VISTA Regulates the Development of Protective Antitumor Immunity

Cancer Microenvironment and Immunology Research

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

Abstract V-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 VISTA relieves negative regulation by hematopoietic cells and enhances protective antitumor immunity. VISTA is highly þ þ expressed on myeloid cells and Foxp3 CD4 regulatory cells, but not on tumor cells within the tumor microenvironment (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 the presence of monocytic myeloid-derived suppressor cells and increasing the presence of activated dendritic cells within the tumor microenvironment. In addition, VISTA blockade impaired the suppressive function and reduced þ þ the emergence of tumor-specific Foxp3 CD4 regulatory T cells. Consequently, VISTA mAb administration as a monotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studies explored a combinatorial regimen using VISTA blockade and a peptide-based cancer vaccine with TLR agonists as adjuvants. VISTA blockade synergized with the vaccine to effectively impair the growth of established tumors. Our study therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or in combination with additional immune-targeted strategies for cancer immunotherapy. Cancer Res; 74(7); 1933–44. 2014 AACR.

Introduction as well as undergoing early-phase trials for other cancers – Immune responses against cancer are negatively regulated (4 6). by multiple checkpoints, including CTLA-4, PD-L1/PD-1, and Programmed death-1 (PD-1) and its ligands PD-L1/PD-L2 B7-H4 pathways. Targeting of these negative immune regula- represent another immune negative checkpoint axis (7, 8). The fi tors has proved to be a clinically effective strategy to enhance PD-1 pathway downregulates tumor-speci c immunity by fi impairing T-cell responses and promoting the induction of tumor-speci c immune responses (1, 2). þ The critical role of T CTLA-4 in suppressing tumor- Foxp3 Tregs in the periphery (1, 9). Blocking the PD-L1/PD-1 specific immunity was demonstrated when antibody-medi- pathway, in conjunction with other immune therapies inhibits – ated CTLA-4 targeting in combination with a cellular vac- tumor progression (10 15). MDX-1106/BMS-936558, the cine induced regression of established, poorly immunogenic human aPD-1 mAb, as well as a human aPD-L1 mAb, have B16 melanoma (3). Ipilimumab, an anti-human CTLA-4 entered clinical trials, and early studies have shown resounding – monoclonal antibody (mAb), as a monotherapy has proved clinical results (16 18). to exert clinical benefit in late-stage melanoma in patients Given the success of immune-checkpoint regulator blockade and has been approved for treating advanced melanoma, in improving both endogenous and vaccine-elicited antitumor immune responses, identification of additional negative checkpoint regulator pathways would likely have important therapeutic implications. We have recently discovered a novel Authors' Affiliations: 1Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth, The Norris Cotton Cancer Center; immunoglobulin (Ig) superfamily ligand, designated V-domain 2ImmuNext Inc., Lebanon, New Hampshire; and 3Medical Research Coun- Ig suppressor of T-cell activation (VISTA; Genbank: JN602184; cil Centre of Transplantation, Guy's Hospital and 4Department of Immune Regulation and Intervention, King's College London, King's Health Part- ref. 19). VISTA bears homology to PD-L1 but displays a distinct ners, London, United Kingdom expression pattern. Within the hematopoietic compartment, VISTA is constitutively and highly expressed on CD11bhigh Note: Supplementary data for this article are available at Cancer Research þ þ Online (http://cancerres.aacrjournals.org/). myeloid cells, and expressed at lower levels on CD4 and CD8 þ Corresponding Author: Li Wang, Geisel school of medicine, Rubin Bldg T cells and Foxp3 Tregs. The human and murine homologs 764, Norris Cotton Cancer Center, Lebanon, NH 03756. Phone: 603-653- share 90% homology, have indistinguishable functional prop- 6094; Fax: 603-653-9902; E-mail: [email protected] erties, and are similar in their lineage restricted expression (20). þ þ doi: 10.1158/0008-5472.CAN-13-1506 VISTA expressed on APCs directly suppresses CD4 and CD8 2014 American Association for Cancer Research. T-cell proliferation and cytokine production (19).

