3C). The inhibition of cytokine release correlated with an inhibition in cell division as CSFE dilution indicated that culture with PI inhibited the percentage of dividing T cells in all culture conditions for Th0 (data not shown), Th1 and Th17 (data not shown) conditions whereas the proliferation of Th2 cells was not strongly affected (Fig. 3D and E). These data indicate that PI inhibits T-cell-dependent cytokine release irrespective of T-cell polarization. To demonstrate that PI inhibits T-cell function through suppression of proliferation

it was assessed whether PI inhibited IL-2 release in Th cell cultures. As shown in Fig. 4A IL-2 release was suppressed independent of the type of T-cell polarization as IL-2 concentrations were equally low in supernatant of PI containing Th0, Th1 and Th2 cultures. To pursue the role of PI as a general suppressor of IL-2 release it was next ABT-737 molecular weight demonstrated that PI acted dose dependently as repeated addition of PI to anti-CD3 anti-CD28 stimulated splenocytes enhanced

inhibition of IL-2 release (Fig. 4B). Next, we assessed whether PI affected both CD4 and CD8 T-cell activation and proliferation. In short, CFSE-labeled murine spleen-derived T cells were stimulated polyclonally in vitro in the presence of a range of PI concentrations and after 72 h IL-2 release as well as kinetics of division were determined. IL-2 release by both CD8+ as well as CD4+ cells was severely suppressed Talazoparib research buy by incubation with PI (Fig. 4C). A concentration of 12.5 μg/mL already exerted suppressive effects. From these experiments it can be concluded that PI effectively inhibited T-cell proliferation, which was associated with reduced IL-2 release. As IL-2 is critical for proliferation and survival of differentiating T cells the subsequent experiments addressed the fate of T cells after activation in the presence of PI. At the concentration of 12.5 μg PI/mL we determined the SPTLC1 kinetics of division to assess whether T-cell inhibition led to deletion or anergy. As illustrated by the measurement of CSFE dilution in Fig. 4D both treatments

yielded cells undergoing five to six divisions. However, during PI treatment fewer cells reached division 4, 5 and 6 and therefore more cells remained in division 1 and 2. This implies that PI does not induce anergy or deletion but rather prevents activated cells from proceeding into later divisions (Fig. 4D). It was excluded that PI exerted its inhibitory effects through cell death by staining with 7AAD (data not shown). Although these data suggest that the inhibition of inflammatory responses during TNBS colitis can be attributed to direct effects of PI on differentiating T cells, it could be hypothesized that PI-mediated inhibition of antigen presentation by DCs indirectly causes T-cell suppression.