VISTA Regulates the Development of Protective Antitumor ......VISTA Regulates the Development of Protective Antitumor Immunity Isabelle Le Mercier1, Wenna Chen1, Janet L. Lines3,4,

Microenvironment and Immunology

VISTA Regulates the Development of Protective AntitumorImmunity

Isabelle Le Mercier1, Wenna Chen1, Janet L. Lines3,4, Maria Day2, Jiannan Li1, Petra Sergent1,Randolph J. Noelle1,3,4, and Li Wang1

AbstractV-domain Ig suppressor of T-cell activation (VISTA) is a novel negative checkpoint ligand that is homologous

to PD-L1 and suppresses T-cell activation. This study demonstrates the multiple mechanisms whereby VISTArelieves negative regulation by hematopoietic cells and enhances protective antitumor immunity. VISTA is highlyexpressed on myeloid cells and Foxp3þCD4þ regulatory cells, but not on tumor cells within the tumormicroenvironment (TME). VISTA monoclonal antibody (mAb) treatment increased the number of tumor-specific T cells in the periphery and enhanced the infiltration, proliferation, and effector function of tumor-reactive T cells within the TME. VISTA blockade altered the suppressive feature of the TME by decreasing thepresence of monocytic myeloid-derived suppressor cells and increasing the presence of activated dendritic cellswithin the tumormicroenvironment. In addition, VISTA blockade impaired the suppressive function and reducedthe emergence of tumor-specific Foxp3þCD4þ regulatory T cells. Consequently, VISTA mAb administration as amonotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studiesexplored a combinatorial regimen usingVISTAblockade and a peptide-based cancer vaccinewith TLRagonists asadjuvants. VISTAblockade synergizedwith the vaccine to effectively impair the growth of established tumors. Ourstudy therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or incombination with additional immune-targeted strategies for cancer immunotherapy. Cancer Res; 74(7); 1933–44.�2014 AACR.

IntroductionImmune responses against cancer are negatively regulated

by multiple checkpoints, including CTLA-4, PD-L1/PD-1, andB7-H4 pathways. Targeting of these negative immune regula-tors has proved to be a clinically effective strategy to enhancetumor-specific immune responses (1, 2).The critical role of T CTLA-4 in suppressing tumor-

specific immunity was demonstrated when antibody-medi-ated CTLA-4 targeting in combination with a cellular vac-cine induced regression of established, poorly immunogenicB16 melanoma (3). Ipilimumab, an anti-human CTLA-4monoclonal antibody (mAb), as a monotherapy has provedto exert clinical benefit in late-stage melanoma in patientsand has been approved for treating advanced melanoma,

as well as undergoing early-phase trials for other cancers(4–6).

Programmed death-1 (PD-1) and its ligands PD-L1/PD-L2represent another immune negative checkpoint axis (7, 8). ThePD-1 pathway downregulates tumor-specific immunity byimpairing T-cell responses and promoting the induction ofFoxp3þ Tregs in the periphery (1, 9). Blocking the PD-L1/PD-1pathway, in conjunction with other immune therapies inhibitstumor progression (10–15). MDX-1106/BMS-936558, thehuman aPD-1 mAb, as well as a human aPD-L1 mAb, haveentered clinical trials, and early studies have shown resoundingclinical results (16–18).

Given the success of immune-checkpoint regulator blockadein improving both endogenous and vaccine-elicited antitumorimmune responses, identification of additional negative check-point regulator pathways would likely have important thera-peutic implications. We have recently discovered a novelimmunoglobulin (Ig) superfamily ligand, designated V-domainIg suppressor of T-cell activation (VISTA; Genbank: JN602184;ref. 19). VISTA bears homology to PD-L1 but displays a distinctexpression pattern. Within the hematopoietic compartment,VISTA is constitutively and highly expressed on CD11bhigh

myeloid cells, and expressed at lower levels on CD4þ and CD8þ

T cells and Foxp3þ Tregs. The human and murine homologsshare 90% homology, have indistinguishable functional prop-erties, and are similar in their lineage restricted expression (20).VISTA expressed on APCs directly suppresses CD4þ and CD8þ

T-cell proliferation and cytokine production (19).

Authors' Affiliations: 1Department of Microbiology and Immunology, TheGeisel School of Medicine at Dartmouth, The Norris Cotton Cancer Center;2ImmuNext Inc., Lebanon, New Hampshire; and 3Medical Research Coun-cil Centre of Transplantation, Guy's Hospital and 4Department of ImmuneRegulation and Intervention, King's College London, King's Health Part-ners, London, United Kingdom

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

Corresponding Author: Li Wang, Geisel school of medicine, Rubin Bldg764, Norris Cotton Cancer Center, Lebanon, NH 03756. Phone: 603-653-6094; Fax: 603-653-9902; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1506

�2014 American Association for Cancer Research.

