immune-checkpoint protein vista regulates antitumor immunity … · cascades. at cellular levels,...

Research Article

Immune-Checkpoint Protein VISTA RegulatesAntitumor Immunity by Controlling MyeloidCell–Mediated Inflammation andImmunosuppressionWenwen Xu1, Juan Dong2, Yongwei Zheng1,3, Juan Zhou1,4, Ying Yuan1, Hieu Minh Ta2,Halli E. Miller1, Michael Olson1, Kamalakannan Rajasekaran3, Marc S. Ernstoff5,Demin Wang1,3, Subramaniam Malarkannan1,3,6,7, and Li Wang2

Abstract

Immune-checkpoint protein V-domain immunoglobulinsuppressor of T-cell activation (VISTA) controls antitumorimmunity and is a valuable target for cancer immunotherapy.This study identified a role of VISTA in regulating Toll-likereceptor (TLR) signaling in myeloid cells and controllingmyeloid cell–mediated inflammation and immunosuppres-sion. VISTA modulated the polyubiquitination and proteinexpression of TRAF6. Consequently, VISTA dampened TLR-mediated activation of MAPK/AP-1 and IKK/NF-kB signalingcascades. At cellular levels, VISTA regulated the effector func-

tions of myeloid-derived suppressor cells and tolerogenicdendritic cell (DC) subsets. Blocking VISTA augmented theirability to produce proinflammatory mediators and dimin-ished their T cell–suppressive functions. These myeloid cell–dependent effects resulted in a stimulatory tumor microenvi-ronment that promoted T-cell infiltration and activation. Weconclude that VISTA is a critical myeloid cell–intrinsicimmune-checkpoint protein and that the reprogramming oftolerogenic myeloid cells following VISTA blockade promotesthe development of T cell–mediated antitumor immunity.

IntroductionThe B7 family of immune-checkpoint receptors are valuable

targets for cancer immunotherapy. Antibodies targeting two B7family coinhibitory receptors, CTLA-4 and PD-1, have eliciteddurable clinical outcomes in previously refractory cancer types,and are considered a breakthrough therapy for cancer (1, 2).Despite this success, the response rate following CTLA-4, PD-L1,or PD-1–blocking monoclonal antibody (mAb) therapies is gen-erally less than 30%, indicating that additional nonredundantimmune-checkpoint pathways hamper antitumor immunity (2).

VISTA (gene Vsir, RIKEN cDNA 4632428N05, also known asGi24, Dies-1, PD-1H, and DD1a) stands for V-domain immu-noglobulin suppressor of T-cell activation and is an inhibitoryB7 family immune-checkpoint molecule (3, 4). VISTA is consti-tutively expressed onmultiple immune cell types such asCD11bþ

myeloid cells, na€�ve CD4þ and CD8þ T cells, Foxp3þCD4þ

regulatory T cells, and TCRgd T cells. Studies from our group andothers have shown that VISTA controls T cell–mediated autoim-munity and antitumor immunity (3, 5). Although other immune-checkpoint regulators such as CTLA-4 and PD-L1/PD-1 controlT-cell activation by recruiting SHP1/2, which antagonizes prox-imal T-cell receptor (TCR) signaling (6, 7), VISTA plays a broaderrole in regulating both myeloid cell–mediated and T cell–mediated immune responses (4). Our previous studies in amurine model of psoriasiform inflammation show that VISTAinhibits TLR7-induced IL23 production inmyeloid dendritic cells(DC) and dampens the IL17 production in TCRgd T cells (8).VISTA expressed on myeloid antigen-presenting cells (APC)engages an unidentified inhibitory receptor on CD4þ and CD8þ

T cells and suppresses their proliferation and cytokine produc-tion (3). VISTA expressed on CD4þ T cells also limits T-cellactivation in a T cell–intrinsic manner (9).

VISTA knockout (KO, Vsir�/�) mice demonstrate the loss ofT cell–mediated peripheral tolerance and develop inflammatoryphenotypes in multiple organs (10). When bred onto an autoim-mune-prone background, VISTA deficiency accelerates diseasedevelopment in the experimental autoimmune encephalomyelitismodelofmultiple sclerosis and the sle1.sle3modelof lupus (10, 11).

The critical role of VISTA in regulating antitumor immunityhas been demonstrated by genetic deletion of VISTA gene(Vsir) or treatment with a VISTA-blocking mAb (4, 10, 12, 13).Blocking VISTA deters tumor growth in multiple preclinical

1Department of Microbiology and Immunology, Medical College of Wisconsin,Milwaukee,Wisconsin. 2Department of Translational Hematology andOncologyResearch, Cleveland Clinic Foundation, Cleveland, Ohio. 3Blood Research Insti-tute, Milwaukee, Wisconsin. 4Department of Immunology, Children's Hospital ofChongqing Medical University, Yuzhong District, Chongqing, P.R. China. 5Ros-well Park Cancer Institute, Buffalo, New York. 6Department of Medicine, MedicalCollege ofWisconsin, Milwaukee,Wisconsin. 7Department of Pediatrics, MedicalCollege of Wisconsin, Milwaukee, Wisconsin.

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

Current address for Y. Yuan: Shanghai University of Traditional Chinese Med-icine, College of Pharmacy, Shanghai, P.R. China; and current address for K.Rajasekaran: Genentech, Inc., South San Francisco, California.

Corresponding Author: Li Wang, Cleveland Clinic Foundation, NE5-217, LernerResearch Building, 2111 East 96th Street, Cleveland, OH 55226. Phone: 216-973-5628; Fax: 216-636-2498; E-mail: [email protected]

Cancer Immunol Res 2019;7:1497–510

doi: 10.1158/2326-6066.CIR-18-0489

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models (10, 12), and use of a VISTA-blocking mAb furthersynergizes with either the PD-L1–blocking mAb or a Toll-likereceptor (TLR) agonistic peptide vaccine and CD40-specificagonistic mAb to optimally control tumor growth and achievelong-term survival (12, 13). The mechanism of synergy has beenattributed to the role of VISTA in directly inhibiting TCR signal-ing and T-cell activation (13). Whether or not VISTA regulates theactivation of myeloid cells that indirectly control tumor-specificT-cell responses has not been elucidated.

This study uncovered a function of VISTA in regulating the TLR/TRAF6-mediated signaling axis and the effector function in mye-loid cells. Blocking VISTA together with a TLR-agonistic vaccineabolished the suppressive functions of myeloid-derived suppres-sor cells (MDSC) and tumorigenic DCs and augmented theproduction of proinflammatory cytokines. These effects collec-tively led to a T cell–permissive tumor microenvironment (TME)that facilitated tumor rejection. The reprogramming of tumor-associated myeloid cells contributes to the protective antitumorimmunity following VISTA inhibition.

Materials and MethodsMice

C57BL/6 (wild-type, WT) mice were purchased from TheCharles River Laboratories. Vsir�/� mice on a fully backcrossedC57BL/6 backgroundwere obtained fromMutantMouse Region-al Resource Centers (www.mmrrc.org; stock # 031656-UCD;refs. 10, 13). Myd88�/� mice were purchased from The JacksonLaboratory.OT-I CD8þ TCR transgenicmicewere purchased fromJax (stock # 003831). Animals were maintained in a specificpathogen-free facility at the Medical College of Wisconsin,Milwaukee, WI. All animal protocols were approved by theinstitutional animal care and use committee of the MedicalCollege of Wisconsin. All methods were performed in accordancewith the relevant guidelines and regulations.

Cell linesB16-BL6 murine melanoma cells were cultured in RPMI-1640

supplemented with 10% FBS, 0.1% b-mercaptoethanol, and pen-icillin (100 U/mL)/streptomycin (100 mg/mL). B16-BL6 (femaleorigin) was originally obtained from I. Fidler (The Universityof Texas MD Anderson Cancer Center, Houston, TX) and wereverified by in vivo growth in syngeneic mice and their expression ofmelanoma antigens TRP1, TRP2, and gp100 but have not beenauthenticated further by other methods. Murine thymoma EG7(female origin), human THP-1 (male origin) monocytes, andhuman HEK293T cells were authenticated and obtained fromATCC and were cultured in the same RPMI-1640 media as above.To generate a VISTA-overexpressing THP-1 cell line, THP-1 cellswere transduced with lentivirus carrying human VISTA gene thatwas cloned into a lentiviral vector pFLRcmv-FH-puro. Transducedcells were stained with VISTA-specific mAb and FACS sorted tohomogeneity. Lentivirus carrying the empty vector was used togenerate the control THP-1 cell line. All cell lines were used at fewerthan five passages and after short-term (less than 1 week) culturetime.Cell lineswere tested forMycoplasma contaminationevery 5 to6 months, using MycoAlert Mycoplasma Detection Kit (Lonza).

Examination of TLR-mediated signalingWT or Vsir–/–mice (two to three of each genotype) were treated

with 3 mL 3% thioglycollate broth (cat. # DF0430-17-1, Thermo

Fisher Scientific). Peritoneal macrophages were harvested fromperitoneal lavage on day þ4 following thioglycollate injection.Cells were rested in RPMI media supplemented with 1% FBS, 2mmol/L L-glutamine, and 50 mmol/L b-mercaptoethanol for1 hour before stimulation with TLR agonists including LPS(1 mg/mL), CpG (1mg/mL; OND1826), Poly (I:C)(25 mg/mL),Pam3cys4 (10mg/mL), andR848 (5mg/mL). All TLRagonistswerepurchased from InvivoGen. mRNA samples were collected after3 hours using the RNeasy Mini Kit (QIAGEN) following themanufacturer's instructions. Expression of genes Il12p35, Il12p40,and Il6 was examined by real-time qRT-PCR as described below.Culture supernatants were harvested after 5 hours and examinedby ELISA to assess the level of cytokines IL6, IL12p40, GM-CSF,IFNg , MIP-1a, and MIP-1b as described below. To determine theeffect of TNFa,macrophageswere stimulatedwithCpG(1mg/mL)in the presence of a TNF receptor–specific blocking mAb (Bio XCell) or isotype control IgG (25 mg/mL) for 6 hours before culturesupernatants were harvested and examined by ELISA.

