Published March 7, 2011 Article VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses Li Wang,1 Rotem Rubinstein,4,5 Janet L. Lines,1 Anna Wasiuk,1 Cory Ahonen,1 Yanxia Guo,1 Li-Fan Lu,1 David Gondek,1 Yan Wang,1 Roy A. Fava,3 Andras Fiser,4,5 Steve Almo,5 and Randolph J. Noelle1,2 1Department of Microbiology and Immunology, Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, NH 03766 2Department of Nephrology and Transplantation, Medical Research Council Centre for Transplantation, King’s College London, Guy’s Hospital, London SE1 9RT, England, UK 3Department of Veterans Affairs Medical Center, White River Junction, VT 05009 4Department of Systems and Computational Biology and 5Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461 Downloaded from The immunoglobulin (Ig) superfamily consists of many critical immune regulators, including the B7 family ligands and receptors. In this study, we identify a novel and structurally distinct Ig superfamily inhibitory ligand, whose extracellular domain bears homology to the B7 family ligand PD-L1. This molecule is designated V-domain Ig suppressor of T cell acti- vation (VISTA). VISTA is primarily expressed on hematopoietic cells, and VISTA expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells. A soluble VISTA-Ig jem.rupress.org fusion protein or VISTA expression on APCs inhibits T cell proliferation and cytokine pro- duction in vitro. A VISTA-specific monoclonal antibody interferes with VISTA-induced suppression of T cell responses by VISTA-expressing APCs in vitro. Furthermore, anti-VISTA treatment exacerbates the development of the T cell–mediated autoimmune disease experi- mental autoimmune encephalomyelitis in mice. Finally, VISTA overexpression on tumor cells interferes with protective antitumor immunity in vivo in mice. These findings show that on March 16, 2011 VISTA, a novel immunoregulatory molecule, has functional activities that are nonredundant with other Ig superfamily members and may play a role in the development of autoimmu- nity and immune surveillance in cancer. CORRESPONDENCE The immune system is tightly controlled by co- autoimmune diseases (Tivol et al., 1995; Randolph J. Noelle: stimulatory and co-inhibitory ligands and re- Waterhouse et al., 1995; Chambers et al., 1997). [email protected] ceptors. These molecules provide not only a The B7 family ligands have expanded to OR The Journal of Experimental Medicine [email protected] second signal for T cell activation but also a bal- include co-stimulatory B7-H2 (inducible T cell anced network of positive and negative signals co-stimulator [ICOS] ligand) and B7-H3, as Abbreviations used: BMDC, to maximize immune responses against infec- well as co-inhibitory B7-H1 (PD-L1), B7-DC BM-derived DC; EAE, experi- mental autoimmune encephalo- tion while limiting immunity to self. (PD-L2), B7-H4 (B7S1 or B7x), and B7-H6 myelitis; ICOS, inducible T cell The best characterized co-stimulatory li- (Greenwald et al., 2005; Brandt et al., 2009). co-stimulator; nTreg cell, natural gands are B7.1 and B7.2, which belong to the Accordingly, additional CD28 family receptors Treg cell; PLP, proteolipid pro- tein; VISTA, V-domain Ig Ig superfamily and are expressed on profes- have been identified. ICOS is expressed on ac- suppressor of T cell activation. sional APCs and whose receptors are CD28 tivated T cells and binds to B7-H2 (Yoshinaga and CTLA-4 (Greenwald et al., 2005). CD28 is et al., 1999). ICOS is a positive coregulator, expressed by naive and activated T cells and is which is important for T cell activation, differen- critical for optimal T cell activation. In contrast, tiation, and function (Yoshinaga et al., 1999; Dong CTLA-4 is induced upon T cell activation and et al., 2001). In contrast, PD-1 (programmed inhibits T cell activation by binding to B7.1/ death 1) negatively regulates T cell responses. B7.2, impairing CD28-mediated co-stimulation. B7.1 and B7.2 KO mice are impaired in adap- © 2011 Wang et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after tive immune response (Borriello et al., 1997), the publication date (see http://www.rupress.org/terms). After six months it is whereas CTLA-4 KO mice cannot adequately available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ control inflammation and develop systemic by-nc-sa/3.0/). The Rockefeller University Press $30.00 Supplemental Material can be found at: J. Exp. Med. Vol. 208 No. 3 577-592 http://jem.rupress.org/content/suppl/2011/03/04/jem.20100619.DC1.html 577 www.jem.org/cgi/doi/10.1084/jem.20100619 Published March 7, 2011 Downloaded from jem.rupress.org on March 16, 2011 Figure 1. Sequence and structural analysis of VISTA. (A) The primary amino acid sequence of mouse VISTA with the Ig-V domain, the stalk segment, 