While 1-D-MT did not induce IDO1 mRNA expression and kyn production in IDO1-negative HeLa cervical carcinoma cells, it increased IDO1 mRNA and kyn production after induction of IDO1 expression and kyn production by IFN-(Fig. escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT. Introduction In recent years tryptophan (trp) degradation has received increasing attention as a Tesevatinib potent immunosuppressive mechanism involved in the maintenance of immunological tolerance. The trp-degrading enzyme indoleamine-2,3-dioxygenase (IDO) has been implicated in maternal tolerance towards allogeneic concepti [1], controlling autoimmune diseases [2], [3] and chronic infection [4], as well as promoting tumor immune escape [5], [6], [7]. IDO-mediated trp degradation is not restricted to tumor cells [7] but is also detected in tumor-draining lymph nodes [8]. In both tumor-draining Tesevatinib Tesevatinib lymph nodes and tumors, IDO1 creates local tolerance by directly suppressing T-cell responses and enhancing immunosuppression mediated by regulatory T cells (TmRNA by siRNA measured by Tesevatinib qRT-PCR. (C) Western blot analysis showing IDO1 protein expression in SKOV-3 cells with siRNA mediated knockdown of IDO1 in comparison to controls. (D) Immunocytochemistry (red, IDO1 staining; blue, DAPI nuclear staining) of control SKOV-3 cells and SKOV-3 cells with IDO1 knockdown. (E) Kyn release of SKOV-3 cells after knockdown of IDO1 in comparison to controls. Experiments were performed at least in triplicate. Data are mean SEM. * (p<0.05). Open in a separate window Figure 2 1-D-MT reduces T cell proliferation in cocultures of SKOV-3 cells with mixed leukocyte reactions.(A) Alloreactive T cell proliferation after addition of 25 M kyn to mixed leukocyte reactions (MLR). (B) Alloreactive T cell proliferation in the presence of 6000 SKOV-3 cells. (C) T cell proliferation in MLR cocultured with 2000 control SKOV-3 cells (white bar) or 2000 SKOV-3 cells with a knockdown of IDO1 (black bar). (D) T cell proliferation in cocultures of MLR with 2000 SKOV-3 cells after addition of increasing concentrations of 1-L-MT. (E) T cell proliferation in Tesevatinib cocultures of MLR with 2000 SKOV-3 cells after addition of increasing concentrations of 1-D-MT. (F) Representative result of MLR/SKOV-3 coculture experiments with PBMC from five different donors and 2000 or 6000 SKOV-3 cells. Cells were treated with or without 1 mM 1-D-MT in combination with or without 250 M trp. Proliferation was measured by 3[H] methylthymidine uptake. Experiments were performed at least in triplicate. Data are mean SEM. * (p<0.05). Open in a separate Ntn1 window Figure 3 1-D-MT does not inhibit the proliferation or cell cycle progression of SKOV-3 cells.(A) 3[H] methylthymidine incorporation of SKOV-3 cells treated with 1 mM 1-D-MT (black bar) or vehicle (white bar) for 6 days. (B) Cell cycle analysis of SKOV-3 cells treated with 1 mM 1-D-MT or vehicle for 48 h. (C) Proliferation analysis of CFSE-stained lymphocytes from 6 day cocultures of MLR with 2000 SKOV-3 cells, treated with indicated concentrations of 1-D-MT (upper panel). Plot of the cell numbers in each generation of the above experiment (lower panel). 1- D-MT increases kyn production in human cancer cells We then tested the effect of 1-MT on the kyn production of SKOV-3 cells. Surprisingly, 1-D-MT concentration-dependently increased kyn formation (Fig. 4A), while its stereoisomer 1-L-MT inhibited kyn formation as expected (Fig. 4A). The racemic mixture of 1-MT, which has been used in many studies, including those that have established IDO1 as an immunosuppressive enzyme, inhibited kyn formation, albeit less than 1-L-MT alone (Fig. 4A). As trp concentrations in the media may have limited the increase in kyn production, we also measured the kyn concentrations produced by SKOV-3 cells in response to 1-D-MT in the presence of increasing trp concentrations. Under these conditions much higher kyn concentrations were reached (Fig. 4B), suggesting that the plateau observed above concentrations of about 250 M 1-D-MT (Fig. 4A) was due.