ido expression by dendritic cells: tolerance and tryptophan ... - Nature

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IDO EXPRESSION BY DENDRITIC CELLS: TOLERANCE AND TRYPTOPHAN CATABOLISM Andrew L. Mellor and David H. Munn Abstract | Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades the essential amino acid tryptophan. The concept that cells expressing IDO can suppress T-cell responses and promote tolerance is a relatively new paradigm in immunology. Considerable evidence now supports this hypothesis, including studies of mammalian pregnancy, tumour resistance, chronic infections and autoimmune diseases. In this review, we summarize key recent developments and propose a unifying model for the role of IDO in tolerance induction.

ANTIGENICITY

The ability to be recognized by the immune system by binding to T- and B-cell receptors, although this might not result in overt immune responses. IMMUNOGENICITY

The ability to provoke overt immune responses. ESSENTIAL AMINO ACIDS

Amino acids that cannot be synthesized by cells and must be acquired in the diet. Tryptophan is energetically unfavourable to synthesize and even organisms that can synthesize tryptophan will take it up in preference to synthesizing it.

Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, Departments of Medicine and Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA. Correspondence to A.L.M. e-mail: [email protected]. edu doi:10.1038/nri1457

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Antigenic challenges do not always provoke effective immune responses. Bacterial antigens can provoke a strong reaction when administered with an adjuvant, yet be tolerated in the gut1. Similarly, strongly immunogenic viral proteins can become tolerizing when expressed by tumour cells2. Therefore, although ANTIGENICITY is necessary for IMMUNOGENICITY, it might not be sufficient. This is an important distinction, because manipulating (enhancing or suppressing) immune responses to improve clinical outcomes is an important challenge in a range of inflammatory diseases, including cancer, and infectious and autoimmune disorders, as well as in improving organ/tissue transplantation success rates. Dendritic cells (DCs) are key regulators of immune outcomes, capable of promoting or suppressing T-cell responses depending on the circumstances3,4. This feature of DCs relates to their ability to integrate a diverse array of incoming signals, and then to direct an appropriate T-cell response5. However, the role of DCs as an ‘information management’ system has been considered mainly from the standpoint of activating the immune system6, with less attention given to signals that prompt DCs to suppress T-cell responses and promote tolerance. Accumulating evidence indicates that DCs can also induce tolerance, rather than immune activation, to the antigens they present7. Similar to the decision to promote immunity, the decision to promote tolerance requires the integration of information that is gathered

| OCTOBER 2004 | VOLUME 4

by DCs from the innate and adaptive immune systems (FIG. 1). This information might be passive — that is, the absence of signals that indicate infection or injured self 8 leading to ‘default’ tolerogenic presentation of antigen by resting or immature DCs9,10. Alternatively, DCs might respond actively to tolerogenic signals, such as interleukin-10 (IL-10) or transforming growth factor-β (TGF-β), that are present in a globally immunosuppressive local milieu11, or to signals that are delivered in an antigen-specific manner by the adaptive immune system through regulatory T cells (TReg cells)12,13. Once a decision has been made in favour of tolerance, it is currently unclear exactly how the pool of DCs functions to create the desired outcome. In this review, we focus on one specific immunosuppressive mechanism: tryptophan catabolism by DCs that express the enzyme indoleamine 2,3-dioxygenase (IDO). IDO is a tryptophan-degrading enzyme that was originally identified in rabbit intestine. It was subsequently found that pro-inflammatory mediators, such as endotoxin and interferon-γ (IFN-γ), induce the expression of IDO in several tissues. Early literature documented the ability of IDO to inhibit the proliferation of facultative intracellular pathogens and tumour cells in vitro through consumption of the ESSENTIAL AMINO ACID tryptophan14. In 1999, we proposed an additional role for IDO, suggesting that IDO-dependent suppression of T-cell responses

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APOENZYME

The protein component of an enzyme that requires additional co-factors to become active (holoenzyme).

might function as a natural immunoregulatory mechanism15, based on data showing that this mechanism inhibited maternal T-cell immunity to fetal tissues during mammalian gestation16. As summarized in TABLE 1, so far, physiological IDO activity has been implicated in T-cell tolerance to tumours17–20; dysfunctional self-tolerance in non-obese diabetic (NOD) mice21; as an effector mechanism for the immunosuppressive reagent cytotoxic T lymphocyte antigen 4 (CTLA4)–immunoglobulin fusion protein22; and as a protective negative regulator in autoimmune disorders23–25 and an induced mouse model of asthma26. IDO activity is certainly not the only immunoregulatory mechanism that occurs in these diverse disorders, and the immune system seems able to create long-term compensation for genetic defects in the IDO mechanism. Nevertheless, acute disruption of IDO activity in all of these models either proved catastrophic to the host or fetus, or markedly enhanced disease pathology. Key new developments in this rapidly advancing field prompt the need for a review focused specifically on regulatory DCs that use the IDO mechanism, and

Tolerogenic factors Innate immune system: TGF-β and IL-10 Adaptive immune system: signals from regulatory T cells

Immunogenic factors Innate immune system: TLR ligands Adaptive immune system: TH1 and TH2 cytokines Integration by the DC pool

Functional plasticity Activating phenotype

Maturation

Specialized DC subsets

Immature DC

Suppressive phenotype

Tolerance

Mature DC

Immunogenic DC

Tolerogenic DC

Immunity

Figure 1 | Pivotal role of dendritic cells in deciding tolerance versus immunity. The heterogeneous pool of dendritic cells (DCs) is centrally positioned to integrate signals from the innate and adaptive immune system. Both systems can contribute signals that bias the outcome either towards immunity or tolerance. The innate system promotes immunity through ligation of Toll-like receptors (TLRs) on DCs or by various other pro-inflammatory signals indicative of infection or injury8. The innate system can promote tolerance by creating a local milieu that is dominated by anti-inflammatory cytokines, such as interleukin-10 (IL-10) or transforming growth factor-β (TGF-β)12, and perhaps through other tolerogenic signals to the DCs, such as apoptotic host cells146. One way that the adaptive system promotes immunity is through CD4+ T helper (TH) cells that condition or ‘license’ DCs to support effector T-cell responses (for example, through CD40 ligation)147,148. Conversely, the adaptive system can promote tolerance through signals from regulatory T cells, such as CD4+CD25+ T cells and TR1 cells. These tolerogenic signals are not yet fully defined, but might include antigen-induced production of IL-10 and TGF-β, and indoleamine 2,3-dioxygenase (IDO)-inducing signals through ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) by CD80/CD86 (REF. 43). The pool of DCs integrates all of the preceding signals to generate a net response of immunity or tolerance. Many mechanisms operate in this integration process, including maturation of stimulatory DCs; selective activation of particular DC subsets that are specialized to induce tolerance or immunity; and functional plasticity of mature DCs, allowing them to adopt either suppressive/tolerogenic or activating/immunogenic phenotypes, depending on the signals received81. These mechanisms are not mutually exclusive and probably operate in combination, depending on the signals and the specific types of DC involved.

