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.
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All data are available from the corresponding author and have been included in the manuscript or Supplementary Information. The VISTA:VISTA.18 Fab co-crystal structure has been deposited into the Protein Data Bank under accession number 6MVL.
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We thank D. Ardourel, C. Bandoski-Gralinski, C. Bee, G. Bolton, M. Broz, I. Chakraborty, C. Connelly, F. Denhez, C. Gao, L. Garrenton, J. Gordon, N. Hammond, S. Hatcher, C. Hollander, M. Han, M. Happer, P. Helbling, V. Jenny, M. Labrecque, D. Myszka, A. Nallakkan, M. A. Pazos, P. Isnard-Petit, R. Lan, T. Metzger, E. Musteata, B. Nichols, M. Rodriguez, F. Sonego, S. Trouttet-Masson, S. Santino, E. Seo, T. Sproul, J. Sung, M. Supe, H. Tang, C. Terragni, J. Toth, S. Walrond and D. Wensel for technical assistance. We thank F. Bahjat, R. Camphausen, G. Cantor, G Chen, P. Chow, R. Das Gupta, A. Dongre, P. Haroldsen, M.-C. Gaudreau, D. Lipovsek, N. Lonberg, J. Muckelbauer, S. Mueller, X. M. Schebye, M. Selby, P. Strop, D. Tenney and M. Wright for discussions. We thank C. Bolger for editorial support.
All authors are or were employees of the companies Bristol-Myers Squibb, Five Prime Therapeutics, and genOway, which develop drugs and research models for profit.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Linda M. Bradley, Gordon Freeman, Christopher Garcia and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Alignment of human VISTA extracellular domain amino acid residues 94–165 with chimpanzee, cynomolgus macaque, dog, rat and mouse equivalents. Histidine residues are highlighted in red. b, Human monocytes (left) and neutrophils (right) labelled with VISTA multimers at pH 6.0 and pH 7.4. Cells labelled with non-VISTA-loaded multimers (control) or left unstained (FMO) are also depicted. These data are representative of two independent experiments. c, VISTA multimer (blue) and non-VISTA-loaded multimer (control, black) binding to the activated human T cells depicted in Fig. 1c. Data are VISTA multimer MFI and are representative of six independent experiments. d, Human VISTA–Fc binding to human PBMC NK cells at pH 6.0 (blue), T cells at pH 6.0 (red) and T cells at pH 7.0 (black). T cells stained at pH 6.0 with the anti-human Fc secondary but not with VISTA–Fc are included as a control (grey filled). These data are representative of ten independent experiments. e, Mouse VISTA–Fc binding at pH 6.0 to wild-type mouse splenic CD8+ T cells (red), CD4+ T cells (orange), B cells (green) and CD11b+ myeloid cells (blue). VISTA–Fc binding at pH 7.0 (black) and isotype-matched human IgG binding at pH 6.0 (grey filled) are included as controls. These data are representative of five independent experiments. f, Competitive SPR epitope binning of VISTA-specific antibodies against VISTA.4 and VISTA.5. Each row represents a unique clone, and for each clone, green indicates no cross-blocking, and red indicates cross-blocking. These data are representative of one experiment. g, h, Antibody blocking of VISTA-multimer binding to T cells as described in Fig. 1. Data are VISTA multimer MFI normalized to control and are representative of one experiment. g, Blocking activity by antibodies from the VISTA.4 epitope bin. VISTA.4 itself is depicted as black squares. h, Blocking activity by antibodies from the VISTA.5 epitope bin. VISTA.5 itself is depicted as black downwards triangles.