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We hypothesize that VISTA is a novel negative checkpoint Materials and Methods are described in detail in the Sup- regulator and a promising new target for cancer immunother- plementary Data. apy. This study has utilized a VISTA-specific blocking mAb to examine the role of VISTA in regulating antitumor immunity. Results Our data show that VISTA mAb treatment impaired the VISTA mAb treatment impairs tumor growth in suppressive character of the tumor microenvironment (TME) immunogenic transplantable tumor models and enhanced protective antitumor immunity. Furthermore, We have previously generated a hamster monoclonal anti- initial studies exploring a combination regimen of VISTA body (clone 13F3) that neutralizes the suppressive activity of blockade together with a peptide vaccine show synergistic VISTA (19). We hypothesized that VISTA mAb-mediated efficacy. As such, our study establishes a foundation for blockade would enhance antitumor immune responses. This designing optimal therapeutic approaches that incorporate hypothesis was tested in murine tumor models. We first VISTA blockade either as a monotherapy or in combination examined the immunogenicity and in vivo clearance of 13F3 with additional immune-targeted therapies. (Supplementary Fig. S1). Our data show that mice developed strong immune response against 13F3 and accumulated high Materials and Methods levels neutralizing antibodies, which presumably leads to fast Mice clearance of 13F3. In fact, after a week of continuous treatment, C57BL/6 mice were from NCI. TRP1 and OTII CD4 trans- we can no longer detect any 13F3 in the serum when blood was genic mice were from the Jackson Laboratory. FoxP3-GFP analyzed 24 hours after each injection. Our data indicates that reporter mice were as described (21) and were generously the most effective window of 13F3-mediated VISTA blockade provided by Dr. Alexander Rudensky (University of Washing- in vivo might be within the first week of treatment. ton School of Medicine, Seattle, WA). The triple transgenic Next, we examined the impact of VISTA mAb treatment in þ mice strain B6.Cg-BrafCA/ Ptenlox5/lox5 Tg (Tyr::Cre/ERT2) was mice bearing melanoma B16OVA, which expresses the chicken obtained from Dr. Bosenberg (Yale School of Medicine, New ovalbumin as a neo tumor antigen. Despite the apparent immu- Haven, CT). All animals were maintained in a pathogen-free nogenicity and short half-life of 13F3 in vivo,13F3treatment facility at Dartmouth Medical School. All animal protocols significantly suppressed tumor growth in the B16OVA model were Institutional Animal Care and Use Committee approved (Fig. 1A). Increased number of IFN-g–producing cells in the at the Dartmouth College. tumor-draining lymph node was detected by ELISPOT assay in response to irradiated tumor cells, indicating that VISTA mAb Tumor models, tumor vaccine, and treatment treatment enhanced tumor-specific T-cell responses. Even MB49 (300,000), B16OVA (120,000), and B16BL6 (18,000) though we cannot detect any expression of VISTA on nonhe- tumor cells were inoculated on the right flank of female mice. matopoietic cells, we examined in vitro cultured tumor cells to For the PTEN/BRAF melanoma model, tumors were induced exclude the possibility that VISTA mAb directly affected tumor by intradermal injection of 10 mL tamoxifen (10 mL dissolved in cell proliferation and apoptosis (Supplementary Fig. S2). dimethyl sulfoxide) on the lower back. Tumor vaccine con- sisted of CD40 agonistic antibody FGK (100 mg), LPS (30 mg), VISTA mAb treatment alters the cellular composition of polyI:C (100 mg), CpG (ODN1826, 30 mg), Gardiquimod (30 mg), the tumor immune microenvironment tumor antigen peptide TRP1 (106–130; 100 mg), and a mutated The TME plays a crucial role in suppressing tumor-specific TRP2 peptide DeltaV-TRP2 (180–188; 100 mg; refs. 22–24). T-cell responses (25, 26). VISTA is found to be highly expressed on tumor-infiltrating myeloid cells, including myeloid DCs Vaccine mixture was applied with split dose subcutaneously þ þ (CD11b CD11c Gr1 ) and myeloid-derived suppressor cells on indicated days. For prophylactic anti-VISTA mAb treat- þ þ ment, mice were treated every 2 days with 300 mg antibody (CD11b Gr1 CD11C ), but is not expressed on B16 tumor subcutaneously, starting on day 0 for the entire duration of the cells (Fig. 1B–D). This expression pattern is in contrast to PD- experiment. For therapeutic treatment, mice were treated with L1, which is expressed on both tumor cells and tumor-infil- subcutaneous injection of 300 mg mAb every day for 10 days on trating leukocytes (TIL; Fig. 1C). dayþ2 or dayþ7 after tumor inoculation, followed by contin- VISTA mAb treatment altered the TIL composition in the B16OVA model. aVISTA administration increased total uous antibody injection every 2 days for the entire duration of þ CD45 leukocytes tumor infiltration from 25.77% 5.95% to the experiment. þ 57.78% 3.97% (Fig. 1E). Within the TIL CD45 population, a þ þ Flow cytometry and analysis decreased percentage of CD11b Gr1 CD11C MDSCs (from Flow cytometry analysis was performed either on FACSCAN 37.74% 2.44% to 25.64% 0.93%), and increased percentages þ using CellQuest software (BD Biosciences) or on MACSquant 7 of tumor-specificCD4 T cells (from 6.38% 0.27% to 11.74% þ color analyzer (Miltenyi). Data analysis was performed using 0.97%) and CD8 T cells (from 9.25% 0.95% to 17.74% FlowJo software (Treestar). 0.41%) were seen with aVISTA treatment. Tumor-infiltrating þ CD8 T cells expressed phenotypes of CD44hiCD62Llow (data Graphs and statistical analysis not shown), produced effector molecules (i.e., IFN-g and All graphs and statistical analysis were generated using Prism granzyme B), and mobilized CD107ab upon restimulation 4 (GraphPad Software, Inc.). A Student t test (two-tailed) was with irradiated tumor cells ex vivo,indicatingthatthese used for the data analyses. , P < 0.005; , P < 0.025; , P < 0.05. tumor-infiltrating T cells contained tumor-specific effector