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We hypothesize that VISTA is a novel negative checkpointregulator and a promising new target for cancer immunother-apy. This study has utilized a VISTA-specific blocking mAb toexamine the role of VISTA in regulating antitumor immunity.Our data show that VISTA mAb treatment impaired thesuppressive character of the tumor microenvironment (TME)and enhanced protective antitumor immunity. Furthermore,initial studies exploring a combination regimen of VISTAblockade together with a peptide vaccine show synergisticefficacy. As such, our study establishes a foundation fordesigning optimal therapeutic approaches that incorporateVISTA blockade either as a monotherapy or in combinationwith additional immune-targeted therapies.

Materials and MethodsMice

C57BL/6 mice were from NCI. TRP1 and OTII CD4 trans-genic mice were from the Jackson Laboratory. FoxP3-GFPreporter mice were as described (21) and were generouslyprovided by Dr. Alexander Rudensky (University of Washing-ton School of Medicine, Seattle, WA). The triple transgenicmice strain B6.Cg-BrafCA/þ Ptenlox5/lox5 Tg (Tyr::Cre/ERT2) wasobtained from Dr. Bosenberg (Yale School of Medicine, NewHaven, CT). All animals were maintained in a pathogen-freefacility at Dartmouth Medical School. All animal protocolswere Institutional Animal Care and Use Committee approvedat the Dartmouth College.

Tumor models, tumor vaccine, and treatmentMB49 (300,000), B16OVA (120,000), and B16BL6 (18,000)

tumor cells were inoculated on the right flank of female mice.For the PTEN/BRAF melanoma model, tumors were inducedby intradermal injection of 10 mL tamoxifen (10 mL dissolved indimethyl sulfoxide) on the lower back. Tumor vaccine con-sisted of CD40 agonistic antibody FGK (100 mg), LPS (30 mg),polyI:C (100 mg), CpG (ODN1826, 30 mg), Gardiquimod (30 mg),tumor antigen peptide TRP1 (106–130; 100 mg), and a mutatedTRP2 peptide DeltaV-TRP2 (180–188; 100 mg; refs. 22–24).Vaccine mixture was applied with split dose subcutaneouslyon indicated days. For prophylactic anti-VISTA mAb treat-ment, mice were treated every 2 days with 300 mg antibodysubcutaneously, starting on day 0 for the entire duration of theexperiment. For therapeutic treatment, mice were treatedwithsubcutaneous injection of 300 mg mAb every day for 10 days ondayþ2 or dayþ7 after tumor inoculation, followed by contin-uous antibody injection every 2 days for the entire duration ofthe experiment.

Flow cytometry and analysisFlow cytometry analysis was performed either on FACSCAN

using CellQuest software (BD Biosciences) or onMACSquant 7color analyzer (Miltenyi). Data analysis was performed usingFlowJo software (Treestar).

Graphs and statistical analysisAll graphs and statistical analysiswere generatedusingPrism

4 (GraphPad Software, Inc.). A Student t test (two-tailed) wasused for the data analyses. ��, P < 0.005; ��, P < 0.025; �, P < 0.05.

Materials and Methods are described in detail in the Sup-plementary Data.

ResultsVISTA mAb treatment impairs tumor growth inimmunogenic transplantable tumor models

We have previously generated a hamster monoclonal anti-body (clone 13F3) that neutralizes the suppressive activity ofVISTA (19). We hypothesized that VISTA mAb-mediatedblockade would enhance antitumor immune responses. Thishypothesis was tested in murine tumor models. We firstexamined the immunogenicity and in vivo clearance of 13F3(Supplementary Fig. S1). Our data show that mice developedstrong immune response against 13F3 and accumulated highlevels neutralizing antibodies, which presumably leads to fastclearance of 13F3. In fact, after aweek of continuous treatment,we can no longer detect any 13F3 in the serumwhen blood wasanalyzed 24 hours after each injection. Our data indicates thatthe most effective window of 13F3-mediated VISTA blockadein vivo might be within the first week of treatment.

Next, we examined the impact of VISTA mAb treatment inmice bearing melanoma B16OVA, which expresses the chickenovalbumin as a neo tumor antigen. Despite the apparent immu-nogenicity and short half-life of 13F3 in vivo, 13F3 treatmentsignificantly suppressed tumor growth in the B16OVA model(Fig. 1A). Increased number of IFN-g–producing cells in thetumor-draining lymph node was detected by ELISPOT assay inresponse to irradiated tumor cells, indicating that VISTA mAbtreatment enhanced tumor-specific T-cell responses. Eventhough we cannot detect any expression of VISTA on nonhe-matopoietic cells, we examined in vitro cultured tumor cells toexclude the possibility that VISTA mAb directly affected tumorcell proliferation and apoptosis (Supplementary Fig. S2).

VISTA mAb treatment alters the cellular composition ofthe tumor immune microenvironment

The TME plays a crucial role in suppressing tumor-specificT-cell responses (25, 26). VISTA is found to be highly expressedon tumor-infiltrating myeloid cells, including myeloid DCs(CD11bþ CD11cþGr1�) and myeloid-derived suppressor cells(CD11bþGr1þCD11C�), but is not expressed on B16 tumorcells (Fig. 1B–D). This expression pattern is in contrast to PD-L1, which is expressed on both tumor cells and tumor-infil-trating leukocytes (TIL; Fig. 1C).