Formeasuring TLR-mediated signaling, macrophages or THP-1cells were stimulated with Pam3 or CpG, respectively, for 0, 15,and 30 minutes. When indicated, cells were pretreated withMG132 (10 mg/mL; Sigma-Aldrich) or DMSO vehicle control for30 minutes before TLR stimulation. Cells were lysed in RIPAbuffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1.0% NP-40,0.5% deoxycholic acid, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/LEGTA, 5 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 10mmol/L b-glycerophosphate) supplemented with 1� Halt Pro-tease and Phosphatase Inhibitor Cocktail (cat. # 78442, ThermoFisher Scientific) at indicated time points. Phosphorylated andtotal levels of proteins including Jnk1/2, Erk1/2, p38, IKB, andIKKa/b were examined by Western blotting as described below.

For denaturing immunoprecipitation (IP) of TRAF6, WT orVsir–/– macrophages were lysed in lysis buffer (1% NP-40, 50mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA)containing 1% SDS and denatured by heating for 5 minutes.Lysates (200 mg) were diluted with SDS-free lysis buffer until theconcentration of SDS reached 0.1%. TRAF6 was immunopreci-pitated using amousemAb (4mg for 200mg lysates; sc-8409) fromSanta Cruz Biotechnology and 20 mL protein G agarose beads(MilliporeSigma). Bound proteins were eluted using 1� SDSsample buffer and analyzed by Western blotting as describedbelow. For HEK293T cells that were transfected with HA-taggedmutant ubiquitin and VISTA-expressing or control vectors, cellswere stimulated with R848 (10 mg/mL) for 15 minutes beforelysed in the denaturing IP buffer, and TRAF6 was immunopreci-pitated as described above.

Electrophoretic mobility shift assays (EMSA) were used tomeasure the DNA binding activity of the transcription factorsAP-1 and NF-kB, using radiolabeled DNA probes as previouslydescribed (14). Briefly,WTorVsir–/–macrophages (2� 106 to 3�106 cells)were lysed in 50mLbuffer containing 20mmol/LHEPESpH 7.9, 350 mmol/L NaCl, 1 mmol/L MgCl2, 0.5 mmol/L EDTA,0.5mmol/LDTT, 20% glycerol, and 1%NP-40. Cell lysates (3 mL)were incubated with [32P]-labeled NF-kB probe (cat. # SC-2505)or AP-1 probe (cat. # SC-2501; 25,000 cpm per reaction; SantaCruz Biotechnology) for 15 minutes at room temperature andthen resolved on a 4% polyacrylamide gel.

Real-time quantitative RT-PCRSingle-cell suspensions of tumor-draining lymph nodes (LN)

were obtained by passing cells through 70-mm cell strainers. For

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peritoneal macrophages, cells (1–2 million) were stimulated withCpG (1 mg/mL) or R848 (5 mg/mL) for 3 hours before RNAisolation. Total RNA of LN cells or macrophages was preparedusing the RNeasy Kit (QIAGEN). Onemicrogram of total RNAwasreverse transcribed in 50 mL of TaqMan reverse transcriptionreagents (Applied Biosystems) using randomhexamer primers andaccording to themanufacturer's instructions. Ten nanogram cDNAand 1mmol/L primerswere used in a 20mL PCR reactionwith SYBRGreenSupermix (Bio-Rad). PCR reactionwas performed in theBio-Rad iQ5 Real-Time PCR System (Bio-Rad). Each sample has trip-licate replication, and the 2–DDCT method was used to determinegene expression. Expression of the target genes was normalized to18s anddisplayedas fold change relative to theWT sample. Primerswere purchased from Integrated DNA Technologies.

Primer sequences are described as follows: Il23-p19 (forward:CCAGCAGCTCTCTCGGAATC; reverse: TCATATGTCCCGCTGGT-GC); IL12 p40 (forward: ACAGCACCAGCT TCT TCA TCA; reverseTCT TCA AAG GCT TCA TCT GCA A); Ifnb (forward: CAGCCC-TCTCCATCAACTATAAG; reverse: TCTCCGTCATCTCCATAGGG);Il6 (forward: GCAGAAAAAGGCAAAGAATC; reverse: CTACATTT-GCCGAAGAGC); Tnfa (forward: AGGCAGTCAGATCATCTTC;reverse: TTATCTCTCAGCTCCACG); and 18s RNA (forward: AACC-CGTTGAACCCCATT; reverse: CCATCCAATCGGTAGTAGCG).

Western blottingForWestern blotting, antibodies specific for K63-linked and K48-

linked polyubiquitin chains, p-Erk1/2 (Thr202/Tyr204), Erk1/2,p-Jnk1/2 (Thr183/Tyr185), Jnk1/2, p-p38, p38, p-IKKa/b, IKKa/b, p-NF-kBp65, NF-kBp65, IkB, TRAF6, pTAK1 (Thr184/187), andTAK1 were purchased from Cell Signaling Technology. Antibodiesspecific for actin were purchased from Santa Cruz Biotechnology.VISTA-blockingmAbwas as described previously (12).HA-taggedmutant ubiquitin pRK5-HA-Ubiquitin-K63 and pRK5-HA-Ubiquitin-K48 were a gift from Ted Dawson (Addgene plasmid#17606 and #17065; ref. 15). All primary antibodies wereused at 1:1,000 dilution. HRP-conjugated secondary antibo-dies (Jackson ImmunoResearch Laboratories) were used at1:5,000 dilution.

THP-1 cells or peritoneal macrophages (5 � 106 to 10 � 106)stimulated with relevant TLR agonists were lysed in RIPA buffer(50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1.0% NP-40, 0.5%deoxycholic acid, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/L EGTA,5 mmol/L sodium pyrophosphate, 50 mmol/L NaF, and 10mmol/L b-glycerophosphate) supplemented with Halt Proteaseand Phosphatase Inhibitor Cocktail (100�; Thermo FisherScientific) at indicated time points. Total lysates were cleared byspinning at 12,000 rpm for 10 minutes. Lysates (20 mg) weresubjected to SDS-PAGE and Western blotting. For detectingTRAF6 in WT or Vsir–/– macrophages and HEK293T cells, 50%of the eluates from the denaturing IP (described above) were usedfor SDS-PAGE and Western blotting.

All primary antibodies were used at 1:1,000 dilution. Appro-priate secondary antibodies were used at 1:5,000 dilution. Pro-teinswere detected usingClarityWestern ECL Substrate (Bio-Rad)and X-ray films. Results were scanned using a digital scanner at600 dpi resolution and quantified using ImageJ software.

ELISAsFor ELISA analyses, culture supernatants from peritoneal

macrophages were diluted 5-fold, homogenized tumor tissueswere diluted 5-fold, plasma samples were diluted 5-fold, and

culture supernatants fromMDSC suppression assays were diluted50-fold. All dilutions were using PBS with 2% BSA as diluent.ELISA kits for detecting murine IL12p40, IL12p70, IL23p19, IL6,TNFa, IFNb, IL27, GM-CSF, MIP-1a, MIP-1b, CCL4, CCL5,CXCL9, and CXCL10 were purchased from BioLegend. OD450

values were read using Typhoon 9400.

Murine tumor models, vaccine treatments, and examination ofcytokines in tumor tissues

EG7 (150,000) or B16-BL6 (30,000) tumor cells were inocu-lated intradermally on the rightflanksof female andmaleC57BL6mice of 7 to 9 weeks of age. A similar number of female andmalemicewere pooled and allocated for each experimental group(n ¼ 10). The peptide vaccine mixture consists of CpG(ODN1826, 50 mg), R848 (100 mg), melanocyte antigen peptideTRP1 (106–130; 50 mg), and a mutated TRP2 peptide DeltaV-TRP2 (180–188; 50 mg) dissolved in PBS (16). All peptides weresynthesized byAtlantic Peptides. The vaccinemixturewas injectedsubcutaneously on dayþ3 following tumor inoculation. Tumor-bearing mice were treated subcutaneously with VISTA-specificmAbs or hamster control IgG (200mg per injection for both) every2 to 3 days until the analytical endpoint or for 5 weeks if miceremained tumor free. Tumor size was measured with a caliperevery 2 to 3 days.

For measuring cytokines within tumor tissues, mice bearingtumors 5 to 6 mm in diameter were treated with CpG (20 mg),R848 (50 mg), and VISTA-specificmAb or control IgG as describedabove. Tumor tissues were harvested after 3 hours and homog-enized using the QIAGEN tissue homogenizer in buffer contain-ing 1� PBS, 0.1% Tween 20, and 1� Halt protease inhibitorcocktail (Thermo Fisher Scientific). The homogenates werecleared by centrifugation at 10,000� g for 20minutes. Cytokinesand chemokines in the supernatant were detected by ELISA asdescribed above. For detecting cytokines in blood, heparinizedblood samples were harvested via cardiac puncture and centri-fuged at 2,500 rpm for 10minutes. Plasmawas collected and alsoexamined by ELISA.