NATURE REVIEWS | IMMUNOLOGY

the role of IDO in tolerance. We review recent data that offer insights into IDO-dependent T-cell suppression and tolerance induction, and propose a unified conceptual model of IDO-dependent tolerogenic processes, highlighting areas of current and future research. Molecular genetics and biochemistry of IDO

In mammals, two genes encode unrelated haemecontaining enzymes that catalyse oxidative degradation of tryptophan: IDO and tryptophan 2,3-dioxygenase (TDO) (BOX 1). Each enzyme catalyses the same reaction, oxidative cleavage of the 2,3 double bond in the indole ring, which is the first, rate-limiting step in tryptophan catabolism by the kynurenine pathway27,28. TDO expression is mainly confined to the liver, and it seems to be a homeostatic or ‘housekeeping’ gene: it is not induced or regulated by signals from the immune system. By contrast, IDO-expressing cells are found in many tissues, and IDO expression is inducible by antigen-presenting cells (APCs) of the immune system and is subject to complex regulation by an array of immunological signals. Regulation of IDO gene expression. The IDO protein (APOENZYME) is encoded by a single gene with 10 exons spread over ~15 kbp of DNA located in a syntenic region of human and mouse chromosome 8 (FIG. 2). The IDO gene has been well conserved29, although IDO effector functions might have been adapted for various applications during evolution (BOX 2). Gene transcription is stringently controlled, responding to specific inflammatory mediators, and confined to a limited range of cell types. The mouse and human IDO gene promoters contain multiple sequence elements that confer responsiveness to type I (IFN-α/β) and, more potently, type II (IFN-γ) interferons14,15,30,31. Various cell types, including certain myeloid-lineage cells (monocyte-derived macrophages and DCs), fibroblasts, endothelial cells and some tumour-cell lines, express IDO after exposure to IFN-γ14,32–35. Signal transducer and activator of transcription 1 (STAT1) and IFN-regulatory factor 1 (IRF1) function cooperatively to mediate the induction of IDO expression by IFN-γ36, and mice that lack either IFN-γ or IRF1 are deficient in IDO expression during infection37. Another inflammatory mediator, peroxynitrite, is overexpressed in diabetes-prone NOD mice, and antagonizes the induction of IDO transcription mediated by the IFN-γ–STAT1 pathway; this might explain in part the defective tolerogenic properties of DCs from these mice21. There is a strong correlation between inflammation, IFN-γ and induced IDO expression, but IFN-γ is not essential for IDO induction. Lipopolysaccharide (LPS) and the inflammatory cytokines IL-1 and tumournecrosis factor (TNF) act synergistically with IFN-γ to enhance IDO expression in vitro38,39. However, in vivo, responsiveness to LPS crucially depends on TNF, but does not require IFN-γ 40, indicating the existence of an IFN-γ-independent pathway for the induction of IDO expression. Additional signalling pathways and

VOLUME 4 | OCTOBER 2004 | 7 6 3 ©2004 Nature Publishing Group

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Table 1 | Mechanistic studies supporting a role for IDO in immune regulation Effect of IDO

IDO+ cell type

Model

Inhibition of T-cell proliferation in vitro (human)*

Monocyte-derived macrophages, monocyte-derived DCs and bone-marrow stromal cells

MLR

Inhibition of T-cell proliferation in vitro

Tumour-cell lines

Transfection with IDO-encoding cDNA

+

References 34,35,45,87,91,92

17

Apoptosis of TH1-cell clones in vitro

CD8α DCs

MLR

57

Inhibition of T-cell proliferation in vitro

Plasmacytoid DCs

MLR

20,46

Protection of allogeneic fetus

Trophoblast cells (?)

Mouse pregnancy

Inhibition of priming to T-cell antigens

IFN-γ-activated DCs

DC vaccine

16,150

Spontaneous tolerance to liver allografts

ND

Orthotopic liver allograft

Delayed destruction of islet cells by NOD T cells

Pancreatic islet cells


74

Tolerance to pancreatic islet-cell allografts

IDO-competent DCs (?)

CTLA4-immunoglobulin in vivo

22

Suppression of alloreactive T cells in vivo

IDO-competent DCs (?)

CTLA4-immunoglobulin in vivo

44

Protection of lung allografts

ND


25

Amelioration of TNBS-induced colitis

ND

Blocking with 1-MT in vivo

24

Amelioration of EAE

ND

Blocking with 1-MT in vivo

23

Inhibition of experimental asthma

Resident lung cells (non-DCs)

OVA-induced asthma

26

Temporal correlation with resistance to autoimmune diabetes

ND

NOD mice

21

Protection of tumour-cell lines against rejection

P815 cell line


19

Induction of CD8+ T-cell anergy in vivo

Plasmacytoid DCs

Adoptive transfer of DCs

20

43,78–80 151

*All data are from mouse systems unless specified as human. CTLA4, cytotoxic T lymphocyte antigen 4; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; IDO, indoleamine 2,3-dioxygenase; IFN-γ, interferon-γ; MLR, mixed leukocyte reaction; 1-MT, 1-methyl-tryptophan; ND, not determined; NOD, non-obese diabetic; OVA, ovalbumin; TH1, T helper 1; TNBS, trinitrobenzene sulphonic acid.

TROPHOBLAST GIANT CELLS

cytokines might modulate IDO expression by specific cell types41,42. So, the control of IDO transcription is complex and cell-type specific. Unexpectedly, soluble CTLA4–immunoglobulin fusion protein was also found to induce IDO expression, through the ligation of cell-surface CD80/CD86 molecules22. This role for CD80/CD86 molecules has subsequently been confirmed in both mouse and human DCs in several model systems43–46. In some, IFN-γ was required for the induction of IDO expression, but in others, ligation of CD80/CD86 induced functional IDO expression by IFN-γ-receptor-deficient DCs, showing that IFN-γ signals are not essential for CD80/CD86-induced upregulation of IDO expression by these DCs46. Although the signal-transduction pathway that connects CD80/CD86 ligation with the induction of IDO expression is not yet fully elucidated, this pathway might have important implications for the mechanisms by which ligands for CD80/CD86 (recombinant CTLA4-immunoglobulin fusion protein and native CTLA4 on TReg cells) suppress T-cell-mediated immunity27,47. This will be discussed further later.

(TGCs). Cells of fetal origin in mouse extra-embryonic tissues that develop into large cells through marked amplification of DNA content coupled with cytoplasmic expansion. Primary TGCs line the outer surfaces of the fetal–placental unit at midgestation in mice. Secondary TGCs migrate into the maternal deciduas at later times during mouse gestation.