a, Representative histograms of CellTrace Violet dilution (left) and supernatant IFN-γ (right) by CD4+ T cells co-cultured with 293T-OKT3-VISTA cells in the presence in VISTA.4 (red), VISTA.5 (blue), a non-VISTA-binding isotype-matched antibody (control, black), or without 293T-OKT3-VISTA cells (grey filled). Data are mean ± s.e.m. with one-way ANOVA and Dunnett's multiple comparisons. *P = 0.0498. n = 3 T cell donors; these data are representative of seven independent experiments. b, Per cent of CD4+ T cells that proliferated following co-culture with 293T-OKT3-VISTA or 293T-OKT3 cells and VISTA.4 or an isotype-matched non-VISTA-binding control antibody. These data are representative of two independent experiments. c, NFκB phosphorylation in human CD4+ T cells following stimulation with plate-coated OKT3 and VISTA–Fc in the presence of the antibodies VISTA.4 (green upward triangles), VISTA.5 (blue downward triangles) and a non-VISTA-binding control (antibody control, red squares) at various pH. Cells stimulated with OKT3 and a plate-coated control antibody (VISTA control, black circles) and without OKT3 (grey diamond) are included as controls. Data are mean ± s.e.m. pNF-κB MFI normalized to control. n = 2 T cell donors; these data are representative of two independent experiments. d, e, Jurkat NFκB-luciferase reporter cells were co-cultured with 293T-OKT3 or 293T-OKT3-VISTA cells and with VISTA.4 or non-VISTA-binding isotype-matched control antibody. These data are a composite of three independent experiments. d, Luciferase signal after culture with 293T-OKT3 cells (blue circles) or without 293T cells (black triangles). e, Per cent increase in the luciferase signal with VISTA.4 treatment during culture with 293T-OKT3-VISTA cells. f, VISTA.4 (red) and VISTA.5 (blue) binding epitopes on the human VISTA extracellular domain. g, Human VISTA SPR binding sensorgrams for the blocking antibody VISTA.4 (left; pH 6.0, light red; pH 6.7, red; pH 7.4, dark red) and the non-blocking antibody VISTA.5 (right; pH 6.0, light blue; pH 6.7, blue; pH 7.4, dark blue). Overlaid sensorgrams are 100 nM VISTA binding responses, normalized to the binding report point. These data are representative of six independent experiments. h, Cell binding of VISTA.4 (pH 6.0, orange downward triangles; pH 7.0, red squares), VISTA.5 (pH 6.0, green diamonds; pH 7.0, blue circles), and a non-VISTA-binding antibody (pH 6.0, unfilled circles; pH 7.0, unfilled upward triangles) to Raji cells ectopically expressing VISTA. Data are VISTA antibody MFI and are representative of five independent experiments. i, VISTA antibody epitope binning against VISTA.4 (centre row) and VISTA.5 (bottom row). Each row represents a unique clone, and for each clone, green indicates a lack of cross-blocking and red indicates cross-blocking. Binding capacity at pH 6.0 relative to binding capacity at pH 7.4 is also depicted (top row). For binding at acidic pH, red indicates a greater than threefold impairment in kd at pH 6.0, green indicates a less than threefold impairment, and white indicates no data. These data are representative of one experiment.
a, Schematic depicting the method by which the VISTA.4 antibody was engineered to identify variants with improved binding at acidic pH. b, Schematic depicting the libraries of VISTA antibody variants used for screening acidic pH-selective variants. c, Schematic depicting the iterative screening strategy for identification of acidic pH-selective VISTA antibody variants. d, Cell binding of the acidic pH-selective antibody VISTA.18 to Raji cells ectopically expressing VISTA at pH 6.0 (red circles), pH 6.4 (orange squares), pH 6.6 (green diamonds), pH 7.0 (blue upward triangles), pH 7.2 (purple downward triangles) and pH 8.1 (black hexagons). Data are VISTA antibody MFI and are representative of three independent experiments.
The VISTA IgV domain (labelled with Alexa Fluor 555) and the VISTA.18 Fab (labelled with Alexa Fluor 488) were co-crystallized as described in Fig. 4 and the Methods. a, Representative bright-field (left), Alexa Fluor 488 fluorescence (centre), and Alexa Fluor 555 fluorescence (right) images of the crystals, indicating the presence of both VISTA and VISTA.18. These data are representative of one experiment. b, c, Superimpositions of the molecular surfaces of the yeast display-defined epitopes for VISTA.18 (purple, b) and VISTA.5 (non-blocking, orange, c) on the VISTA IgV domain (green). d, 2m|FO − DFC| electron density map (blue mesh) contoured to 1σ about the VISTA histidine triad (green sticks) and VISTA.18 HCDR3 (grey sticks). e, Human VISTA SPR binding data for the acidic pH-selective antibody VISTA.18 and variants of VISTA.18 in which the indicated residues have been reverted back to their identity in VISTA.18’s non-acidic-pH-selective parent, VISTA.16. VISTA.16 and a non-VISTA binding isotype-matched control antibody are included as controls. These data are representative of one experiment.
