3C), PD-L1 is an interesting tool to manipulate immune responses. It has been shown that the PD-1/PD-L1 pathway controls graft versus host reactive T cells 44 and that PD-L1 knockout mice have a stronger allostimulatory reactivity compared to WT mice 45. Hence, we were especially interested
in the regulation of PD-L1 expression. We identified a MAPK-dependent production of IL-6 and IL-10 that cause a long-lasting STAT-3 activation as a central buy Quizartinib hallmark of TLR-APCs and accordingly to PD-L1 expression. The TLR-stimulus led to the production of two cytokines that mainly signal via STAT-3: IL-6 and IL-10 (Fig. 4A and B). Both cytokines are able to alter the phenotype of iDCs toward the TLR phenotype: no CD1a expression, retained CD14 expression and high expression levels of PD-L1 (Supporting Information Fig. 4). To verify the importance
of IL-6 and IL-10 we compared the activation of different STAT molecules (Fig. 7). As expected, TLR-APCs show an almost constitutive STAT-3 activation. In contrast, STAT-5 was activated in iDCs and diminished in TLR-APCs. Therefore, TLR-APCs and iDCs show clear differences in STAT-3 and STAT-5 activation pattern. Our results indicate that TLR agonists added at an early time point of iDC differentiation block STAT-5 activation and shift the STAT activation pattern toward STAT-3. Indeed, blocking of STAT-3 signal transduction had an I BET 762 eminent effect on the TLR-APC phenotype. STAT-3 inhibition repressed CD14 and PD-L1 (Fig. 8A and B). In accordance with our data, Barton et al. 11 suggested that stimulatory or tolerogenic function of APCs depends on their STAT-3 activation level. To further support the role of STAT-3, we performed ChIP assays and detected that STAT-3 binds to the PD-L1 promoter (Fig. 8C). STAT-1 was also able to bind PD-L1, Ureohydrolase but less effectively (Fig. 8D). There were only few quantitative differences in the magnitude of STAT-1-binding between iDCs and
TLR-APCs, indicating a minor role for STAT-1 in the initial differentiation process of TLR-APCs. Induction of cytokine expression can be regulated by different mechanisms controlled by the stimulus. For TLR signaling, NF-κB and MAPKs have been described as major signaling pathways. We revealed that IL-6 and IL-10 were not released after blocking p38 (SB) and p44/42 (UO) MAPKs (Fig. 5B and C) and that CD14 and PD-L1 expression was reduced (Fig. 6A and B). Blocking p38 (SB) alone influenced the production of IL-10 but had no effect on IL-6 production. In contrast, the inhibition of p44/42 (UO) affected IL-6 expression. Similar preferences were also discernible in regulation of CD14 and PD-L1 surface expression: inhibition of p44/42 affects to a greater extent expression of CD14, while the inhibition of p38 is related more to the expression of PD-L1.