Regulation of functional IDO activity. IDO is an intracellular enzyme; there is no known secreted or extracellular form. IDO activity is found constitutively at the maternal–fetal interface, expressed by human extravillous trophoblast cells48–51. At a corresponding interface in mice, immunohistochemistry showed that primary TROPHOBLAST GIANT CELLS of fetal origin express IDO52. In another study, using a different IDO-specific antibody, cells located in the maternal

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metrial glands were reported to express IDO53; it is unclear how these distinct results can be reconciled. Outside of the placenta, functional IDO expression (assessed as tryptophan catabolizing activity in tissue homogenates) was reported to be highest in the mouse epididymis, gut (distal ileum and colon), lymph nodes, spleen, thymus and lungs54. IDO activity in these locations was markedly increased by treatment with LPS in vivo55,56. The function of IDO in the epididymis is unknown, but the other tissues all have extensive mucosal surfaces and/or large lymphoid compartments. The presence of constitutive and inducible IDO expression in these tissues might function as an anti-inflammatory/immunosuppressive mechanism. IDO protein can be expressed without functional enzymatic activity. Isolated mouse splenic CD8α+ DCs were found to catabolize tryptophan when exposed to IFN-γ, whereas other (CD8α–) DCs did not, even though both subsets expressed comparable amounts of IDO protein as analysed by western blot57. Similarly, human DCs can constitutively express immunoreactive IDO protein as shown by flow cytometry and western blot, yet it does not have functional enzymatic activity until these cells are activated by IFN-γ and CD80/CD86 ligation45. As IDO would probably be toxic if it were constitutively active (as it consumes an essential nutrient), it is understandable that enzyme activity must be tightly regulated. It is not yet clear how post-translational regulation occurs, but possible mechanisms include controlling the supply of enzyme co-factors and substrates (for example, haeme and reactive oxygen species), post-translational


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EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

An experimental model for the human disease multiple sclerosis. Autoimmune disease is induced in experimental animals by immunization with myelin antigen or peptides derived from myelin. The animals develop a paralytic disease with inflammation and demyelination in the brain and spinal cord.

modification of the enzyme, alternative splicing, regulation of protein stability and the presence of inhibitors of the enzyme active site. Incorporation of the haeme prosthetic group into the active site is required for IDO activity, and inhibitors of haeme biosynthesis inhibit functional activity without affecting protein levels58. Antioxidants and the cellular redox potential also affect enzyme activity58. Nitric oxide directly inactivates the haeme active site59, and promotes proteasome-mediated degradation of the IDO protein60. There are no published data on alternative splicing or post-translational modification. More research will be required to identify the post-translational factors that regulate IDO enzyme function and stability, particularly in DCs. IDO and the immune system

Historically, IDO has been considered to be part of the host defence against pathogens14. It is now clear that IDO serves more than one role in the immune system. However, it is not clear whether these roles are always beneficial to the host. IDO and host defence. IDO forms part of the innate host defence against certain infections. Although most microorganisms can synthesize their own tryptophan, some depend on exogenous tryptophan (auxotrophs). Such organisms are sensitive to the tryptophan-depleting activity of IDO. Examples include Chlamydia pneumoniae, Toxoplasma gondii and certain bacteria, such as group B streptococci and mycobacteria61–65. These are pathogens that are either intracellular or else live in intimate association with a host cell, and induction of IDO expression by the host cell inhibits pathogen replication in vitro. Similarly, during viral infection IDO inhibits the replication of cytomegalovirus and herpes simplex

Box 1 | Tryptophan metabolism Tryptophan is one of several amino acids that are essential in mammals (that is, that cannot be synthesized de novo). It is the rarest of all amino acids, accounting for ~1% of total amino acids in cellular proteins. Incorporation of tryptophan into protein is initiated by tryptophanyl-transfer RNA synthetase (WRS). WRS is the only aminoacyl synthetase that responds to inflammatory mediators, such as interferon-γ (IFN-γ)133, and overexpression of WRS has been postulated to help cells that express indoleamine 2,3-dioxygenase (IDO) compensate for the reduction in intracellular tryptophan. It might therefore be important that T-cell lines do not show induction of WRS expression in response to IFN-γ134. The basal level of tryptophan in the serum is mainly controlled by the enzyme tryptophan 2,3-dioxygenase (TDO). TDO is expressed in the liver and seems to be mainly a homeostatic or housekeeping enzyme. TDO gene transcripts have also been detected in mouse endometrium and conceptus, but the biological significance of this observation is unknown135,136. TDO does not respond to immunological signals. By contrast, IDO is highly responsive to signals from the immune system and can be induced by such signals in various cell types. However, expression of IDO, and factors that regulate it are highly cell-type specific. IDO catalyses the conversion of tryptophan to N-formyl-kynurenine, which is then converted into various downstream metabolites28. IDO catalyses the initial, ratelimiting step in this pathway, but the pattern of subsequent metabolites is determined by the particular set of downstream enzymes that are expressed by the various cell types137. IDO enzyme activity is blocked by the competitive inhibitor 1-methyltryptophan, whereas TDO is not136, thereby assisting in differentiating the biological effects of these two enzymes.


virus in vitro66,67. In all of these examples, the effect of IDO was found to be due specifically to its ability to deplete tryptophan, because adding supra-physiological levels of exogenous tryptophan restored pathogen or viral replication. Although IDO therefore has an effect on pathogen replication in vitro, the biological relevance of IDO in controlling infections in vivo remains unclear. IFN-γdeficient mice, which fail to induce IDO during infection, are less able to control C. pneumoniae and T. gondii infection in vivo37,68,69; however, it is unknown whether this is due specifically to the lack of IDO expression or to one of the many other effects of IFN-γ. Further studies are therefore required to address the specific contribution of IDO to host defence against infection. This question is particularly relevant, given that one (highly undesirable) consequence of inducing IDO expression by the innate system might be the suppression of T-cell responses by the adaptive system15,70. The fact that IDO slows pathogen replication in vitro does not necessarily mean that it would be a useful host defence in vivo (particularly in the case of chronic, slow-growing infections such as those described). So, like the T helper 2 (TH2)-cell bias in chronic leishmaniasis71, it is possible that IDO could benefit the pathogen more than the host. IDO-dependent suppression of adaptive immunity. Mechanistic studies from a range of mouse models demonstrate that IDO regulates adaptive T-cell immunity (TABLE 1). In many of these studies, 1-methyltryptophan (1-MT), a pharmacological inhibitor of IDO72, was used to inhibit IDO activity in vivo. Administration of 1-MT to pregnant mice resulted in uniform rejection of allogeneic fetuses — a process that required both an intact maternal immune system and paternal alloantigen16,73. In autoimmune models, 1-MT treatment exacerbated symptoms of EXPERIMENTAL 23 AUTOIMMUNE ENCEPHALOMYELITIS , and markedly increased disease severity and lethality in a model of T-cell mediated colitis24. Administration of 1-MT also abrogated the tolerogenic properties of CTLA4–immunoglobulin fusion protein in an islet-cell transplant model 22. DCs from autoimmune-prone NOD mice spontaneously exhibited a transient defect in IFN-γ-inducible IDO activity and tolerance induction in vivo, specifically during the autoimmune-prone pre-diabetic phase21. By contrast, overexpression of IDO results in immunosuppression and tolerance. IDO-transfected cell lines suppressed antigen-specific T-cell responses in vitro17, and IDO overexpression in mouse tumourcell lines rendered them resistant to immune rejection in vivo19. Adenoviral-mediated IDO gene transfer into pancreatic islet cells prolonged their survival in allogeneic hosts74, and transfection with IDO protected allogeneic lung transplants from rejection25. Pre-treatment of mice with CTLA4–immunoglobulin fusion protein (to induce IDO expression) suppressed the rejection of pancreatic islet allografts22 and completely blocked clonal expansion of alloreactive T-cell receptor


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GRAFT-VERSUS-HOST-DISEASE

(GVHD). An immune response mounted against the recipient of an allograft by immunocompetent T cells that are derived from the graft. Typically, it is seen in the context of allogeneic bonemarrow transplantation.