1934 Cancer Res; 74(7) April 1, 2014 Cancer Research

T-cell populations (Fig. 1F–H). VISTA mAb administration expressed higher level of MHCII and CD80, as well as inflam- enhanced the proliferation and the effector function of matory cytokines IL-12 and TNF-a (Fig. 3F). Taken together, þ tumor-infiltrating CD8 T cells, as evidenced by increased these results demonstrated the ability of VISTA mAb to alter þ number of Ki67 cells, enhanced effector molecule produc- the suppressive signature of the TME and promote tumor- tion (i.e., IFN-g and granzyme B), and CD107 mobilization specific effector T-cell function, which likely contributed to (Fig. 1F–H and Supplementary Fig. S1). There are no signif- reduced tumor growth. icant alterations of myeloid cells and T-cell lineages in the þ spleen or tumor-draining lymph nodes upon VISTA mAb VISTA regulates the induction of Foxp3 iTregs from þ treatment (data not shown), indicating that VISTA mAb did na€ve CD4 T cells as well as the suppressive activity of þ not induce antibody-mediated deletion of VISTA cells. nTregs VISTA blockade therefore appears to act predominantly on Our previous study showed that VISTA-Ig fusion protein altering the TME by enhancing the frequency of tumor- directly suppressed T-cell activation by inhibiting T-cell infiltrating effector T cells as well as their effector functions, proliferative responses and cytokine production (19). Our resulting in enhanced control of tumor growth. current data show that VISTA-Ig promoted the induction of þ þ Similar analysis was performed in an immunogenic bladder Foxp3 CD4 regulatory T cells (iTregs) in the presence of tumor model MB49 (27). Consistent with that observed in the TGF-b in vitro (Fig. 4C). This effect is also seen on human þ melanoma model, VISTA mAb treatment significantly sup- CD4 T cells treated with human-VISTA-Ig fusion protein in pressed MB49 tumor growth (Fig. 2A). Similar to the B16OVA vitro (manuscript submitted). Tregs induced by VISTA-Ig model, VISTA is highly expressed on tumor-infiltrating mye- obtained similar suppressive activity in vitro when compared loid cell populations (Fig. 2B). Unlike the B16OVA tumors, with control iTregs (Fig. 4C). To validate the role of VISTA on VISTA mAb treatment in MB49 tumors did not reduce the promoting the differentiation of iTregs, we examined the þ relative percentage of MDSCs within the CD45 TILs. Further effect of VISTA mAb treatment on the induction of tumor- analysis indicated that more than 90% of the MDSC popula- specific iTregs, as previously described (9). Na€ve OVA- þ tions infiltrating MB49 tumors were of the granulocytic phe- specificOTIICD4 T cells (purified from Foxp3GFP reporter þ þ notype (Cd11b Gr1hiLy6G Ly6C-int), which is in contrast to mice as CD25 CD62Lhi Foxp3GFP ) were adoptively trans- þ the predominant monocytic MDSC populations (Cd11b Gr1in- ferred into sublethally irradiated host bearing the B16OVA tLy6G Ly6Chi)infiltrating B16 melanoma (Supplementary Fig. tumor. Mice were treated with either control-Ig or VISTA þ S3). Despite the unaltered presence of MDSCs, VISTA mAb mAb. The induction of Foxp3GFP OTII iTreg cells in the þ enhanced the activation status of tumor-associated CD11c tumor-draining lymph node and within the tumor tissue was DCs, which showed higher expression of MCHII and CD80, and examined when tumors reached >8 mm diameter (approx- higher production of interleukin (IL)-12 and TNF-a (Fig. 2D). In imately day 20). As shown in Fig. 4D, VISTA blockade þ aVISTA–treated mice, tumor-specific T-cell responses were significantly diminished the percentage of Foxp3GFP þ significantly enhanced, both in the tumor-draining lymph node iTregs within the tumor-infiltrating OTII transgenic CD4 and within the tumor tissue (Fig. 2). T-cell population (from 61.51% 5.71% to 35.75% 7.09%). Because both B16OVA and MB49 tumors express neoanti- Similarreductionwasseeninthetumor-draininglymph gens that elicit strong immune responses, we sought to eval- node (from 3.51% 0.49% to 1.75% 0.25%). uate whether VISTA blockade might impact on the growth of VISTA expression level on tumor-infiltrating Tregs is poorly immunogenic tumors, such as the B16BL6 melanoma. higher than Tregs from peripheral lymph nodes (Fig. 4B), VISTA mAb in this model treatment significantly delayed indicating that VISTA expressed on Tregs within the TME tumor growth (Fig. 3A). Similar to the B16OVA model, reduc- might play a role in suppressing tumor-specific immunity. tion of tumor-infiltrating monocytic MDSCs and increase of Studies were developed to functionally assess whether þ þ tumor-infiltrating CD4 and CD8 T cells were observed upon VISTA mAb-mediated blockade impaired the suppressive VISTA mAb treatment (Fig. 3B). To determine whether VISTA function of Tregs. Thymus-derived natural Tregs (nTregs) mAb could directly promote T-cell infiltration to tumor tissues, contain different subsets that are distinguished by surface we tracked the tumor-infiltration of TRP1 TCR transgenic markers. Our analysis show that VISTA is more highly þ CD4 T cells that are specific for the melanoma antigen expressed on CD62L and ICOS Treg subsets, when com- þ þ tyrosinase-related protein-1 (TRP1), in B16 tumor-bearing paredwithCD62L and ICOS Treg subsets (Fig. 4A; refs. 28, mice. Our data show that VISTA mAb treatment significantly 29). No difference of VISTA expression was observed on þ enhanced tumor infiltration of TRP1 transgenic CD4 T cells other Treg subsets based on surface markers such as CD25, (Fig. 3C). Phenotypic analysis of tumor-infiltrating polyclonal GITR, CD73, Folate receptor 4, and IL-7 receptor (data not þ þ populations of CD4 and CD8 T cells demonstrated height- shown). Treg subsets were sorted from na€ve mice based on ened activation status upon VISTA blockade, which was evi- markers ICOS and CD62L and tested for their suppressive þ denced by enhanced Ki67 cells and heightened frequency of activity in vitro. VISTA mAb enhanced naive target T-cell cells bearing a CD44hiCD62Llow surface phenotype (Fig. 3D). proliferation in the presence of all the Treg subsets tested. When restimulated with BMDCs pulsed with tumor antigens in This data indicates that VISTA blockade might directly þ þ vitro, TIL CD4 and CD8 T cells produced enhanced levels of impair the suppressive activity of Tregs. Notably, VISTA effector molecules (IFN-g and/or granzyme B; Fig. 3E). Con- mAb also enhanced T-cell proliferation in the absence þ sistent with the MB49 model, tumor-infiltrating CD11c DCs of Tregs, indicating an alternative possibility that VISTA