a–c, ELISA binding curves of human PSGL-1 19-mer–Fc proteins produced with (blue lines) or without (red lines) FUT7 and Core2 co-transfection. Binding curves for isotype-matched control IgG are also shown (green lines). Data are absorbance at 450 nm and are representative of three independent experiments. a, Binding to the anti-human PSGL-1 antibody KPL1. b, Binding to the anti-sialyl-Lewis X moiety antibody HECA452. c, Binding to recombinant human P-selectin–Fc. d, Extracted ion chromatograms of the peptide YLDY in PSGL-1 19-mer–Fc proteins produced with or without FUT7 and Core2 co-transfection and with or without fractionation as indicated. The percentage of total YLDY that was sulfated is indicated for each sample. These data are representative of one experiment.
a–d, Isothermal titration calorimetry (ITC) measurements of the interaction between PSGL-1 and VISTA. Top plots depict the raw calorimetric data of the titrations, and the bottom plots depict the integrated data corrected for the heat of dilution. The one set of sites model was used for data fitting. These data are representative of one experiment. a, Titration of 130 μM PSGL-1–Fc into 10 μM VISTA–Fc at 25 °C and pH 6.0. b, Titration of 130 μM PSGL-1–Fc into 10 μM VISTA–Fc at 37 °C and pH 6.0. c, Titration of 200 μM PSGL-1–Fc into 10 μM VISTA–His at 25 °C and pH 6.0. d, Titration of 130 μM PSGL-1–Fc into 10 μM VISTA–Fc at 25 °C and pH 7.4. The thermodynamic parameters determined by ITC are listed in Supplementary Table 2. e, Effects of PSGL-1–Fc (red circles) and P-selectin–Fc (blue squares) recombinant proteins on VISTA multimer binding to activated human CD4+ T cells. A non-binding antibody (black triangles) is included as a control. Data are VISTA multimer MFI normalized to control and are representative of two independent experiments. f, Effects of the indicated VISTA antibodies on PSGL-1 19-mer–Fc fusion protein Octet binding to VISTA–Fc fusion protein at pH 6.0. A non-VISTA-binding antibody is included as a control. Data are BLI binding magnitudes and are representative of two independent experiments. g, Effects of the PSGL-1 antibody KPL1 and recombinant P-selectin on human PSGL-1 19-mer–Fc fusion protein Octet binding to VISTA–Fc fusion protein at pH 6.0. A non-PSGL-1-binding antibody (control) and no added antibody are included as controls. Data are BLI binding magnitudes and are representative of two independent experiments. h, Effects of KPL1 (blue circles) on human VISTA–Fc binding to human PBMC monocytes at pH 6.0. An isotype-matched non-PSGL-1-binding antibody is included as a control. Data are VISTA-Fc MFI and are representative of two independent experiments. i, Percentage of T cells with no PSGL-1 expression after CRISPR using guides against PSGL-1, CD4 or a scrambled control. These data are representative of five independent experiments. j, VISTA multimer (blue circle) binding to CHO cells expressing PSGL-1 at various pH. Non-VISTA-loaded multimer (black square) binding is included as a control. Data are VISTA multimer MFI normalized to control and are representative of two independent experiments. k, Human VISTA–Fc binding to wild-type and heparan sulfate-deficient CHO-K1 cells (red and orange, respectively) at pH 6.0. Isotype-matched control antibody binding to wild-type and heparan sulfate-deficient CHO-K1 cells (blue and green, respectively) at pH 6.0 is also shown. These data are representative of two independent experiments.