(TCR)-transgenic T cells in a model of GRAFT-VERSUS-HOST 44 DISEASE ; use of IDO-deficient mice in the latter model showed that this effect was IDO dependent. So, studies in various models confirm that IDO can be a potent regulator of adaptive immune responses (TABLE 1).

Interferons and CD80/CD86 ligation

+

2 kb

IDO gene 1

23

4 5 6

7 8 9 10

ISRE/GAS

APC-mediated T-cell suppression by IDO

The studies mentioned earlier demonstrate that artificial overexpression of IDO in various settings is immunosuppressive, and that IDO can have an important immunoregulatory role in vivo. But they do not elucidate which cell types in the immune system are the normal, biologically relevant sites of expression for IDO. Some information on this issue is now emerging. Human monocyte-derived DCs. IDO-mediated suppression of human T-cell proliferation was initially shown using monocyte-derived macrophages34. Subsequent human studies have focused on the more physiologically relevant monocyte-derived DCs35,45,75. Although artificial culture models do not capture the diversity of myeloid and lymphoid-lineage DCs in vivo76,77, they do provide accessible models for studying IDO-mediated immune regulation. These in vitro studies showed that the culture conditions used to differentiate and mature monocytederived DCs were crucially important in determining the degree of IDO-mediated suppression. Relevant factors included the technique used for isolating the monocytes, the culture medium, the cytokine and maturation regimen, and the ability of the readout assay itself to detect IDO-mediated suppression45. This sensitivity to in vitro culture conditions raises the question of whether IDOmediated suppression is truly a biologically relevant property of human DCs or merely an artefact of the culture system. We suspect that it is a real attribute, because the immunoregulatory properties of human monocyte-derived DCs correspond in several important respects to studies using ex vivo-derived mouse DCs (for example, their responsiveness to the IDOmediated inhibitory activity of CD80/CD86 ligation45). Given that mouse studies indicate that even a minor population of IDO-expressing DCs can induce systemic immunosuppression in vivo20,78, the admittedly artificial human culture model might nevertheless yield clinically relevant insights. IDO-competent DCs in vivo. The sensitivity of monocytederived DCs to subtle alterations in culture conditions might reflect the fact that only certain types of DC would normally express IDO in vivo. This hypothesis was originally proposed by mouse studies from Grohmann and colleagues78 showing that IDO activity seemed to preferentially segregate with the CD8α+ subset of DCs after IFN-γ treatment in vitro. This was subsequently confirmed by the same group in further studies79,80, although it was also possible to induce IDO expression by CD8α– DCs, by exposing them to CTLA4–immunoglobulin or TReg cells43,81. However, these assays were carried out on bulk populations; therefore, the finding that both CD8α+ and CD8α– DCs could express IDO did not necessarily mean that

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JAK–STAT signalling


– IDO protein (~45 kDa apoenzyme)

? Haeme

IDO holoenzyme (inactive) Post-translational modifications?

IDO enzyme (active)

Oxygen (2 x O2–)

Tryptophan (other indole compounds)

–

Metabolites such as kynurenine and quinolinate (vary depending on cell type)

1-methyl-tryptophan and nitric oxide

Figure 2 | Indoleamine 2,3-dioxygenase molecular genetics and enzyme activity. Known inducers of indoleamine 2,3-dioxygenase (IDO) transcription act through JAK–STAT signalling pathways on interferon stimulatory response elements (ISREs) and γ-activating sequences (GAS) in the IDO promoter. Although transcriptional repressors of IDO are not defined, cis- and perhaps trans-regulatory factors are inferred because of the highly cell-type specific pattern of IDO inducibility. No specific post-translational modifications of IDO have yet been identified, but they might explain the existence of enzymatically inactive enzyme protein. Functional activity might also be regulated at the level of holoenzyme assembly (that is, incorporation of the haeme prosthetic group), cofactor availability, cellular redox potentials or inhibitors of the active site (either endogenous inhibitors, such as nitric oxide, or pharmacological inhibitors, such as 1-methyl-tryptophan)58,149. JAK, Janus activated kinase; STAT, signal transducer and activator of transcription.

all DCs were expressing IDO — it merely proved that some subpopulation within each group was able to do so57,78. In studies using immunohistochemistry to detect IDO protein expression in vivo, treatment with CTLA4–immunoglobulin fusion protein was found to upregulate the levels of immunoreactive IDO only by certain subsets of mouse APC in the spleen. This response was restricted mainly to cells in the B220+ (plasmacytoid) and CD8α+ populations of splenic DCs44. Studies with APC fractions that were isolated from mice exposed to CTLA4–immunoglobulin confirmed that IDO-dependent T-cell suppression was confined to specific DC subsets that express these markers46. Whether these outcomes identify a single ‘IDO-competent’ population of cells that expresses both markers (that is, B220+CD8α+ DCs82) or several different populations within the complex mixture of DC subtypes in the


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Box 2 | IDO during evolution of the immune system The structural gene that encodes indoleamine 2,3-dioxygenase (IDO) is evolutionarily ancient (far pre-dating adaptive immunity), yet the promoter is highly responsive to signals from the adaptive immune system. So, IDO might be an example of the immune system’s ability to ‘recycle’ effector mechanisms as it evolves. Competition for nutrients. Bacteria and fungi compete vigorously for scarce resources (for example, iron and other nutrients). Successful uptake by one organism might inhibit the growth of neighbouring organisms. In addition, microorganisms also secrete various toxins to keep competitors in check. The primitive mononuclear phagocyte system. The vertebrate macrophage system evolved from the ancient invertebrate mononuclear phagocyte system138. Macrophages use some primitive antimicrobial strategies, including competition for nutrients (for example, iron chelation) and secretion of toxins. Although not proven, IDO might have evolved initially as a host-defence strategy against infectious pathogens. Professional antigen-presenting cells (APCs) and T cells. As the adaptive immune system evolved, professional APCs (macrophages and dendritic cells) were faced with the new challenge of regulating another population of ‘hostile’ cells — autoreactive T cells. It might be that IDO, which was already important as an antimicrobial strategy, was adapted for such a use. Although IDO might have functioned initially through a simple strategy of nutrient competition or production of toxins, the stage could have been set for T cells to co-evolve heightened responsiveness to biochemical changes that are mediated by IDO. Regulation of maternal T cells by trophoblast cells. Gestation in placental mammals introduced a more complex challenge in immune regulation: acquired tolerance to paternal antigens. Trophoblast cells have been noted to share expression of several genes with macrophages139, and they have been suggested to function as a component of the innate immune system during pregnancy140. So, expression of IDO by trophoblast cells and uterine macrophages might represent the most recent evolutionary recycling of IDO as a regulatory mechanism in the immune system.