VISTA mAb treatment altered the TIL composition in theB16OVA model. aVISTA administration increased totalCD45þ leukocytes tumor infiltration from 25.77% � 5.95% to57.78%� 3.97% (Fig. 1E). Within the TIL CD45þ population, adecreased percentage of CD11bþGr1þCD11C� MDSCs (from37.74%� 2.44% to 25.64%� 0.93%), and increased percentagesof tumor-specific CD4þ T cells (from 6.38%� 0.27% to 11.74%� 0.97%) and CD8þ T cells (from 9.25% � 0.95% to 17.74% �0.41%) were seen with aVISTA treatment. Tumor-infiltratingCD8þ T cells expressed phenotypes of CD44hiCD62Llow (datanot shown), produced effector molecules (i.e., IFN-g andgranzyme B), and mobilized CD107ab upon restimulationwith irradiated tumor cells ex vivo, indicating that thesetumor-infiltrating T cells contained tumor-specific effector

Le Mercier et al.

Cancer Res; 74(7) April 1, 2014 Cancer Research1934



T-cell populations (Fig. 1F–H). VISTA mAb administrationenhanced the proliferation and the effector function oftumor-infiltrating CD8þ T cells, as evidenced by increasednumber of Ki67þ cells, enhanced effector molecule produc-tion (i.e., IFN-g and granzyme B), and CD107 mobilization(Fig. 1F–H and Supplementary Fig. S1). There are no signif-icant alterations of myeloid cells and T-cell lineages in thespleen or tumor-draining lymph nodes upon VISTA mAbtreatment (data not shown), indicating that VISTA mAb didnot induce antibody-mediated deletion of VISTAþ cells.VISTA blockade therefore appears to act predominantly onaltering the TME by enhancing the frequency of tumor-infiltrating effector T cells as well as their effector functions,resulting in enhanced control of tumor growth.Similar analysis was performed in an immunogenic bladder

tumor model MB49 (27). Consistent with that observed in themelanoma model, VISTA mAb treatment significantly sup-pressed MB49 tumor growth (Fig. 2A). Similar to the B16OVAmodel, VISTA is highly expressed on tumor-infiltrating mye-loid cell populations (Fig. 2B). Unlike the B16OVA tumors,VISTA mAb treatment in MB49 tumors did not reduce therelative percentage of MDSCs within the CD45þ TILs. Furtheranalysis indicated that more than 90% of the MDSC popula-tions infiltrating MB49 tumors were of the granulocytic phe-notype (Cd11bþGr1hiLy6GþLy6C-int), which is in contrast tothe predominantmonocyticMDSCpopulations (Cd11bþGr1in-tLy6G�Ly6Chi) infiltrating B16 melanoma (Supplementary Fig.S3). Despite the unaltered presence of MDSCs, VISTA mAbenhanced the activation status of tumor-associated CD11cþ

DCs, which showed higher expression ofMCHII and CD80, andhigher production of interleukin (IL)-12 andTNF-a (Fig. 2D). InaVISTA–treated mice, tumor-specific T-cell responses weresignificantly enhanced, both in the tumor-draining lymphnodeand within the tumor tissue (Fig. 2).Because both B16OVA and MB49 tumors express neoanti-

gens that elicit strong immune responses, we sought to eval-uate whether VISTA blockade might impact on the growth ofpoorly immunogenic tumors, such as the B16BL6 melanoma.VISTA mAb in this model treatment significantly delayedtumor growth (Fig. 3A). Similar to the B16OVA model, reduc-tion of tumor-infiltrating monocytic MDSCs and increase oftumor-infiltrating CD4þ and CD8þ T cells were observed uponVISTA mAb treatment (Fig. 3B). To determine whether VISTAmAbcould directly promoteT-cell infiltration to tumor tissues,we tracked the tumor-infiltration of TRP1 TCR transgenicCD4þ T cells that are specific for the melanoma antigentyrosinase-related protein-1 (TRP1), in B16 tumor-bearingmice. Our data show that VISTA mAb treatment significantlyenhanced tumor infiltration of TRP1 transgenic CD4þ T cells(Fig. 3C). Phenotypic analysis of tumor-infiltrating polyclonalpopulations of CD4þ and CD8þ T cells demonstrated height-ened activation status upon VISTA blockade, which was evi-denced by enhanced Ki67þ cells and heightened frequency ofcells bearing a CD44hiCD62Llow surface phenotype (Fig. 3D).When restimulatedwith BMDCs pulsedwith tumor antigens invitro, TIL CD4þ and CD8þ T cells produced enhanced levels ofeffector molecules (IFN-g and/or granzyme B; Fig. 3E). Con-sistent with the MB49 model, tumor-infiltrating CD11cþ DCs

expressed higher level of MHCII and CD80, as well as inflam-matory cytokines IL-12 and TNF-a (Fig. 3F). Taken together,these results demonstrated the ability of VISTA mAb to alterthe suppressive signature of the TME and promote tumor-specific effector T-cell function, which likely contributed toreduced tumor growth.

