDF membranes(Thermo Fisher; #LC2002) in transfer buffer (Thermo Fisher;#NP0006), blocked by 5% BSA (for antibodies from Cell Signal-ing Technology; Sigma, #A7906) or 5% nonfat milk (for anti-bodies from Abcam and Santa Cruz Biotechnology) at roomtemperature for 1 hour, and immunoblotted with primary anti-bodies at 4�C overnight. Tris-buffered saline with 0.1% Tween-20(Sigma; #P9416; TBST) was used as washing buffer in the wholeprocedure, followed by incubation with appropriate horseradishperoxidase–conjugated secondary antibody (Abcam; #ab97051and #ab6741) at room temperature for 1 hour, and detection ofthe blots was performed by the ECL detection system (ChemiDocXRSþ imaging system; Bio-Rad). Antibodies against b-tubulin(#ab6046) and PTEN (#ab32199) were purchased from Abcam.

Statistical analysesP values were calculated by GraphPad Prism 6. The P values

for comparison among groups were obtained by one-wayANOVA with Dunnett multiple comparisons test or unpairedt test with Welch correction. The Kaplan–Meier method andlog-rank test were used to compare survival among groups. P <0.05 was considered statistically significant. Data are presentedas mean � SEM.

ResultsCombination of AMD3100 and VIC-008 augments tumorcontrol and mouse survival

We established two i.p. malignant mesothelioma models inimmunocompetent C57BL/6mice, separately using the syngeneic40L and AE17 cell lines (30). Here, we tested the effect ofAMD3100 and VIC-008, used alone or in combination, on tumorgrowth and animal survival in mesothelioma-bearing mice. Inanimals treated with VIC-008 alone (20 mg per mouse), the totalweight of i.p. tumors collected 1 week after the last treatment wasnot significantly reduced (Fig. 1A and B), but animal survival was

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significantly prolonged (Fig. 1C and D) compared with salinecontrol treatment in both the 40L and AE17 models (P < 0.01and P < 0.01, respectively). AMD3100 alone at 1 mg/kg ofmouse body weight did not confer significant benefit to survivalin both 40L and AE17 mouse malignant mesothelioma modelscompared with saline control treatment. However, the combina-tion treatment with VIC-008 and AMD3100 significantlyenhanced tumor control (P < 0.0001 and P < 0.001, respectively)and prolonged animal survival (P < 0.0001 and P < 0.0001,respectively) compared with saline control in both 40L and AE17models. The combination treatment showed further significantlyimproved antitumor efficacy on inhibition of tumor growth (P <0.001 and P < 0.05, respectively) and prolongation of mousesurvival (P < 0.0001 and P < 0.001, respectively) compared withVIC-008 monotherapy in both 40L and AE17models. These dataindicate that when combined, AMD3100 significantly enhancesthe antitumor effect of VIC-008 in both 40L and AE17 malignantmesothelioma mouse models compared with monotherapy witheither agent.

VIC-008 increases lymphocyte infiltration in spleens, lymphnodes, and tumors

Spleens, axillary and inguinal lymph nodes, and i.p. tumorswere collected from tumor-bearing mice 1 week after the lasttreatment. Single cells were prepared from these tissues andanalyzed by flow cytometry (Fig. 2A). The proportion of CD8þ

T cells in the total live cells recovered from spleens (P < 0.05

and P < 0.05, respectively) and tumors (P < 0.01 and P < 0.01,respectively) for both the 40L and AE17 models, and fromlymph nodes (P < 0.01) in AE17 model, was significantlyincreased in the VIC-008 treatment group compared withthat in the saline control group (Fig. 2B–F). In the VIC-008and AMD3100 combination treatment group, the proportionof CD8þ T cells in the total live cells recovered from spleens (P <0.05 and P < 0.05, respectively) and tumors (P < 0.01 and P <0.05, respectively) for both the 40L and AE17 models, and fromlymph nodes (P < 0.001) in AE17 model, were significantlyincreased compared with that in the saline control group, andAMD3100 treatment did not increase the proportion of CD8þ Tcells in these tissues. However, no difference in the proportionof CD8þ T cells between the VIC-008 and combination treat-ment groups was seen, indicating that VIC-008 increased lym-phocyte infiltration of these tissues.

