The signaling function of IDO1 incites the malignant progression of mouse B16 melanoma.

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene (Ido1WT ) or a mutated variant lacking the catalytic, but not signaling activity (Ido1H350A ). As compared to the Ido1WT -transfected counterpart (B16WT), B16-F10 cells expressing Ido1H350A (B16H350A) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16H350A cells, also detectable in vivo, were found to be accompanied by a reduction in tumor-infiltrating CD8+ T cells and an increase in Foxp3+ regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule.

Identification of a 2-propanol analogue modulating the non-enzymatic function of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3 dioxygenase 1 (IDO1) is a metabolic enzyme that catalyzes the conversion of the essential amino acid tryptophan (Trp) into a series of immunoactive catabolites, collectively known as kynurenines. Through the depletion of Trp and the generation of kynurenines, IDO1 represents a key regulator of the immune responses involved in physiologic homeostasis as well as in neoplastic and autoimmune pathologies. The IDO1 enzyme has been described as an important immune checkpoint to be targeted by catalytic inhibitors in the treatment of cancer. In contrast, a defective expression/activity of the enzyme has been demonstrated in autoimmune diseases. Beside its catalytic activity, the IDO1 protein is endowed with an additional function associated with the presence of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which, once phosphorylated, bind SHP phosphatases and mediate a long-term immunoregulatory activity of IDO1. Herein, we report the screening of a focused library of molecules bearing a propanol core by a protocol combining microscale thermophoresis (MST) analysis and a cellular assay. As a result, the combined screening identified a 2-propanolol analogue, VIS351, as the first potent activator of the ITIM-mediated function of the IDO1 enzyme. VIS351 displayed a good dissociation constant (Kd = 1.90 µM) for IDO1 and a moderate cellular inhibitor activity (IC50 = 11.463 µM), although it did not show any catalytic inhibition of the recombinant IDO1 enzyme. Because we previously demonstrated that the enzymatic and non-enzymatic (i.e., ITIM-mediated) functions of IDO1 reside in different conformations of the protein, we hypothesized that in the cellular system VIS351 may shift the dynamic conformational balance towards the ITIM-favoring folding of IDO1, resulting in the activation of the signaling rather than catalytic activity of IDO1. We demonstrated that VIS351 activated the ITIM-mediated signaling of IDO1 also in mouse plasmacytoid dendritic cells, conferring those cells an immunosuppressive phenotype detectable in vivo. Thus the manuscript describes for the first time a small molecule as a positive modulator of IDO1 signaling function, paving the basis for an innovative approach to develop first-in-class drugs acting on the IDO1 target.

T cell apoptosis by tryptophan catabolism.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.

IL-6 inhibits the tolerogenic function of CD8 alpha+ dendritic cells expressing indoleamine 2,3-dioxygenase.

The outcome of dendritic cell (DC) presentation of tumor and/or self peptides, including P815AB (a tumor peptide of murine mastocytoma cells) and NRP-A7 (a synthetic peptide mimotope recognized by diabetogenic T cells), may depend on a balance between the activities of immunogenic (CD8alpha(-)) and tolerogenic (CD8alpha(+)) DC. By virtue of their respective actions on CD8(-) and CD8(+) DC, IL-12 and IFN-gamma have functionally opposing effects on peptide presentation by the CD8(-) DC subset, and IFN-gamma-activated CD8(+) DC mediate tolerogenic effects that prevail over the adjuvant activity of IL-12 on CD8(-) DC. We have previously shown that CD40 ligation abrogates the tolerogenic potential of CD8(+) DC, an effect associated with an impaired capacity of the CD40-modulated and IFN-gamma-treated DC to degrade tryptophan and initiate T cell apoptosis in vitro. We report here that IL-6 may both replace (upon administration of the recombinant cytokine) and mediate (as assessed by the use of neutralizing Abs) the effect of CD40 ligation in ablating the tolerogenic activity of CD8(+) DC. The activity of IL-6 includes down-regulation of IFN-gammaR expression in the CD8(+) DC subset and correlates to a reduced ability of these cells to metabolize tryptophan and initiate T cell apoptosis in vitro.

Positive regulatory role of IL-12 in macrophages and modulation by IFN-gamma.

Similar to myeloid dendritic cells, murine macrophages and macrophage cell lines were found to express a surface receptor for IL-12. As a result, peritoneal macrophages could be primed by IL-12 to present an otherwise poorly immunogenic tumor peptide in vivo. Using binding analysis and RNase protection assay, we detected a single class of high affinity IL-12 binding sites (K(d) of approximately 35 pM) whose number per cell was increased by IFN-gamma via up-regulation of receptor subunit expression. Autocrine production of IL-12 was suggested to be a major effect of IL-12 on macrophages when the cytokine was tested alone or after priming with IFN-gamma in vitro. In vivo, combined treatment of macrophages with IFN-gamma and IL-12 resulted in synergistic effects on tumor peptide presentation. Therefore, our findings suggest a general and critical role of IL-12 in potentiating the accessory function of myeloid APC.

Interleukin-9 induces 24P3 lipocalin gene expression in murine T cell lymphomas.