Examination of tumor-associated myeloid cellsThirty to 40 B16-BL6 tumors (7–8 mm in diameter) were har-

vested from untreated tumor-bearing C57BL6 WT mice. Tumorswere pooled, cut into smaller pieces, and digested for 20minutes at37�C in RPMI buffer containing Liberase TL (150 mg/mL) andDNase I (120 mg/mL; Sigma-Aldrich). Single-cell suspensions weregenerated by passing cells through 70-mm cell strainers. Myeloidcells, including polymorphonuclear MDSCs (PMN-MDSC;CD11bþCD11C�LY6Cint LY6Gþ), monocytic MDSCs (M-MDSC; CD11bþCD11C�LY6ChiLY6G�), myeloid conventionalDCs (cDC; CD11bþCD11CþMHCIIþLY6C�F4/80�CD103�),inflammatory DCs (Inf-DC; CD11bþCD11CþMHCIIþLY6Cþ

LY6G�FceRIþ), and CD103þ DCs (CD103þCD11CþMHCIIþ F4/80�), were purified from dissociated tumor tissues by FACS usingBD FACSAria II, with 99% purity.

To test TLR-mediated cytokine production, purified myeloidcell subsets (10,000 cells) were stimulatedwith TLR9 agonist CpG(1 mg/mL) in the presence of VISTA-blocking mAb (20 mg/mL) orisotype control IgG (20mg/mL) as indicated. Culture supernatantswere harvested after 24 hours. Secreted cytokines (IL12p40, IL6,and TNFa) were quantified by ELISA as described above.

For testing the T cell–suppressive function of MDSCs, sortedmyeloid cDCs and Inf-DCs (10,000)were coculturedwith 30,000

Immune-Checkpoint Protein VISTA Regulates Myeloid Cells

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whole splenocytes fromOT-I CD8þ TCR transgenic mice togetherwith ovalbumin peptide 257–264 (1 mg/mL; GenScript), CpG(1mg/mL), andVISTA-blockingmAbor control IgG (20mg/mL) asindicated. Culture supernatants were harvested after 48 hours,and secreted IFNg was quantified by ELISA. For testing the T cell–stimulatory function of CD103þ DCs, sorted CD103þ DCs(10,000) were cocultured with 30,000 purifiedOT-I CD8þ T cells,ovalbumin peptide 257–264 (1 mg/mL), CpG (1 mg/mL), andVISTA-blocking mAb or control IgG (20 mg/mL) as indicated.Culture supernatants were harvested after 48 hours, and secretedIFNg was quantified by ELISA.

Flow cytometry and data analysisFor flow cytometry, antibodies specific for CD4 (GK1.5),

CD8 (53-6.7), CD16/CD32, and IFNg (XMG1.2) were pur-chased from BioLegend. Single-cell suspension tumor tissuesand spleens were stained with lineage-specific antibodies (CD4,CD8, CD11b, CD11c, Ly6C, Ly6G, MHCII, F4/80, and FceRI) toidentify CD4þ and CD8þ T cells and myeloid cell populations.To detect intracellular cytokines in T cells, single-cell suspen-sions of tumor tissues (2–3 million) were stimulated for5 hours in RPMI medium containing phorbol 12-myristate13-acetate (PMA; 50 mg/mL), ionomycin (1 mg/mL), 10% FBS,2 mmol/L L-glutamine, 50 mmol/L 2-mercaptoethanol, 1%penicillin–streptavidin, 1� monensin, and 1� Brefeldin A(BioLegend). Cells were stained with the LIVE/DEAD FixableViolet Dead Cell Stain Kit first (Thermo Fisher Scientific), thenstained with T-cell–lineage markers in the presence of pan-CD16/CD32 blocking antibody before fixation with 1% para-formaldehyde, and then permeabilized with 0.5% saponin andstained for intracellular IFNg . Cells were analyzed on an LSR IIflow cytometer (BD Biosciences). Data were analyzed withFlowJo version 9.9.6 analysis software (Tree Star).

Graphs and statistical analysisAll graphs and statistical analysis were generated using Prism 7

(GraphPad Software). Unpaired two-tailed t tests were used forcomparingWT andVsir�/� cells ormice, or comparing treatmentsusing VISTA-blocking mAb or control IgG. Survival differences oftumor-bearingmicewere assessedusingKaplan–Meier curves andanalyzed by log-rank testing. A P value less than 0.05 wasconsidered as statistically significant. �, P < 0.05; ��, P < 0.025;��, P < 0.005; ��, P < 0.0001.

ResultsAbsence of VISTA augments TLR-mediated proinflammatorycytokine production

TLR stimulation initiates MyD88- or TRIF-dependent signal-ing pathways to induce the expression of proinflammatorycytokines, chemokines, and type I interferons (17). Owing tothe high expression of multiple TLRs on peritoneal macro-phages, we chose this cellular system as a model to dissect therole of VISTA in regulating TLR-mediated signaling pathways.Peritoneal macrophages were isolated from WT and Vsir�/�

mice following thioglycollate treatment and stimulated ex vivowith TLR agonists CpG (TLR9) and R848 (TLR7; Fig. 1A–C).The expression of cytokines IL12 and IL6 was examined by real-time quantitative RT-PCR (qRT-PCR) and ELISA. CpG andR848 significantly upregulated gene expression and proteinproduction of IL12 and IL6 in Vsir�/� macrophages (Fig. 1A

and B). The expression of multiple cytokines and chemokines,including GM-CSF, IFNg , MIP-1a, and MIP-1b, was also sig-nificantly increased in Vsir�/� macrophages (Fig. 1C). In addi-tion to CpG and R848, Vsir�/� macrophages were hyperrespon-sive toward additional TLR agonists, including Pam3Cys4(TLR2), LPS (TLR4), and poly (I:C) (TLR3; Fig. 1D).

Although the results above indicated that VISTA directlyregulated TLR-mediated signaling, we wanted to assess ifcell-extrinsic factors such as TNFa or additional soluble factorsmay have contributed to the hyperresponses of Vsir�/� cells inan autocrine manner. We first tested a TNFR-specific neutral-izing mAb and found minimal effects (SupplementaryFig. S1A). Next, we harvested supernatants from CpG-treatedWT and Vsir�/� cell cultures (designated as WT and KO sups,respectively) and tested their effects on freshly isolated perito-neal macrophages (Supplementary Fig. S1B). The residual CpGin the supernatants directly stimulated cells, and adding CpG tothe culture further boosted cytokine production. Under alltested conditions, WT and KO supernatants stimulated cellsto similar levels. These data support the conclusion that thehyperresponsiveness in Vsir�/� macrophages was due to cell-intrinsic TLR signaling rather than secondary responses tosoluble factors.

VISTA inhibits the activation of MAP kinases and NF-kBsignaling cascades

Transcription factors AP-1 and NF-kB are critical for the geneexpression of many inflammatory cytokines and chemo-kines (18). Previous studies have established the role of MAPkinases (MAPK Erk1/2, Jnk1/2, and p38) in the activation ofAP-1 (19, 20). The activation of NF-kB requires the phosphory-lation of IkB kinase IKKa/b and degradation of the inhibitorIkB (21). We, therefore, examined the activation of MAPKs/AP-1and IKK/NF-kB signaling cascades following TLR stimulation.Cell lysates from WT and Vsir�/� macrophages were stimulatedwith CpG or R848. Phosphorylation of MAPKs and IKKa/b , aswell as total IkB, was examined by Western blotting. Both CpG(Fig. 2A) and R848 (Fig. 2B) induced greater phosphorylation ofErk1/2, Jnk1/2, and p38 in Vsir�/� macrophages. Phosphoryla-tion of IKKa/b and degradation of IkB were enhanced in Vsir�/�

macrophages following CpG stimulation but were not signifi-cantly increased following R848 stimulation.

Next, we carried out EMSAs to measure the DNA bindingactivity of AP-1 andNF-kB. As shown in Fig. 2C, CpG stimulationinduced higher activation of AP-1 and NF-kB in Vsir�/� macro-phages than in WT cells. These results indicate that VISTA inhib-ited TLR-mediated activation of MAPKs/AP-1 and IKK/NF-kBsignaling cascades.

Previous studies show thatVsir�/�mice develop systemic tissueinflammation (10). It is possible that Vsir�/� macrophages werepartially activated due to exposure to the existing tissue inflam-mation. To rule out this possibility and validate the cell-intrinsicrole of VISTA, we examined the effects of VISTA overexpression inTHP-1 human monocyte cells. VISTAþ and control THP-1 cellswere stimulated with TLR2 agonist Pam3Cys4 (10 mg/mL) for theindicated time, and total cell lysates were harvested and analyzedby Western blotting (Fig. 3). VISTA expression in THP-1 cellssuppressed the phosphorylation of MAPKs (Jnk1/2, Erk1/2, andp38; Fig. 3A and C), the phosphorylation of IKKa/b and NF-kBp65, and the degradation of IkB (Fig. 3B and C) following TLR2stimulation.

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VISTA controls the activation of MAPKs and NF-kB byregulating TRAF6

TLRsutilize eitherMyD88-dependent (TLR2/4/5/7/8/9) or TRIF-dependent (TLR3/4) signaling pathways (22). Following TLR2/4/5/7/8/9 engagement, the MyD88/IRAK1/4 complex recruits andactivates the RING-domain E3 ubiquitin ligase TRAF6, which isrequired for the activation of MAPKs and NF-kB (22). TRAF6 alsomediates the activation of NF-kB downstream of TLR3 by bindingto TRIF (23). Because Vsir�/� macrophages are hyperresponsive toall TLR agonists (Figs. 1–3), we hypothesized that VISTA targeted ashared signaling adaptor such as TRAF6 (24).Wefirst examined thetotal protein level of TRAF6 and observed increased TRAF6 expres-

sion in freshly isolated Vsir�/� macrophages compared with WTcells (Fig. 4A). Because K48-linked polyubiquitination could targetproteins to proteasomes for degradation and K63-linked polyubi-quitination activates TRAF6 (25), we analyzed the ubiquitinationstatus of TRAF6 after immunoprecipitating TRAF6 under denatur-ing conditions (Fig. 4B).Western blots showed that VISTA deletionresulted in lower K48-linked polyubiquitination and higher K63-linked polyubiquitination of TRAF6 following CpG stimulation(Fig. 4B).