spleen remains to be determined. However, an important point is that many more cells expressed the target ligands for the CTLA4–immunoglobulin fusion protein than actually showed upregulation of IDO expression. Recently, a detailed analysis of IDO-mediated T-cell suppression in tumour-draining lymph nodes20 showed that although a significant fraction of DCs expressed detectable levels of IDO protein by immunohistochemistry, the functional IDO-mediated suppression was mediated almost entirely by a small, well-defined CD19+ subset among the B220+ plasmacytoid DCs. Whether this particular DC phenotype is a specialized feature of tumour-draining lymph nodes or is a more general phenomenon, remains to be determined, but these data emphasize that the biologically relevant population of IDO-expressing DCs might be a minor subset. Even within the population of IDO-competent DCs there can be a considerable degree of functional plasticity — for example, certain pro-inflammatory signals might downregulate the expression of IDO by cells that would normally express it80,81. Conversely, different tolerogenic stimuli might induce IDO expression by different populations of DCs43,81. When carrying out phenotypic analyses of DC subsets, it is becoming evident that single broad markers, such as CD8α or B220, are no longer sufficiently precise to define the specific IDO-expressing population. These markers group together several different subsets (for example, CD8α is expressed by subsets of both plasmacytoid and


non-plasmacytoid DCs83). Also, expression of some markers, such as CD8α, might change during DC maturation/activation82. However, the key conceptual point remains that not all DCs are competent to express functional IDO, and that the biological effects of IDO expression might be mediated by a relatively small subset of the total DC pool. Induction of IDO expression by TReg cells. As mentioned earlier, IDO seems to function as the downstream mediator for certain tolerogenic effects of CTLA4– immunoglobulin fusion protein22. However, recombinant CTLA4–immunoglobulin is an artificial means of inducing IDO expression, and it has been suggested that the physiological signal that normally fulfils this role might be a CTLA4-expressing subset of TReg cells27,47. This hypothesis is strengthened by the observation that mouse CD4+CD25+ TReg cells and a CTLA4-transfected cell line both induced IDO activity in mouse DCs in vitro, in a CTLA4-dependent manner43. Also, a cloned CD4+CD25+ T-cell line expressing surface CTLA4 induced IDO expression by B220+ and CD8α+ DC subsets, and in this particular model, the regulatory functions of these T cells completely depended on their ability to induce functional IDO expression by the DCs46. Further studies will be required to confirm that this mechanism occurs in vivo, and under which circumstances, but it seems reasonable to speculate that certain CTLA4+ TReg cells can induce IDO expression. TReg cells therefore become an important upstream mechanism for the induction of IDO expression; and conversely, IDO becomes one of the potential downstream effector mechanisms by which TReg cells can mediate their inhibitory effects. From this perspective, it might be relevant that maternal TReg cells have recently been shown to be required for the maintenance of tolerance to the allogeneic fetus84. As pharmacological inhibition of IDO activity similarly disrupts maternal tolerance, it is possible that maternal TReg cells might be a required upstream inducer of IDO expression during pregnancy. In FIG. 3, the concept of IDO-competent DC subsets is combined with the concept of immunogenic versus tolerogenic signal integration illustrated in FIG. 1. Under this model, IDO-mediated immune regulation first requires an immature (unactivated) DC that can express IDO. (It is not yet clear whether this would be a specific DC subset or a certain developmental stage of many types of DC.) During — or perhaps even after — maturation, the IDO-competent cell can receive two types of ‘conditioning’ signal that lead to different phenotypes. Tolerogenic signals, such as CD80/CD86 ligation by CTLA4+ TReg cells, would induce IDO expression, thereby eliciting the full, functionally suppressive (regulatory) phenotype. By contrast, immunogenic signals, such as CD40 ligation by TH cells or exposure to pro-inflammatory cytokines, would promote a non-suppressive phenotype and downregulate IDO expression79,80. In FIG. 3, we tentatively label the ‘maturation’ step as separate from the process termed ‘functional plasticity’81 that determines


REVIEWS

Immature IDO-competent DC

Maturation

Functional plasticity Pro-inflammatory signals such as CD40 ligand

Tolerogenic signals such as CTLA4–CD80/CD86

Mature immunogenic DC (IDO–)

Mature regulatory DC (IDO+)

Figure 3 | Maturation of indoleamine 2,3-dioxygenasecompetent dendritic cells. Dendritic-cell (DC) maturation has traditionally been viewed as a linear process leading from an immature (tolerogenic) to a mature (immunogenic) phenotype. However, some IDO-expressing DCs seem to be both mature and tolerogenic, suggesting that maturation and the decision to express indoleamine 2,3-dioxygenase (IDO) might be separate processes, or (producing the same outcome) that there might be two different maturation pathways for IDO-competent DCs. We hypothesize that pro-inflammatory maturation signals, such as CD40 ligation, might act to downregulate functional IDO expression. By contrast, tolerogenic signals, such as ligation of CD80/CD86 by cytotoxic T lymphocyte antigen 4 (CTLA4) on regulatory T cells (probably acting in combination with other maturation signals), result in differentiation of mature but IDO+ regulatory DCs that actively suppress T-cell responses.

IDO expression. We hypothesize that both pathways lead to fully mature APCs. It is a surprising feature of IDO-expressing DCs that potent IDO-mediated suppression can be maintained even while the underlying APC function undergoes maturation45. Indeed, in some studies using the IDO inhibitor 1-MT and IDOdeficient mice20, it seems that the only difference between an immunogenic and a tolerogenic phenotype is IDO expression. Although this is most probably an oversimplification, blocking IDO often reveals marked underlying T-cell-stimulatory capacity. Therefore, we hypothesize that both the immunogenic and the tolerogenic phenotypes are fully competent, mature APCs, but they are specialized for opposing functions.

NITRIC OXIDE SYNTHASE

An inducible haeme-containing enzyme that produces nitric oxide in response to inflammatory signals.