VIC-008 enhances tumor antigen–specific CD8þ T-cellresponses

We next restimulated single cells isolated from the spleens andlymph nodes of tumor-bearing mice with recombinant MSLN exvivo and analyzed intracellular IFNg in CD8þ T cells. MSLN-specific IFNg expressions in splenic CD8þ T cells, both in 40L(P < 0.001) and AE17 (P < 0.001) models, and in lymph nodeCD8þ T cells in the AE17 model (P < 0.01) were significantlygreater in mice treated with VIC-008 alone compared with that inmice treated with saline (Fig. 3). In VIC-008 and AMD3100

Figure 1.

Tumor growth and survival in different treatment groups. All i.p. tumorswere collected at day7 after the last treatment andweighed in (A) 40L (n¼ 10 per group) and(B) AE17 (n¼ 10 per group) mice. The survival of (C) 40L (n¼ 12 per group) and (D) AE17 (n¼ 12 per group) tumor–bearing mice. Numbers on the x axis representdays of mouse survival after inoculation of tumor cells. NS, not significant; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; and �� , P < 0.0001. Data were presentedasmean� SEM; representative of three independent experiments. Statistical method: (A andB) One-wayANOVAwith Dunnett multiple comparisons and (C andD)Kaplan–Meier method and log-rank test.

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combination treatment group,MSLN-specific IFNg expressions insplenic CD8þ T cells, both in 40L (P < 0.01) and AE17 (P < 0.01)models, and in lymph node CD8þ T cells in the AE17 model (P <0.001) were significantly greater compared with that in micetreated with saline. AMD3100 treatment by itself did not enhanceantigen-specific IFNg secretion in CD8þ T cells. No difference inthe IFNg expression in CD8þ T cells between the VIC-008 andcombination treatment groups was seen. Together, these datasupport the view that VIC-008 treatment enhanced antitumorCD8þ T-cell responses in both the 40L and AE17 mesotheliomamouse models.

AMD3100 decreases PD-1 expression on CD8þ T cellsWe next evaluated expression of PD-1 on CD8þ T cells in

spleen, tumor, and lymph nodes. No significant differencebetween the VIC-008 treatment group and the saline controlgroup in the proportion of PD-1–expressing CD8þ T cells inspleens in both the 40L and AE17 tumor–bearing mice and inlymph nodes for AE17 mice was seen (Fig. 4A–C). In contrast,significantly more intratumoral CD8þ T cells in the VIC-008–treated group expressed PD-1 in the 40L tumors (P < 0.05) andAE17 tumors (P < 0.01) compared with saline-treated controlsand AMD3100 treatment (Fig. 4D and E). The percentage of PD-

Figure 2.

VIC-008 facilitated intratumoral lymphocyte infiltration. A, Gating strategy for tumor infiltrating lymphocytes. The proportion of CD8þ T cells in totallive splenocytes in (B) 40L (n ¼ 6 per group) and (C) AE17 (n ¼ 5 per group) mice. D, The proportion of CD8þ T cells in total live cells in the lymph nodes ofAE17 mice (n ¼ 5 per group). The proportion of CD8þ T cells in total live cells in the tumors of (E) 40L (n ¼ 6 per group) and (F) AE17 (n ¼ 5 per group)mice. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001. Data are presented as mean � SEM; representative of two independent experiments. Statisticalmethod: (B–F) One-way ANOVA with Dunnett multiple comparisons.






1–expressing CD8þ T cells ranged between 43%–76% and 28%–

47% in the 40L tumors and AE17 tumors, respectively, comparedwith only 5% to 10% in spleen and lymph nodes. These dataindicate that the antitumor activity of the CD8þ T cells in thetumor environment induced by VIC-008 treatment could beobstructed by activation of the PD-1/PD-L1 pathway. Similarly,we found that significantly more intratumoral CD4þ T cells inVIC-008–treated mice expressed PD-1 in the 40L tumors (P <0.01) and AE17 tumors (P < 0.05) compared with saline-treatedcontrols and AMD3100 treatment (Fig. 4F and G). Given thatothers have reported the impact of CXCL12 on PD-1 and PD-L1expression (22, 31, 32), we examined the levels of CXCL12 in theintratumoral microenvironment. We found that the concentra-tion of intratumoral CXCL12measured by ELISAwas significantlyhigher in VIC-008–treated AE17 tumor–bearing mice (2,962 �351 pg/mg) than that in saline-treated (1,444 � 261 pg/mg; P <0.01) and AMD3100-treated mice (1,044 � 172 pg/mg; P <0.01; Fig. 4H). In the VIC-008–treated mice, the concentrationof CXCL12 was significantly higher in tumors than that in spleens(P < 0.001) and lymph nodes (P < 0.001; Fig. 4I). These dataindicated that high CXCL12 in intratumoral microenvironmentmay be associated with the upregulation of PD-1 expression ontumor-infiltrating T cells.

