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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 (IC 50  = 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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Indoleamine 2,3-dioxygenases (IDOs) - belonging in the heme dioxygenase family and degrading tryptophan - are responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD + ). As such, they are expressed by a variety of invertebrate and vertebrate species. In mammals, IDO1 has remarkably evolved to expand its functions, so to become a prominent homeostatic regulator, capable of modulating infection and immunity in multiple ways, including local tryptophan deprivation, production of biologically active tryptophan catabolites, and non-enzymatic cell-signaling activity. Much like IDO1, arginase 1 (Arg1) is an immunoregulatory enzyme that catalyzes the degradation of arginine. Here, we discuss the possible role of amino-acid degradation as related to the evolution of the immune systems and how the functions of those enzymes are linked by an entwined pathway selected by phylogenesis to meet the newly arising needs imposed by an evolving environment., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To investigate the effect of cyclooxygenase inhibition in experimental Gram-negative sepsis, indomethacin was administered to mice at different times (1 or 5 days, or 1 h) before sublethal infection with an intravenous inoculum of Pseudomonas aeruginosa Early indomethacin exposure did not alter the outcome of infection, yet treatment at the time of bacterial challenge resulted in a high mortality rate. Polymerase chain reaction-assisted mRNA amplification in the spleens of infected mice revealed that tumor necrosis factor alpha (TNF-alpha) messenger was selectively expressed by the drug-treated and infected mice during the 24 h preceding death. Higher TNF-alpha levels were found in sera from these mice, whose macrophages produced increased levels of nitric oxide in vitro. Both pentoxifylline, an inhibitor of TNF-alpha synthesis, and an inhibitor of nitric oxide production improved survival in the indomethacin-treated and infected mice, although no such effect followed the administration of TNF-neutralizing antibodies. These data support the notion that cyclooxygenase inhibitors may exert both positive and negative effects in Gram-negative sepsis, the latter presumably involving overproduction of TNF-alpha.

Murine tumor cells treated with triazene compounds (TZC), in vivo or in vitro, are capable of eliciting specific transplantation resistance in syngeneic hosts, and T-cell-mediated proliferative and cytotoxic responses, directed against novel drug-induced antigen(s). Since this phenomenon, referred to as chemical xenogenization (CX) could open up new perspectives in the immunochemotherapy of human neoplasias, it was of interest to investigate whether CX could also occur in human tumors. However, established human tumor cell lines along with fully immunocompetent autologous lymphocytes, are seldom available. Therefore studies were carried out to test whether parental or TZC-treated tumor cells could be differentially recognized by allogeneic lymphocytes. Experiments were performed in both human and murine models, using a lung adenocarcinoma line treated in vitro with TZC, or an established xenogenized mouse lymphoma, respectively. The results indicate that allogeneic cytotoxic T-lymphocytes (CTL) recognize specifically murine TZC-treated tumor cells. This was supported by the finding that antisera directed against the drug-treated cells abrogated the generation and the cytolytic activity of allogeneic CTL reactive against the TZC-treated tumor. In addition it was found that changes of the antigenic pattern of cell membrane recognizable by cloned allogeneic CTL occur in the TZC-treated human carcinoma cell line.

We have investigated the tumor-specific reactivity of different T-cell subsets from mice primed with clonal variants of L5178Y and P815 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In both tumor systems, anti-parental tumor immunity and protection against non-immunogenic clones were only induced by vaccinating the hosts with highly immunogenic cell variants, and the effect correlated with the detection of TATA-specific delayed-type hypersensitivity (DTH) reactions. The footpad reaction was transferable with spleen cell populations from immunized mice, and enrichment of splenic lymphocytes in L3T4+ but not Lyt-2+ lymphocytes increased the footpad swelling. Unfractionated spleen cell populations from immunized mice released high amounts of IL-2 and IFN-gamma in vitro in response to parental antigens. Purified L3T4+ and Lyt-2+ lymphocytes also produced IFN-gamma when incubated in vitro with the parental tumors and accessory cells. It is suggested that the mechanisms of anti-parental tumor immunity induced by MNNG-treated variants may be similar to those described previously for triazene-xenogenized L5178Y/DTIC cells, and may involve induction of a tumor-specific DTH reaction and IFN-gamma-mediated stimulation of non-specific tumoricidal effects.

To determine whether a novel pattern of lymphokine production might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a panel of murine tumors xenogenized by DTIC for production of soluble factors with lymphokine-like activity and induction of lymphokine release from naïve or specifically sensitized lymphocytes. In the L5178Y tumor system, a majority of xenogenized but not parental clones produced an IL-1-like factor, and this was associated, as a rule, with class II antigen expression and antigen-presenting ability. However, no such properties were exhibited by the xenogenized variants of P815 and L1210Ha cells, which nevertheless occasionally expressed other lymphokine (GM-CSF, IL-3) activities. On examining the ability of xenogenized and parental tumors to cause release of IL-1, IL-2, IL-3, IFN-gamma, TNF/LT and GM-CSF from T-cells, we found, as a rule, an increased lymphokine production when lymphocytes primed in vivo to a xenogenized tumor were restimulated in vitro with the same or parental cells.

To develop monoclonal antibodies (MAbs) recognizing drug-mediated tumor antigens on a chemically xenogenized murine lymphoma, hybridomas were constructed with splenocytes from histocompatible mice hyperimmunized with L5178Y cells antigenically altered by triazene treatment in vivo (clone D, derived from a polyclonal L5178Y/DTIC subline). Screening of supernatants with parental and xenogenized cells showed that nine MAbs displayed exclusive or preferential reactivity with clone D cells as detected by immunofluorescence, and failed, as a rule, to bind normal or unrelated malignant cells of the same or different haplotype. Moreover, no reactivity was displayed to the triazene-xenogenized variants of antigenically unrelated tumors. All nine MAbs, however, were capable of binding a panel of L5178Y/DTIC clones in addition to clone D. When the ability of these antibodies to interfere with the development of cell-mediated immunity to clone D cells in vitro was tested, it was found that the proliferative reaction and generation of cytolytic activity by syngeneic lymphocytes were inhibited by addition of several MAbs to the tumor--lymphocyte co-cultures.