VSIG-3/IGSF11 is a Ligand of VISTA/PD-1H and Inhibits Human T Cell Function Jinghua Wang, Guoping Wu, Brian Manick, Vida Hernandez, Mark Renelt, Ming Bi, Jun Li, and Vassilios Kalabokis | R&D Systems, 614 McKinley Place NE, Minneapolis, MN 55413 Abstract Results A B A B B7 family members and their receptors play a central role in the regulation of 3.0 80 1.0 500 ) ) 0 T cell responses through T cell co-stimulatory and co-inhibitory pathways that 0 70 4 4 2.5 . constitute very attractive targets for the development of immunotherapeutic . 0.8 400 10 µg/mL VSIG-3 IgG -Fc 60 Mouse Anti-hVISTA 1 5 µg/mL VSIG-3 IgG1-Fc 2.0 + VISTA IgG -Fc drugs. In this study, we report that VSIG-3/IGSF11 is a ligand of the B7 family Sheep Anti-hVISTA 1 + Sh x hVISTA VSIG-3 IgG -Fc 50 VSIG-3 IgG -Fc member VISTA/PD-1H that inhibits human T cell functions through a novel 0.6 Mouse IgG Control 1 1 1.5 Sheep IgG Control 300 VSIG-3/VISTA pathway. An extensive functional ELISA binding screening 40 1.0 0.4 (pg/mL) IL-17 assay reveals that VSIG-3 binds the new B7 family member VISTA, but does 30 200 CCL/RANTS (pg/mL) VISTA Binding (Mean .D VISTA not interact with other known members of the B7 family. Furthermore, Binding (Mean .D VSIG-3 0.5 0.2 20 VSIG-3 inhibits human T cell proliferation in the presence of T cell receptor 100 10 -2 -1 0 1 -1 0 1 signaling. In addition, VSIG-3 significantly reduces cytokine and chemokine 10 10 10 10 10 10 10 -2 -1 0 1 10 10 10 10 10-2 10-1 100 101 102 VSIG-3 IgG -Fc (g/mL) Anti-human VISTA or isotype antibodies (g/mL) production by human T cells including IFN-g, IL-2, IL-17, CCL5/RANTES, 1 VSIG-3 IgG -Fc or VISTA IgG -Fc (g/mL) 1 1 VSIG-3 IgG1-Fc or Sh x hVISTA (g/mL) Figure 1. VSIG-3 binds to VISTA. (A) Recombinant human VSIG-3 protein specifically binds to recombinant human VISTA in a functional ELISA binding as- Figure 4. VSIG-3 is a specific ligand of VISTA. (A)The extracellular domain of VISTA attenuates VSIG-3 functions. Pretreatment of immobilized Recom- CCL3/MIP-1a, and CXCL11/I-TAC. Anti-VISTA neutralization antibodies say. Recombinant Human VISTA IgG -Fc (R&D Systems, Catalog # 7126-B7) was immobilized at 2 μg/mL (100 μL/well), and the indicated concentrations 1 binant Human VSIG-3 IgG -Fc (10 μg/mL) with the indicated concentrations of Recombinant Human VISTA IgG -Fc for 1 hour significantly attenuated the of Recombinant Human VSIG-3 IgG -Fc (R&D Systems, Catalog # 9229-VS) were added. The concentration of VSIG-3 IgG -Fc that produced 50% of the op- 1 1 1 1 ability of VSIG-3 to inhibit CCL5/RANTES secretion from anti-CD3 activated PBMCs. (B) Anti-Human VISTA Antibody attenuates VSIG-3 inhibitory functions. attenuate the binding of VSIG-3 to VISTA, as well as VSIG-3-induced T cell timal binding response was approximately 0.25 μg/mL (3.4 nM). (B) Anti-human VISTA antibodies neutralize the binding of VSIG-3 and VISTA. Biotinylated Human PBMCs were treated with immobilized Anti-Human CD3 (1 μg/ml), and various concentrations of Recombinant Human VSIG-3 IgG -Fc, or 5 μg/ Recombinant Human VISTA IgG -Fc (1 µg/mL) was pretreated with the indicated concentrations of Anti-Human VISTA Antibodies (R&D Systems, Catalog # 1 1 mL of Recombinant Human VSIG-3 IgG -Fc plus the indicated concentrations of a Sheep Anti-Human VISTA Antibody (R&D Systems, Catalog #AF7126), or inhibition. Thus, we have identified a novel B7 pathway that is able to inhibit AF7126 or # MAB71261) or Isotype Controls (R&D Systems, Catalog # 5-001-A or # MAB0041) and then added to ELISA plates containing immobilized Re- 1 Sheep IgG (R&D Systems, Catalog # 5-001-A) as a control. VSIG-3 significantly inhibited anti-CD3 induced IL-17 production in a dose-dependent manner. combinant Human VSIG-3 IgG -Fc (2 µg/mL). After washing away any unbound proteins, streptavidin-HRP (R&D Systems, Catalog # DY998) and substrate 1 Anti-human VISTA Antibody attenuated the inhibitory effect of VSIG-3 on IL-17 secretion from anti-CD3-activated PBMCs. The Sheep IgG control did not solution (R&D Systems Cat# DY999) were added to the wells. The color development was stopped and the intensity of the color was measured using an human T cell proliferation and cytokine production. This unique VSIG-3/VISTA alter the inhibitory effect of VSIG-3 on IL-17 secretion (data not shown). Results are representative of three independent experiments. co-inhibitory pathway may provide new strategies for the treatment of human ELISA plate reader. 