In concert with this, we found that AMD3100 reduced PD-1expression on CD8þ T cells. AMD3100 treatment alone led tosignificantly fewer PD-1–expressing CD8þ T cells in spleens (P <0.05 and P <0.01, respectively) and tumors (P< 0.05 and P< 0.05,respectively) in both the 40L and AE17 tumor–bearing mice andin lymph nodes (P < 0.01) from AE17 mice than in mice treatedwith saline. In the AMD3100 and VIC-008 combination treat-ment group, significantly fewerCD8þT cells expressedPD-1 in thespleen (P < 0.05 and P < 0.01, respectively) and tumor (P < 0.05and P < 0.05, respectively) in both the 40L and AE17 tumor–

bearing mice and in lymph nodes (P < 0.01) for AE17 micecompared with the saline-treated controls. No significant differ-ence in the proportion of PD-1–expressing CD8þ T cells in thesetissues between the AMD3100monotherapy and AMD3100-VIC-008 combination therapy groups was found. In contrast, we didnot observe significant inhibition of PD-1 on CD4þ T cells byAMD3100 in tumors (Fig. 4F and G).

In order to address whether AMD3100-driven PD-1 down-regulation on tumor-infiltrating CD8þ T cells could be medi-ated in isolated single cells, CD3þCD8þ T cells were sortedfrom tumor-infiltrating lymphocytes in untreated AE17 tumor–bearing mice and exposed in vitro to AMD3100 (5 mg/mL).AMD3100 significantly decreased the expression of PD-1 onCD8þ T cells (P < 0.0001; Fig. 4J and K). Together, these datashowed that AMD3100 can inhibit CD8þ T cells from expres-sing PD-1 in spleens, lymph nodes, and tumors in vivo andmediate the downregulation of PD-1 on CD8þ T cells in vitro atthe single-cell level. These data are consistent with previousstudies that support the view that the CXCR4/CXCL12 axis andits antagonism modulate intratumoral immune checkpointmolecule expression (22, 31, 32).

AMD3100 reduces tumor-infiltrating TregsWe next evaluated the impact of AMD3100 on Tregs. AMD3100

did not alter the proportion of Tregs found in the spleens of either40L or AE17 tumor–bearingmice (Fig. 5A andB). In AE17 tumor–bearingmice, AMD3100 alone did not significantly reduce Tregs inthe lymph nodes (Fig. 5C), and AMD3100 alone or in combina-tion with VIC-008 significantly increased the cell ratio of CD8þ

T cells to Tregs (P < 0.01 and P < 0.0001, respectively) comparedwith saline treatment (Fig. 5D). In tumors from both the 40Land AE17 models, AMD3100 applied as monotherapy signifi-cantly decreased the proportion of Tregs (P < 0.05 and P < 0.01,

Figure 3.

VIC-008 promoted CD8þ T-cell IFNg secretion. A, Representative dot plots of IFNg-secreting cells in different treatment groups. The proportion of IFNg-secreting cells in CD8þ T cells in the spleens of (B) 40L (n¼6 per group) and (C) AE17 (n¼ 5 per group)mice.D, The proportion of IFNg-secreting cells in CD8þT cellsin the lymph nodes of AE17 mice (n ¼ 5 per group). �� , P < 0.01 and �� , P < 0.001. Data are presented as mean � SEM; representative of two independentexperiments. Statistical method: (B–D) One-way ANOVA with Dunnett multiple comparisons.

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Figure 4.