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Induction of IDO expression by IFN-γ. As mentioned earlier, IFN-γ is a potent inducer of IDO expression, and the IFN-γ pathway is required for the normal upregulation of IDO expression during infection37,68,69. Such a role for IFN-γ is understandable when IDO functions as a host-defence mechanism, but what is the role of IFN-γ when the outcome of IDO expression is tolerogenic? IFN-γ is often thought of as a prototypical pro-inflammatory cytokine, so it might seem paradoxical


that IFN-γ should also activate an immunosuppressive mechanism. However, it is a recurring theme in the immune system that inflammation frequently elicits counter-regulatory (anti-inflammatory) pathways. Induction of IDO expression by IFN-γ might constitute one such pathway, helping to limit excessive T-cell activation at local sites of inflammation. In this local antiinflammatory role, IDO would not necessarily have to be expressed by professional APCs; consistent with this, inflammation-inducible IDO expression has been described in intestinal epithelial cells85, skin fibroblasts86, resident (non-DC) lung cells26 and bone-marrow stromal cells87. More fundamentally, however, it might be a misinterpretation to consider IFN-γ as purely proinflammatory as many reports have shown that IFN-γ has anti-inflammatory and tolerogenic roles in certain settings27. So, the effect of T-cell-derived IFN-γ might be to signal the presence of an activated T cell; it is then up to the responding cell — the IDO-competent DC, in our case — to determine whether the response should be pro-inflammatory or immunosuppressive. Mechanisms of IDO-dependent suppression

The downstream molecular mechanisms by which IDO affects immune outcomes are still a subject of active investigation. Conceptual possibilities include direct effects on T cells, mediated either by tryptophan depletion or by downstream tryptophan metabolites, or effects of IDO on the APC. These are not mutually exclusive possibilities; and other regulatory biochemical pathways (such as the cyclooxygenase and NITRIC OXIDE SYNTHASE systems) characteristically trigger multiple downstream pathways. Direct effects on T cells. Historically, the first mechanism that was recognized for IDO was tryptophan depletion, following the observation that adding excess tryptophan reversed the antimicrobial effects of IDO (described earlier). Several studies have now shown that excess tryptophan similarly reverses IDO-mediated inhibition of T cells23,34,45,46,88. This argues for a mechanistic role for tryptophan depletion, at least in these particular models, which include human polyclonal mixed leukocyte cultures (MLRs) and activation of TCR-transgenic mouse CD8+ T cells. Consistent with this possibility, in vitro-activated human and mouse T cells undergo cell-cycle arrest when deprived of tryptophan34,89, and arrested cells might be rendered more sensitive to apoptosis89. In other models, however, toxic metabolites of tryptophan seem to have a prominent role in mediating the immunosuppressive effects of IDO. Mouse thymocytes, and mouse CD4+ T-cell clones, were sensitive to apoptosis induced by tryptophan metabolites, such as quinolinic acid and 3-hydroxy-anthranilic acid90. In this study, TH1-cell clones but not TH2-cell clones were sensitive to metabolite-induced apoptosis, raising the possibility that IDO might alter the TH1/TH2-cell balance. Human T cells were also found to be sensitive to the antiproliferative and cytotoxic effects of exogenously added kynurenine, picolinic acid and 3-hydroxy-anthranilic acid91,92. The mechanisms by which these downstream


REVIEWS

LINKED SUPPRESSION

The phenomenon of suppressing responses to a specific antigen by co-presenting it simultaneously with another antigen, against which tolerance has previously been established.

metabolites in the kynurenine pathway affect T cells are currently unclear; they might be directly toxic or might bind to a receptor that triggers T-cell death, as has been described for other small molecules93. The possible immunoregulatory activities of metabolites of the kynurenine pathway have been reviewed in detail recently27,28,94. For both of these possible mechanisms — tryptophan depletion and toxic metabolites — the effects have been mainly shown in vitro. Precisely how either would function in vivo remains speculative. The degree of tryptophan depletion that is required to inhibit T-cell proliferation is lower than that normally found in the plasma. In the case of tryptophan metabolites, although some T cells seem to be relatively sensitive to their effects90, in other systems the toxic concentrations of metabolites far exceed the total available concentration of tryptophan in vivo91,92. So, whichever mechanism is proposed, the model must make the assumption that the tissue microenvironment that surrounds the APC is

Box 3 | Potential pathways in T cells responding to the effects of IDO Indoleamine 2,3IDO dioxygenase (IDO) decreases the local ↓ Free tryptophan ↑ Downstream metabolites concentration of free – tryptophan, while increasing Selective ↑ Uncharged Other TSC1/ the concentration of apoptosis tryptophanyl-tRNA effects? TSC2 downstream metabolites. + – These metabolites are known to be biologically Proliferation arrest GCN2 mTOR active27, but the specific molecular mechanisms by which they mediate their Stress Translational effects are currently unclear. response block There are two known amino-acid-responsive signal-transduction pathways by which T cells might sense decreased levels of free tryptophan. Essential amino acid deficiency antagonizes signalling through the mTOR (mammalian target of rapamycin) kinase pathway97,98. mTOR signalling is required for normal initiation of ribosomal translation. This pathway is important for growth-factor signalling, and T cells are particularly sensitive to inhibitors of mTOR, such as the immunosuppressant drug rapamycin141. A second amino-acid responsive pathway is initiated by the GCN2 kinase, which contains a binding domain that is specific for the uncharged form of transfer RNA (tRNA)95,96. Deprivation of any amino acid causes activation of the kinase domain, resulting in phosphorylation of eukaryotic translation-initiation factor 2α and repression of translation. However, a limited set of mRNA transcripts for stress-response genes have internal ribosomal entry site (IRES) sequences that allow them to bypass this generalized translational repression, and these genes show selective upregulation in response to GCN2 (REF. 142). Precisely how activating this stress-response pathway or inhibiting the mTOR pathway might affect the programme of T-cell activation is currently unknown. However, translational control is a fundamental regulatory process, and alterations can affect cellular activation and cell-cycle progression in marked ways143,144. Such translational control mechanisms are distinct from simple starvation and can be highly gene specific — for example, tryptophan deprivation blocks interferon-γ-induced expression of nitric oxide synthase by macrophages, but expression of tumour-necrosis factor by the same cells is unaffected145. So, it is possible that a period of tryptophan deprivation early in T-cell activation might fundamentally alter the proliferation and differentiation programme of the responding cells. TSC, tuberous sclerosis.


