The Negative Checkpoint Regulator VISTA in and Tolerance

Isabelle Le Mercier, PhD Randolph Noelle Lab Presenter Disclosure Information

Isabelle Le Mercier

The following relationships exist related to this presentation:

In part supported by a grant from ImmuNext, Inc. and Janssen Biotech, Inc.

2 What are the Immune Checkpoints ? IG SUPERFAMILY TNF/TNFR SUPERFAMILY

B7-1/CD80 CTLA-4 -2/CD86 CD28 B7-1/CD80 OX40 OX40L PD-L1/B7-H1 PD-1

PD-L2/B7-DC RGMb CD40 CD154/CD40L B7-H2/ICOSL ICOS

B7-H3 CD27 CD70 APC B7-H4 APC VISTA VISTA 4-1BB 4-1BBL

MHC TCR LAG3 GITR GITRL Gal-9 TIM-3 HVEM BTLA HVEM LIGHT CD226 PVR TIGIT VISTA (PD-1H, DD1a)

V-region of Immunoglobulin containing Suppressor of T cell Activation

Negative Checkpoint Regulator

• Membrane of the Ig SF • 92 Aa cytoplasmic domain • Homologous to PD-L1 () or PD-1 ()

• Hematopoietically-restricted * VISTA is expressed on: • Myeloid cells • T cells • In the TME on MDSCs, human TILs

 Controls inflammation through the suppression of T cell and myeloid cell mediated activities  It is likely both a ligand and a receptor VISTA Biology VISTA is expressed on T cells and myeloid cells

100 Total CD4 CD8 CD11b high B cells spleen80 T cells T cells cells cells 60 Murine

% % of Max 40 spleen 20

0 10 0 10 1 10 2 10 3 10 4 VISTA

Human PBMCs

VISTA

Li Wang et al. J Exp Med 2011 Louise Lines et al. Cancer Res. 2014 VISTA is expressed on T cells and myeloid cells

Anti-VISTA on WT Anti-VISTA on KO Ham Ig on WT

Marginal Zone T cell zone SPLEEN

CD11b CD3 VISTA Over expression of VISTA on APCs inhibits T cell activation A20 B cell line + OVA peptide + OVA specific (D011) TCR Tg T cells

VISTA on APCs inhibits T cell proliferation VISTA on APCs inhibits activation markers upregulation A20-RFP A20-VISTA

CFSE

Li Wang et al. J Exp Med 2011 Jiannan Li Over expression of VISTA on tumor cell prevents antitumor immunity

2 weeks MCA105-VISTA MCA105-RFP Immunization Tumor challenge Irradiated tumor cells

Li Wang et al. J Exp Med 2011 Activated T cell and myeloid cell phenotype in VISTA deficient mice

 Mild splenomegaly/lymphadenopathy in aged mice

 Increased in myeloid populations

 Gradual accumulation of spontaneously activated T cells (CD44+ CD62L low)

 Enhanced T-cell responsiveness

 Increased infiltration in specific organs

 No systemic or organ specific autoimmune disease VISTA deficiency exacerbates EAE, model of MS

Myelin specific (2D2) TCR Tg WT Myelin specific (2D2) TCR Tg x VISTA KO - No disease - Spontaneous EAE disease (MS model) - Ascending paralysis - Reduced survival

Li Wang et al. PNAS 2014 VISTA deficiency on the host exacerbates passive EAE

Passive EAE, MS model Immune deficient

Myelin specific T cells Host KO Host KO Host WT T cells KO T cells WT T cells KO

HostHost WTWT TT cellscells WTWT Disease score Disease

Li Wang et al. PNAS 2014 VISTA deficiency in SLE prone mice exacerbates disease

Sle1/Sle3 WT Sle1/Sle3 x VISTA KO - Late incidence lupus - Early incidence lupus (starts 7 weeks) - Reduced survival (< 20 weeks)

100 500 B6 VISTA -/- 400 sle1,3 300 sle1,3xVISTA -/- 50 200 Protein (mg/dL) Protein 100 survival Percent 0 0 5 10 15 20 25 0 0 10 20 30 40 50 Weeks Weeks

Sabrina Ceeraz Delong

o

k

A

T

S

I V

**

6 B 0

750 500 250

1000 # IFNg spots in dLN in spots IFNg # eg function suppressive / conversion Allogeneic skin skin Graft Allogeneic 1 week Time (anti-CD40L) Costimulatory Blockade VISTA deficiency prevents allograft tolerance allograft prevents deficiency VISTA Donor Specific Transfusion Donor Transfusion Specific 0 20 40 60 80 0

75 50 25

100

Graft survival survival Graft Graft No defect in graft specific T cell deletion and Tr and deletion cell T specific graft in defect No Loss of VISTA from the host is responsible for enhanced graft T cell response

