1E). Levels of IL-10 were below the detection limit in both groups of mice (data not shown). Finally, analysis of the OVA-stimulated LNC cultures for the proportion of activated T cells showed similar frequency of CD3+CD4+CD44hi T cell in stimulated LNs from WT and PD-1−/− mice (Fig. 1F). Taken together, these results demonstrate that
during breakdown of tolerance and induction of autoimmunity, the absence of PD-1 expression on T cells results in aberrant activation and proliferation of these cells and more severe disease. To identify the potential involvement of microRNAs in PD-1-mediated breakdown of tolerance, we screened the expression of 365 microRNAs by microarray analysis of WT and PD1−/− lymphocytes, isolated from draining LNs of OVA-primed mice, before and after stimulation with OVA (Fig. 2A).
Five microRNAs (miR-21, miR-20a, miR-16, Selleck Sirolimus miR-155, and miR-375) differentially expressed after OVA stimulation in WT and PD1−/− cells. MiR-21 was statistically upregulated (2.3-fold) in unstimulated PD1−/− https://www.selleckchem.com/products/VX-765.html compared with WT cells. OVA stimulation induced miR-21 expression to a higher degree in PD-1−/− than WT cells. The effect of PD-1 on miR-21 expression was also validated by real-time PCR analysis (Fig. 2B). To further assess the role of PD-1 as an miR-21 regulator, we inhibited PD-1 by siRNA treatment (Fig. 2C) and tested miR-21 expression. PD-1 inhibition resulted in >11-fold upregulation in miR-21 expression levels, thus confirming the role of PD-1 as negative regulator of miR-21 (Fig. 2D). We next sought to identify whether this regulation occurs at the transcriptional PDK4 or post-transcriptional level. The observation that PD-1 inhibition by siRNA resulted in upregulation of the primary transcript miR-21 (Fig. 3A) suggests that PD-1 regulates miR-21 transcriptional levels. The previous studies have shown that PD-1 regulates the expression and phosphorylation of STAT5 17. Western blot analysis showed that siRNA inhibition of PD-1 in Jurkat cells resulted in upregulation of STAT5 protein expression and phosphorylation (Fig. 3B). We next analyzed the
known putative promoter area of miR-21 18 for STAT5-binding sites. To this end, we used the TRANSFAC bioinformatic program and identified an evolutionary conserved STAT5 binding site on the miR-21 precursor sequence (Fig. 3C). In support of this, PD-1 inhibition resulted in enrichment of STAT5 binding in miR-21 promoter area (Fig. 3D) and resulted in upregulation of pri-miR-21. Furthermore, concurrent inhibition of PD-1 and STAT5 did not upregulate miR-21 expression (Fig. 3E), suggesting that PD-1 regulates miR-21 expression through STAT5. MicroRNAs exert their function through post-transcriptional inhibition of gene targets 14. Bioinformatic algorithm prediction analysis revealed programmed cell death 4 (PDCD4) as a potential miR-21 gene target.