VISTA regulates the induction of Foxp3þ iTregs fromna€�ve CD4þ T cells as well as the suppressive activity ofnTregs

Our previous study showed that VISTA-Ig fusion proteindirectly suppressed T-cell activation by inhibiting T-cellproliferative responses and cytokine production (19). Ourcurrent data show that VISTA-Ig promoted the induction ofFoxp3þCD4þ regulatory T cells (iTregs) in the presence ofTGF-b in vitro (Fig. 4C). This effect is also seen on humanCD4þ T cells treated with human-VISTA-Ig fusion protein invitro (manuscript submitted). Tregs induced by VISTA-Igobtained similar suppressive activity in vitro when comparedwith control iTregs (Fig. 4C). To validate the role of VISTA onpromoting the differentiation of iTregs, we examined theeffect of VISTA mAb treatment on the induction of tumor-specific iTregs, as previously described (9). Na€�ve OVA-specific OTII CD4þ T cells (purified from Foxp3GFP reportermice as CD25�CD62Lhi Foxp3GFP�) were adoptively trans-ferred into sublethally irradiated host bearing the B16OVAtumor. Mice were treated with either control-Ig or VISTAmAb. The induction of Foxp3GFPþ OTII iTreg cells in thetumor-draining lymph node and within the tumor tissue wasexamined when tumors reached >8 mm diameter (approx-imately day 20). As shown in Fig. 4D, VISTA blockadesignificantly diminished the percentage of Foxp3GFPþ

iTregs within the tumor-infiltrating OTII transgenic CD4þ

T-cell population (from 61.51% � 5.71% to 35.75% � 7.09%).Similar reduction was seen in the tumor-draining lymphnode (from 3.51% � 0.49% to 1.75% � 0.25%).

VISTA expression level on tumor-infiltrating Tregs ishigher than Tregs from peripheral lymph nodes (Fig. 4B),indicating that VISTA expressed on Tregs within the TMEmight play a role in suppressing tumor-specific immunity.Studies were developed to functionally assess whetherVISTA mAb-mediated blockade impaired the suppressivefunction of Tregs. Thymus-derived natural Tregs (nTregs)contain different subsets that are distinguished by surfacemarkers. Our analysis show that VISTA is more highlyexpressed on CD62L� and ICOS� Treg subsets, when com-pared with CD62Lþ and ICOSþ Treg subsets (Fig. 4A; refs. 28,29). No difference of VISTA expression was observed onother Treg subsets based on surface markers such as CD25,GITR, CD73, Folate receptor 4, and IL-7 receptor (data notshown). Treg subsets were sorted from na€�ve mice based onmarkers ICOS and CD62L and tested for their suppressiveactivity in vitro. VISTA mAb enhanced naive target T-cellproliferation in the presence of all the Treg subsets tested.This data indicates that VISTA blockade might directlyimpair the suppressive activity of Tregs. Notably, VISTAmAb also enhanced T-cell proliferation in the absenceof Tregs, indicating an alternative possibility that VISTA

Anti-VISTA Cancer Immunotherapy

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Figure 1. VISTA mAb treatment suppressed tumor growth and altered the tumor microenvironment in B16OVA melanoma model. A, B16OVA tumor-bearingmice (n ¼ 8 for control group; n¼ 10 for 13F3 group) were treated with anti-VISTA mab (13F3) or control-Ig starting from day 0, every 2 days throughout theduration of the experiment. Tumor size was measured with a caliper. IFN-g ELISPOT was performed using tumor-draining lymph node cells on day 14.Cells were restimulated in vitrowith irradiated tumor cells for 20 hours. IFN-g–producing cells were visualized and counted. B andC, cells were harvested fromperipheral lymph node, tumor-draining lymph node, and tumor tissues around day 20 when tumors reached approximately 8–10 mm diameter. VISTAexpression onmyeloid-derived suppressor cells (MDSC, CD11bhi CD11c�Gr1þ) andmyeloid DCs (CD11bþCD11cþ), and a comparison of VISTA and PD-L1expression on tumor cells and tumor-infiltrating leukocytes (TIL) was shown by flow cytometry. D, VISTA expression within B16 melanoma tumor tissue wasexamined by immunofluorescence. 40, 6-diamidino-2-phenylindole (DAPI), blue; Cd11b, green; VISTA or control-IgG, red. (Continued on the following page.)

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blockade might enhance the resistance of na€�ve T cells toTreg-mediated suppression. Either mechanism might ulti-mately contribute to the therapeutic efficacy of VISTA mAb-mediated blockade in tumor models.