Next, we utilized HA-tagged mutant ubiquitin containing onlyK48 (HA-Ub-K48) or K63 (HA-Ub-K63) to determine the effect oftransient VISTA expression on TRAF6 ubiquitination. HEK293T

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200

400

600

MIP

-1β

(pg/

mL)

* P = 0.09

Figure 1.

Genetic deletion of VISTA augments the expression of inflammatory cytokines in macrophages in response to TLR stimulation. A–C,WT and Vsir�/� peritonealmacrophages were isolated and pooled from 3mice of each genotype. Cells were stimulated ex vivowith PBS, R848 (5 mg/mL; TLR7/8), and CpG (1 mg/mL;TLR9). RNAwas extracted and supernatants were collected after 3 and 6 hours of culture, respectively. A,Gene expression of Il12p35, Il12p40, and Il6wasexamined by real-time quantitative RT-PCR (qRT-PCR). B, Secreted IL12p40 and IL6 were examined by ELISA. C, Additional cytokines (GM-CSF and IFNg) andchemokines (MIP-1a and MIP-1b) were also examined by ELISA. D,WT and Vsir�/�macrophages were stimulated with TLR agonists LPS (1 mg/mL; TLR4),Pam3Cys4 (Pam3, 10mg/mL; TLR2), and poly (I:C)(25 mg/mL; TLR3) for 6 hours. Secreted IL6 and IL12p40 were quantified by ELISA. Statistical significance wasdetermined by unpaired two-tailed t test. Error bars, SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001. Shown are representative results from at leastthree independent repeats.






cells were cotransfected with mutant ubiquitin together with aVISTA-expressing vector or empty control vector and analyzed at24 hours after transfection (Fig. 4C). Transient VISTA expression

significantly augmented K48-linked polyubiquitination of TRAF6and simultaneously decreased K63-linked polyubiquitinationlevels (Fig. 4C).

A

B

p-Jnk1/2

p-Erk1/2

p-p38

p-IKKα/β

Jnk1/2

Erk1/2

p38

IkB

IKKα/β

WT Vsir -/-

R848

0 5 15 30 0 5 15 30 (min)

WT Vsir-/-

CpG

0 5 15 30 0 5 15 30 (min)

p-Jnk1/2

p-Erk1/2

p-p38

p-IKKα/β

Jnk1/2

Erk1/2

p38IkB

IKKα/β

WTVsir−/−

WTVsir−/−

0 5 15 300

5

10

Rat

io(p

Jnk1

/2/to

tal J

nk1/

2)

(min) 0 5 15 300

10

20

Rat

io(p

Erk

1/2/

tota

l Erk

1/2)

(min) 0 5 15 300

10

20

Rat

io(p

-p38

/tota

l p38

)

(min)

0 5 15 300.0

2.5

5.0

Rat

io(p

IKKα

/β/to

tal I

KK

α/β)

(min) 0 5 15 300

20

40

60

(Blo

t int

ensi

ty ×

1,0

00)

(min)

0 5 15 300

8

16

24

Rat

io(p

Jnk1

/2/to

tal J

nk1/

2)

(min) 0 5 15 300

5

10

Rat

io(p

Erk

1/2/

tota

l Erk

1/2)

(min) 0 5 15 300

2

4

6

Rat

io(p

-p38

/tota

l p38

)

(min)

0 5 15 300.0

2.5

5.0

Rat

io(p

IKKα

/β/to

tal I

KK

α/β)

(min) 0 5 15 300

20

40

60

Tota

lIκB

Tota

lIκB

(blo

t int

ensi

ty ×

1,0

00)

(min)

0 5 15 30 0 5 15 30 (min)

WT

AP-1

NF-κB

Actin

Vsir −/−C

WT

0 5 15 300.0

0.5

1.0

1.5

Rat

io(A

P-1

/act

in)

(min)

0 5 15 300.0

0.5

1.0

1.5

Rat

io(N

F-κB

/act

in)

(min)

Vsir −/−

Figure 2.

Genetic deletion of VISTA enhances the activation of MAPKs/AP-1 and IKKa/b/NF-kB pathways following TLR7 and TLR9 stimulation. WT and Vsir�/� peritonealmacrophages pooled from 2 to 3 mice of each genotype were stimulated with CpG (1 mg/mL; A) or R848 (10 mg/mL; B) for the indicated amount of time beforetotal cell lysates were assessed. Phosphorylated (p-) and total Jnk1/2, Erk1/2, p38, and IKKa/b, and total IkB were examined byWestern blotting and quantifiedusing ImageJ software. Ratios of phosphorylated vs. total protein were shown. C,WT and Vsir�/� peritoneal macrophages were stimulated with CpG (1 mg/mL)as indicated. DNA binding activity of AP-1 and NF-kB in total cell lysates was measured using an EMSA and visualized by autoradiography. Actin in the totallysates was determined byWestern blotting. The amount of bound probes was normalized to actin in the total lysates. Shown are representative results from atleast three independent repeats. min, minutes.

Xu et al.





The TAK1/TAB1/TAB2 complex is recruited to the MyD88/IRAK/TRAF6 complex by binding to the K63-polyubiquitinchains of TRAF6 and mediates the activation of the Jnk1/2/AP-1 and IKKa/b/NF-kB signaling pathways (25). Consistentwith the higher protein level and K63 polyubiquitination ofTRAF6, phosphorylation of TAK1 at Thr184/187 in Vsir�/�

macrophages was significantly enhanced compared withWT cells following CpG stimulation (Fig. 4D). Reducedprotein expression of TRAF6 and diminished TAK1 activationwere also seen in VISTA-expressing THP-1 cells comparedwith control THP-1 cells following Pam3Cys4 treatment(Fig. 4E and F).

These results prompted us to test whether proteasomal inhi-bition reversed the inhibitory effects of VISTA. As shownin Fig. 5A, pretreatment with proteasome inhibitor MG132 effec-tively reversed the inhibitory effects of VISTA in THP-1 cells byrescuing the phosphorylation of Jnk1/2, Erk1/2, p38, and IKKa/b.MG132 pretreatment also enhanced TLR signaling in WT but notVISTA-deficient macrophages (Fig. 5B). These results, therefore,support the conclusion that VISTA downregulates TLR-mediatedsignaling by promoting TRAF6 protein degradation and restrict-ing the activation of MAPKs/AP-1 and IKKa/b/NF-kB signalingpathways.

Blocking VISTA augments the TLR/MyD88-mediatedproinflammatory responses

We have previously shown that treatment with a VISTA-blocking mAb deters tumor growth in preclinical models (12).Our current study indicated that the proinflammatory responsesinduced by VISTA blockade promote the development of antitu-mor immunity. Supporting this hypothesis, treatment with theVISTA-blocking mAb led to a significant accumulation ofIL12p40, IL12p70, TNFa, and IL27 in the serum of mice(Fig. 6A). This cytokine response was absent in tumor-bearingMyD88 KO hosts (Fig. 6B). Consistent with the lack of cytokineinduction, the therapeutic efficacy of theVISTA-blockingmAbwasdiminished inMyD88 KO hosts (Fig. 6C). These results indicatedthat blocking VISTA elicited an endogenous TLR/MyD88-mediated proinflammatory response that contributed to thedevelopment of antitumor immune responses.

TLR agonists are promising vaccine adjuvants for cancer ther-apy (26, 27). We hypothesized that VISTA inhibitors wouldsynergize with TLR-agonistic vaccines owing to an augmentedproinflammatory response. We tested this hypothesis in theB16-BL6 melanoma model (Fig. 6C). Mice bearing 3-day estab-lished B16-BL6 tumors were treated with either VISTA-blockingmAb, or a vaccine containing TLR7/8/9 agonists (CpG and R848)

THP-1Ctrl

0 15 30 0 15 30 (min)

p-Jnk1/2

p-Erk1/2

p-p38

p-IKKa/bJnk1/2

Erk1/2

p38

IkB

IKKa/b

THP-1VISTA

pIkB

NF-κBp65

pNF-κBp65

0 15 30 0 15 30 (min)

A

C

150 300

1

2

Rat

io(p

Jnk1

/2/to

tal J

nk1/

2)

(min) 150 300.0

0.5

1.0

Rat

io(p

Erk

1/2/

tota

l Erk

1/2)

(min) 150 300

1

2

Rat

io(p

-p38

/tota

l p38

)

(min)

150 300

4

8

Rat

io(p

IKKα

/β/to

tal I

KK

α/β)

(min)150 300

5

10

15

Rat

io(p

-IκB

/tota

l IκB

)

(min) 150 300.0

0.5

1.0

1.5

Rat

io(p

-p65

/tota

l p65

)(min)

B THP-1Ctrl THP-1VISTA

THP-1Ctrl

THP-1VISTA

Figure 3.