AMD3100 decreased PD-1 expression onCD8þT cells. The proportion of PD-1–expressing cells in CD8þ T cells in the spleens of (A) 40L (n¼ 6per group) and (B) AE17(n ¼ 5 per group) mice. C, The proportion of PD-1–expressing cells in CD8þ T cells in the lymph nodes of AE17 mice (n ¼ 5 per group). The proportion ofPD-1–expressing cells in CD8þ T cells in the tumors of (D) 40L (n¼ 6 per group) and (E) AE17 (n¼ 5 per group)mice. The proportion of PD-1–expressing cells in CD4þ

T cells in the tumors of (F) 40L (n ¼ 6 per group) and (G) AE17 (n ¼ 5 per group) mice. H, The concentration of CXCL12 was measured by ELISA in AE17tumors in the differently treated mice (n¼ 5 per group). I, CXCL12 in tumors, lymph nodes, and spleen in VIC-008–treated mice. J, Representative dot plots of PD-1expression presented as mean fluorescence intensity (MFI) on intratumoral CD8þ T cells from AE17 tumors with or without AMD3100 treatment in vitro, and (K)statistical difference was analyzed (n ¼ 5 per group). � , P < 0.05; �� , P < 0.01; and �� , P < 0.001. Data are presented as mean � SEM; representative of twoindependent experiments. Statistical method: (A–I) One-way ANOVA with Dunnett multiple comparisons and (J) unpaired t test with Welch correction.






respectively) and increased the ratio of CD8þ T cells to Tregs (P <0.001 and P < 0.01, respectively) compared with saline treatment(Fig. 5E–H). In the AMD3100 and VIC-008 combination treat-ment group, the proportion of Tregs was significantly decreased(P < 0.05 and P < 0.05, respectively), and the ratio of CD8þ T cellsto Tregs increased (P < 0.001 and P < 0.0001, respectively)compared with saline treatment in both the 40L and AE17models. In these two murine mesothelioma models, AMD3100reduced intratumoral Treg infiltration.

AMD3100 modulates Tregs toward a T helper phenotypeWe observed that AMD3100, alone or in combination with

VIC-008, significantly increased the ratio of CD25– cells toCD25þ cells within the Foxp3þ population in both 40L(Fig. 6A; P < 0.01 and P < 0.001, respectively) and AE17 tumors(Fig. 6B; P < 0.01 and P < 0.05, respectively), as well asin the lymph nodes in the AE17 model (Fig. 6C; P < 0.001and P < 0.01, respectively). Among the Foxp3þCD25– Tregpopulation, significantly more cells were phenotypicallyIL2þCD40Lþ (Fig. 6D–G) after AMD3100 monotherapy andcombination therapy with VIC-008, suggesting a change tohelper-like cells that have lost CD25 without the loss of Foxp3(33), andmay have lost their immunosuppressive function (34,35). No differences in the proportion of IL2þCD40Lþ cells inthe Foxp3þCD25– Treg population between AMD3100 mono-therapy and combination therapy groups were seen, indicatingthat AMD3100 treatment may be the major driver of repro-gramming Tregs into helper-like cells.

The T helper phenotype is a result of TCR activation and PTENoxidation by AMD3100

We next addressed whether AMD3100-driven Treg modulationcould be initiated in isolated Tregs and whether we could define itsmolecular basis. Cells expressingGFP-Foxp3 inCD4þ splenocytesfromT-Red/FoxP3-GFP transgenicmicewere sorted and treated invitro with AMD3100. AMD3100 treatment alone did not changethe proportional ratio of CD25– cells to CD25þ cells in theFoxp3þCD4þ population (Fig. 7A and B) or the proportion ofIL2þCD40Lþ cells in Foxp3þ CD25– Tregs (Fig. 7C and D).However, in the presence of stimulation by anti-CD3/CD28antibodies to trigger TCR activation, AMD3100 treatment signif-icantly increased the proportional ratio of CD25– cells to CD25þ

cells in the Foxp3þCD4þ population (Fig. 7E and F; P ¼ 0.0017)and converted more Foxp3þCD25– Tregs into IL2þCD40Lþ cells(Fig. 7G and H; P ¼ 0.0015). Sharma and colleagues (33, 36)demonstrated that the inhibition of PTEN by a small-moleculeinhibitor, VO-OHpic, can cause Tregs to lose CD25 without loss ofFoxp3, which disrupted their immune-suppressive function. Inview of this, we next examined the impact of AMD3100 on theexpression of CD25 and PTEN at the single Treg level by flowcytometry. We found simultaneous downregulation of CD25 andPTEN as a result of AMD3100 treatment (Fig. 7I). This indicatedthat the initiation of the conversion from CD4þFoxp3þCD25þ toCD4þFoxp3þCD25– Tregs was associatedwith the loss of PTEN. Inview of previous reports demonstrating that the loss of PTEN canbe associated with oxidative inactivation (37), we examined thisaspect in the context of Treg function. UsingWestern blot analysis,we further demonstrated that the loss of PTEN in Tregs was due tooxidative inactivation after AMD3100 treatment (Fig. 7J). Togeth-er, these data demonstrated that the conversion of Tregs into

helper-like cells can be mediated by AMD3100 treatment uponTCR activation and PTEN oxidation.