VISTAmAb treatment suppresses tumorprogression in agenetic model of melanoma

Encouraged by results seen in the transplantable tumormodels, we tested the therapeutic impact of VISTA mAb

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Figure 2. Effects of VISTA mAb treatment on the growth and the tumor microenvironment of MB49 bladder tumors. MB49 tumor-bearing mice (n ¼ 14per group) were treated with control-Ig or 13F3 every 2 days starting from day 0. Tumor size was measured with a caliper. A, IFN-g ELISPOT wasperformed using tumor-draining lymph node cells on day 14 as described for the B16OVA tumor model in Fig. 1. B, when MB49 tumors reached 9–10 mmdiameter (�day 20), cells were harvested from peripheral lymph node, tumor-draining lymph node, and tumor tissues. VISTA expression on myeloid-derived suppressor cells (MDSC, CD11bhi CD11c� Gr1þ) and myeloid DCs (CD11bþ CD11cþ) was analyzed by flow cytometry. C–E, VISTA mAb treatmentaltered the TME and improved the effector function of tumor-infiltrating T cells. MB49-bearing mice were treated with 13F3 or control Ig from day 0until tumors in the control group reached 9–10 mm diameter. Tumors were harvested and total CD45þ TILs were quantified. Tumor-infiltrating DCs wereanalyzed by flow cytometry for their surface expression of MHCII and CD80 and cytokine production (i.e., IL12p40 and TNF-a). Tumor-infiltrating CD4þ andCD8þ T cells were stimulated with irradiated tumor cells for 20 hours. Cytokine production (i.e., IFN-g and TNF-a) was analyzed by flow cytometry andquantified. Data are representative of 2–3 independent experiments.

(Continued.) E–H, VISTA mAb treatment altered the TME and improved the effector function of tumor-infiltrating CD8þ T cells. B16OVA-bearing mice weretreated with 13F3 or control Ig from day 0 until tumors in the control group reached 9–10 mm diameter (�dayþ19). Tumors were harvested and CD45þ

TILswereanalyzedE.VariousTILpopulations, includingCD4þandCD8þTcells, andMDSCwere identifiedbyflowcytometry.Proliferationof tumor-infiltratingCD8þ T cells was analyzed on the basis of Ki67 expression (F). G and H, TIL CD8þ cells were stimulated with irradiated tumor cells for 20 hours. Cytokineproduction (IFN-g ), surface mobilization of CD107ab, and granzyme B expression were quantified. Data are representative of 2–3 independent experiments.





administration in an inducible melanoma model. This modelrelies on the interbreeding of three transgenicmouse lines (30):(i) BrafCA, which carries a conditional BrafV600E allele. (ii)Ptenlox5, which carries a conditional allele of Pten, permit-ting Cre-mediated deletion. (iii) Tyr::Cre/ERT2, which carriesa 4-hydroxytamoxifen (4-OHT)-inducible CreERT2 allele,allowing melanocyte-specific inducible expression of Cre.The triple mutant mice, B6.Cg-BrafCA/þ Ptenlox5/lox5 Tg (Tyr::Cre/ERT2), develop pigmented skin lesions upon topicaltreatment of 4-OHT within 21 days, which quickly progressto malignant melanoma (Fig. 5A). Analysis of tumor-infil-trating leukocytes reveals high levels of VISTA expression ontumor-associated myeloid cells (Fig. 5B). No VISTA expres-sion was observed on tumor cells, whereas some level of PD-L1 expression was detected (Fig. 5C). Importantly, VISTAmAb treatment significantly delayed tumor progression (Fig.

5A). Further analysis of TIL populations demonstrated sim-ilar alterations of TME as shown in the transplantable B16tumor models, including increased tumor infiltration of Tcells, decreased tumor-infiltration of myeloid cells, andenhanced IFN-g production of tumor-infiltrating CD8þ Tlymphocytes (Fig. 5D–F).

VISTA mAb synergizes with a tumor vaccine to achieveoptimal therapeutic outcome

Although VISTA mAb as a single-agent therapy delayedtumor progression, it was not curative under the conditionstested. In an attempt to increase the magnitude of the tumor-specific immune response, a peptide-based cancer vaccine wasused in combination with VISTA blockade. Based on previousmelanoma vaccine studies from our lab (R.J. Noelle) and others(31–34), we applied amodified vaccine platform containing the

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Figure 3. Effects of VISTA mAb treatment on the growth and the tumor microenvironment of B16-BL6 melanoma. A, mice (n ¼ 12 per group) wereinoculatedwithB16BL6 tumor cells (18,000) on the right flank.Micewere treatedwith anti-VISTAmAbevery 2days, starting fromdayþ2 for the entire durationof the experiment. Tumor growth was measured by a caliper every 2 days. B, tumors were harvested when control tumors reached �9–10 mmdiameter, which typically corresponded to day19–20 posttumor inoculation. TIL populations, including CD4þ and CD8þ T cells, and MDSCs (CD11bþ

CD11C� Gr1þ) were analyzed by flow cytometry. C, purified TRP1 transgenic CD4þ T cells (25,000) were adoptively transferred into congenic B16-tumorbearing mice that were treated with anti-VISTA mAb or control Ig. The number of tumor-infiltrating TRP1 cells was analyzed dayþ10 post transfer.D, the proliferative marker (Ki67 expression) and activation status (CD44 and CD62L expression) of tumor-infiltrating polyclonal CD4þ and CD8þ T cells wereanalyzed. E, the expression level of effector molecules (i.e., IFN-g and granzyme B) by TIL CD8þ T cells was analyzed after overnight stimulation byantigen-loaded BMDCs. F, expression levels of surfacemarkers (i.e., MHCII andCD80) and cytokine production (i.e., IL12p40 and TNF-a) of tumor-infiltratingCD11Cþ DCs were analyzed. Data are representative of 2–3 independent experiments.