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A P = 0.027 B MDSC 120 Control (n=8) 175 13F3 (n=10)

) 100 2 150 P = 0.04 80 125 60 100

40 75 100 101 102 103 104 100 101 102 103 104 50

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Figure 1. VISTA mAb treatment suppressed tumor growth and altered the tumor microenvironment in B16OVA melanoma model. A, B16OVA tumor-bearing mice (n ¼ 8 for control group; n ¼ 10 for 13F3 group) were treated with anti-VISTA mab (13F3) or control-Ig starting from day 0, every 2 days throughout the duration 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 vitro with irradiated tumor cells for 20 hours. IFN-g–producing cells were visualized and counted. B and C, cells were harvested from peripheral lymph node, tumor-draining lymph node, and tumor tissues around day 20 when tumors reached approximately 8–10 mm diameter. VISTA þ þ þ expression on myeloid-derived suppressor cells (MDSC, CD11bhi CD11c Gr1 ) and myeloid DCs (CD11b CD11c ), and a comparison of VISTA and PD-L1 expression on tumor cells and tumor-infiltrating leukocytes (TIL) was shown by flow cytometry. D, VISTA expression within B16 melanoma tumor tissue was examined by immunofluorescence. 40, 6-diamidino-2-phenylindole (DAPI), blue; Cd11b, green; VISTA or control-IgG, red. (Continued on the following page.)

1936 Cancer Res; 74(7) April 1, 2014 Cancer Research

P = 0.0007 MDSC A 120 Control-Ig (n=14) B 100 175 13F3 (n=14) 80 100 P 150 ) = 0.001 2 80 125 60 100 40 60 75 20 40 50 0 0 1 2 3 4 Spots/250k cells Tumor size (mm 20 10 10 10 10 10 25 VISTA 0 0 TIL 04812162024283236 Control 13F3 MDSC Nondrain LN Days Myeloid DC Drain LN Isotype Isotype

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0 0 Control 13F3 Control 13F3 Control 13F3 Control 13F3 + + CD4+ CD8+ CD4 CD8

Figure 2. Effects of VISTA mAb treatment on the growth and the tumor microenvironment of MB49 bladder tumors. MB49 tumor-bearing mice (n ¼ 14 per 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 was performed 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 mm diameter (day 20), cells were harvested from peripheral lymph node, tumor-draining lymph node, and tumor tissues. VISTA expression on myeloid- þ þ þ derived suppressor cells (MDSC, CD11bhi CD11c Gr1 ) and myeloid DCs (CD11b CD11c ) was analyzed by flow cytometry. C–E, VISTA mAb treatment altered the TME and improved the effector function of tumor-infiltrating T cells. MB49-bearing mice were treated with 13F3 or control Ig from day 0 until tumors in the control group reached 9–10 mm diameter. Tumors were harvested and total CD45þ TILs were quantified. Tumor-infiltrating DCs were þ analyzed by flow cytometry for their surface expression of MHCII and CD80 and cytokine production (i.e., IL12p40 and TNF-a). Tumor-infiltrating CD4 and þ CD8 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 and quantified. Data are representative of 2–3 independent experiments. blockade might enhance the resistance of na€ve T cells to VISTA mAb treatment suppresses tumor progression in a Treg-mediated suppression. Either mechanism might ulti- genetic model of melanoma mately contribute to the therapeutic efficacy of VISTA mAb- Encouraged by results seen in the transplantable tumor mediated blockade in tumor models. models, we tested the therapeutic impact of VISTA mAb

þ (Continued.)E–H, VISTA mAb treatment altered the TME and improved the effector function of tumor-infiltrating CD8 T cells. B16OVA-bearing mice were þ treated with 13F3 or control Ig from day 0 until tumors in the control group reached 9–10 mm diameter (dayþ19). Tumors were harvested and CD45 þ þ TILs were analyzed E. Various TIL populations, including CD4 and CD8 T cells, and MDSC were identified by flow cytometry. Proliferation of tumor-infiltrating þ þ CD8 T cells was analyzed on the basis of Ki67 expression (F). G and H, TIL CD8 cells were stimulated with irradiated tumor cells for 20 hours. Cytokine production (IFN-g), surface mobilization of CD107ab, and granzyme B expression were quantified. Data are representative of 2–3 independent experiments. www.aacrjournals.org Cancer Res; 74(7) April 1, 2014 1937

