Search

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement. V-domain immunoglobulin suppressor of T cell activation (VISTA) selectively engages P-selectin glycoprotein ligand-1 (PSGL-1) and suppresses T cells at acidic pH similar to those in tumour microenvironments, thereby mediating resistance to anti-tumour immune responses.

Background: Therapeutic blockade of the immune checkpoints CTLA-4 and PD-1/PD-L1 has provided durable survival benefits in multiple malignancies. However, additional treatment options are often required to maximally reverse immune dysfunction. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an orphan B7 family ligand that is highly expressed on immunosuppressive myeloid cells and has been shown to inhibit T-cell responses in vitro and in preclinical models of cancer. Here we report that VISTA, a ligand for the receptor P-selectin glycoprotein ligand-1 (PSGL-1), uses a histidine-rich interface to engage PSGL-1 and suppress immune responses selectively in acidic environments, such as tumor beds. Methods: Recombinant VISTA multimers were used to assess binding to cells and recombinant PSGL-1 over a range of pH values (6.0-7.4). Antibodies against human and mouse VISTA were used to map binding and functional epitopes. Acidic pH receptor-based ligand capture was used to identify PSGL-1 as a VISTA receptor. X-ray crystallography was used to resolve the VISTA structure in complex with an anti-VISTA antigen-binding fragment. The MC38 mouse tumor model was used to assess the effects of VISTA deficiency and the effects of VISTA antibody blockade alone and combined with anti-PD-1 in vivo. Results: Recombinant VISTA bound leukocytes at pH 6.0 but was not detectable at pH 7.4. Antibodies in a single epitope bin blocked VISTA binding and reversed VISTA suppression of T cells. VISTA-mediated inhibition of T cells was detectable at pH 7.4 but was more pronounced below pH 7.0, suggesting that VISTA functions selectively in acidic conditions. VISTA’s structure was resolved at 1.6 Å and characterized by a histidine-rich extension of the immunoglobulin V domain central β-sheet. VISTA blocking antibodies, but not nonblocking antibodies, bound this β-sheet region. Engineered antibodies could distinguish this epitope in its active and inactive states at acidic and neutral pH, respectively. Receptor capture on T cells at acidic pH identified PSGL-1 as a VISTA receptor. T-cell PSGL-1 CRISPR ablated VISTA binding, whereas PSGL-1 expression on CHO cells conferred VISTA binding at acidic pH. Thus, an antibody that blocks mouse VISTA binding to mouse T cells at acidic pH combined with a PD-1 blocking antibody was shown to enhance anti-tumor T-cell responses and drive MC38 tumor rejection in vivo. Conclusions: VISTA is a highly pH-selective ligand for PSGL-1. VISTA antibody blockade reverses immune suppression in vitro and in vivo, especially when combined with PD-1 antibody blockade. Our results identify acidic pH as a direct regulator of VISTA engagement with PSGL-1 and suggest new strategies to enhance anti-tumor T-cell responses. Citation Format: Robert J. Johnston, Linhui Julie Su, Jason Pinckney, David Critton, Arathi Krishnakumar, Martin Corbett, Andrew Rankin, Rose A. DiBella, Lynne Campbell, Xiaodi Deng, Haibin Chen, Alexander Kozhich, Jim Holloway, Zheng Yang, Ginger Rakestraw, Michael Quigley, Alan J. Korman. Acidic pH selective binding of VISTA to PSGL-1 and anti-tumor activity of combined VISTA and PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1548.

The nuclear receptor protein superfamily is a large group of transcription factors involved in many aspects of animal development, tissue differentiation, and homeostasis in the higher eukaryotes. A subfamily of receptors, ERRα and β (estrogen receptor-related receptor α and β), closely related to the ER, were among the first orphan nuclear receptors identified. These receptors can bind DNA as monomers and are thought to activate transcription constitutively, unaffected by β-estradiol. Studies of the expression patterns of ERRα and gene disruption experiments of ERRβ indicate that they play an important role in the development and differentiation of specific tissues in the mouse. In this work we demonstrate the existence in humans of a third member of this subfamily of receptors, termed ERRγ, which is highly expressed in a number of diverse fetal and adult tissues including brain, kidney, pancreas, and placenta. The ERRγ mRNA is highly alternatively spliced at the 5′-end, giving rise to a number of tissue-specific RNA species, some of which code for protein isoforms differing in the N-terminal region. Like ERRα andβ , ERRγ binds as a monomer to an ERRE. A GAL4-ERRγ fusion protein activates transcription in a ligand-independent manner in transfected HEK293 cells to a greater degree than either the GAL4-ERRα or -β fusion proteins.