24 hours 4 hours A B cancers, autoimmune disorders, infection, and transplant rejection, and may CCL5/RANTES MIP-1α/β IL-17A CXCL11/I-TAC CCL5/RANTES MIP-1α/β IL-17A CXCL11/I-TAC help to design better vaccines. 100 1300 90 Control 10 g/mL IgG1-Fc 80 1200 Introduction 70 (pg/mL) γ 60 CCL5/RANTES MIP-1α/β IL-17A CXCL11/I-TAC CCL5/RANTES MIP-1α/β IL-17A CXCL11/I-TAC IFN- 1100 Critical modulators of the immune system, also referred to as immune IL-2 (pg/mL) checkpoint regulators, generate co-stimulation or co-inhibition of T cell 50 10 g/mL responses.1 Checkpoint blockage can generate potent anti-tumor responses 40 1000 VSIG-3 IgG1-Fc by enhancing the immune system’s ability to seek and destroy cancer cells. 30 -1 0 1 Members of the B7 family have emerged as important checkpoint regulators 10-1 100 101 10 10 10 VSIG-3 IgG -Fc (g/mL) VSIG-3 IgG1-Fc (g/mL) and recently the United States Food and Drug Administration has approved 1 Figure 2. VSIG-3 inhibits cytokine and chemokine production by PBMCs. Human PBMCs were stimulated with a combination of plate-bound Mouse Anti- C D 2,3 Human CD3 antibody (1 μg/mL; R&D Systems, Catalog # MAB100) and either plate-bound Recombinant Human VSIG-3 IgG -Fc (10 μg/mL) or Control the use of anti-PD-1 and anti-CTLA-4 antibodies for cancer immunotherapy. 1 IgG -Fc (10 μg/mL; R&D Systems, Catalog # 110-HG) for 24 or 48 hours. Cytokine levels in the cell culture supernatants were measured using the 120 1 2800 There is an urgent need for more agents to enter clinical use. Proteome Profiler™ Human XL Cytokine Array Kit (R&D Systems, Catalog # ARY022B). 2600 100 300 V domain-containing Ig suppressor of T cell activation (VISTA), also termed 550 2400 80 Differentiation of Embryonic Stem Cells 1 (Dies1), Gi24, and PD-1 homolog 250 * * 2200 (PD-1H), is a 55- to 65-kDa type I Ig membrane protein with an extracellular * 500 2000 60 (RF) Cell Proliferation high + domain homologous to PD-L1. VISTA is highly expressed on mature CD11b 200 (pg/mL) IL-17 VSIG-3 IgG -Fc + + VSIG-3 IgG1-Fc 450 1 1800 myeloid-derived APCs and to a lesser extent on CD4 T cells, CD8 T cells, * IgG -Fc 40 IgG1-Fc 1 4 150 1600 and regulatory T cells, and is also found on tumor infiltrating lymphocytes. It (pg/mL) IL-17 Human CD3 Human + 400 has been demonstrated that VISTA is a co-inhibitory receptor on CD4 T cells CCL/RANTS (pg/mL) * 20 1400 100 * * -1 0 1 -1 0 1 or a co-inhibitory ligand for T cells. In vitro, a VISTA-Ig fusion protein inhibits * 10 10 10 10 10 10 350 * VSIG-3 IgG -Fc (g/mL) VSIG-3 IgG1-Fc (g/mL) CD3-stimulated T cell activation, proliferation, and production of cytokines, 1 50 Figure. 5. VSIG-3 negatively regulates human T cell activation. (A-C) VSIG-3 inhibits anti-CD3 induced IL-2, IFN-g, and IL-17 production by human CD3+ 5 −/− + + + g -1 0 1 10-1 100 101 T cells in a dose-dependent manner. Human CD3 T cells were isolated from PBMCs using a MagCellect™ Human CD3 T Cell Isolation Kit (R&D Systems, such as IL-2 and IFN- . Another group reported that VISTA CD4 T cells 10 10 10 Catalog # MAGH101). Human T cells were then incubated with an immobilized Mouse Anti-Human CD3 Monoclonal Antibody (1 μg/mL) and the indicat- g/mL g g/mL ed concentrations of Recombinant Human VSIG-3 IgG1-Fc or IgG1-Fc for 24 hours. The levels of IL-2, IFN- , and IL-17 in the cell culture supernatants were showed stronger Ag-specific proliferation and cytokine production than wild- g ® measured using the Human IL-2, IFN- , and IL-17 Quantikine ELISA Kits (R&D Systems, Catalog # D2050, # DIF50, and # D1700). IgG1-Fc controls did not + g 600 170 alter anti-CD3-induced IL-2, IFN- , or IL-17 secretion (data not shown). Results are representative of three independent experiments. (D) VSIG-3 inhibits type CD4 T cells, which supports the thesis that VISTA functions as an anti-CD3-induced human CD3+ T cell proliferation in a dose-dependent manner. Human T cells were incubated with an immobilized Mouse Anti-Human + CD3 Monoclonal Antibody (1 μg/mL) and the indicated concentrations of Recombinant Human VSIG-3 IgG -Fc or IgG -Fc for 72 hours.