not in equilibrium with the blood, and that local concentrations of tryptophan or its metabolites can vary markedly from that observed in the plasma. At the molecular level, the signalling pathways by which T cells might detect and respond to the local conditions that are generated by IDO require further investigation. It is not known how T cells sense toxic metabolites, but low tryptophan might be detected by either of two amino-acid sensitive signalling pathways (BOX 3): the GCN2 stress-kinase pathway, which is activated by amino-acid withdrawal95,96; and the mTOR (mammalian target of rapamycin)-signalling pathway, which is inhibited by amino-acid withdrawal97,98. In the case of mTOR, T cells seem particularly sensitive to inhibition of this pathway, as shown by the clinical use of rapamycin (an mTOR inhibitor) as a T-cell immunosuppressant. Indirect effects through altered APC function. A third possibility is that IDO might alter the biology of the IDO-expressing APCs. Grohmann et al. 78 showed that pre-activation of CD8α+ DCs in vitro with IFN-γ rendered them tolerogenic when subsequently injected in vivo. This effect required IDO expression during the in vitro pre-activation, because addition of 1-MT during that time blocked the development of tolerogenic activity. However, the effect of IDO seemed to be exerted on the DCs themselves (cellautonomous), rather than on the responding T cells, as 1-MT was not present when the DCs were subsequently transferred into the recipient hosts. The nature of this effect is unknown, but it might be mediated either by intracellular tryptophan depletion (sensed through the pathways discussed in BOX 3) or by some effect of intracellular tryptophan metabolites. IDO might therefore functionally alter the DC, either by decreasing its APC function or by upregulating expression of suppressive ligands (for example, B7-H1 or CD95 ligand), or by triggering the secretion of immunoregulatory cytokines (for example, IL-10 or TGF-β). Although these notions are speculative in the case of DCs, IDO has been shown to mediate cellautonomous regulation of gene expression and cell biology in other cell types. For example, endogenous IDO alters metalloprotease expression and prostaglandin synthesis in transfected cell lines99; stabilizes cytokine mRNAs in airway epithelial cells100, and downregulates MHC class I expression by keratinocytes101. Therefore, it is possible that IDO might alter the basic biology of the IDO-expressing DCs — in effect, functioning as an intracellular second-messenger system. Bystander suppression. It has been observed that IDOmediated suppression can act in a dominant manner — that is, IDO can suppress T-cell responses to antigens that are presented by neighbouring, IDO– APCs. This effect can be dramatic in vivo, where small populations of IDO+ DCs efficiently suppress all T-cell responses to a particular antigen, despite the fact that the same antigen was presented by many other IDO– APCs20,44,46. In vitro, LINKED SUPPRESSION (or bystander suppression) has been


REVIEWS

a

Direct suppression of effector T-cell development

IDO+ DC

Naive T cell Macrophage B cell Bystander Suppression Regulatory cytokines

New (adaptive) regulatory T cells?

Indirect suppression of neighbouring T-cell activation

IDO– DC

b CTLA4

Regulatory T cell IDO-competent naive DC

IDO

Naive T cell IDO+ regulatory DC

Figure 4 | Potential downstream mechanisms of indoleamine 2,3-dioxygenase. a | Potential mechanisms for the ‘bystander’ effects of indoleamine 2,3-dioxygenase (IDO)expressing cells. In vitro and in vivo studies indicate that even small numbers of IDO+ dendritic cells (DCs) can have widespread suppressive effects on T cells that recognize antigen presented by IDO– DCs. The figure depicts hypothetical mechanisms by which suppression of adjacent T cells might occur. There could be bystander suppression mediated by the IDO-expressing DC itself — for example, through toxic metabolites, widespread local tryptophan depletion or IDO-induced regulatory cytokines. Neighbouring cells (for example, macrophages, regulatory T or B cells) might similarly be induced by IDO-responsive signalling pathways to secrete regulatory cytokines. Naive T cells (CD4+ or CD8+) might be biased by IDO+ DCs to adopt a regulatory phenotype. Any of these effects could then suppress nearby T cells responding to IDO– antigen-presenting cells (APCs). The effect of IDO would therefore be to convert the local tissue microenvironment into a tolerizing milieu, even for antigens presented by other, normally immunogenic APCs. b | Model of a self-amplifying regulatory network involving interactions between IDO-competent DCs and regulatory T cells. The generation of regulatory T cells by IDO-expressing DCs could be a potent mechanism, as these regulatory T cells might in turn create other tolerogenic DCs through the induction of IDO expression by cytotoxic T lymphocyte antigen 4 (CTLA4) interactions. This could provide one possible molecular mechanism for epitope spreading and ‘infectious’ tolerance, as described in other systems132.

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described using defined IDO+ and IDO– APC populations, simultaneously presenting two different antigens in the same MLR20. It is important to emphasize, however, that these effects occur in a defined, local context (that is, affecting two antigens presented by the same APC or by closely adjacent APCs). Therefore, the dominant suppression described for IDO is not a global, non-specific effect. Rather, it is reminiscent of the antigen-driven linked suppression model described by Waldmann and colleagues13,102 in which the suppression triggered by T cells of one specificity might dominantly affect T cells responding to other antigens, but only when they are encountered in the same physical context. Indeed, such a mechanism would almost have to exist for IDO to serve as a downstream mechanism of suppression for TReg cells. Dominant (bystander) suppression might (at least in theory) be due to local effects of IDO — depletion of tryptophan or production of diffusible soluble factors, such as toxic tryptophan metabolites or immunoregulatory cytokines. Any of these might affect T cells responding to antigens that are presented by nearby APCs. Alternatively, it is theoretically possible that IDO expression by the DC might affect nearby (non-APC) cell types, causing them to produce factors that indirectly regulate local T-cell responses. Lymphoid tissues contain macrophages, which are potential immunoregulatory cells103, as well as TReg cells and regulatory B cells104,105, all of which are capable of producing immunosuppressive cytokines (FIG. 4a). There are as yet no published data to favour any one of the preceding possible mechanisms. As we currently do not understand the remarkable immunosuppressive potency that is mediated by small numbers of IDO+ DCs, it is probably wise to consider all potential mechanisms that might amplify the direct regulatory effects of IDO. Do IDO-expressing APCs generate new TReg cells? Several studies have indicated that a relatively small number of IDO+ DCs can suppress potent T-cell responses in vivo46 and promote systemic tolerance78,79. It is difficult to envision that the IDO+ DCs in these models directly contacted every antigen-specific T cell. So it seems probable that the original DCs somehow recruited secondary mechanisms that indirectly enhanced suppression. The nature of such mechanisms is unknown, but one possibility is that IDO+ DCs might promote TReg-cell development. So far, no published data address this hypothesis with respect to IDO+ DCs, but there is considerable evidence that some types of regulatory DC can induce TReg cells, including CD4+CD25+ T cells106,107, IL-10-producing TR1 cells108 and CD8+ TReg cells109,110. If IDO+ DCs are found to have a similar role, it would raise the interesting possibility that IDO-competent DCs might collaborate with CTLA4-expressing TReg cells in a self-reinforcing network (FIG. 4b). We think it is unlikely that IDO participates in development of the self-directed TReg cells that arise in the thymus111, as IDO-deficient mice do not develop spontaneous lethal autoimmunity44 (unlike mice deficient in these TReg cells112). However, IDO+ www.nature.com/reviews/immunol

©2004 Nature Publishing Group

REVIEWS cells might have a role in generating acquired (‘adaptive’) TReg cells that arise extrathymically and drive acquired peripheral tolerance111. Relevant places to look for such a process might include sites of known IDO expression that are also sites for generating TReg cells. This might include mucosal surfaces in the gut113 and possibly tumour-draining lymph nodes114. IDO in human disease