Le Mercier et al.




agonistic CD40 antibody FGK, TLR agonists, and tumor anti-gen peptides. Both a MHCI-restricted mutant TRP2 peptideand a MHCII-restricted TRP1 peptide were incorporated (22–24). Early therapeutic intervention on 2-day tumors using acombinatorial therapy of aVISTA mAb and a single dose ofpeptide vaccine effectively eradicated tumors in a majority ofmice, whereas either reagent alone only transiently impairedtumor growth without significant survival benefit (Fig. 6A).Combinatorial treatment of 7-day established tumors (withaverage tumor diameter �3mm) showed significant suppres-sion of tumor growth and led to approximately 30% long-termsurvival, whereas monotherapy had little effect (Fig. 6B). Aprime-boost vaccine regimen was applied on dayþ7 and þ14for treating 7-day established tumors, and showed better long-term survival (from 10% to 30%) than a single vaccine dose inthis setting (Fig. S6). Tumor-infiltrating T cells showed syner-

gistically enhanced responses against immunizing peptide,indicating that the development of T-cell–mediated immuneresponses might be critical for the efficacy of the combinationtherapy (Fig. 6C). Additional evidence supporting this hypoth-esis is provided by the loss of therapeutic efficacy uponpredepletion of CD8þ T cells, or when treating immune-deficient tumor-bearing Rag�/� hosts (Supplementary Fig.S7). This data is consistent with the mechanisms of actionassociated with CD40/TLR-based vaccines (31–34). A secondtumor challenge of survivors at day þ60 post-treatmentshowed delayed tumor growth (8/12) or no tumor growth(4/12), indicating that certain levels of T-cell memory wasdeveloped during the combination therapy (Fig. 6D). Theprotection rate for the rechallenge did not improve regardlesswhether a second boost vaccine was introduced during theprimary tumor challenge (data not shown). These studies set

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Figure 4. VISTA regulates the suppressive function of Foxp3þCD4þ nTregs, as well as the de novo induction of iTregs. A, VISTA expression on nTreg subsets.nTreg subsets from spleens of 7–8-week-old naïve mice were distinguished based on markers CD62L and ICOS. B, higher levels of VISTA expressionon Tregs within the TME, when compared with Tregs from the peripheral LN. C, VISTA-Ig promotes the de novo induction of Foxp3þCD4þ iTregs.Naïve murine CD4þ T cells were stimulated with plate-bound aCD3 (2.5 mg/mL) together with either control-Ig or VISTA-Ig (5 mg/mL). ExogenousTGF-b (2.5 mg/mL) was added as indicated. Cells were examined for the induction of Foxp3 after 72 hours. Foxp3GFPþ iTregs were sorted ondayþ5 and their suppressive activity was examined as described in Materials and Methods. D, VISTA mAb treatment diminished tumor-mediatedinduction of iTregs. Congenic naïve OTII CD4þ T cells were adoptively transferred into B16OVA tumor-bearing mice (n ¼ 14 for control, n ¼ 17for 13F3 group), which were treated with control-Ig or VISTA mab 13F3 every 2 days starting from day 0. Mice were analyzed around day 20 whentumor size in control group reached 9–10 mm diameter. When necessary, small size tumors (diameter less than 5 mm) were pooled within thegroup to obtain sufficient cell number for analysis. Conversion of naïve OTII to Foxp3þ iTregs in tumor-draining lymph node and within the tumortissue was quantified and shown as the percentages of Foxp3þ cells among gated OTII cells. E, the suppressive activity of Treg subsets is partiallyimpaired by VISTA mAb. Foxp3þCD4þ nTreg subsets were sorted from spleen based on expression level of ICOS and CD62L. ThesenTregs were cocultured with naïve CD4þ T cells in indicated ratios, in the presence of VISTA mAb or control IgG. The proliferation of T cellswas measured by tritium incorporation. Data are representative of 2–3 independent experiments.





the stage for extensive efforts in the future to define successfulcombination therapies with aVISTA mAb as a platform.

DiscussionOur study introduces a new negative immune-checkpoint

regulator, VISTA, whose expression within the TME plays acritical role in regulating protective immunity to cancer. Ourdata show thataVISTAmonotherapy impairs tumor growth inmultiple tumor models. We have dissected the multiple

mechanisms whereby VISTA mAb-mediated blockade en-hances antitumor immune responses.