DiscussionMSLN is overexpressed on the surface of a number of com-

mon epithelial cancers, including epithelial malignant meso-thelioma, although expressed only at relatively low levels innormal mesothelial cells lining the pleura, pericardium, andperitoneum in healthy individuals (38–40). MtbHsp70 is wellcharacterized and functions as a potent immune adjuvant (41).It stimulates monocytes and dendritic cells (DC) to produceCC-chemokines (42, 43), which attract antigen processing andpresenting macrophages, DCs, and effector T and B cells (44).Antigenic peptides linked to MtbHsp70 can elicit both MHCclass I–restricted CD8þ and MHC class II–restricted CD4þ T-cellresponses (45–49). The fusion of the anti-MSLN scFv andMtbHsp70 takes advantage of the immune-activating actionof MtbHsp70 and the tumor-targeting activity of the scFv topromote antitumor responses against the broadest profile oftumor antigens. Consistent with our previous findings in amouse model of ovarian cancer, here we demonstrated thatVIC-008 enhanced tumor-specific CD8þ T-cell–mediated cyto-toxic responses and facilitated lymphocyte intratumoral infil-tration in mouse models of malignant mesothelioma, resultingin significant prolongation of animal survival. However, wealso found that PD-1 expression was significantly upregulatedon tumor-infiltrating T cells in VIC-008–treated mice and thatthis was associated with high CXCL12 expression in the intra-tumoral microenvironment. These data indicated that theCXCL12–CXCR4 axis in the intratumoral microenvironment,itself, may modulate the PD-1/PD-L1 pathway and that theantitumor activity of tumor-infiltrating T cells could be com-promised by PD-1/PD-L1 pathway activation (50). This isconsistent with previous findings that CXCR4 inhibition orblockade of CXCL12 in the tumor microenvironment facilitatesPD-1 blockade immunotherapy in hepatocellular carcinoma(22), pancreatic cancer (31), and colorectal cancer (32).

CXCL12 and its cognate receptor, CXCR4, constitute a che-mokine–receptor axis that is known to be overexpressed in thetumor microenvironment of various cancers, and activation ofthe CXCL12–CXCR4 axis is associated with disease progression(19, 20). In vitro and in vivo models have revealed thatAMD3100, an antagonist of CXCR4, can inhibit tumor growth,metastasis, and angiogenesis by blockade of CXCL12–CXCR4interaction and subsequent inhibition of PI3K-Akt or Ras/Raf-Erk1/2 signaling (22, 23). The CXCL12–CXCR4 axis is alsoknown to mediate trafficking and retention of various immunecells at specific anatomic sites (51, 52). Tregs are thought tomodulate antitumor immune responses through selectivemigration to and accumulative retention at tumor sites, therebyplaying an important role in the immunopathogenesis oftumors (53). For example, basal-like breast cancers behavemore aggressively despite the presence of dense lymphoidinfiltration due to Treg recruitment driven by hypoxia-inducedupregulation of CXCR4 in Tregs (54). We have reported that inan environment of elevated CXCL12 in an ovarian cancermodel, blockade of the CXCL12/CXCR4 axis with AMD3100elicited multimodal effects on tumor pathogenesis, includingselective reduction of intratumoral Tregs and release of antitu-mor effector T cells from immunosuppression, which conferred

Li et al.





a significant survival advantage to the ovarian tumor–bearingmice (23). In our current study, we demonstrated thatAMD3100 promoted the conversion of phenotypically suppres-sive CD25þFoxp3þ Treg cells to CD25–Foxp3þIL2þCD40Lþ

helper-like cells (33), which may result in the loss of immu-nosuppressive function of intratumoral Tregs (34, 35). In a

similar scenario, a study on renal cell carcinoma demonstratesthat CXCR4 antagonism elicited by a new peptidic antagonist,Peptide-R29, reverted the suppressive activity of Tregs (50).Meiron and colleagues demonstrate, in opposition, thatCXCL12 redirected the polarization of Th1 cells into IL10-producing Tregs (55).

Figure 5.

