Several lines of evidence indicate an important role of miRNAs during innate and adaptive immune responses 14. MiR-146 has been shown to regulate Selleck p38 MAPK inhibitor TLR-mediated inflammatory responses, whereas miR-155 and miR-181a have been implicated in B- and T-cell-mediated immune responses 14–16. To date, most of the information regarding the role of the miRNAs during an immune response has been obtained through either genetic ablation or overexpression. However, how individual miRNAs are altered during breakdown of tolerance and initiation of an autoimmune response at the cellular level remains unclear. In this study, using PD-1−/− mice, we demonstrate that Ag-primed PD-1−/− T cells

upregulate microRNA 21 (miR-21) expression upon TCR ligation. In vitro inhibition of miR-21 activity results in reduced proliferation and cytokine secretion

by Ag-specific T cells. Importantly, PD-1 inhibition results in phosphorylation of STAT5, which binds in the promoter area of miR-21 and upregulates miR-21 expression. Collectively, our findings suggest that PD-1 through a microRNA signaling cascade regulates the balance Seliciclib between tolerance and immune activation. To assess the breakdown of tolerance and development of AIA in the absence of PD-1 pathway, PD-1−/− and WT control mice were subcutaneously (s.c.) immunized with ovalbumin (OVA)/CFA followed Tangeritin by an intra-articular injection of OVA/PBS. One day after the intra-articular challenge, both group of mice developed an acute inflammatory reaction and severe arthritis (Fig. 1A). The extend of joint swelling decreased in WT mice starting day 2 after AIA induction in contrast to PD-1−/− mice where disease severity remained high during the entire period of monitoring. As shown in Fig. 1B, on day 8 after challenge the affected paw of PD-1 KO mice was observed to have severe swelling and loss in the range of motion, in contrast to WT mice in which the inflammatory reaction had diminished. To characterize the T-cell immune responses

in OVA-immunized PD-1−/− mice, draining lymph nodes (dLNs) were isolated 9–10 days after antigenic challenge and cells were cultured in vitro in the presence of varying concentrations of OVA. T cells from PD-1−/− mice exhibited higher proliferation than T cells from WT mice in response to Ag (stimulation index=18 for PD-1−/− mice versus 5.5 for WT at 50 μg/mL OVA) (Fig. 1C). The T-cell proliferation profile correlated with the capacity of OVA to elicit IFN-γ and IL-17 production by OVA-primed T cells. Thus, the analysis of culture supernatants revealed that OVA-primed LN cells from PD-1−/− mice secrete increased amounts of IFN-γ upon stimulation as compared with WT T cells (2400±76 pg/mL for PD-1−/− versus 1790±5 pg/mL for WT) (Fig. 1D).