First, VISTA mAb enhanced tumor-specific T-cell response,both in the periphery and within the TME. VISTA is constitu-tively highly expressed on myeloid cells, with even higherdensities observed within the TME. This data suggests thatVISTAmay be abundantly present within the TME of any solidtumor types that are infiltrated with myeloid cells and T cells,indicating a broad clinical applicability for VISTA-blockadetherapy. The high expression of VISTA on myeloid cells

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Figure 5. VISTA monoclonal antibody treatment suppressed tumor growth and altered the tumor microenvironment in the PTEN/BRAF induciblemelanoma model. Tumors were induced by application of tamoxifen on the back skin. Mice were treated with VISTA mAb 13F3 (n ¼ 10) or control hamsterIg (n ¼ 8) every 2 days throughout the duration of the experiment. A, representative pictures of melanomas grown on mice, as well as dissected tumorsare shown. Because these inducible tumors grew with irregular and diverse height, which is in contrast to the relatively uniform shape and height oftransplantable B16 tumors, tumor size was measured with a caliper and is shown as mm3 (length � width � height). B, high levels of VISTA expression ontumor-infiltratingmyeloid cells. C, expression of VISTA and PD-L1 on CD45þ TILs andCD45� tumor cells is shown. D–F, analysis of tumor-infiltrating CD8þ Tcells (D), MDSCs (CD11bhi CD11c� Gr1þ; E), and cytokine (IFN-g ) production of tumor-infiltrating CD8þ T cells upon in vitro stimulation with anti-CD3 (F) isshown. Data are representative of 2–3 independent experiments.

Le Mercier et al.




within the TME suggests that VISTA might directly suppresstumor-infiltrating effector T cells. This hypothesis is sup-ported by enhanced proliferation, activation, and effectorfunction of tumor-infiltrating T cells in VISTA mAb-treatedmice (Figs. 1–3). We have not detected VISTA expression ontumor cells investigated. As such, we predict that VISTAtargeted immunotherapy will be effective independent of itsexpression on tumors. Similarly restricted hematopoieticexpression of human VISTA has been observed in humanmelanoma, colorectal cancer, and lung cancer (data notshown). In contrast, PD-L1 is known to be expressed athigh levels on nonhematopoietic tumor cells (Fig. 1;refs. 2, 35). It is therefore clinically relevant that VISTA mAbdemonstrated efficacy despite the high levels of PD-L1within the TME. Our future studies will compare the efficacy

of VISTA-blockade with PD1 blockade and define synergistictreatment regimens for the remission of established tumors.

Second, VISTA blockade impaired the suppressive effect ofnatural Tregs and the differentiation of tumor-specific iTregs(Fig. 4). It remains to be determinedwhether VISTA expressionon Tregs directly contributes to their suppressive function,even though VISTA neutralization in vitro failed to show anydirect effect on Treg proliferation, apoptosis, and Foxp3 sta-bility on in vitro cultured Tregs (Supplementary Fig. S4).Alternatively, it is possible that VISTA neutralization mightenhance na€�ve T-cell proliferation, making them more resis-tant to Treg-mediated suppression. Future studies will uti-lize VISTAKO mice to tease out the underlying mechanismswhereby VISTA regulates the suppressive function of Tregs.In this context, both CTLA-4 and PD-1 impact on either the

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Figure 6. VISTA mAb synergizedwith tumor vaccine to impair thegrowth of established B16-BL6tumors. Naïve mice (n ¼ 13 pergroup for A and n¼ 8–10 per groupfor B) were inoculated with B16-BL6 tumor cells (18,000) on theright flank. On dayþ2 (A) or dayþ7(B) postinoculations, mice weretreated with peptide vaccinecomplex (1 dose) or anti-VISTAmAb 13F3 (continuous treatmentfor the entire duration of theexperiment) or the combination asindicated. C, to determine whetherthe combination therapy inducedoptimal T-cell responses whencompared with single treatment,tumor-bearing mice were treatedon dayþ7 with vaccine or 13F3 orthe combination as indicated.Tumor-infiltrating lymphocyteswere harvested �dayþ21,restimulated with the immunizingpeptides TRP1/deltaV-TRP2, andanalyzed for their cytokineproduction (i.e., IFN-g ) by flowcytometry as described inMaterials and Methods. D, micethat survived the first B16BL6tumor challenge (n ¼ 12) aftercombination treatment wererechallenged with B16BL6 cells(18,000) on the same flank ondayþ60 post primary tumorrejection. Naïve mice were used asthe control group. Tumor size wasmeasured every 2–3 days with acaliper. Data are representative of2–3 independent experiments.





suppressive function and/or the peripheral induction ofFoxp3þ Tregs (9, 36–38).

Third, in the B16 melanoma model, VISTA blockadealtered the suppressive cellular signature of the TME, byreducing the tumor-infiltrating monocytic MDSCs whileincreasing the frequency of infiltrating effector T cells.Because there is no apparent deletion of both myeloidlineages and T-cell lineages in the periphery upon VISTAmAb treatment (data not shown), such alterations within thetumor tissue likely reflect either the impaired migration ofmonocytic MDSCs into tumor tissues, or impaired infiltra-tion of immature myeloid progenitor cells, which mightdifferentiate into MDSCs at tumor site (39, 40). In contrastto melanoma, VISTA blockade did not significantly alter theinfiltration of the granuolytic MDSC population within theMB49 bladder tumors. This intriguing result might indicatedifferent mechanisms whereby VISTA regulates monocyticand granuolytic myelopoiesis during tumor development.

