#CAP STABLE PVP PRODEUS 2017 SKIN#

( B) Titration of soluble VISTA.COMP (blue) or VISTA-Fc (red) on 2.10 cells activated as described in A. Data are representative of at least three independent experiments. As observed with primary CD4 + T cells, pentameric VISTA.COMP suppresses proliferation when immobilized or when added soluble in culture media, whereas VISTA-Fc only exhibits suppressive activity when immobilized (data represent mean ± SEM, *** P < 0.01 relative to anti-CD3 stimulated control by Student’s t test, n = 3). ( A) 2.10 clonal T cells were activated with immobilized anti-CD3 antibody (3 μg/ml) in the presence of immobilized or soluble VISTA-Fc or VISTA.COMP (10 μg/ml), and proliferation was measured by pulsing cells with 3H-thymidine in the last 6 hours of a 24-hour culture. All data shown are representative of at least 3 independent experiments. 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). 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. Soluble VISTA.COMP suppresses T cell expansion (top, forward scatter and side scatter profiles) and proliferation (bottom, CFSE dilution). ( 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). Reduced VISTA.COMP migrates as a single band approximately 50 kDa, while the disulfide-stabilized pentamer has an apparent mass of 250 kDa. ( 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). VISTA-Fc suppresses the proliferation of CD4 + T cells when immobilized but not when added as a soluble ligand in the culture media. ( 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. Collectively, we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses.

#CAP STABLE PVP PRODEUS 2017 SKIN#

Furthermore, we show that VISTA.COMP, but not VISTA-Fc, functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. In contrast to VISTA-Fc, VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Herein, we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP).

However, this effect required its immobilization on a solid surface, suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped, largely due to the inability to derive agonists targeting its unknown receptor.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity.

Cap stable pvp prodeus 2017