Interleukin-9 (IL-9) is a Th2 cytokine whose overexpression is associated with asthma and T cell lymphomagenesis. All the IL-9 activities studied so far are mediated by a specific hemopoietin receptor that activates a Jak/STAT pathway. Searching for genes specifically modulated by IL-9, we observed that the 24P3 mRNA is strongly upregulated in BW5147 T lymphoma cells upon IL-9 stimulation. 24P3 is a member of the lipocalin family, and has been reported to bind N-formyl-Met-Leu-Phe, a potent neutrophil chemoattractant, and possibly other lipophilic mediators of inflammation. A similar 24P3 induction was observed in other T cell lymphomas (EL4 and TH201) in response to IL-9, as well as in EL4 cells stimulated with IL-6 or IL-1. By contrast, other IL-9-responsive cells such as mast cell line MC9 and B cell lymphoma A20 showed no 24P3 induction upon IL-9 stimulation. Experiments using IL-9R mutants indicated that STAT transcription factors, particularly STAT3, are involved in this process. However, 24P3 gene induction was slow, reaching a plateau from 36 to 72 hours after stimulation and was inhibited if cells were treated with cycloheximide during the first 8 hours of IL-9 stimulation, suggesting an indirect induction requiring new protein synthesis.

IL-12 induces SDS-stable class II alphabeta dimers in murine dendritic cells.

We assessed the effect of rIL-12 on the expression of class II molecules and on the ratio between SDS-stable and unstable alphabeta dimers in dendritic cells. We found that in vitro exposure of the cells to IL-12 increased their surface expression of mature class II molecules, despite a marked decline in class II biosynthesis. This effect was accompanied by a striking increase in the overall proportion of SDS-stable heterodimers.

Immunogenicity of tumor peptides: importance of peptide length and stability of peptide/MHC class II complex.

Nonameric P815AB, a cytotoxic-T-lymphocyte-defined minimal core peptide encoded by the murine mastocytoma gene P1A, fails to initiate CD4+ cell-dependent reactivity in vivo to class-I-restricted epitopes when mice are administered peptide-pulsed dendritic cells. Effective immunization requires T helper effects, such as those mediated by coimmunization with class-II-restricted (helper) peptides or by the use of recombinant interleukin-12 (rIL-12). Although P815AB does possess class-II-restricted epitopes, they are likely suboptimal, resulting in poor affinity and/or stability of MHC/P815AB complexes and inadequate activation of the antigen-presenting cell function of dendritic cells. The present study has examined a series of longer, P815AB-centered peptides (11-14 amino acids in length, all P1A-encoded) for their ability to initiate CD4+ and CD8+ cell-mediated responses to the nonamer in vivo, their ability to bind class II MHC in vitro, and their ability to assemble class II molecules stably. By means of a class-I-restricted skin test assay in mice receiving peptide-pulsed dendritic cells, we found that a 12-mer and a 13-mer effectively immunized against the core P815AB peptide, and that this correlated with IL-2 production in vitro by CD4+ cells in response to the nonamer. In vitro studies, involving affinity-purified class II molecules, showed that the capacity to assemble class II molecules stably, more than the affinity for class II MHC, correlated with the ability of the different P815AB peptides to prime the host to the core peptide seen by the T cells.

IL-12 acts directly on DC to promote nuclear localization of NF-kappaB and primes DC for IL-12 production.

We analyzed the expression of an IL-12 receptor by fresh dendritic cells (DC) and a DC line. Using RT-PCR, RNAse protection, and electrophoretic mobility shift assay analysis, we found that DC possess an IL-12 receptor with beta1 subunit (downstream box 1)-related differences from that on T cells. IL-12 signaling through this receptor involved members of the NF-KB but not STAT family. The unique properties of the IL-12 receptor on DC, characterized by a single class of binding sites with a Kd of about 325 pM, may underlie rather unique effects, such as IFNgamma-independent augmentation of class II antigen expression and priming for LPS-induced production of IL-12.

Circulating levels of IL-10 are critically related to growth and rejection patterns of murine mastocytoma cells.

Previously tumorigenic P815 tumor cells are rejected by histocompatible mice after transfection with a mutated retroviral gene, and the host is made resistant to subsequent challenge with tumorigenic (control) cells transfected with the nonmutated sequence. To functionally characterize the class I-restricted response to the tumor cell vaccine, we have assessed the in vitro (by CD8+ cells) and in vivo production of type 1 or type 2 cytokines in mice injected with either type of transfected P815 derivative. IL-12 and IL-10 were selectively or preferentially expressed by the regressor mice, and this correlated with the detection of functional type 1 reactivity in vivo (i.e., delayed-type hypersensitivity). Other cytokines were produced by the regressor mice only in vitro (IFN-gamma) or were not detected at all with either type of tumor recipient (IL-4). By means of monoclonal antibody-mediated neutralization or enhancement of endogenous cytokine levels, IL-10 was found to serve an important role in the growth and rejection patterns of the transfected P815 derivatives. In addition to previous evidence for an IL-12 requirement in promoting anti-P815 reactivity, these data establish IL-10 as an important cytokine in permitting optimal expression of this reactivity, which apparently develops in the absence of a strong bias toward a type 1 or type 2 cytokine response.