A *** B 150 *** *** *** Control 50 35

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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) were inoculated with B16BL6 tumor cells (18,000) on the right flank. Mice were treated with anti-VISTA mAb every 2 days, starting from day þ2 for the entire duration of the experiment. Tumor growth was measured by a caliper every 2 days. B, tumors were harvested when control tumors reached 9–10 mm þ þ þ diameter, which typically corresponded to day19–20 posttumor inoculation. TIL populations, including CD4 and CD8 T cells, and MDSCs (CD11b þ þ CD11C Gr1 ) were analyzed by flow cytometry. C, purified TRP1 transgenic CD4 T cells (25,000) were adoptively transferred into congenic B16-tumor bearing mice that were treated with anti-VISTA mAb or control Ig. The number of tumor-infiltrating TRP1 cells was analyzed dayþ10 post transfer. þ þ D, the proliferative marker (Ki67 expression) and activation status (CD44 and CD62L expression) of tumor-infiltrating polyclonal CD4 and CD8 T cells were þ analyzed. E, the expression level of effector molecules (i.e., IFN-g and granzyme B) by TIL CD8 T cells was analyzed after overnight stimulation by antigen-loaded BMDCs. F, expression levels of surface markers (i.e., MHCII and CD80) and cytokine production (i.e., IL12p40 and TNF-a) of tumor-infiltrating þ CD11C DCs were analyzed. Data are representative of 2–3 independent experiments.

administration in an inducible melanoma model. This model 5A). Further analysis of TIL populations demonstrated sim- relies on the interbreeding of three transgenic mouse lines (30): ilar alterations of TME as shown in the transplantable B16 (i) BrafCA, which carries a conditional BrafV600E allele. (ii) tumor models, including increased tumor infiltration of T Ptenlox5, which carries a conditional allele of Pten, permit- cells, decreased tumor-infiltration of myeloid cells, and þ ting Cre-mediated deletion. (iii) Tyr::Cre/ERT2, which carries enhanced IFN-g production of tumor-infiltrating CD8 T a 4-hydroxytamoxifen (4-OHT)-inducible CreERT2 allele, lymphocytes (Fig. 5D–F). allowing melanocyte-specific inducible expression of Cre. þ The triple mutant mice, B6.Cg-BrafCA/ Ptenlox5/lox5 Tg (Tyr:: VISTA mAb synergizes with a tumor vaccine to achieve Cre/ERT2), develop pigmented skin lesions upon topical optimal therapeutic outcome treatment of 4-OHT within 21 days, which quickly progress Although VISTA mAb as a single-agent therapy delayed to malignant melanoma (Fig. 5A). Analysis of tumor-infil- tumor progression, it was not curative under the conditions trating leukocytes reveals high levels of VISTA expression on tested. In an attempt to increase the magnitude of the tumortumor-associated myeloid cells (Fig. 5B). No VISTA expres- specific immune response, a peptide-based cancer vaccine was sion was observed on tumor cells, whereas some level of PD- used in combination with VISTA blockade. Based on previous L1 expression was detected (Fig. 5C). Importantly, VISTA melanoma vaccine studies from our lab (R.J. Noelle) and others mAb treatment significantly delayed tumor progression (Fig. (31–34), we applied a modified vaccine platform containing the

1938 Cancer Res; 74(7) April 1, 2014 Cancer Research

A 100 100 B 80 80 60 60 40 40 52.2 43.9 % of Max % of Max

20 20 CD4 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 Foxp3 VISTA VISTA VISTA Foxp3+ Tregs LN CD62L– Treg ICOS– Treg Foxp3+ Tregs tumor + + CD62L Treg ICOS Treg Foxp3– T cells tumor Isotype control Isotype control Isotype control

CD30 100 Control-iTreg ** ** 80 8 80 VISTA-iTreg % + 20 60 60 6

40 % OTII cells 40 % OTII cells 4

10 + + Foxp3GFP 20 20 2 CFSE-low cells% Foxp3 0 Foxp3 0 0 0 VISTA-Ig + – – 2-1 1-1 1-2 1-4 noTreg Control 13F3 Control 13F3 TGFβ – ++ Ratio Tregs:T naïve TIL Draining LN

E ICOS+ CD62L– ICOS– CD62L+ ICOS– 60,000 75,000 75,000 50,000 62,500 62,500 Control 40,000 50,000 50,000 13F3 30,000 37,500

cpm 37,500 cpm cpm 20,000 25,000 25,000 10,000 12,500 12,500 0 0 0 1-1 1-2 1-4 notreg 2-1 1-1 1-2 notreg 2-1 1-1 1-2 notreg Ratio Treg:T naïve Ratio Treg:T naïve Ratio Treg:T naïve