VISTA overexpression in humanmonocyte THP-1 cells inhibits theactivation of MAPKs and NF-kBfollowing TLR2 stimulation. Humanmonocyte THP-1 cells overexpressingVISTA (THP-1VISTA) and control cells(THP-1Ctrl) were stimulated withPam3Cys4 (10 mg/mL) for theindicated amount of time. A and B,Total cell lysates were generated andexamined byWestern blotting.Phosphorylated (p-) Jnk1/2, Erk1/2,p38, IKKa/b, and NF-kBp65, andtotal IkBwere quantified by ImageJsoftware and shown in C. Shown arerepresentative results from at leastthree independent repeats. min,minutes.






A B

E

D

0 5 15 30 (min)0 5 15 30

TAK1

pTAK1

WT Vsir −/− macrophages

WT

CpG − + − + (15 min)

K63-pUb

K48-pUb

TRAF6

Vsir −/−

0 15 30 (min)0 15 30

TAK1

pTAK1 (Thr184/187)

F THP-1VISTA THP-1Ctrl

0 15 30 (min)0 15 30

TRAF6

Actin

THP-1VISTA THP-1Ctrl

TRAF6

Actin

0 15 30 0 15 30

WT Vsir −/−

0 15 30 0 15 30

WT Vsir −/−

MG132DMSO

0 15 300.0

0.4

0.8

Rat

io(T

RA

F6/a

ctin

)

(min) 0 15 300.00

0.75

1.50

Rat

io(T

RA

F6/a

ctin

)

WT

(min)

+MG132+DMSO

Vsir −/−

Ctrl

R848 − + − + (15 min)

VISTA

WB:Anti-HA

HA-Ub-K48

TRAF6

Ctrl VISTA

TRAF6

R848 − + − + (15 min)

HA-Ub-K63

WB:Anti-HA

C

0 5 15 300

40

80

Rat

iopT

AK

1/to

talT

AK

1 WTVsir-/-

(min)

0 15 300.0

0.4

0.8

Rat

iopT

AK

1/to

talT

AK

1

(min)0 15 300.0

0.5

1.0

Rat

io(T

RA

F6/a

ctin

)

THP-1Ctrl

THP-1VISTATHP-1Ctrl

THP-1VISTA

Figure 4.

VISTAmodulates the K48-linked and K63-linked polyubiquitination of TRAF6 and inhibits the activation of TAK1 following TLR stimulation. A,WT and Vsir�/�

peritoneal macrophages (pooled from 2–3 mice of each genotype) were pretreated with MG132 (10 mg/mL) or vehicle control DMSO for 30 minutes before beingstimulated with CpG (1 mg/mL) for 0, 15, and 30minutes. TRAF6 and actin in total cell lysates were determined byWestern blotting. B, To examine theubiquitination status, WT and Vsir�/� peritoneal macrophages (pooled from 2–3 mice of each genotype) were stimulated with CpG (1 mg/mL) for 15 minutes.TRAF6 protein was immunoprecipitated from total lysates under denaturing conditions. Both K63-linked and K48-linked polyubiquitination (pUb) wereexamined byWestern blotting using antibodies specific for K48-linked or K63-linked polyubiquitin chains. C, TLR7-expressing HEK293T cells were transfectedwith HA-tagged mutant ubiquitin HA-Ub-K48 or HA-Ub-K63, together with a VISTA-expressing plasmid or vector control (Ctrl) as indicated. Cells werestimulated with R848 at 24 hours after transfection. TRAF6 was immunoprecipitated as in B and examined byWestern blotting (WB) using anti-HA. D,WT andVsir�/� peritoneal macrophages were stimulated by CpG (1 mg/mL) for indicated amount of time. Phosphorylated (p-) and total TAK1 in cell lysates wereexamined byWestern blotting and quantified using ImageJ software. The ratio of pTAK1/TAK1 is shown. E and F, VISTA-expressing THP-1 cells (THP-1VISTA) andcontrol cells (THP-1Ctrl) were stimulated with Pam3Cys4 (10 mg/mL) for indicated amount of time. TRAF6, actin, pTAK1, and total TAK1 in total cell lysates wereexamined byWestern blotting and quantified using ImageJ. Ratios of TRAF6/actin and pTAK1/TAK1 are shown. Shown are representative results from at leastthree independent repeats. min, minutes.

Xu et al.





and peptides derived from melanoma antigens TRP1 and TRP2,or both. Combining VISTA-blocking mAb with the TLR vaccineresulted in tumor-free long-term survival in �50% mice,whereas either monotherapy transiently delayed tumor growthwithout long-lasting effects (Fig. 6C). This stronger therapeuticeffect correlated with significantly increased numbers ofIFNg-expressing tumor-infiltrating CD8þ and CD4þ T cells(Fig. 6D and E).

To dissect the inflammatory TME, we examined the expres-sion of inflammatory mediators within tumor tissues follow-ing CpG/R848 treatment (Fig. 6F and H). The VISTA-blockingmAb significantly enhanced the expression of cytokines(IL12p40, IL12p70, IL23p19, IL6, IFNb, and TNFa) andchemokines (CCL4, CCL5, CXCL9, and CXCL10). To excludethe possibility that this inflammatory response may be due toadditional effects of the antibody (i.e., Fc receptor–mediatedactivation of myeloid cells), we examined WT and Vsir�/�

tumor-bearing mice treated with CpG/R848 (Fig. 6G and I).Consistent with the antibody treatment, Vsir�/� mice pro-duced higher amounts of inflammatory mediators. In addi-tion to tumor tissues, enhanced cytokine production wasdetected in tumor-draining LNs (Supplementary Fig. S2).These results indicated that VISTA blockade augments theproinflammatory responses in the periphery and withinthe TME, which are critical for the development of antitumorT-cell responses.

VISTA regulates the effector functions of tumor-associatedmyeloid cells

Tumor tissues are enriched with immunosuppressiveMDSCs and dysfunctional DCs (28, 29). Importantly, VISTA isexpressed on tumor-associated myeloid cDCs (CD11bþCD11Cþ

MHCIIþLY6C�), Inf-DCs (CD11bþCD11CþMHCIIþLy6Cþ-Ly6G�FceRIþ), PMN-MDSCs (CD11bþCD11C�Ly6CintLy6Gþ),and M-MDSCs (CD11bþCD11C�Ly6CþLy6G�), as well as onCD103þ DCs (Fig. 7A). This result led us to examine how VISTAdirectly regulated the effector function of these myeloid cell typesin tumor-bearing hosts.

We first analyzed the accumulation of MDSCs and DC subsetsin tumor-bearing mice treated with CpG/R848 and VISTA-blocking mAb (Fig. 7B). VISTA inhibition reduced the accumu-lationofM-MDSCs andPMN-MDSCs and concurrently expandedInf-DCs within tumor tissues (Fig. 7B). A similar reduction ofPMN-MDSCs and expansion of Inf-DCs was observed in thespleen, whereas the number of M-MDSCs in the spleen remainedunaltered (Supplementary Fig. S3). The number of CD103þ DCsand myeloid cDCs within tumor tissues and spleen was notsignificantly altered.

Next, we assessed whether VISTA blockade augmented theability of tumor-associated myeloid cells to produce immune-stimulatory cytokines, such as IL12 (30). We purifiedMDSCs andDC subsets from tumor tissues and examined their cytokineexpression following CpG/TLR9 stimulation. Our results showed

A

0 15 30 0 15 30

BMG132

0 15 30 (min)0 15 30

DMSO

pJnk1/2

Jnk1/2

pErk1/2

Erk1/2

p-p38

p38

pIKKα/β

IKKα/β

pJnk1/2

Jnk1/2

pErk1/2

Erk1/2

p-p38

p38

pIKKα/β

IKKα/β

Vsir −/− macrophages

0 15 30 0 15 30

WT

MG132

0 15 30 (min)0 15 30

DMSO

Vsir −/− THP-1VISTATHP-1VISTATHP-1Ctrl THP-1CtrlWT

1.6 1.9 1.4 2.1 3.9 3.0 1.7 3.2 2.6 1.2 2.2 1.9

0.0 0.3 0.4 0.0 0.9 0.5 0.5 1.3 0.9 0.7 1.1 0.8

0.2 0.4 0.2 0.5 1.2 0.6 0.6 0.9 0.8 0.4 0.6 0.6

0.1 0.2 0.2 0.3 1.4 0.7 0.2 0.9 0.3 0.2 0.8 0.4

0.5 1.6 0.8 0.4 0.8 0.4 0.3 0.6 0.4 0.3 1.0 0.8

0.4 1.8 1.4 0.4 0.7 1.2 0.6 0.8 0.6 0.4 0.7 0.5

0.5 1.5 0.9 0.3 0.7 0.4 0.3 0.8 0.7 0.5 1.2 0.9

0.3 0.9 0.3 0.3 0.5 0.3 0.2 0.7 0.3 0.2 1.0 0.3

Figure 5.

The proteasome inhibitor MG132 reverses the inhibitory effect of VISTA on TLR signaling. A, VISTA-expressing THP-1 cells (THP-1VISTA) and control cells (THP-1Ctrl) were pretreated with DMSO solvent control or MG132 (10 mg/mL) for 30minutes prior to stimulation with Pam3Cys4 (10 mg/mL). Phosphorylation (p) ofJnk1/2, Erk1/2, TAK1, and IKKa/bwas examined byWestern blotting. The ratios of phosphorylated proteins to total protein were quantified using ImageJsoftware and are shown numerically under each time point. B,WT and Vsir�/� peritoneal macrophages (pooled from 2–3mice of each genotype) werepretreated with DMSO or MG132 as in A before being stimulated with CpG (1 mg/mL). Total lysates were generated and analyzed as in A. Shown arerepresentative results from three independent repeats. min, minutes.






