VISTA, a novel immune checkpoint protein ligand that suppresses anti-tumor T cell responses Li Wang Dartmouth Medical School The B7 Immunoglobulin Super-Family immune regulators APC T cell B7.1/2 - CD28 Co-stimulatory: B7-H2 - ICOS MHC TCR B7 – CTLA-4 Co-inhibitory PD-L1/2 – PD-1 Checkpoint Pathways: B7-H3 – TLT2 B7-H4 – ? etc…….. Immune checkpoint-mediated T cell suppression during tumorigenesis Tumor PDL-1 PD-1 CTLA-4 B71/2 DC T effector CD28 -1 cells D PD-1 PDL-1 P -1 L D P Myeloid Suppressor CTLA-4 Foxp3+ cells Tregs Release the brake: Immune checkpoint blockade in cancer immunotherapy Antibody-mediated CTLA-4 blockade in combination with a cellular vaccine (Gvax) induced regression of established poorly immunogenic B16 melanoma. ● Elsas et.al. 1999, J Exp Med 190:355-366 Ipilimumab, the human aCTLA-4 mab, was approved by the FDA in March 2011 to treat patients with late-stage melanoma. Ipilimumab has also undergone early phase trials for other cancers, such as lung cancer and prostate cancer. ● Calabro et.al. 2010, Semin Oncol 37:460-467 Release the brake: Immune checkpoint blockade in tumor immunotherapy Blocking the PD-L1:PD-1 pathway, in conjunction with other immune therapies, inhibits tumor progression. ● Blank et.al. 2005, Cancer Immunol Immunother 54:307-314 ● Hirano et.al. 2005, Cancer Res 65:1089-1096 ● Geng et.al. 2006, Int J Cancer 118:2657-2664 ● Li et.al. 2009, Clin Cancer Res. 15: 1632-4 ● Pilon-Thomas et.al. 2010 J Immunol. 184: 3442-9 ● Weber J, 2010, Semin Oncol 37(5):430-9 MDX-1106, the human anti-PD-1 mab has entered clinical trials. ● Brahmer JR et.al. 2010, J Clin Oncol. 28(19):3167-75 Phase I study of anti-programmed death-1 (MDX-1106) as single-agent in refractory solid tumors is well tolerated and demonstrates clinical anti-tumor activity. The B7 Immunoglobulin Super-Family immune regulators APC T cell B7.1/2 - CD28 Co-stimulatory: B7-H2 - ICOS MHC TCR B7 – CTLA-4 Co-inhibitory PD-L1/2 – PD-1 Checkpoint Pathways: B7-H3 – TLT2 B7-H4 – ? VISTA VISTA: a new checkpoint protein, and a V-domain Immunoglobulin Suppressor of T cell Activation Ig-v domain TM cytoplasmic Ig-v domain structual model of VISTA, using PD-L1 as template Sequence of the Ig-v domain loop VISTA is highly expressed on CD11bhi myeloid cells Monocytes/macrophages DCs VISTA is expressed on T cells Peripheral LN Spleen 100 100 80 80 60 60 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 VISTA CD8+ cells CD4+ cells isotype + Foxp3 nTregs CD4+ naive CD4+ memory 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 VISTA VISTA expression on human PBMC cells CD11c+ CD8+ CD4+ Isotype CD11b+ Human VISTA Data contributed by Janet L. Lines Immobilized VISTA-Ig fusion protein inhibits T cell activation T cell proliferation Plate-bound CD3 + VISTA-Ig or control Ig CD3 : VISTA or control Ig 1:1 1:4 100 100 80 80 CD4+ 60 60 T cells 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 Control Ig 10 10 10 10 10 10 10 10 10 10 FL1-H: CFSE FL1-H: CFSE VISTA-Ig 100 100 80 80 + CD8 60 60 T cells 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL1-H: CFSE FL1-H: CFSE Immobilized VISTA-Ig fusion protein inhibits T cell activation Plate-bound CD3 + VISTA-Ig or control Ig ► Inhibit proliferation, but do not enhance apoptosis. ► Inhibit activation markers: CD69, CD44, CD62L. ► Inhibit T cell cytokine production (IL2, IFN etc). ► Suppression can be partially rescued by exogenous IL-2. ► PD-1 KO T cells are also inhibited. VISTA promotes the induction of adaptive Tregs Plate-bound CD3 + VISTA-Ig or control-Ig +/- TGF human mouse 30 40 % % + + 30 20 20 10 10 Foxp3GFP Foxp3GFP 0 0 VISTA-Ig - 2.5 5 ug/mL VISTA-Ig - - + TGF - -- TGF - + + VISTA expression on APC suppress T cell proliferation APC (A20 cells) + T cells (OTII) + peptide OVA peptide: 5 ng/mL 10 ng/mL A20-RFP A20-VISTA 100 100 70 80 80 60 60 60 50 40 40 40 30 20 20 20 0 0 CFSE-low% 100 101 102 103 104 100 101 102 103 104 10 CFSE 0 5 10 A20-VISTA A20-RFP OVA peptide (ng/ml) ● Similar results are obtained using BM-derived DCs that are transduced with VISTA-expressing retrovirus VISTA expression on tumors impairs protective tumor immunity Day-14 Day 0 Measure tumor growth (mm2) Irradiated Live tumor MCA105 Challenge: MCA105-RFP MCA105-VISTA MCA105-control MCA105-VISTA 125 ) 2 100 75 50 P = 0.0011 25 Tumor size (mm size Tumor 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (days) VISTA expression on tumors impairs protective tumor immunity Day-14 Day 0 Measure tumor growth (mm2) Irradiated Live tumor MCA105 Challenge: MCA105-RFP MCA105-VISTA 150 ) 2 T-depleted, ns 100 50 P= 0.0011 Tumor size (mm size Tumor 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Time (days) MCA105-control MCA105-VISTA MCA105-control T-depletion MCA105-VISTA T-depletion VISTA monoclonal antibody treatment enhances inflammatory disease Day0 treat with control-Ig or VISTA SJL Monitor EAE progression Adoptive transfer primed pathogenic CD4+ T cells 3.0 (n=7) 13F3 100 control Ig(n=8) 90 2.5 p<0.0001 80 13F3 e r 2.0 70 control-Ig p<0.0001 60 1.5 50 40 1.0 Disease sco 30 20 0.5 Disease incidenceDisease (%) 10 0.0 0 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 7 9 11 13 15 17 19 21 23 25 27 29 31 33 Time (days) Time (days) VISTA-specific antibody controls tumor growth Bladder tumor IFN ELISPOT MB49 (s.c) 175 *** 150 125 100 75 50 25 spots/250k cells 0 control 13F3 VISTA-specific antibody controls tumor growth Fibrosarcoma Thymoma MCA105 (s.c) EG7 (s.c) VISTA-specific antibody controls tumor growth Ovarian tumor ID8-luciferase (peritoneal) Control-Ig 13F3 Day30 Day55 p value:0.03 p= 0.0167 ) ) 5 4 (n=8) 120 60 (n=8) 100 50 80 40 60 30 40 20 20 10 Total photon flux (X 10 (X flux photon Total Total photon flux (X 10 (X flux photon Total 0 0 control +13F3 control +13F3 VISTA-specific antibody controls tumor growth Skin tumor B16F10-OVA Vaccine: CD40 agonist + LPS + OVA control 13F3 control + vaccine 13F3+vaccine 175 ) 2 150 125 100 75 50 tumor size (mm 25 0 11 13 15 17 19 21 23 25 Days VISTA-specific antibody controls tumor growth B16F10 (day-2 prophylactic treatment) 175 150 125 100 *** control (n=10) 13F3 (n=18) 75 MIH5 50 tumor size (mm2) 25 0 6 8 10 12 14 16 18 20 22 Days Combinatorial blockade of VISTA and PD-L1/PD-1 results in better tumor control B16F10 (day+4 therapeutic treatment) ) 2 200 150 control (n=10) MIH5 (n=10) 100 *** 113F3 (n=16) combo (n=16) 50 tumor size (mm 0 6 8 10 12 14 16 18 20 Days VISTA and PD-L1/PD-1 synergize to suppress T cell proliferation VISTA-Ig fusion protein + PD-L1-Ig fusion protein + CD3/CD28 CD4+ T cells CD8+ T cells 100 100 75 75 50 50 25 25 CFSE-low cells% 0 CFSE-low cells% 0 -Ig -Ig bo -Ig -Ig bo NA m NA m VISTA PDL1 co VISTA PDL1 co The inducible melanoma model Tyr::Cre/ERT2: Tyrosinase promoter driven expression of Cre-ERT2, permitting tamoxifen-inducible, melanocyte-specific cre expression. BrafCA: carrying a conditional BrafV600E allele, permitting cre-mediated expression of BrafV600E Pten1ox5: carrying a conditional allele of Pten, permitting cre-mediated deletion of exon 5 Dankort et al 2009 Nature Immunology High VISTA expression within the tumor microenvironment gated CD11c+ CD11b+ 19.8 3.18 33 50.2 TIL naïve LN dLN isotype VISTA 6.59 40.6 38.7 7.85 CD11C CD11b VISTA VISTA blockade inhibited the growth of the inducible melanoma Conclusions VISTA functions as a novel immune checkpoint protein ligand: controls inflammation and autoimmunity. impairs the generation of anti-tumor immunity. VISTA antibody-mediated blockade either alone, or in combination with other checkpoint blockade might provide a novel therapeutic strategy for cancer immunotherapy. J Exp Med, 2011, 208(3):577-92 ACKNOWLEDGEMENTS Noelle Lab Almo Lab (Dartmouth Medical School, NH) (Albert Einstein College of Medicine) (King’s college in London, UK) Steve Almo Randy Noelle Rotem Rubinsteine Janet Louise Lines Cory Ahonen Andras Fiser Petra Sergent (Albert Einstein College of Medicine).