1D), which implies that they were newly recruited and activated

monocytes. In contrast, most Mψ in the cancer nests were negative for HLA-DR. Confocal microscopic analysis showed that most CD68+ Temsirolimus nmr cells in HCC tumors were also stained positive for CD14, but negative for CD3 (Supporting Fig. 2). We recently observed that tumor environments can alter the normal development of Mψ that is intended to trigger transient early activation of monocytes in the peritumoral region.8 To investigate whether such a mechanism is also responsible for the selective accumulation of Th17 in peritumoral stroma, we purified circulating monocytes (CD14high cells), nontumor-and tumor-infiltrating monocytes from the same HCC patient, and then cultured those cells with purified autologous T cells. The result showed that tumor monocytes expressed higher levels of HLA-DR, secreted larger amounts of inflammatory cytokines IL-1β, IL-6, and IL-23 (Fig. 2A,B), and were more potent in promoting the expansion of Th17 cells with phenotypic features similar to those isolated from HCCs (Fig. 2C). To further elucidate the effect of tumor monocytes/Mψ on Th17 expansion, we incubated monocytes with TSN from hepatoma

cells to generate tumor-activated monocytes or immunosuppressive tumor-associated Mψ (TAMs) (Fig. 3A),8 and then cultured those cells with purified autologous T cells. The results showed that tumor-activated monocytes were Maraviroc manufacturer significantly MCE superior to TAMs in inducing Th17 expansion, whereas the effects of control monocytes or Mψ were negligible. Notably, almost half of the Th17 cells generated from tumor-activated

monocytes were able to produce both IL-17 and IFN-γ (Th17/Th1), whereas most of the TAM-induced Th17 cells were negative for IFN-γ (Fig. 3B). We used commercial kits to further purify naive and memory T cells. Tumor-activated monocytes effectively promoted the expansion of Th17 cells (45.4% ± 7.2%, n = 4) from memory CD4+ T cells, and most of these memory IL-17+ cells (65.7% ± 11%, n = 4) were also able to produce IFN-γ (Fig. 3C). These results indicate that, compared to immunosuppressive TAMs, monocytes activated by a short exposure to a tumor environment play a more prominent role in the development of memory T cells into IL-17-producing T cells that have phenotypic features similar to those of tumor-infiltrating Th17 cells. Recent studies have shown that proinflammatory cytokines released by activated APC can facilitate the differentiation and expansion of Th17.13–15, 24 Therefore, we examined cytokine profiles of TSN-exposed monocytes/Mψ at an early and a late stage of differentiation. Consistent with the results from tumor-infiltrating monocytes (Fig. 2B), TSN-exposed monocytes secreted significant amounts of TNF-α, IL-1β, IL-6, IL-12, IL-23, and IL-10, and that pattern was dramatically reduced in Mψ that had been incubated in TSN for 7 days, with the exception of IL-6 and IL-10 (Fig. 3D).