ELISPOT Recall

OVA specific T cells OVA skin Graft

2.0×10 4 ** *** Host WT host VISTA KO host 4 T cell 1.5×10

WT WT  WT WT  KO 1.0×10 4 T cell Normal Elevated response response 5.0×10 3 # IFNg spots in dLN in spots IFNg # KO  WT KO  KO VISTA KO 0.0 Normal Elevated T cell t t s s response response o o h h host T host W KO WT KO WT T cells KO T cells VISTA intrinsically regulates innate immunity

Listeria Spleen IV infection Bug (CFU) count

Early infection control Control in T cell deficient in VISTA KO RAG KO x VISTA KO

WT ** 6 KO 6 10 *** 10 10 5 10 5

10 4 10 4

10 3 10 3 CFU/spleen CFU / spleen / CFU

10 2 10 2 0 1 2 3 4 5 RAG Days post-infection

Louise Lines RAG x VISTA VISTA negatively regulates T cells and myeloid cells activities

 Negatively regulates T cell responses

- acts as a ligand expressed on APCs (host)

 Negatively regulates innate immunity

- acts as a receptor

 Prevents autoimmunity in various suceptible models

 Is critical for tolerance induction VISTA in Cancer VISTA is highly expressed in tumor infiltrating MDSCs

PD-L1 VISTA B16 OVA Melanoma B16 OVA CD45 TILs Tumor cells Isotype

Bladder carcinoma Melanoma MB49 B16 OVA MDSCs CD11b hi CD11c - Gr1 +

TILs DAPI CD11b VISTA dLN CD11b+/VISTA+ cells Non dLN Isotype

VISTA Le Mercier et al. Cancer Res. 2014 Hypoxia upregulates VISTA expression in the TME

HRE1 HRE2 Bladder carcinoma MB49 ACGTG GCGTG CD45 TILs VISTA -1200 -800 -400

21% O 2 1% O 2

Pimonidazole

Gr1 Non hypoxic

Relative Luciferase Units Luciferase Relative Hypoxic Empty HRE1 Mut-HRE1 HRE2 Mut-HRE2 Isotype Vector

CD11b Jie Deng Rodwell Mabaera VISTA Anti -VISTA monotherapy controls tumor growth in multiple models

Melanoma Bladder carcinoma Fibrosarcoma B16-OVA MB49 MCA105

Le Mercier et al. Cancer Res. 2014 Anti -VISTA mediated changes in TME and antitumor immune responses

Melanoma B16-OVA

Le Mercier et al. Cancer Res. 2014 Anti -VISTA synergizes with anti -PD-1 to control tumor growth CT26 Colon Carcinoma

Early intervention (2-3 mm) Late intervention (5-6 mm)

Control Ig 1500 control 2500 ) ) anti-VISTA13F3 3 3 2000 anti-PD-1PD-1 1000 Combocombo 1500 co ntro l 1000 500 500 Tumor volume (mm volume Tumor Tumor volume (mm volume Tumor 0 0 0 10 20 30 5 7 1012141719212426293133 Days post-tumor inoculation Days post-tumor inoculation Combo : 50% CR Other groups 0% Louise Lines Anti-VISTA mediated changes in cancer immunity

 VISTA is highly expressed in the TME: - Expression on MDSCs - Transcriptionally regulated by hypoxia

 Anti-VISTA mediated myeloid changes: - Decrease in MDSCs - Increase in macrophages and activated DCs

 Anti-VISTA mediated lymphoid changes: - Increase in T cells infiltration and recall responses

 Anti-VISTA synergizes with anti-PD-1 Therapy VISTA is a potential target in lung cancer

Tumor cells

TILs VISTA regulation of immunity

VISTA is a negative regulator ligand of TUMOR T cell activation 1 Naïve/early activated Tumor T cell VISTA infiltrating MDSCMDSC VISTA T cell VISTA VISTA is a negative APC regulator receptor VISTA of T cell activation 3 VISTA is a receptor that regulates the biology of myeloid cells 2

1. Wang (J Exp Med 2011), Wang (PNAS 2014), Le Mercier (Cancer Res 2014); Lines (Cancer Immunol 2014); 2. Bharaj (PLoS One 2014) and unpublished data 3. Flies (J Clin Invest 2014), Flies (J Immunol 2015), Flies (J Immunol 2011) Geisel School of Medicine at Dartmouth

Janssen Pharmaceutical Companies of Johnson & Johnson

Randolph Noelle Team ImmuNext Rodwell Mabaera, M.D. Ph.D. Dave DeLucia Louise Lines, Ph.D. Jay Rothstein Sabrina Ceeraz Delong, Ph.D Catherine Carriere Anna Kuta, Ph.D. Tse Dan Elizabeth Nowak, Ph.D. Phil Gobeil Tommy Broughton David Blair Jie Deng Mike Molloy Yuchi Lee Yalin Guo Jiannan Li Xin Huang Aurelien Sarde Dongxu Son Petra Sergent Dov Pechenick Maria Day Rheumatology Ron Hirte Christopher Burns , M.D. Nick Schwertner Mike Nelson