D

200 40 600

2,000

4,000

Ctrl-Ig(0/9)

Days

Tum

or s

ize

(mm

3 )

0 20 40 600

1,000

2,000

3,000

aVISTA mAb(0/10)

Days

Tum

orsi

ze(m

m3 ) 3

0 20 40 600

1,000

2,000

Vaccine(0/9)

Days

Tum

orsi

ze(m

m)

0 20 40 600

1,000

2,000

Combo(5/10)

Days

Tum

orsi

ze(m

m3 )

C

E

0 30 60 90 1200

50

100

Days

Per

cent

surv

ival Vaccine

Combo

Ctrl-IgαVISTA

* ****

*

F

Ctrl-Ig

VISTA mAb

Ctrl-IgVISTA mAb

0

10

20

30

IFN

γ%

CD

8+TI

Ls

P = 0.01

0

200

400

600

IFN

γC

D8+

TILs

/mill

ion

tum

orce

lls

P = 0.02

0

5

10

15

IFN

γ%

CD

4+TI

Ls

P = 0.008

0

50

100

150

IFN

γC

D4+

TILs

/mill

ion

tum

orce

llsP = 0.02

0

100

200

TNFα

(pg/

mL)

P = 0.025

0

4,000

8,000

IL12

p40

(pg/

mL)

P = 0.027

0

2,000

4,000

IL6

(pg/

mL)

P = 0.037

0

20

40

IFN

β (p

g/m

L)

P = 0.037

0

40

80

IL23

p19

(pg/

mL)

P = 0.033

0

50

100

150

IL12

p70

(pg/

mL)

P = 0.047

0

300

600

900

IL27

(pg/

mL)

P = 0.01

0

125

250

TNFα

(pg/

mL)

P = 0.018

0

1,250

2,500

IL12

p40

(pg/

mL)

P = 0.03

0

125

250

IL6

(pg/

mL

× 10

0)

P = 0.006

0

50

100

150

IL12

p70

(pg/

mL)

P = 0.013

0

50

100

150

IL23

p19

(pg/

mL)

P = 0.013

0

20

40

IFN

β (p

g/m

L)

P = 0.006

WTVsir −/−

0

200

400

600

IL27

(pg/

mL)

P = 0.03G

H I

Ctrl-Ig VISTA mAb WT Vsir −/−

0

20

40

60

80

CC

L5 (p

g/m

L ×

1,00

0) P = 0.027

0

50

100

150

CC

L4 (p

g/m

L ×

1,00

0) P = 0.006

0

500

1,000

1,500

2,000

CX

CL9

(pg/

mL)

P = 0.001

0

100

200

300

CX

CL1

0 (p

g/m

L ×

1,00

0)

P = 0.022

0

20

40

60

CC

L5 (p

g/m

L ×

1,00

0) P = 0.079

0

20

40

60

80

100

CC

L4 (p

g/m

L ×

1,00

0)

P = 0.006

0

500

1,000

1,500

2,000

CX

CL9

(pg/

mL)

P = 0.0008

0

1,000

2,000

3,000

CX

CL1

0 (p

g/m

L)

P = 0.006

B

A

128 16 20 240

1,000

2,000

3,000

Days

Tum

orsi

ze(m

m3 )

WT Ctrl-IgGWT αVISTAMyD88 KO Ctrl-IgGMyD88 KO αVISTA

ns

**

0

2,000

4,000

IL12

p40

(pg/

mL)

P < 0.0001

0

400

800

IL27

(pg/

mL)

P < 0.0001 nsCtrl-IgGVISTA mAbMyD88 KO Ctrl-IgGMyD88 KO VISTA mAb

0

75

150

TNFα

(pg/

mL)

P = 0.002 ns

20

40

60

IL12

p70

(pg/

mL)

P < 0.0001 ns

Figure 6.

TLR/MyD88-mediated inflammation promotes the therapeutic efficacy of VISTA blockade.A,WT or MyD88 KOmice were inoculated with B16-BL6 melanomacells (200,000) on the flank on day 0. On dayþ7, mice bearing established tumors (�5mm in diameter) were treated with VISTA-blocking mAb or isotypecontrol IgG (n¼ 10/group). Three hours later, serumwas harvested and examined by ELISA for cytokines. B,WT and MyD88 KOmice (n¼ 6–7/group) wereinoculated with EG7 thymoma cells (150,000). Beginning on day 2 and repeated every 2 to 3 days, tumor-bearing mice were treated with VISTA-blocking mAb orcontrol IgG. Tumor size was measured by a caliper. Shown are representative results from three independent repeats. C–E,Na€�veWTmice (n¼ 10) wereinoculated with B16-BL6 melanoma cells (30,000) on day 0. On dayþ3, tumor-bearing mice were treated with a peptide vaccine containing CpG, R848, andpeptides (details in Materials and Methods). Mice were also treated with VISTA-blocking mAb or control IgG every 2 to 3 days. C, Tumor size was recorded. In aseparate group of mice (n¼ 10), tumor tissues (�6–8mm in diameter) were harvested. Tumor-infiltrating CD4þ and CD8þ T cells were stimulated with anti-CD3.Percentage and number of IFNg-expressing CD8þ T cells (D) and CD4þ T cells (E) were examined by flow cytometry. F and H, To examine the inflammatory TME,we treated B16-BL6 tumor–bearing mice (n¼ 5–7/group) with the peptide vaccine and VISTA-blocking mAb or control IgG. Tumor tissues were harvested after3 hours and homogenized. Cytokines (IL12p40, IL12p70, IL23p19, IL6, TNFa, and IFNb) and chemokines (CCL4, CCL5, CXCL9, and CXCL10) in the homogenateswere quantified by ELISA. G and I, B16-BL6 tumors were harvested fromWT and Vsir�/�mice (n¼ 5–7/group) following treatment with the peptide vaccine asdescribed above. Cytokines and chemokines in tumor tissue homogenates were examined by ELISA. Ctrl, control. Statistical significance of all results wasdetermined by an unpaired two-tailed t test. Error bars, SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001; ns, not significant. Shown are representativeresults from three independent repeats.

Xu et al.





A

B

C

0

20,000

40,000

PMN-MDSCCpG

********

ns

********

ns

VISTA mAb

PMN-MDSC

D

F G H

0

4,000

8,000

Myd88 M-MDSC

M-MDSCCpG

VISTA mAb

****ns

****ns

0

25,000

50,000

M-MDSC

M-MDSCCpG

VISTA mAb

******

****

0

30,000

60,000

Inf-DC

Inf-DCCpG

VISTA mAb

********

********

0

12,500

25,000

Myeloid cDC

myeloid cDCCpG

VISTA mAb

*****

******

0

5,000

10,000

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)IF

Nγ

(pg/

mL)

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)

CD103+ DC

CpGVISTA mAb

*** ****

E

Isotype controlCD103+ DCsMyeloid cDCsInf-DCsPMN-MDSCM-MDSC

0 102 103 104 1050

20

40

60

80

100

% o

f Max

0

1,500

3,000

IL12

p40

(pg/

mL)

** ***ns

Ctrl-IgGVISTA mAb

Inf-DCPMNMDSC

MMDSC

myeloidcDC

CD103+

DC

* **

Ctrl-IgGVISTA mAb

VISTA mAbCtrl-IgG

0

10

20

30

M-M

DSC

%am

ong

CD

45+

TILs

P = 0.0003

0

1,000

2,000

3,000

4,000

M-M

DS

Cnu

mbe

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r1m

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Figure 7.

VISTA regulates the accumulation and effector functions of tumor-associated MDSCs and DC subsets. A, Tumor tissues were harvested from 5 B16-BL6 tumor–bearing mice and pooled. VISTA expression on tumor-infiltrating MDSCs and DC subsets were examined by flow cytometry. B,Mice bearing established B16-BL6tumors (4–5 mm in diameter) were treated with TLR agonists CpG and R848 together with the VISTA-blocking mAb or isotype control IgG. Numbers of tumor-infiltrating M-MDSCs, PMN-MDSCs, and Inf-DCs were examined by flow cytometry 48 hours after treatment. P values are shown. C, Tumor-associated PMN-MDSCs, M-MDSCs, myeloid cDCs, Inf-DCs, and CD103þ DCs were sorted from pooled B16-BL6 tumor tissues (7–8mm in diameter) harvested from 20 to 30tumor-bearing mice. Each type of purified myeloid cell (10,000 cells each) was stimulated ex vivowith CpG (1 mg/mL) in the presence of VISTA-blockingmAb orcontrol IgG. Culture supernatants were collected after 24 hours, and IL12p40 was quantified by ELISA. M-MDSCs (WT and MyD88 KO;D), PMN-MDSCs (E),Inf-DCs (F), and myeloid cDCs (G) were sorted from B16-BL6 tumor tissues (pooled from 20–30 tumor-bearing mice) and cocultured with whole splenocytesfrom the OT-I transgenic mice in the presence of OVA peptide. CpG and VISTA-blocking mAb or control IgG were added as indicated. Culture supernatants wereharvested after 48 hours, and IFNg was examined by ELISA. H, Tumor-associated CD103þ DCs were sorted from B16-BL6 tumor tissues and incubated withpurified na€�ve OT-I CD8þ T cells in the presence of OVA peptide and VISTA-blocking mAb or control IgG. After 48 hours, secreted IFNg was examined by ELISA.Ctrl, control. Statistical significance of all results was determined by an unpaired two-tailed t test. Error bars represent SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001; ns, not significant. Results from three independent experiments are shown.






that, with the exception of PMN-MDSCs, VISTA blockade signif-icantly augmented the expression of IL12p40 in M-MDSCs, Inf-DCs, myeloid cDCs, and CD103þ DCs (Fig. 7C).