In addition to affecting MDSCs, VISTA blockade directlyenhanced the migration of tumor-specific effector T cells (i.e.,TRP1 Tg CD4þ T cells) into tumor tissue (Fig. 3). Furthermore,VISTA blockade enhanced the immune-stimulatory phenotypeof TIL DCs, which showed higher expression levels of MHCIIand CD80, and cytokine production (i.e., IL-12 and TNF-a; Figs.2 and 3). Collectively, these multiple effects of VISTA mAbtreatment facilitate the establishment of an immune-stimula-tory TME, which lead to enhanced antitumor immunity.

It is clear that combination immunotherapy for cancerusing multiple biologics will be critical for improving thetherapeutic outcome. In addition to the monotherapeutictargeting of negative checkpoint regulators, combining mul-tiple checkpoint blockades, or combining checkpoint block-ade with chemotherapy or cancer vaccines is moving intoclinical trials. Some examples of such combinatorialapproaches include CTLA4 blockade combined with GVaxor Flt3vax (3, 41), CTLA4 blockade combined with PD-L1/PD1 blockade (42, 43), PD1 blockade combined with vaccine(44, 45). Our initial studies explore a combination regimenusing VISTA blockade together with a peptide vaccine (22–24, 33). Our data show promising synergistic effects fortreating both 2-day and 7-day established tumors. Futurestudies will focus on elucidating the cellular and molecularmechanisms of such synergy, as well as determining thelimitations of this combination platform and strategies forimprovement. For example, the use of defined peptidesmight lead to loss-of-antigen escape variants (46). Thedelivery of vaccine component might be optimized throughthe use of nanoparticles or other particulate formulations orTLR-peptide conjugates (47–49). Additional immune-sup-pressive pathways (i.e., other immune checkpoint pathwayssuch as PD-1, and immune suppressive cytokines) might be

blocked to achieve better long-term tumor-specific memoryT-cell responses. Furthermore, the immunogenicity and theFcR binding activity of the VISTA mAb might be criticallimiting factors for achieving optimal target neutralizationand therapeutic efficacy, thus warrant future studies (50, 51).

Recent breakthroughs using CTLA-4 and PD-1 checkpointblockade have reinvigorated the field of cancer immunother-apy (2, 35). Multiple checkpoints control the development ofantitumor immunity, each with their own unique signatureand mechanisms. Our studies provide compelling evidencesupporting the role of VISTA as a negative immune check-point regulator that controls immunity against cancer. Takentogether with the findings that VISTA expression and functionon human leukocytes and within the human tumors haverecapitulated the murine data (20), we present VISTA as apromising new target for cancer immunotherapy, either as asingle target or in combination with other immunotherapeuticstrategies.

Disclosure of Potential Conflicts of InterestL. Wang is a consultant/advisory board member of Immunext. J.L. Lines is a

consultant/advisory board member of Immunext. M. Day is a research associateand has ownership interest (including patents) in ImmuNext. R.J. Noelle is CSO,has commercial research grant, other commercial research support, ownershipinterest (including patents), and is a consultant/advisory board member ofImmuNext. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Wang, R.J. NoelleDevelopment of methodology: L. Wang, W. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Wang, I. LeMercier, J.L. Lines, P. SergentAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Wang, I. LeMercier, W. Chen, J. Li, R.J. NoelleWriting, review, and/or revision of themanuscript: L. Wang, P. Sergent, R.J.NoelleAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Wang, M. Day, J. Li, P. Sergent

AcknowledgmentsThe authors thank Dr. Alexander Rudensky (University ofWashington School

ofMedicine, Seattle, WA) for providing the Foxp3-GFP reportermice, Dr. MarcusBosenberg (Yale School of Medicine) for providing the B6.Cg-BrafCA/þ

Ptenlox5/lox5 Tg (Tyr::Cre/ERT2)-inducible melanoma model, Dr. Mary Jo Turk(Geisel School of Medicine) for maintaining the inducible melanoma model andproviding insightful discussions, and Dartlab Flow Cytometry Core Facility fortechnical support.

Grant SupportThis work is supported by NIH grant CA164225 (L. Wang), Melanoma

Research Foundation Career Development Award (L. Wang), HitchcockFoundation Research grant (L. Wang), AI048667 (to R.J. Noelle), Wellcome Trust(R.J. Noelle), and the Medical Research Council Centre for Transplantation andBiomedical Research Center at King's College London (R.J. Noelle).

The costs of publication of this article were defrayed in part 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 May 29, 2013; revised December 10, 2013; accepted December 19,2013; published online April 1, 2014.

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2014;74:1933-1944. Cancer Res Isabelle Le Mercier, Wenna Chen, Janet L. Lines, et al. ImmunityVISTA Regulates the Development of Protective Antitumor

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VISTA Regulates the Development of Protective Antitumor ......VISTA Regulates the Development of Protective Antitumor Immunity Isabelle Le Mercier1, Wenna Chen1, Janet L. Lines3,4,

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