þ þ Figure 4. VISTA regulates the suppressive function of Foxp3 CD4 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 expression þ þ on Tregs within the TME, when compared with Tregs from the peripheral LN. C, VISTA-Ig promotes the de novo induction of Foxp3 CD4 iTregs. þ Naïve murine CD4 T cells were stimulated with plate-bound aCD3 (2.5 mg/mL) together with either control-Ig or VISTA-Ig (5 mg/mL). Exogenous þ TGF-b (2.5 mg/mL) was added as indicated. Cells were examined for the induction of Foxp3 after 72 hours. Foxp3GFP iTregs were sorted on dayþ5 and their suppressive activity was examined as described in Materials and Methods. D, VISTA mAb treatment diminished tumor-mediated þ induction of iTregs. Congenic naïve OTII CD4 T cells were adoptively transferred into B16OVA tumor-bearing mice (n ¼ 14 for control, n ¼ 17 for 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 when tumor size in control group reached 9–10 mm diameter. When necessary, small size tumors (diameter less than 5 mm) were pooled within the þ group to obtain sufficient cell number for analysis. Conversion of naïve OTII to Foxp3 iTregs in tumor-draining lymph node and within the tumor þ tissue was quantified and shown as the percentages of Foxp3 cells among gated OTII cells. E, the suppressive activity of Treg subsets is partially þ þ impaired by VISTA mAb. Foxp3 CD4 nTreg subsets were sorted from spleen based on expression level of ICOS and CD62L. These þ nTregs were cocultured with naïve CD4 T cells in indicated ratios, in the presence of VISTA mAb or control IgG. The proliferation of T cells was measured by tritium incorporation. Data are representative of 2–3 independent experiments. agonistic CD40 antibody FGK, TLR agonists, and tumor anti- gistically enhanced responses against immunizing peptide, gen peptides. Both a MHCI-restricted mutant TRP2 peptide indicating that the development of T-cell–mediated immune and a MHCII-restricted TRP1 peptide were incorporated (22– responses might be critical for the efficacy of the combination 24). Early therapeutic intervention on 2-day tumors using a therapy (Fig. 6C). Additional evidence supporting this hypoth- combinatorial therapy of aVISTA mAb and a single dose of esis is provided by the loss of therapeutic efficacy upon þ peptide vaccine effectively eradicated tumors in a majority of predepletion of CD8 T cells, or when treating immune- mice, whereas either reagent alone only transiently impaired deficient tumor-bearing Rag / hosts (Supplementary Fig. tumor growth without significant survival benefit (Fig. 6A). S7). This data is consistent with the mechanisms of action Combinatorial treatment of 7-day established tumors (with associated with CD40/TLR-based vaccines (31–34). A second average tumor diameter 3mm) showed significant suppres- tumor challenge of survivors at day þ60 post-treatment sion of tumor growth and led to approximately 30% long-term showed delayed tumor growth (8/12) or no tumor growth survival, whereas monotherapy had little effect (Fig. 6B). A (4/12), indicating that certain levels of T-cell memory was prime-boost vaccine regimen was applied on dayþ7 and þ14 developed during the combination therapy (Fig. 6D). The for treating 7-day established tumors, and showed better long- protection rate for the rechallenge did not improve regardless term survival (from 10% to 30%) than a single vaccine dose in whether a second boost vaccine was introduced during the this setting (Fig. S6). Tumor-infiltrating T cells showed syner- primary tumor challenge (data not shown). These studies set

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n A 500 Control =8 *** 13F3 n=10 ) 3 400 ***

300 ***

200 +13F3

Tumor size (mm 100 Control 0 15 17 19 21 23 25 27 29 31 Days B C CD11c+ CD11b+ 100 100 19.8 3.18 80 80 33 50.2 60 60

40 40

20 20 VISTA 0 0 6.59 0 1 2 3 4 40.6 38.7 7.85 10 10 10 10 10 0 200 400 600 800 1,000 VISTA PD-L1 CD11C CD11b VISTA + TIL CD45 TIL Naïve LN Tumor cells dLN Isotype Isotype

Tumor-infiltrating + TIL CD8+ T cell% TIL myeloid% TIL IFNg CD8 % D CD45+ % EF * *** 10.0 * 40 60 12 50 7.5 9 30

% 40 + 6 5.0 20 30 CD8 T cell % T CD8 cell + CD45 γ

T cell% of TIL 20 3 + 2.5 10 MDSC% of TIL

within tumor mass 10 CD8

0 0.0 0 IFN- TIL 0 Control 13F3 Control 13F3 Control 13F3 Control 13F3

Figure 5. VISTA monoclonal antibody treatment suppressed tumor growth and altered the tumor microenvironment in the PTEN/BRAF inducible melanoma model. Tumors were induced by application of tamoxifen on the back skin. Mice were treated with VISTA mAb 13F3 (n ¼ 10) or control hamster Ig (n ¼ 8) every 2 days throughout the duration of the experiment. A, representative pictures of melanomas grown on mice, as well as dissected tumors are shown. Because these inducible tumors grew with irregular and diverse height, which is in contrast to the relatively uniform shape and height of transplantable B16 tumors, tumor size was measured with a caliper and is shown as mm3 (length width height). B, high levels of VISTA expression on þ þ tumor-infiltrating myeloid cells. C, expression of VISTA and PD-L1 on CD45 TILs and CD45 tumor cells is shown. D–F, analysis of tumor-infiltrating CD8 T þ þ cells (D), MDSCs (CD11bhi CD11c Gr1 ; E), and cytokine (IFN-g) production of tumor-infiltrating CD8 T cells upon in vitro stimulation with anti-CD3 (F) is shown. Data are representative of 2–3 independent experiments.

