Enzymatic Properties, Tissue Distribution, and Regulation of Expression

IDO is a heme-containing intracellular enzyme that catalyzes the initial and rate-limiting step of tryptophan catabolism by cleavage of the pyrrole ring of L-tryptophan (Fig. 1). While only a small amount of tryptophan from food is converted to serotonin and further converted into melatonin, most (more than 95%) dietary tryptophan is metabolized by IDO along the kynurenine pathway, leading finally to the biosynthesis of nicotinamide adenine dinucleotide (NAD).

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IDO is expressed in a wide range of tissues such as the lung, intestine, brain, and placenta, although within these tissues, expression occurs only in a limited range of cell types. In contrast, in the liver, tryptophan is catabolized by a structurally distinct liver-specific enzyme, tryptophan 2,3-dioxygenase (TDO; EC 1.13.1.12), but not by IDO.

A cDNA encoding human IDO has been cloned and its deduced primary structure is obtained. Human IDO cDNA encodes a protein of 403 amino acids with a molecular weight of approximately 45 kDa. The IDO protein is encoded by a tightly regulated gene that responds to inflammatory mediators such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and lipopolysaccharide (LPS). In humans, IDO is encoded by the INDO gene, which is comprised of ten exons spanning approximately 15 kb at chromosome site 8p11-p12. IFN-γ is a major inducer of IDO expression. Transcriptional induction of the INDO gene is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, specifically JAK1 and STAT1α. NF-κB also contributes to IDO induction. It has been shown that IFN-γ and  TNF act synergistically through NF-κB activation to induce expression of interferon regulatory factor (IRF)-1, which leads to upregulation of IDO expression. In addition,  prostaglandin E₂ (PGE₂) strongly upregulates IDO.

IDO and Immune Suppression

Accumulating evidence indicates immunosuppressive function of IDO. First, IDO is expressed in placental trophoblasts and macrophages during pregnancy and prevents rejection of the allogeneic fetus, thereby suggesting involvement of IDO in fetal–maternal tolerance. This is based on the findings that a pharmacological inhibition of IDO activity by 1-methyl-tryptophan (1-MT) results in rejection of the fetus in pregnant mice. Subsequent studies clarified the mechanism of IDO  immunosuppression by locally depleting tryptophan and by producing toxic tryptophan metabolites (e.g., kynurenine, see Fig. 1), which causes the arrest of proliferation and the apoptosis of alloreactive T-cells. It has been similarly demonstrated that enzymatic activity of IDO correlates with reduced T-cell-mediated immune responses in several experimental systems, including models of inflammatory diseases, autoimmune diseases, and organ/tissue transplant rejection.

Secondly, IDO is expressed in antigen-presenting cells (APCs), especially certain subsets of  dendritic cells (DCs), and it regulates immune response and induces tolerance. For example, exposure of monocyte-derived DCs to a cocktail of cytokines for the purpose of maturation results in strong upregulation of IDO expression. Mature DCs that express functional IDO can be potent suppressors of  T-cell response and promote tolerance in vivo and in vitro. Expression of functional IDO requires ligation of B7-1/B7-2 (CD80/CD86) molecules on DC cell membranes by cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expressed by CD4 + CD25- regulatory T cells. This binding of CTLA-4 to B7 receptors on DCs activates IFN-α-mediated STAT1-dependent IDO upregulation. Similarly, the synthetic soluble fusion protein CTLA-4 immunoglobulin (CTLA-4 Ig) is a potent inducer of IDO expression by DCs. IDO-expressing DCs may also promote the development of regulatory T cells. These findings therefore suggest that IDO-expressing DCs and regulatory T cells may cooperate to induce antigen-specific T-cell anergy and create a state of peripheral immune tolerance.

While the mechanisms of IDO-dependent inhibition of T-cell function have been elucidated, less is known about the possible role of IDO in regulating natural killer cell activity. NK cell function is also suppressed by IDO, and IDO inhibits the proliferation of NK cells in vitro. Furthermore, L-kynurenine, a tryptophan-derived catabolite produced by IDO activity, inhibits the cytokine-mediated expression of the specific triggering receptors, NKp46 and NKG2D, which are responsible for the induction of NK cell-mediated killing. As a consequence, L-kynurenine-treated NK cells display an impaired ability to kill target cells recognized via NKp46 and NKG2D. These findings suggest that IDO can suppress both T-cell and NK cell responses.

IDO and Cancer

Although many tumor-associated antigens have been identified in various tumor cells, the reason why tumor antigen-specific host T-cells fail to control tumor progression remains obscure. Tumors are known to successfully escape the host immune system by two possible mechanisms; a loss of surface antigens that renders them invisible to immune cells or an attack on the immune cells that disables their antitumor functions. The accumulated evidence that IDO is physiologically important for establishing peripheral tolerance to alloantigens prompts a hypothetical mechanism that tumors could create a state of tolerance through tryptophan catabolism carried out by IDO. If IDO is actually present in tumor tissues, it could induce local immune suppression, resulting in the protection of tumor cells from host T-cell-mediated rejection and/or attack by NK cells, and subsequently leading to tumor growth and progression.

IDO is expressed in various human cancer tissues, and that it is expressed not only by immune cells, but also by the tumor cells themselves. Tumors expressing IDO can resist immune rejection by tumor-associated antigen-specific host cytotoxic T-cells in mouse models. This effect is accompanied by a lack of accumulation of T-cells at the tumor site, resulting from arrest of T-cell proliferation caused by IDO-mediated local tryptophan depletion. When the IDO inhibitor 1-MT is dissolved in the drinking water given to tumor-bearing mice, the growth of IDO-expressing tumors is significantly reduced.

In contrast, it is shown that IDO is expressed by CD19 + plasmacytoid DCs in tumor-draining lymph nodes in mice, and these specific IDO-expressing DCs potently suppress host antitumor T-cell responses and induce tolerance to tumor-derived antigens. In humans, IDO + cells of host origin are also present in draining lymph nodes of patients with  melanoma,  breast cancer, and other tumors.

Taken together, two possible mechanisms for the immunosuppressive action of IDO in tumor-bearing hosts are proposed. IDO expressed by the tumor cells themselves can create a localized immunosuppressive status within the tumor microenvironment either by suppressing effector T-cell function and proliferation due to tryptophan depletion, or by directly killing tumor-infiltrating T-cells and NK cells using toxic metabolites of tryptophan. Alternatively, host DCs expressing IDO can pick up tumor-derived antigens and migrate into tumor-draining lymph nodes, where these IDO-expressing APCs cannot effectively prime naïve T-cells, resulting in T-cell deletion, failure of clonal expansion, or perhaps induction of regulatory T cells.

Clinical Relevance