No abstract

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme that contributes to tolerance in a number of biological settings. In cancer, IDO activity may help promote acquired tolerance to tumor antigens. The IDO inhibitor 1-methyltryptophan is being developed for clinical trials. However, 1-methyl-tryptophan exists in two stereoisomers with potentially different biological properties, and it has been unclear which isomer might be preferable for initial development. In this study, we provide evidence that the D and L stereoisomers exhibit important cell type-specific variations in activity. The L isomer was the more potent inhibitor of IDO activity using the purified enzyme and in HeLa cell-based assays. However, the D isomer was significantly more effective in reversing the suppression of T cells created by IDO-expressing dendritic cells, using both human monocyte-derived dendritic cells and murine dendritic cells isolated directly from tumor-draining lymph nodes. In vivo, the D isomer was more efficacious as an anticancer agent in chemo-immunotherapy regimens using cyclophosphamide, paclitaxel, or gemcitabine, when tested in mouse models of transplantable melanoma and transplantable and autochthonous breast cancer. The D isomer of 1-methyl-tryptophan specifically targeted the IDO gene because the antitumor effect of D-1-methyl-tryptophan was completely lost in mice with a disruption of the IDO gene (IDO-knockout mice). Taken together, our findings support the suitability of D-1-methyl-tryptophan for human trials aiming to assess the utility of IDO inhibition to block hostmediated immunosuppression and enhance antitumor immunity in the setting of combined chemo-immunotherapy regimens. [Cancer Res 2007;67(2):792-801]

IntroductionRegulatory T cells (Tregs) represent a critical barrier to immunotherapy of tumors. Established tumors suppress immune responses against their own antigens, and Tregs are emerging as a key mechanism contributing to this state of functional unresponsiveness. 1 In murine models, host Tregs become activated within days of tumor implantation. 2 Once activated, Tregs are difficult to eliminate and serve to potently and dominantly inhibit otherwise effective immune responses against the tumor. 3 We have shown that Foxp3 ϩ Tregs in the draining lymph nodes of mouse tumors become highly activated by exposure to the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). 4,5 In tumor-draining lymph nodes (TDLNs), IDO is expressed by a specific subset of IDO-competent plasmacytoid dendritic cells (DCs). 6 The combination of these IDO-expressing pDCs and IDO-activated Tregs renders the local milieu in the TDLNs profoundly inhibitory for T-cell activation. 7 Tregs can be suppressive, but this is not a fixed and immutable attribute. Resting Tregs are not spontaneously suppressive, and require an activation step before they become functionally inhibitory. 8 Conversely, the suppressive phenotype of Tregs is plastic. When Foxp3 is artificially ablated in mature Tregs, the suppressor phenotype is converted to a proinflammatory, T helper-like phenotype that can participate in autoimmunity. 9 Likewise, Tregs exposed to certain inflammatory signals (eg, from activated DCs or TLR ligands) can lose their suppressor activity 10 and may alter their phenotype (be "reprogrammed") to resemble proinflammatory effector cells. [11][12][13] Thus, at least in these experimental models, Tregs show a significant degree of phenotypic plasticity and are susceptible to both activation and deactivation (reprogramming) by signals from their local microenvironment.However, it is not known whether this apparent plasticity of Tregs is of biologic relevance for tumor immunology. In the current study, we test the hypothesis that, under conditions of antigendriven T-cell response to tumors, IDO functions as a critical molecular "switch" in TDLNs, regulating the phenotype and functional activity of Tregs. We show that, when IDO is active, Tregs are maintained in their normal potently suppressive state; but when IDO is blocked, Tregs undergo an inflammation-induced, interleukin-6 (IL-6)-dependent conversion into a nonsuppressive, proinflammatory phenotype similar to T-helper-17 (TH17) cells. These findings position IDO as a previously unsuspected key molecular regulator of Treg phenotype and function in TDLNs. Methods Reagents, cell lines, and mouse strainsA complete list of reagents, 1-methyl-D-tryptophan (1MT) preparation, tumor cell lines, and all transgenic and knockout mouse strains is given in supplemental materials (available on the Blood website; see the Supplemental Materials link at the top of the online article). Animal studies were approved by the Institutional Animal Care and Use Committee of the Medical College of Georgia. Detail...

SUMMARY Foxp3+ regulatory T (Treg) cells can undergo reprogramming into a phenotype expressing proinflammatory cytokines. However, the biologic significance of this conversion remains unclear. We show that large numbers of Treg cells undergo rapid reprogramming into activated T-helper cells following vaccination with antigen plus Toll-like receptor 9 (TLR-9) ligand. Helper activity from converted Treg cells proved essential during initial priming of CD8+ T cells to a new cross-presented antigen. Help from Treg cells was dependent on CD40L, and (unlike help from conventional non-Treg CD4+ cells) did not require pre-activation or prior exposure to antigen. In hosts with established tumors, Treg cell reprogramming was suppressed by tumor-induced indoleamine 2,3-dioxygenase (IDO), and vaccination failed due to lack of help. Treg reprogramming, vaccine efficacy and anti-tumor CD8+ T cell responses were restored by pharmacologic inhibition of IDO. Reprogrammed Treg cells can thus participate as previously unrecognized drivers of certain early CD8+ T cell responses.

Inflammation stimulates immunity but can create immune privilege in some settings. Here, we show that cutaneous Leishmania major infection stimulated expression of the immune regulatory enzyme indoleamine 2,3 dioxygenase (IDO) in local lymph nodes. Induced IDO attenuated the T cell stimulatory functions of dendritic cells and suppressed local T cell responses to exogenous and nominal parasite antigens. IDO ablation reduced local inflammation and parasite burdens, as did pharmacologic inhibition of IDO in mice with established infections. IDO ablation also enhanced local expression of proinflammatory cytokines and induced some CD4(+) T cells to express interleukin (IL) 17. These findings showed that IDO induced by L. major infection attenuated innate and adaptive immune responses. Thus, IDO acts as a molecular switch regulating host responses, and IDO inhibitor drugs are a potential new approach to enhance host immunity to established leishmania infections.