the stage for extensive efforts in the future to define successful mechanisms whereby VISTA mAb-mediated blockade en- combination therapies with aVISTA mAb as a platform. hances antitumor immune responses. First, VISTA mAb enhanced tumor-specific T-cell response, both in the periphery and within the TME. VISTA is constitu- Discussion tively highly expressed on myeloid cells, with even higher Our study introduces a new negative immune-checkpoint densities observed within the TME. This data suggests that regulator, VISTA, whose expression within the TME plays a VISTA may be abundantly present within the TME of any solid critical role in regulating protective immunity to cancer. Our tumor types that are infiltrated with myeloid cells and T cells, data show that aVISTA monotherapy impairs tumor growth in indicating a broad clinical applicability for VISTA-blockade multiple tumor models. We have dissected the multiple therapy. The high expression of VISTA on myeloid cells

1940 Cancer Res; 74(7) April 1, 2014 Cancer Research

Control A 250 13F3 100 ) 2 200 Vaccine 80 Combo Control Figure 6. VISTA mAb synergized 150 with tumor vaccine to impair the 60 13F3 growth of established B16-BL6 100 40 Vaccine tumors. Naïve mice (n ¼ 13 per Combo group for A and n ¼ 8–10 per group 50 20 Tumor size (mm for B) were inoculated with B16- Percentage survival BL6 tumor cells (18,000) on the 0 0 010203040506070 right ﬂank. On dayþ2 (A) or dayþ7 10 13 16 19 22 25 28 31 34 37 40 43 (B) postinoculations, mice were Days Days treated with peptide vaccine B 250 Control complex (1 dose) or anti-VISTA )

2 Vaccine Control mAb 13F3 (continuous treatment 100 200 Vaccine for the entire duration of the 13F3 13F3 experiment) or the combination as 150 Vac+13F3 75 indicated. C, to determine whether Vac+13F3 the combination therapy induced 100 50 optimal T-cell responses when compared with single treatment, Tumor size (mm 50 25

tumor-bearing mice were treated Percentage survival þ on day 7 with vaccine or 13F3 or 0 0 the combination as indicated. 9 121518212427 020406080 Tumor-inﬁltrating lymphocytes Days Days þ were harvested day 21, C 4 8 restimulated with the immunizing TILs TILs peptides TRP1/deltaV-TRP2, and +

3 + 6 analyzed for their cytokine production (i.e., IFN-g)byﬂow 2 4 % CD4 % CD8

cytometry as described in + + g Materials and Methods. D, mice 1 g 2 ﬁ IFN-

that survived the rst B16BL6 IFN- tumor challenge (n ¼ 12) after 0 0 Control Vaccine 13F3 Combo Control Vaccine 13F3 Combo combination treatment were rechallenged with B16BL6 cells D 250 Control 100 Control (18,000) on the same ﬂank on

) Rechallenge dayþ60 post primary tumor 2 200 Rechallenge 75 rejection. Naïve mice were used as the control group. Tumor size was 150 measured every 2–3 days with a 50 caliper. Data are representative of 100 2–3 independent experiments. 50 25 Tumor size (mm Percentage survival 0 0 10 15 20 25 0 20406080 Days Days

within the TME suggests that VISTA might directly suppress of VISTA-blockade with PD1 blockade and define synergistic tumor-infiltrating effector T cells. This hypothesis is sup- treatment regimens for the remission of established tumors. ported by enhanced proliferation, activation, and effector Second, VISTA blockade impaired the suppressive effect of function of tumor-infiltrating T cells in VISTA mAb-treated natural Tregs and the differentiation of tumor-specific iTregs mice (Figs. 1–3). We have not detected VISTA expression on (Fig. 4). It remains to be determined whether VISTA expression tumor cells investigated. As such, we predict that VISTA on Tregs directly contributes to their suppressive function, targeted immunotherapy will be effective independent of its even though VISTA neutralization in vitro failed to show any expression on tumors. Similarly restricted hematopoietic direct effect on Treg proliferation, apoptosis, and Foxp3 sta- expression of human VISTA has been observed in human bility on in vitro cultured Tregs (Supplementary Fig. S4). melanoma, colorectal cancer, and lung cancer (data not Alternatively, it is possible that VISTA neutralization might shown). In contrast, PD-L1 is known to be expressed at enhance na€ve T-cell proliferation, making them more resis- high levels on nonhematopoietic tumor cells (Fig. 1; tant to Treg-mediated suppression. Future studies will uti- refs. 2, 35). It is therefore clinically relevant that VISTA mAb lize VISTAKO mice to tease out the underlying mechanisms demonstrated efficacy despite the high levels of PD-L1 whereby VISTA regulates the suppressive function of Tregs. within the TME. Our future studies will compare the efficacy In this context, both CTLA-4 and PD-1 impact on either the

