4C). Altogether, our results demonstrate that B7-H4 on activated HSC inhibits the generation of cytokine-producing T cells. We investigated the influence of B7-H4-expressing HSC on recall GPCR Compound Library in vivo responses of previously primed effector CD8+ T cells. Effector T cells upon re-encountering cognate antigen are capable of producing IFNγ. We generated CD8+ T cell blasts

or effector CD8+ T cells by pulsing P14 splenocytes with 1 μg/mL gp33 peptide and subsequently purifying them after 6 days of expansion. They were then cocultured with gp33 peptide-pulsed B7-H4 knockdown and control AHSC for 6 hours. As shown, IFNγ production is reduced after stimulation with B7-H4-expressing AHSC compared to B7-H4 knockdown AHSC (Fig. 5A). CD8+ T cell blasts or effector CD8+ T cells were also generated using anti-CD3/CD28 and restimulated with anti-CD3/CD28 in the presence of B7-H4-Ig or control-Ig. The addition of B7-H4-Ig reduces IFNγ production in a dose-dependent manner, whereas high levels of IFNγ +CD8+ T cells are seen with treatment with control-Ig (Fig. 5B). These results indicate

that B7-H4 on AHSC attenuates the recall effector T cell response. CD8+ T cells that are stimulated by B7-H4 silenced HSC exhibit a highly activated phenotype with a high frequency of CD44hi cells, even before cell division has ensued (Fig. 6A). Thus, in concordance with previous reports,22, 23 B7-H4 inhibits or delays T cell activation. To show whether HSC were capable of 3-mercaptopyruvate sulfurtransferase processing and presenting antigen and whether inhibition of T cells by B7-H4 on infected HSC still occurred, AHSC were infected with 5 plaque-forming Doxorubicin in vivo units (pfu)/mL of vaccinia virus expressing gp33 epitope of LCMV for 24 hours. Subsequent to vaccinia infection expressing gp33, AHSC were then transfected with B7-H4 siRNA or control siRNA. Two days post-transfection, P14 transgenic CD8+ T cells were added and the level of activation based on CD44 expression was evaluated. Our results demonstrate that in the absence of B7-H4, the vaccinia-gp33-infected HSC induce T cell

activation efficiently as compared to the control siRNA treatment (Fig. 6B). However, it is important to note that similar to stimulation with peptide pulsed HSC, the effect of B7-H4-mediated inhibition of T cell activation and proliferation is dose-dependent. To elucidate the mechanism of B7-H4 inhibition of T cells, we assessed the early signaling pathway and phosphorylation state of various signal transduction molecules on T cells. T cell activation is associated with mitogen activated protein kinase (MAPK) signaling through extracellular signal-regulated kinase (ERK)1/2. We evaluated the initiation of MAPK signaling by way of the presence of phosphorylated ERK molecules in T cells with and without B7-H4-Ig. Phosphorylated ERK1/2 could be detected in T cells with control-Ig as early as 5-15 minutes after stimulation.