578 A new Ig superfamily inhibitory ligand | Wang et al. Published March 7, 2011 Article PD-1 KO mice developed lupus-like autoimmune disease diseases such as autoimmunity and cancer (Keir et al., 2008; or autoimmune dilated cardiomyopathy (Nishimura et al., Zou and Chen, 2008). In the context of extending our under- 1999, 2001). The autoimmunity most likely results from the standings of immune regulation, this study identifies a novel loss of signaling by both ligands PD-L1 and PD-L2. Recently, immune regulatory ligand, referred to as V-domain Ig sup- CD80 was identified as a second receptor for PD-L1 that trans- pressor of T cell activation (VISTA). We demonstrate that the duces inhibitory signals into T cells (Butte et al., 2007). extracellular Ig domain of VISTA shares significant sequence The two inhibitory B7 family ligands, PD-L1 and PD-L2, homology with the B7 family ligands PD-L1 and PD-L2, al- have distinct expression patterns. PD-L2 is inducibly ex- beit with unique structural features that distinguish it from pressed on DCs and macrophages, whereas PD-L1 is broadly the B7 family members. Together with its distinctive expres- expressed on both hematopoietic cells and nonhematopoietic sion pattern and functional impact on T cell activation, it is cell types (Okazaki and Honjo, 2006; Keir et al., 2008). Con- concluded that VISTA represents a novel immune regulatory sistent with the immune-suppressive role of PD-1 receptor, a ligand within the Ig superfamily. study using PD-L1/ and PD-L2/ mice has shown that both ligands have overlapping roles in inhibiting T cell prolif- RESULTS eration and cytokine production (Keir et al., 2006). PD-L1 Cloning and sequence and structural analysis of VISTA deficiency enhances disease progression in both the nonobese Affymetrix analysis of activated versus resting mouse + + diabetic model of autoimmune diabetes and the mouse model CD25 CD4 natural Treg cells (nTreg cells) revealed the ex- Downloaded from of multiple sclerosis (experimental autoimmune encephalo- pression of a gene product (RIKEN cDNA 4632428N05 or myelitis [EAE]; Ansari et al., 2003; Salama et al., 2003; Latchman 4632428N05Rik) with unknown function but with sequence et al., 2004). PD-L1/ T cells produce elevated levels of the homology to the Ig superfamily. A 930-bp gene product was proinflammatory cytokines in both disease models. In addi- cloned from the mouse CD4+ T cell cDNA library, which tion, BM chimera experiments have demonstrated that the matched the predicted size and sequence. Silico sequence and tissue expression of PD-L1 (i.e., within pancreas) uniquely structural analysis predicts a type I transmembrane protein of contributes to its capacity of regionally controlling inflamma- 309 aa upon maturation. Its extracellular domain contains a jem.rupress.org tion (Keir et al., 2006, 2007; Grabie et al., 2007). PD-L1 is also single extracellular Ig-V domain of 136 aa, which is linked to highly expressed on placental syncytiotrophoblasts, which a 23-aa stalk region, a 21-residue transmembrane segment, critically control the maternal immune responses to alloge- and a 97-aa cytoplasmic domain (Fig. 1 A). The cytoplasmic neic fetus (Guleria et al., 2005). tail of 4632428N05Rik does not contain any signaling do- Consistent with its immune-suppressive role, PD-L1 po- mains. Based on the structural feature of the Ig-V domain and tently suppresses antitumor immune responses and helps tu- its immune-suppressive function that is shown in this study, on March 16, 2011 mors evade immune surveillance. PD-L1 can induce apoptosis this molecule is named VISTA. of infiltrating cytotoxic CD8+ T cells, which express a high A BLAST (Altschul et al., 1990) sequence search with the level of PD-1 (Dong et al., 2002; Dong and Chen, 2003). VISTA Ig-V domain identified PD-L1 of the B7 family as the Studies have shown that blocking the PD-L1–PD-1 signaling closest evolutionarily related protein with a borderline signif- pathway, in conjunction with other immune therapies, pre- icant e-value score of 104 and with a sequence identity of vents tumor progression by enhancing antitumor CTL activ- 24%. These relationships were confirmed by computationally ity and cytokine production (Iwai et al., 2002; Blank et al., threading the sequence of the VISTA Ig-V domain through 2004, 2005; Geng et al., 2006). More recently, we have shown all known structures. This threading exercise indicated with a that PD-L1 expression on DCs promotes the induction of high but not certain confidence (P < 0.001) that the structure + + adaptive Foxp3 CD4 regulatory T cells (aTreg cells), and PD- of VISTA is most similar to the Ig-V domains of PD-L1 (Pro- L1 is a potent inducer of aTreg cells within the tumor micro- tein Data Bank accession no.