Furthermore, our previous study showed that hyaluronan fragments constitute a common factor produced by several types of human tumors, including hepatoma, to stimulate the activation of monocytes.8 Here we found that the production of proinflammatory cytokines by monocytes and the expansion-promoting effect of monocytes on Th17 cells Daporinad molecular weight was significantly impaired when monocyte activation was attenuated either by adding a hyaluronan-specific blocking peptide (Pep-1) or by silencing hyaluronan synthase 2 in tumor cells to reduce hyaluronan levels in TSN (Fig. 3E).8, 22 Our next endeavor was to determine

whether soluble factors secreted from TSN-activated monocytes could suffice to induce the expansion of Th17 and Th17/Th1 cells. Purified T cells were cultured in conditioned medium Cabozantinib chemical structure from control monocytes (CCM) or in conditioned medium from TSN-activated monocytes (TCM). We found that TCM, but not CCM, effectively induced the development of both Th17 and Th17/Th1 cells in a time-dependent manner that reached a maximum or a plateau within 9 days (Fig. 4A). Such generation of Th17 cells was associated with a parallel reduction in Th2 cells (Fig. 4B). To determine the proliferation of

Th17 cells, we labeled T cells with CFSE and then cultured them in one of the conditioned media. As the T cells proliferated, the frequency of Th17 cells exposed to TCM gradually increased and reached a maximum on day 9; in contrast, only a small percentage (4.1% ± 0.6%, n = 4) of the cells treated with CCM were Th17 cells on day 9 (Fig. 4C). In agreement with that, we

found that TCM elicited robust production of IL-17 and IFN-γ by T cells (Fig. 4D). Taken together, these results suggest that activated monocytes play a critical role in maintaining functional Th17 and Th17/Th1 pools in tumor environments in humans. In both mice and humans, phosphorylation of signal transducer and activator of transcription 3 (STAT3) and Temsirolimus induction of RORγt expression are essential for Th17 development, whereas STAT1 and T-bet are selective for Th1.13, 15, 25 Accordingly, we performed immunoblotting to determine whether those molecules were also involved in the Th17 and Th17/Th1 expansions observed in our study. Although activation of STAT1 and expression of T-bet gradually increased over time in both the CCM and TCM culture systems, expression of RORγt and phosphorylation of STAT3 were markedly up-regulated in T cells exposed to TCM (Fig. 4E). The coexistence of RORγt and T-bet proteins in T cells in situ was further confirmed by confocal microscopic analysis of frozen tumor tissues (Supporting Fig. 3) and TCM-cultured T cells (data not shown). TSN-activated monocytes secreted several key cytokines, including IL-1β, IL-6, IL-23, and TNF-α, which have been shown to regulate the development of Th17 cells.