AMD3100 reduced tumor-infiltrating Tregs.The proportion of Tregs in total livesplenocytes in (A) 40L (n ¼ 6 per group)and (B) AE17 (n ¼ 5 per group) mice.C, The proportion of Tregs in total live cellsin the lymph nodes of AE17mice (n¼ 5 pergroup).D, The ratio of CD8þ T cells to Tregsin the lymph nodes of AE17mice (n¼ 5 pergroup). E, The proportion of Tregs intotal live cells in the tumors of 40Lmice (n ¼ 6 per group). F, The ratio ofCD8þ T cells to Tregs in the tumors of 40Lmice (n ¼ 6 per group). G, The proportionof Tregs in total live cells in the tumors ofAE17 mice (n ¼ 5 per group). H, Theratio of CD8þ T cells to Tregs in the tumorsof AE17 mice (n ¼ 6). � , P < 0.05;�� , P < 0.01; �� , P < 0.001; and�� , P < 0.0001. Data are presented asmean � SEM; representative of twoindependent experiments. Statisticalmethod: (A–H) One-way ANOVA withDunnett multiple comparisons.






CD25–Foxp3þIL2þCD40Lþ helper-like cells have been dem-onstrated to have rapid-acting, supportive roles in priming CD8þ

T-cell responses, but they need initial signals fromactivated CD8þ

T cells to initiate the reprogramming (33, 36). Our data robustlydemonstrated that AMD3100-mediated conversion of Tregs intohelper-like cells was TCR activation–dependent. PTEN is a criticalregulator that is capable of stabilizing the immune-suppressivefunction of Tregs (33, 36, 56–58). In brief, it has been shown thatTregs have high expression of PTEN, which abrogates PI3K/mTORC2-Akt pathway signaling and, thereby, maintains theexpression of CD25 and Foxp3 (36, 56–58). The inhibition ofPTEN results in the loss of CD25 expression, despite the main-tenance of Foxp3 (33) and, thereby, the disruption of theirimmune-suppressive function (34, 35). In this current study, we

further demonstrated that AMD3100-mediated conversion ofTregs into IL2þCD40Lþ helper-like cells was characteristic ofdownregulation of CD25 and associated with the loss of PTENdue to oxidative inactivation. We, thus, hypothesize that in thecombinatorial treatment setting, VIC-008-activated CD8þ T-cellresponses further facilitate AMD3100-mediated Treg reprogram-ming, contributing to the observed enhanced antitumor efficacyof the combination therapy.

Studies demonstrate that inhibition of CXCR4 signalingrestores sensitivity to CTLA-4 and PD-1 checkpoint inhibitors(21, 22), suggesting a mechanism by which AMD3100 maymodulate immune responses. Indeed, in this study, wefound that AMD3100 was associated with suppression of PD-1expression on CD8þ T cells, consistent with the abrogation of

Figure 6.

AMD3100 reprogrammed Tregs to helper-like cells. The ratio of CD25– to CD25þ cells in the CD4þFoxp3þ T-cell population from the tumors of (A) 40L (n ¼ 6per group) and (B) AE17 (n¼ 5 per group)mice, aswell as in (C) the lymph nodes of AE17 (n¼ 5 per group)mice.D,Representative density plots of IL2þCD40Lþ cellsin the Foxp3þCD25– Treg population in different treatment groups. The proportion of IL2þCD40Lþ cells in Foxp3þCD25– Treg population in the tumors of (E)40L (n¼ 6 per group) and (F) AE17 (n¼ 5 per group) mice, as well as in (G) the lymph nodes of AE17 (n¼ 5 per group) mice. � , P < 0.05; �� , P < 0.01; �� , P < 0.001;and �� , P < 0.0001. Data are presented as mean � SEM; representative of two independent experiments. Statistical method: (A–C and E–G) One-wayANOVA with Dunnett multiple comparisons.

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Figure 7.

