VSIG-IGSF11, VISTA-PD-1Hの新規リガンド

(A) Recombinant human VSIG-3 protein specifically binds to recombinant human VISTA in a functional ELISA binding assay. Recombinant Human VISTA IgG1-Fc (#7126-B7) was immobilized at 2μg/ml (100μl/well), and the indicated concentrations of Recombinant Human VSIG-3 IgG1-Fc (#9229-VS) were added. The concentration of VSIG-3 IgG1-Fc that produced 50% of the optimal binding response was approximately 0.25μg/ml (3.4 nM).

(B) Anti-human VISTA antibodies neutralize the binding of VSIG-3 and VISTA. Biotinylated Recombinant Human VISTA IgG1-Fc (1μg/ml) was pretreated with the indicated concentrations of Anti-Human VISTA Antibodies (#AF7126, #MAB71261) or Isotype Controls (#5-001-A, #MAB0041) and then added to ELISA plates containing immobilized Recombinant Human VSIG-3 IgG1-Fc (2μg/ml). After washing away any unbound proteins, streptavidin-HRP (#DY998) and substrate solution (#DY999) were added to the wells. The color development was stopped and the intensity of the color was measured using an ELISA plate reader.

Human PBMCs were stimulated with a combination of plate-bound Mouse Anti-Human CD3 antibody (1μg/ml; #MAB100) and either plate-bound Recombinant Human VSIG-3 IgG1-Fc (10μg/ml) or Control IgG1-Fc (10μg/ml; #110-HG) for 24 or 48 hours. Cytokine levels in the cell culture supernatants were measured using the Proteome Profiler Human XL Cytokine Array Kit (#ARY022B).

Array results were further confirmed by measuring individual cytokines in the cell culture supernatants from anti-CD3-activated human PBMCs using the Human IL-17, CCL5/RANTES, CXCL11/I-TAC, and CCL3/MIP-1a Quantikine ELISA Kits (#D1700, #DRN00B, #DCX110, and #DMA00). (*) indicates p<0.01, compared with the IgG1-Fc controls. Results are representative of three independent experiments.

(A) The extracellular domain of VISTA attenuates VSIG-3 functions. Pretreatment of immobilized Recombinant Human VSIG-3 IgG1-Fc (10μg/ml) with the indicated concentrations of Recombinant Human VISTA IgG1-Fc for 1 hour significantly attenuated the ability of VSIG-3 to inhibit CCL5/RANTES secretion from anti-CD3 activated PBMCs.

(B) Anti-Human VISTA Antibody attenuates VSIG-3 inhibitory functions. Human PBMCs were treated with immobilized Anti-Human CD3 (1 μg/ml), and various concentrations of Recombinant Human VSIG-3 IgG1-Fc, or 5μg/ml of Recombinant Human VSIG-3 IgG1-Fc plus the indicated concentrations of a Sheep Anti-Human VISTA Antibody (#AF7126), or Sheep IgG (#5-001-A) as a control. VSIG-3 significantly inhibited anti-CD3 induced IL-17 production in a dose-dependent manner. 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 alter the inhibitory effect of VSIG-3 on IL-17 secretion (data not shown). Results are representative of three independent experiments.

Human CD3+ T cells were isolated from PBMCs using a MagCellect Human CD3+ T Cell Isolation Kit (#MAGH101). Human T cells were then incubated with an immobilized Mouse Anti-Human CD3 Monoclonal Antibody (1μg/ml) and the indicated concentrations of Recombinant Human VSIG-3 IgG1-Fc or IgG1-Fc for 24 hours. The levels of IL-2, IFN-g, and IL-17 in the cell culture supernatants were measured using the Human IL-2, IFN-g, and IL-17 Quantikine ELISA Kits (#D2050, #DIF50, and #D1700). IgG1-Fc controls did not alter anti-CD3-induced IL-2, IFN-g, or IL-17 secretion (data not shown). Results are representative of three independent experiments. (D) VSIG-3 inhibits 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 IgG1-Fc or IgG1-Fc for 72 hours. Cell proliferation was assessed by a fluorometric assay using the redox-sensitive dye Resazurin (#AR002). IgG1-Fc controls did not alter anti-CD3-induced CD3+ cell proliferation (data not shown). Results are representative of three independent experiments.

RNAscope staining of VSIG-3 mRNA in human colon tumor adenocarcinoma and colon normal tissue. VSIG-3 transcript levels were detected using RNAscope 2.0 HD Red Detection Kit (Advanced Cell Diagnostics, a Bio-Techne brand). A custom-designed human VSIG-3 RNAscope probe was used to stain following the instructions contained in the RNAscope 2.0 HD Red Detection Kit.