a, BLI binding magnitudes for GP1BA–His (blue squares) and PSGL-1 19-mer–Fc (red circles) to captured VISTA–Fc at the indicated pH. These data are representative of one experiment. b, VISTA multimer binding histograms to human platelets. Binding was performed in the presence of non-VISTA-binding control antibodies (pH 7.4, blue; pH 6.0, red) or the VISTA.4 blocking antibody (pH 6.0, green). Unstained platelets (grey filled histogram) are included as controls. These data are representative of two independent experiments. c, BLI binding magnitudes for VSIG-3–Fc binding to captured VISTA–Fc at the indicated pH. These data are representative of two independent experiments. d, Left, anti-VISTA stained (red), control stained (blue) or unstained (black) parental HEK293 (top) and VISTA-expressing HEK293 cells (bottom). Right, VSIG-3–Fc (red) or control–Fc (blue) binding to the same cells at the indicated pH. These data are representative of two independent experiments. e, Left, anti-VSIG-3 stained (red), control stained (blue) or unstained (black) parental CHO (top) and VSIG-3-expressing CHO cells (bottom). Right, VISTA–Fc (red) or control–Fc (blue) binding to the same cells at the indicated pH. These data are representative of two independent experiments. f, BLI binding magnitudes of VISTA–Fc binding to captured PSGL-1 19-mer–Fc at pH 6.0. Competition was provided at the indicated concentrations by a non-binding control antibody (grey bars), the VISTA blocking antibody VISTA.16 (yellow bars), the VISTA non-blocking antibody VISTA.5 (blue bars) or human VSIG-3–Fc fusion protein (purple bars). Darker bars depict the BLI binding magnitudes of competitors without VISTA. These data are representative of one experiment. g, BLI binding magnitudes of VSIG-3–Fc at the indicated concentrations binding to captured VISTA–Fc at pH 6.0. Competition was provided by buffer alone (blue bars), human PSGL-1 19-mer–Fc (green bars), non-binding isotype matched control antibody (red bars), VISTA.16 (yellow bars), VISTA.18 (purple bars), or VISTA.5 (orange bars). These data are representative of one experiment. h, VSIG-3–Fc binding to activated human PBMC T cells at pH 6.0 (green circles) or pH 7.4 (blue squares). Binding of isotype-matched control antibody at pH 6.0 (black diamond) and pH 7.4 (grey triangle) is included as a control. Data are VSIG-3–Fc MFI and are representative of two independent experiments. i, BLI binding magnitudes for VISTA–Fc (left) and PSGL-1 19-mer–Fc (right) binding to captured VISTA–Fc at the indicated pH. These data are representative of one experiment.
a, BLI binding magnitudes of anti-PSGL-1 clone KPL1 to captured total, sulfotyrosine-poor, and sulfotyrosine-rich fractions of PSGL-1 19-mer–Fc at pH 6.0 (green) and pH 7.4 (blue). These data are representative of one experiment. b, BLI binding magnitudes of wild-type PSGL-1 19-mer–Fc (WT, blue) and tyrosine to alanine mutant PSGL-1 19-mer–Fc (Y→A, green) to captured VISTA–Fc at the indicated pH. These data are representative of one experiment. c, BLI binding magnitudes for VISTA.5 (a non-blocking antibody, left) and VISTA.16 (a blocking antibody, right) binding to captured wild-type (WT, black), 153/154/155 histidine to alanine mutant (H→A, red), 153/154/155 histidine to aspartic acid mutant (H→D, blue) and 153/154/155 histidine to arginine mutant (H→R, green) VISTA–Fc proteins at pH 6.0 and pH 7.4. These data are representative of one experiment. d, SPR binding %Rmax values for VISTA.18 Fab binding to captured wild-type, H→D mutant, H→R mutant and H→A mutant VISTA proteins at pH 6.0 (left, green) and pH 7.4 (right, blue). Binding at 25, 50 and 100 nM are indicated by light, medium and dark coloured bars respectively. These data are representative of one experiment. e, Wild-type and mutant VISTA–Fc binding to CHO-PSGL-1 cells at pH 6.0. Data are VISTA–Fc MFI and are representative of two independent experiments. f, Wild-type and mutant VISTA–Fc suppression of primary T cell NF-kB phosphorylation at pH 6.8. Data are pNF-kB MFI normalized to control. n = five T cell donors; these data are representative of two independent experiments. g, Additional human VISTA–Fc recombinant proteins were produced with the histidine residues at positions 98 and 100, or a 98, 100, 153, 154 and 155 (quintuple) replaced by arginine. Wild-type and H→R mutant VISTA–Fc binding to CHO-PSGL-1 cells at pH 7.4. Data are VISTA–Fc MFI and are representative of two independent experiments.
