Figure 1. Pentameric VISTA.COMP suppresses T cell activation and proliferation as a soluble ligand in vitro.

(A) CFSE-labeled purified murine CD4⁺ T cells were activated by plate-bound anti-CD3 antibody (2.5 μg/ml) in the presence (blue) or absence (red) of immobilized (left) or soluble (right) VISTA-Fc (10 μg/ml) for 48 hours. VISTA-Fc suppresses the proliferation of CD4⁺ T cells when immobilized but not when added as a soluble ligand in the culture media. (B) Recombinant VISTA.COMP was expressed as described in the Methods, and the purity and pentameric status was confirmed by SDS-PAGE and Western blot in the presence or absence of a reducing agent (DTT). Reduced VISTA.COMP migrates as a single band approximately 50 kDa, while the disulfide-stabilized pentamer has an apparent mass of 250 kDa. (C) Proliferation assay of CD4⁺ T cells undergoing activation in the presence of coated (9 μg/ml, left) or soluble (12 μg/ml, right) VISTA.COMP (blue) or COMP (red). Soluble VISTA.COMP suppresses T cell expansion (top, forward scatter [FSC] and side scatter [SSC] profiles) and proliferation (bottom, CFSE dilution). Culture medium was harvested from CD4⁺ T cells 48 and 72 hours after anti-CD3 activation in the presence of COMP or VISTA.COMP (10 μg/ml), and IL-2 (D) and IFN-γ (E) secretion was quantified by ELISA. In addition to proliferation, VISTA.COMP was found to significantly suppress IL-2 and IFN-γ secretion from T cells (data represent mean ± SEM, ***P < 0.005 relative to COMP control by Student’s t test, n = 3). All data shown are representative of at least 3 independent experiments.