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suppressive function and/or the peripheral induction of blocked to achieve better long-term tumor-specificmemory þ Foxp3 Tregs (9, 36–38). T-cell responses. Furthermore, the immunogenicity and the Third, in the B16 melanoma model, VISTA blockade FcR binding activity of the VISTA mAb might be critical altered the suppressive cellular signature of the TME, by limiting factors for achieving optimal target neutralization reducing the tumor-infiltrating monocytic MDSCs while and therapeutic efficacy, thus warrant future studies (50, 51). increasing the frequency of infiltrating effector T cells. Recent breakthroughs using CTLA-4 and PD-1 checkpoint Because there is no apparent deletion of both myeloid blockade have reinvigorated the field of cancer immunother- lineages and T-cell lineages in the periphery upon VISTA apy (2, 35). Multiple checkpoints control the development of mAb treatment (data not shown), such alterations within the antitumor immunity, each with their own unique signature tumor tissue likely reflect either the impaired migration of and mechanisms. Our studies provide compelling evidence monocytic MDSCs into tumor tissues, or impaired infiltra- supporting the role of VISTA as a negative immune check- tion of immature myeloid progenitor cells, which might point regulator that controls immunity against cancer. Taken differentiate into MDSCs at tumor site (39, 40). In contrast together with the findings that VISTA expression and function to melanoma, VISTA blockade did not significantly alter the on human leukocytes and within the human tumors have infiltration of the granuolytic MDSC population within the recapitulated the murine data (20), we present VISTA as a MB49 bladder tumors. This intriguing result might indicate promising new target for cancer immunotherapy, either as a different mechanisms whereby VISTA regulates monocytic single target or in combination with other immunotherapeutic and granuolytic myelopoiesis during tumor development. strategies. In addition to affecting MDSCs, VISTA blockade directly fi enhanced the migration of tumor-speci c effector T cells (i.e., Disclosure of Potential Conflicts of Interest TRP1 Tg CD4þ T cells) into tumor tissue (Fig. 3). Furthermore, L. Wang is a consultant/advisory board member of Immunext. J.L. Lines is a VISTA blockade enhanced the immune-stimulatory phenotype consultant/advisory board member of Immunext. M. Day is a research associate and has ownership interest (including patents) in ImmuNext. R.J. Noelle is CSO, of TIL DCs, which showed higher expression levels of MHCII has commercial research grant, other commercial research support, ownership and CD80, and cytokine production (i.e., IL-12 and TNF-a; Figs. interest (including patents), and is a consultant/advisory board member of fl 2 and 3). Collectively, these multiple effects of VISTA mAb ImmuNext. No potential con icts of interest were disclosed by the other authors. treatment facilitate the establishment of an immune-stimula- Authors' Contributions tory TME, which lead to enhanced antitumor immunity. Conception and design: L. Wang, R.J. Noelle It is clear that combination immunotherapy for cancer Development of methodology: L. Wang, W. Chen using multiple biologics will be critical for improving the Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Wang, I. LeMercier, J.L. Lines, P. Sergent therapeutic outcome. In addition to the monotherapeutic Analysis and interpretation of data (e.g., statistical analysis, biostatistics, targeting of negative checkpoint regulators, combining mul- computational analysis): L. Wang, I. LeMercier, W. Chen, J. Li, R.J. Noelle Writing, review, and/or revision of the manuscript: L. Wang, P. Sergent, R.J. tiple checkpoint blockades, or combining checkpoint block- Noelle ade with chemotherapy or cancer vaccines is moving into Administrative, technical, or material support (i.e., reporting or orga- clinical trials. Some examples of such combinatorial nizing data, constructing databases): L. Wang, M. Day, J. Li, P. Sergent approaches include CTLA4 blockade combined with GVax or Flt3vax (3, 41), CTLA4 blockade combined with PD-L1/ Acknowledgments The authors thank Dr. Alexander Rudensky (University of Washington School PD1 blockade (42, 43), PD1 blockade combined with vaccine of Medicine, Seattle, WA) for providing the Foxp3-GFP reporter mice, Dr. Marcus þ Bosenberg (Yale School of Medicine) for providing the B6.Cg-BrafCA/ (44, 45). Our initial studies explore a combination regimen lox5/lox5 T2 – Pten Tg (Tyr::Cre/ER )-inducible melanoma model, Dr. Mary Jo Turk using VISTA blockade together with a peptide vaccine (22 (Geisel School of Medicine) for maintaining the inducible melanoma model and 24, 33). Our data show promising synergistic effects for providing insightful discussions, and Dartlab Flow Cytometry Core Facility for treating both 2-day and 7-day established tumors. Future technical support. studies will focus on elucidating the cellular and molecular mechanisms of such synergy, as well as determining the Grant Support This work is supported by NIH grant CA164225 (L. Wang), Melanoma limitations of this combination platform and strategies for Research Foundation Career Development Award (L. Wang), Hitchcock improvement. For example, the use of defined peptides Foundation Research grant (L. Wang), AI048667 (to R.J. Noelle), Wellcome Trust (R.J. Noelle), and the Medical Research Council Centre for Transplantation and might lead to loss-of-antigen escape variants (46). The Biomedical Research Center at King's College London (R.J. Noelle). delivery of vaccine component might be optimized through The costs of publication of this article were defrayed in part by the payment of the use of nanoparticles or other particulate formulations or page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. TLR-peptide conjugates (47–49). Additional immune-suppressive pathways (i.e., other immune checkpoint pathways Received May 29, 2013; revised December 10, 2013; accepted December 19, such as PD-1, and immune suppressive cytokines) might be 2013; published online April 1, 2014.

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Isabelle Le Mercier, Wenna Chen, Janet L. Lines, et al.

Cancer Res 2014;74:1933-1944.

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