AMD3100-driven Treg reprogramming required TCR activation. A, Representative dot plots of the Foxp3þCD25– and Foxp3þCD25þ population with or withoutAMD3100 treatment under no anti-CD3/CD28 stimulation and (B) statistical analysis of the dataset (n¼ 4per group).C,Representative density plots of IL2þCD40Lþ

cells in the Foxp3þCD25– Treg population with or without AMD3100 treatment under no anti-CD3/CD28 stimulation and (D) statistical analysis of the dataset(n ¼ 4 per group). E, Representative dot plots of the Foxp3þCD25– and Foxp3þCD25þ population with or without AMD3100 treatment under anti-CD3/CD28stimulation and (F) statistical analysis of the dataset (n ¼ 4 per group). G, Representative density plots of IL2þCD40Lþ cells in the Foxp3þCD25– Treg populationwith or without AMD3100 treatment under anti-CD3/CD28 stimulation and (H) statistical analysis of the dataset (n ¼ 4 per group). I, Representative dot plotsof CD25 and PTEN on CD4þFoxp3þ T cells with and without AMD3100 treatment in vitro under anti-CD3/CD28 stimulation. J, Western blot analysis ofPTEN in CD4þFoxp3þ T cells with and without AMD3100 treatment in vitro. Statistical difference was analyzed using unpaired t test with Welch correction.Data are presented as mean � SEM; representative of two independent experiments.






PD-1–mediated immunosuppression. Our findings demonstrat-ed that the augmentation of tumor-specific CD8þ T-cell responsesinduced by VIC-008, together with the abrogation of immuno-suppression mediated by AMD3100, both through inhibition ofPD-1 expression on intratumoral CD8þ T cells and throughconversion of suppressive Treg to helper-like cells, conferredsignificant benefits on tumor control and animal survival inmalignantmesothelioma.A clear next stepwouldbe to investigatehow AMD3100 affects other checkpoint molecules, includingLAG-3, TIM-3, and TIGIT.

Taken together, these data revealed the therapeutic potentialof AMD3100, highlighting its potential to act as a potentimmune modulator that can complement the activity of tar-geted cancer immunotherapies, such as DC- or T-cell–basedvaccines. These data also highlighted that vaccine-based cancerimmunotherapy may be further improved if Tregs are quanti-tatively reduced or functionally impaired. In this way, our studymay offer a therapeutic approach for significantly prolongingthe survival of patients with malignant mesothelioma andexpand the potential clinical efficacy of both novel and estab-lished DC- or T-cell–based vaccines and immunotherapies forthis difficult-to-treat disease.

Disclosure of Potential Conflicts of InterestJ.A. Gelfand is a consultant/advisory board member for Supportive

Therapeutics and Henry Schein, Inc. T.A. Brauns has ownership interest(including patents) in AperiSys, Inc. M.C. Poznansky is a scientific consul-tant at AperiSys, Inc. and has ownership interest (including patents) andfounder's equity. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: B. Li, P.M. Reeves, J.A. Gelfand, T.A. Brauns, H. Chen,M.C. PoznanskyDevelopment of methodology: B. Li, Y. Zeng, P.M. Reeves, P.R. Leblanc, Z. Ye,J.A. Gelfand, H. Chen, M.C. PoznanskyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Li, P.M. Reeves, C. Ran, Q. Liu, X. Qu, Y. Liang,Z. Liu, J.A. GelfandAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Li, Y. Zeng, J.A. Gelfand, H. Chen, M.C. PoznanskyWriting, review, and/or revision of the manuscript: B. Li, Y. Zeng, C. Ran,Q. Liu, P.R. Leblanc, A.E. Sluder, J.A. Gelfand, T.A. Brauns, H. Chen,M.C. PoznanskyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Zeng, Q. Liu, Z. Liu, J. Yuan, A.E. Sluder,H. Chen, M.C. PoznanskyStudy supervision: H. Chen, M.C. Poznansky

AcknowledgmentsThis work was supported by DOD CDMRP (W81XWH-14-1-0206), the

VIC Mesothelioma Research Program, VIC Innovation Fund, and theEdmund C. Lynch, Jr. Cancer Fund. Cytometric findings reported herewere performed in the MGH Department of Pathology Flow and ImageCytometry Research Core, which was supported by the NIH SharedInstrumentation grants 1S10OD012027-01A1, 1S10OD016372-01,1S10RR020936-01, and 1S10RR023440-01A1.

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 September 18, 2017; revisedNovember 25, 2017; accepted February28, 2018; published first March 6, 2018.

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2018;6:539-551. Published OnlineFirst March 6, 2018.Cancer Immunol Res Binghao Li, Yang Zeng, Patrick M. Reeves, et al. Intratumoral ImmunosuppressionImmune-Activating VIC-008 in Mesothelioma by Modulating AMD3100 Augments the Efficacy of Mesothelin-Targeted,

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