In addition to cytokine production, we sought to determinewhether VISTA regulated the immunosuppressive functions oftumor-associated myeloid cells. Tumor-associated MDSCs(Fig. 7D and E), Inf-DCs (Fig. 7F), and myeloid cDCs(Fig. 7G) inhibited IFNg production by OT-I CD8þ T cellsstimulated with splenic APCs. The VISTA-blocking mAb or TLRstimulation partially reversed the suppressive functions ofM-MDSCs, myeloid cDCs, and Inf-DCs, whereas combinedtreatments maximally abolished suppression (Fig. 7D–F).MyD88 KO M-MDSCs failed to respond to the VISTA-blocking mAb, thus validating the role of MyD88 in mediatingthe myeloid-intrinsic function of VISTA (Fig. 7D). On the otherhand, PMN-MDSCs were not responsive to VISTA inhibition orTLR stimulation (Fig. 7E).

In contrast to the tolerogenic tumor-infiltrating myeloid cDCsand Inf-DCs, Batf3-dependent CD103þ DCs are the most potentcross-presentingDCsthatprimeCTLsat thetumorsite(29,31,32).We showed that VISTA was expressed on CD103þ DCs (Fig. 7A),and treatment with VISTA-blocking mAb significantly aug-mented IFNg production from purified OT-I CD8þ T cellscocultured with CD103þ DCs, whereas CpG treatment hadonly minimal impact (Fig. 7H). Combining VISTA blockadewith CpG treatment led to optimal T-cell activation and IFNgproduction (Fig. 7H). Together, these results indicated thatblocking VISTA promotes the development of antitumorimmunity by reprogramming tumor-associated tolerogenicMDSCs and DCs.

DiscussionPrevious studies have shown that VISTA is expressed on

myeloid cells at steady state and in tumor-bearing hosts, butthe myeloid-intrinsic functions of VISTA have not been eluci-dated (3, 12, 33, 34). This study demonstrated that VISTAdownregulated the TLR/TRAF6/TAK1 signaling pathway bymodulating the polyubiquitination and protein expression ofTRAF6. At cellular levels, VISTA regulated the effector functionsof MDSCs and DC subsets. Targeting VISTA in tumor-associatedmyeloid cells induced inflammatory cytokines and promotedantitumor T-cell responses.

TLRs expressed on DCs and other APCs maintain antitumorimmunosurveillance by recognizing endogenous danger-associatedmolecular patterns derived from commensal/invadingmicrobes or necrotic tumors (35, 36). Our study showed that inthe absence of a TLR-agonistic vaccine, the efficacy of theVISTA-blocking mAb relied upon endogenous TLR/MyD88-mediated signaling and correlated with the production ofimmune-stimulatory cytokines such as IL12, IL27, and TNFa.Thus, VISTA inhibited antitumor immunosurveillance at thesteady state.

Synthetic TLR agonists such as imiquimod (a TLR7 agonist)and PF-3512676 (CpG ODN for TLR9) are promising vaccineadjuvants (27, 37). We showed that in conjunction with theTLR7/8/9 agonistic vaccine, VISTA blockade augmented theexpression of chemokines (CXCL9/10 and CCL4/5) and cyto-kines (IFNb, IL12, IL27, IL23, IL6, and TNFa) within tumortissues. Intratumor CXCL9/10 and CCL4/5 are critical forrecruiting Th1 CD4þ T cells and CD8þ cytotoxic T cells and

are associated with better clinical outcomes in variouscancer types (38–40). Although IL12, IL27, TNFa, and IFNbplay well-established roles in promoting antitumor T-cellresponses (30, 41, 42), IL6 impairs antitumor immunity byinducing STAT3 activation in tumor cells and MDSCs (43).IL23 can both promote and inhibit tumor growth, dependingupon the stage of tumor development (44). It remains to bedetermined whether neutralizing IL6 and IL23 will improve theantitumor efficacy of VISTA inhibitors and reduce immune-related adverse events (irAE).

Tumor tissues are enriched with dysfunctional and immu-nosuppressive MDSCs and myeloid DCs (29, 45). MDSCsutilize diverse mechanisms such as the production of reactiveoxygen species, nitric oxide (NO), and the secretion ofarginase I, IL10, and TGFb to inhibit T-cell responses (45).Tumor-associated myeloid DCs impair T-cell activation byexpressing PD-L1 or secreting soluble mediators such asgalectin-1 (46, 47), and the frequencies of circulating MDSCshave been correlated with resistance to ipilimumab therapy inmelanoma patients (45, 48). Myeloid-inflamed TME profileassociates with poor clinical outcomes in pancreatic cancer andhead and neck cancer following GVAX vaccine therapy (49).Therefore, effective strategies that reduce the accumulation andsuppressive functions of tumorigenic myeloid cells are urgentlyneeded.

Our study showed that VISTA was an immune-checkpointprotein that regulated the immunosuppressive functions oftumor-associated myeloid cells. Blocking VISTA togetherwith TLR stimulation abolished the suppressive function ofM-MDSCs and myeloid DCs and induced IL12 production.These myeloid cell–dependent effects converted the immuno-suppressive TME into a T cell–stimulatory one that maysensitize tumors to additional immunotherapies. Supportingthis notion, we have previously shown that blockingVISTA synergizes with PD-L1 inhibitors in murine tumormodels (13).

In contrast to M-MDSCs, the suppressive function of PMN-MDSCs was not reversed by the VISTA-blocking mAb despitehigh VISTA expression on these cells. This result indicatedestablished that tumors enriched with PMN-MDSCs maybe resistant to VISTA-targeted inhibitors. Previous studieshave identified approaches to reduce the accumulation andsuppressive functions of PMN-MDSCs, including treatmentwith sildenafil (50), gemcitabine (51), inhibitors of DNAmethyltransferase or histone deacetylase (52), and PI3Kd/ginhibitor (53). However, it remains to be determined ifcombining VISTA inhibitors with these reagents will maxi-mally block both PMN-MDSCs, M-MDSCs, and other tumor-igenic myeloid DCs, thereby achieving optimal therapeuticoutcomes.

In conclusion, this study established VISTA as an immune-checkpoint protein that regulates the functions of myeloid cellsand established that myeloid cell activation contributes to theoverall antitumormechanisms of VISTA inhibitors. Unlike VISTA,immune-checkpoint proteins CTLA-4 and PD-1 do not directlyregulate the function of myeloid cells (28). Targeting VISTA maybe a valuable approach for patients resistant to the existingimmune-checkpoint inhibitors. Results from this study provideinsights for monitoring myeloid cell–dependent inflammatoryresponses that precede T cell–mediated antitumor responsesfollowing VISTA-targeted therapies.

Xu et al.





Disclosure of Potential Conflicts of InterestD. Wang is Adjunct Professor at Medical College of Wisconsin and Fujian

Normal University. L. Wang has ownership interest (including stock,patents, etc.) in ImmuNext Inc. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: W. Xu, M.S. Ernstoff, D. Wang, S. Malarkannan,L. WangDevelopment of methodology: W. Xu, Y. Zheng, L. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Xu, J. Dong, Y. Zheng, J. Zhou, Y. Yuan,H.E. Miller, M. OlsonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Xu, M. Olson, K. Rajasekaran, D. Wang,S. Malarkannan, L. WangWriting, review, and/or revision of the manuscript: W. Xu, H.M. Ta,H.E. Miller, M.S. Ernstoff, S. Malarkannan, L. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):H.M. Ta, H.E. Miller, M.Olson, S.Malarkannan,L. WangStudy supervision: D. Wang, S. Malarkannan, L. WangOther (performed experiments and analyzed data): K. Rajasekaran

AcknowledgmentsThis study was supported by research funding from NCI R01 CA164225

and NCI R01 1R01CA223804 (L. Wang), Advancing A Healthier Wiscon-sin Research and Education Program (AHW REP) fund (L. Wang), theOffice of the Assistant Secretary of Defense for Health Affairs through thePeer Reviewed Cancer Research Program under Award No. W81XWH-14-1-0587 (L. Wang), Worldwide Cancer Research Foundation (UK) researchgrant 16-1161 (L. Wang), and a Research Scholar Grant, RSG-18-045-01 -LIB, from the American Cancer Society (L. Wang). This work is alsosupported by NIH R01 AI102893 (S. Malarkannan), NCI R01 CA179363(S. Malarkannan), Nicholas Family Foundation (S. Malarkannan),Gardetto Family (S. Malarkannan and D. Wang), PO1 CA 206980 andP30 CA 016056 (M.S. Ernstoff), and NIH grants AI079087 and HL130724(D. Wang).

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 July 19, 2018; revised January 4, 2019; accepted July 19, 2019;published first August 1, 2019.

References1. Sharma P, Allison JP. The future of immune checkpoint therapy. Science

2015;348:56–61.2. Postow MA, Callahan MK, Wolchok JD. Immune checkpoint blockade in

cancer therapy. J Clin Oncol 2015;33:1974–82.3. Wang L, Rubinstein R, Lines JL, Wasiuk A, Ahonen C, Guo Y, et al. VISTA, a

novel mouse Ig superfamily ligand that negatively regulates T cellresponses. J Exp Med 2011;208:577–92.

4. Xu W, Hieu T, Malarkannan S, Wang L. The structure, expression, andmultifaceted role of immune-checkpoint protein VISTA as a critical regu-lator of anti-tumor immunity, autoimmunity, and inflammation. CellMolImmunol 2018;15:438–46.

5. Flies DB, Wang S, Xu H, Chen L. Cutting edge: a monoclonal antibodyspecific for the programmed death-1 homolog prevents graft-versus-hostdisease in mouse models. J Immunol 2011;187:1537–41.

6. Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4function. Annu Rev Immunol 2006;24:65–97.

7. Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, et al. T cellcostimulatory receptor CD28 is a primary target for PD-1-mediated inhi-bition. Science 2017;355:1428–33.

8. Li N, Xu W, Yuan Y, Ayithan N, Imai Y, Wu X, et al. Immune-checkpointprotein VISTA critically regulates the IL-23/IL-17 inflammatory axis.Sci Rep 2017;7:1485.

9. FliesDB,HanX,Higuchi T, Zheng L, Sun J, Ye JJ, et al. Coinhibitory receptorPD-1H preferentially suppresses CD4(þ) T cell-mediated immunity. J ClinInvest 2014;124:1966–75.

10. Wang L, LeMercier I, Putra J, ChenW, Liu J, Schenk AD, et al. Disruption ofthe immune-checkpoint VISTA gene imparts a proinflammatory pheno-type with predisposition to the development of autoimmunity. Proc NatlAcad Sci U S A 2014;111:14846–51.

11. Ceeraz S, Sergent PA, Plummer SF, Schned AR, Pechenick D, Burns CM,et al. VISTA deficiency accelerates the development of fatal murine lupusnephritis. Arthritis Rheumatol 2017;69:814–25.

12. Le Mercier I, Chen W, Lines JL, Day M, Li J, Sergent P, et al. VISTA regulatesthe development of protective antitumor immunity. Cancer Res 2014;74:1933–44.

13. Liu J, Yuan Y, Chen W, Putra J, Suriawinata AA, Schenk AD, et al.Immune-checkpoint proteins VISTA and PD-1 nonredundantly reg-ulate murine T-cell responses. Proc Natl Acad Sci U S A 2015;112:6682–7.

14. Chen Y, Zheng Y, You X, Yu M, Fu G, Su X, et al. Kras Is Critical for B CellLymphopoiesis. J Immunol 2016;196:1678–85.

15. Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, et al. Parkinmediates nonclassical, proteasomal-independent ubiquitination of syn-

philin-1: implications for Lewy body formation. J Neurosci 2005;25:2002–9.

16. Ahonen CL, Wasiuk A, Fuse S, Turk MJ, Ernstoff MS, Suriawinata AA, et al.Enhanced efficacy and reduced toxicity of multifactorial adjuvants com-paredwith unitary adjuvants as cancer vaccines. Blood 2008;111:3116–25.

17. Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol2014;5:461.

18. Janeway CA Jr., Medzhitov R. Innate immune recognition. Annu RevImmunol 2002;20:197–216.

19. Liu W, Ouyang X, Yang J, Liu J, Li Q, Gu Y, et al. AP-1 activated by toll-likereceptors regulates expression of IL-23 p19. J Biol Chem 2009;284:24006–16.

20. Whitmarsh AJ. Regulation of gene transcription by mitogen-activatedprotein kinase signaling pathways. Biochim Biophys Acta 2007;1773:1285–98.

21. Napetschnig J, Wu H. Molecular basis of NF-kappaB signaling. Annu RevBiophys 2013;42:443–68.

22. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76.

23. Jiang Z, Mak TW, Sen G, Li X. Toll-like receptor 3-mediated activationof NF-kappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-beta. Proc Natl Acad Sci U S A2004;101:3533–8.

24. Kawai T, Akira S.The role of pattern-recognition receptors in innateimmunity: update on Toll-like receptors. Nat Immunol 2010;11:373–84.

25. Adhikari A, Xu M, Chen ZJ. Ubiquitin-mediated activation of TAK1 andIKK. Oncogene 2007;26:3214–26.

26. Lu H. TLR agonists for cancer immunotherapy: tipping the balancebetween the immune stimulatory and inhibitory effects. Front Immunol2014;5:83.

27. Dowling JK, Mansell A. Toll-like receptors: the swiss army knife of immu-nity and vaccine development. Clin Transl Immunol 2016;5:e85.

28. Kumar V, Patel S, Tcyganov E, Gabrilovich DI. The nature of myeloid-derived suppressor cells in the tumormicroenvironment. Trends Immunol2016;37:208–20.

29. Veglia F, Gabrilovich DI. Dendritic cells in cancer: the role revisited.Curr Opin Immunol 2017;45:43–51.

30. Xu M, Mizoguchi I, Morishima N, Chiba Y, Mizuguchi J, Yoshimoto T.Regulation of antitumor immune responses by the IL-12 family cytokines,IL-12, IL-23, and IL-27. Clin Dev Immunol 2010;2010:832454.

31. Spranger S, Dai D, Horton B, Gajewski TF. Tumor-residing Batf3 dendriticcells are required for effector T cell trafficking and adoptive T cell therapy.Cancer Cell 2017;31:711–23.






32. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, et al.Dissecting the tumormyeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 2014;26:938.

33. Lines JL, Pantazi E, Mak J, Sempere LF, Wang L, O'Connell S, et al. VISTA isan immune checkpoint molecule for human T cells. Cancer Res 2014;74:1924–32.

34. Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. VISTA isan inhibitory immune checkpoint that is increased after ipilimumabtherapy in patients with prostate cancer. Nat Med 2017;23:551–5.

35. Connolly DJ, O'Neill LA. New developments in Toll-like receptor targetedtherapeutics. Curr Opin Pharmacol 2012;12:510–8.

36. Marabelle A, Filatenkov A, Sagiv-Barfi I, Kohrt H. Radiotherapy and toll-like receptor agonists. Semin Radiat Oncol 2015;25:34–9.

37. Goutagny N, Estornes Y, Hasan U, Lebecque S, Caux C. Targeting patternrecognition receptors in cancer immunotherapy. TargetOncol2012;7:29–54.

38. Groom JR, Luster AD. CXCR3 ligands: redundant, collaborative andantagonistic functions. Immunol Cell Biol 2011;89:207–15.

39. Gonzalez-Martin A,GomezL, Lustgarten J,Mira E,Manes S.Maximal T cell-mediated antitumor responses rely upon CCR5 expression in both CD4(þ) and CD8(þ) T cells. Cancer Res 2011;71:5455–66.

40. Liu JY, Li F, Wang LP, Chen XF, Wang D, Cao L, et al. CTL- vs Treglymphocyte-attracting chemokines, CCL4 and CCL20, are strong recipro-cal predictive markers for survival of patients with oesophageal squamouscell carcinoma. Br J Cancer 2015;113:747–55.

41. Murugaiyan G, Saha B. IL-27 in tumor immunity and immunotherapy.Trends Mol Med 2013;19:108–16.

42. Corrales L, Matson V, Flood B, Spranger S, Gajewski TF. Innate immunesignaling and regulation in cancer immunotherapy. Cell Res 2017;27:96–108.

43. Bromberg J, Wang TC. Inflammation and cancer: IL-6 and STAT3 completethe link. Cancer Cell 2009;15:79–80.

44. Teng MW, Bowman EP, McElwee JJ, Smyth MJ, Casanova JL, CooperAM, et al. IL-12 and IL-23 cytokines: from discovery to targeted

therapies for immune-mediated inflammatory diseases. Nat Med2015;21:719–29.

45. Veglia F, Perego M, Gabrilovich D. Myeloid-derived suppressor cellscoming of age. Nat Immunol 2018;19:108–19.

46. Curiel TJ, Wei S, DongH, Alvarez X, Cheng P, Mottram P, et al. Blockade ofB7-H1 improves myeloid dendritic cell-mediated antitumor immunity.Nat Med 2003;9:562–7.

47. Tesone AJ, Rutkowski MR, Brencicova E, Svoronos N, Perales-PuchaltA, Stephen TL, et al. Satb1 overexpression drives tumor-promotingactivities in cancer-associated dendritic cells. Cell Rep 2016;14:1774–86.

48. Weber R, Fleming V, Hu X, Nagibin V, Groth C, Altevogt P, et al. Myeloid-derived suppressor cells hinder the anti-cancer activity of immune check-point inhibitors. Front Immunol 2018;9:1310.

49. Tsujikawa T, Kumar S, Borkar RN, Azimi V, Thibault G, Chang YH, et al.Quantitative multiplex immunohistochemistry reveals myeloid-inflamedtumor-immune complexity associatedwithpoor prognosis. Cell Rep2017;19:203–17.

50. Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W, et al.Phosphodiesterase-5 inhibition augments endogenous antitumor immu-nity by reducingmyeloid-derived suppressor cell function. J ExpMed2006;203:2691–702.

51. Clark CE, Hingorani SR, Mick R, Combs C, Tuveson DA, Vonderheide RH.Dynamics of the immune reaction to pancreatic cancer from inception toinvasion. Cancer Res 2007;67:9518–27.

52. Kim K, Skora AD, Li Z, Liu Q, Tam AJ, Blosser RL, et al. Eradication ofmetastatic mouse cancers resistant to immune checkpoint blockade bysuppression of myeloid-derived cells. Proc Natl Acad Sci U S A 2014;111:11774–9.

53. Davis RJ,Moore EC, Clavijo PE, Friedman J, CashH, Chen Z, et al. Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid-derived suppressorcells with a selective inhibitor of PI3Kdelta/gamma. Cancer Res 2017;77:2607–19.


Xu et al.




2019;7:1497-1510. Published OnlineFirst July 24, 2019.Cancer Immunol Res Wenwen Xu, Juan Dong, Yongwei Zheng, et al. Immunosuppression

Mediated Inflammation and−by Controlling Myeloid Cell Immune-Checkpoint Protein VISTA Regulates Antitumor Immunity

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