Contributions of IDO-expressing cells to specific disease states have not yet been mechanistically defined. However, based on data from several mouse and human systems, IDO might have an aetiological (pathogenic) role in certain disorders in which the immune system unexpectedly fails to respond in a protective manner and pathological tolerance occurs to certain antigens, leading to chronic disease. IDO and cancer. It is increasingly clear that tumours can induce tolerance to their own antigens2,115,116. They can also evade local immune destruction, despite the systemic presence of tumour-reactive T cells116. IDO might participate in these processes in two ways: as a tolerogenic mechanism expressed by host APCs that cross-present tumour antigens, or by directly suppressing immune effector cells in the tumour microenvironment. Relevant to the latter scenario, Uyttenhove and colleagues19 showed that transfecting tumour cells with IDO rendered a normally immunogenic tumour-cell line resistant to immune rejection, even in primed hosts that were fully protected against the untransfected tumours. As IDO is expressed by various human tumours in vivo14,19,117, this mechanism might serve as a defence against activated effector T cells that infiltrate the tumour. This assumes, of course, that the T cells are more sensitive to the antiproliferative effects of IDO than the tumour cells; and the data from Uyttenhove and colleagues would indicate that this is indeed a possibility. However, IDO expression by tumour cells themselves would not affect T-cell responses to tumour antigens that are cross-presented by host APCs. This probably occurs in tumour-draining lymph nodes, and constitutes an important route by which the immune system initially becomes aware of tumours118,119. Constitutive IDO expression has been observed in a population of host APCs in tumour-draining lymph nodes of both humans and mice18,20,75,120. It is not yet known whether this reflects preferential recruitment of IDO-expressing cells to these nodes or upregulation of IDO expression by a preexisting population of APCs. In mice, we have recently shown that these IDO+ cells are plasmacytoid DCs that potently suppress T-cell responses in vitro, and induce antigen-specific T-cell anergy following adoptive transfer in vivo 20. It is not yet known whether the IDO-expressing APCs in human tumour-draining lymph nodes are also plasmacytoid DCs; however, plasmacytoid DCs are well documented in human tumours121–123, and the IDOexpressing cells in tumour-draining lymph nodes have morphological features that are characteristic of plasmacytoid DCs120.


Mechanistically, we hypothesize that IDO-expressing host APCs (whatever their subtype) might contribute to a state of local immunosuppression in tumour-draining lymph nodes, and might help to induce systemic tolerance to tumour antigens as well. Expression of IDO by tumour cells and expression of IDO by host APCs would be reinforcing strategies, rather than mutually exclusive. Expression by either or both cell types might be relevant in a given tumour. Fortunately from a therapeutic standpoint, drugs that inhibit IDO, such as 1-methyl-D-tryptophan (NSC-721782), seem to inhibit IDO activity in both tumour cells and host APCs19,20. IDO and HIV. It has been proposed that IDO might also have a role in the pathogenesis of HIV infection. HIV triggers high levels of IDO expression when it infects human macrophages in vitro124, and simian immunodeficiency virus (SIV) infection of the brain in vivo induces IDO expression by cells of the macrophage lineage125. Infection with HIV or SIV increases functional IDO enzymatic activity in the brain, and it has been suggested that neurotoxic metabolites that are produced by IDO might contribute to HIV-associated neuropathology125–127. From the standpoint of immunity and tolerance, however, the most intriguing possibility is that IDO might actively suppress immune responses to HIV antigens. This hypothesis has been suggested by Fuchs and colleagues70, who were one of the first groups to point out that patients infected with HIV have chronically reduced levels of plasma tryptophan and increased levels of kynurenine, consistent with widespread activation of IDO128,129. Little is known about the sites of IDO expression in patients with HIV infection, but we have observed that monkeys infected with simian/human immunodeficiency virus show widespread IDO-expressing macrophages and other mononuclear cells in peripheral lymphoid tissues, sometimes in high numbers. Based on other studies showing that overexpression of IDO renders various cell types resistant to eradication by the immune system (TABLE 1), it is possible that IDO-expressing APCs could form a protected reservoir of HIV. This might occur either because IDO-expressing cells were resistant to attack by T cells, similar to IDO-transfected tumour cells19; or more speculatively, because IDOexpressing APCs actually helped induce acquired, antigen-specific tolerance to HIV antigens. The latter process would represent a true subversion of the immune system. Therapeutic induction of IDO. As discussed earlier, the efficacy of CTLA4–immunoglobulin fusion protein in a mouse islet-cell transplant model was found to depend on its ability to induce IDO expression22. CTLA4–immunoglobulin and related compounds might therefore function in part as IDO-inducing agents, although the relative contribution of induction of IDO expression versus co-stimulatory blockade to the clinical efficacy of these reagents remains to be determined130. However, recent encouraging results


REVIEWS

INFECTIOUS TOLERANCE

The ability of a tolerized population of T cells to induce tolerance in a new, naive population of T cells. Tolerance might be to the same antigens or to new antigens that are encountered in the same context (linked suppression). Newly tolerized T cells can, in turn, induce tolerance of other T cells.

from phase II clinical trials of recombinant CTLA4– immunoglobulin fusion protein in autoimmune rheumatoid arthritis131 imply that it might be possible to induce IDO expression in settings where immunosuppression would be clinically beneficial. Less controversial is the possibility that IDO transfection of tissue allografts might delay or prevent rejection, as has already been demonstrated for mouse lung transplants25 and pancreatic islet cells74. Whether IDO would need to be delivered to the entire graft or (more intriguingly) just to a population of tolerogenic APCs requires further investigation. Summary and future directions

This review summarizes new insights that are emerging from recent studies, focusing on the links between IDO activity, immunosuppression and tolerance. One key new concept is that only certain subsets of DCs seem competent to express IDO, at least at the level of functional immunoregulation. The factors that influence the development and tissue distribution of these IDO-competent DCs have yet to be determined. Key unanswered issues include whether the different IDOcompetent subsets arise from one or multiple lineages; and which signals during differentiation determine

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their ability to express IDO. Following differentiation, it is becoming clear that functional IDO expression is further regulated by additional signals. Some of the IDOinducing signals, such as upregulation by CD80/CD86 ligation and downregulation by CD40 ligation, have already been shown; we predict that there will be further signals that induce and downregulate IDO expression, perhaps specific to different contexts. Clearly, many opportunities exist for the immune system to integrate incoming information and decide whether the IDO system becomes functionally active. A second emerging concept is that TReg cells and IDO+ regulatory DCs might interact to form a selfreinforcing network, capable of suppressing local T-cell responses and promoting systemic tolerance. A role for a population of TReg cells in inducing IDO expression now seems probable, and IDO might constitute a downstream effector mechanism for certain types of TReg cell. The more speculative possibility that IDOexpressing DCs might also give rise to new TReg cells remains under investigation. If verified, this might offer mechanistic insight into the phenomena of linked suppression and INFECTIOUS TOLERANCE132, as well as acquired tolerance to antigens presented at mucosal surfaces or in the lymph nodes that drain tumour sites.

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Acknowledgements We acknowledge the National Institutes of Health for supporting the research described in this review.

Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene CD8α | CD80 | CD86 | CTLA4 | GCN2 | IDO | IFN-γ | IL-1 | IL-10 | IRF1 | mTOR | STAT1 | TDO | TGF-β | TNF Access to this interactive links box is free online.