a, VISTA.10 antibody blockade of mouse VISTA–Fc binding to activated mouse CD4+ T cells at pH 6.0. Binding with no blocking antibody is included as a control. Data are mouse VISTA–Fc MFI and are representative of two independent experiments. b–d, MC38 tumour-bearing wild-type mice were treated with PD-1 and VISTA blocking antibodies as described in Fig. 4. n = 5 per group; these data are representative of three (b) or two (c, d) independent experiments. b, The frequency of intratumoural CD4+ T cells seven days after the start of treatment. ***P = 0.0001. c, LAG-3 and TIM-3 MFI on intratumoral CD8+ T cells. ***P < 0.0001. d, The frequencies of intratumoral leukocytes (CD45+, first plot from the left), macrophages (CD11b+MHCII+LyClowLyGlow), monocytes (CD11b+LyChighLyGlow) and granulocytes (CD11b+LyClowLy6Ghigh). Per cent CD45+, ***P = 0.0001; per cent macrophages, **P = 0.0071; ***P = 0.0009. e, MC38 tumour-bearing VISTA-knockout mice and their wild-type littermates were treated with control or PD-1 blocking antibodies. Frequencies of intratumoral CD8+ (left) and CD4+ (right) T cells seven days after the start of treatment. Per cent CD8+, *P = 0.0285; per cent CD4+, *P = 0.0330. n = 4 (KO mice treated with anti-PD-1) or 5 mice per group; these data are representative of two independent experiments. f, Schematic of the endogenous (top) and humanized (bottom) VISTA sequence. g, Representative Southern blot of 4 heterozygous mice and 1 wild-type mouse (WT) for the humanized VISTA allele (7.1 kb) and the endogenous Vista allele (6.1 kb). These data are representative of three independent experiments. h, Expression of mouse and human VISTA on leukocytes from wild-type (blue) and homozygous human VISTA knock-in mice (red). Non-Treg CD4+ T cell (CD3+CD4+FoxP3−), CD4+ Treg cell (CD3+CD4+FoxP3+), CD8+ T cell (CD3+CD8+) and myeloid cell (CD3−B220−CD11b+) subsets were assessed. Data are per cent VISTA-expressing and are representative of four independent experiments. i, Wild-type mice were treated with a single intravenous injection of 200 μg of an anti-mouse VISTA antibody (red downward triangles) or an isotype-matched control antibody (blue squares). Data are serum antibody concentrations and are representative of two independent experiments. Statistics depict mean ± s.e.m. and one-way ANOVA with Dunnett’s multiple comparisons.
a, Quantitative tissue biodistribution of fluorescently labelled VISTA.16 (left) and VISTA.18 (right) at 2.5 h (red), 24 h (green) and 51 h (blue) after injection into MC38 tumour-bearing human VISTA knock-in mice. n = 5 (VISTA.16 at 51 h) or 3 mice per group. Data are radiant efficiency mean ± s.e.m. and are representative of two independent experiments. b–e, Human and cynomolgus macaque sensorgrams for the antibodies VISTA.4 and VISTA.18 at pH 7.4 (blue, left) and pH 6.0 (red, right). These data are representative of two independent experiments. b, Human VISTA sensorgrams for VISTA.4. c, Human VISTA sensorgrams for VISTA.18. d, Cynomolgus macaque VISTA sensorgrams for VISTA.4. e, Cynomolgus macaque VISTA sensorgrams for VISTA.18. f, MC38 tumour-bearing human VISTA knock-in mice were treated as described in Fig. 4. Tumour growth in mice treated with VISTA.16 only (left) or with VISTA.18 only (right). n = 16 mice per group. Data are tumour volumes and are a composite of two independent experiments.
Supplementary Table.Table 2: Thermodynamic parameters for PSGL1-VISTA interactions determined by ITC. Supplemental information on the isothermal titration calorimetry data presented in the manuscript.
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Johnston, R.J., Su, L.J., Pinckney, J. et al. VISTA is an acidic pH-selective ligand for PSGL-1. Nature 574, 565–570 (2019). https://doi.org/10.1038/s41586-019-1674-5
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