1 Introduction

Recent work has provided new support to the idea that the interaction between immune system and tumor tissue can lead to the development of specific escape mechanisms of certain tumor cells which might predict outcome in cancer patients. These findings are summarized by the cancer immunosurveillance hypothesis that takes a broader view of immune system-tumor interactions. Basically tumor cells are either rejected due to tumor associated antigens (TAA) or they can induce immunological resistance by secretion of anti-inflammatory cytokines such as transforming growth factor-ß (TGF-β) and finally tumors can induce T-cell anergy and thereby leading to immunological tolerance.

Indoleamine 2,3-dioxygenase (IDO) is the key enzyme in the extrahepatic degradation of the essential amino acid tryptophan via the kynurenine pathway. IDO has been implicated in the pathophysiology of inflammation, host immune defence and maternal tolerance. Recently it has been shown that local tryptophan depletion upon IDO activation by pro-inflammatory cytokines such as interferon-γ (IFN-γ) is an important mechanism for suppressing T-cell responses and ultimately leading to T-cell unresponsiveness. However, the role of IDO in tumor immunology and its role in tumorigenesis and tumor growth has not been clarified yet (Fig. 27.1).

Fig. 27.1
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Essential amino acid tryptophan is precursor for 3 major biochemical pathways: (1) for the biosynthesis of proteins, (2) for the biosynthesis of neurotransmitter 5-hydroxytrypt- amin (serotonin) by enzyme tryptophan 5-hydroxylase (T5H) and (3) for the conversion to N-formyl-kynurenine and kynurenine by hepatic tryptophan 2,3-dioxygenase (TDO, tryptophan pyrrolase) and by its extrahepatic isoform indoleamine 2,3-dioxygenase (IDO). Kynurenine serves as a precursor for further catabolites such as quinolinic acid and the end products NAD/NADH

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Recent studies from our laboratory have shown that colon-cancer cells are able to express IDO upon stimulation with exogenous IFN-γ and thereby lead to profound inhibition of T-cell activation in the tumor microenvironment.

In tissue specimens from patients with colorectal cancer, analysed by immunohistochemistry, a high IDO expression score correlated significantly with the invasiveness and the frequency of liver metastasis and poor overall survival. In addition IDO expression showed an inverse correlation with the number of CD3+ T-cells within the tumor stroma. IDO over-expression was found to be associated with a significant reduction of CD3+ tumor-infiltrating T-cells.

In conclusion, IDO over-expression within colorectal cancer specimens results in impaired local T-cell infiltration and thereby contributes to different pattern of invasiveness and the prevalence of metastasis, by actively reducing the number and power of anti-tumoral T-cell attacks. IDO-high expression by certain colorectal tumor subtypes enables such tumors to initially avoid immune attack and to defeat the invasion of T-cells via local tryptophan depletion and the production of pro-apoptotic tryptophan catabolites. Thus, IDO significantly contributes to and predicts disease progression, frequency of metastases and overall survival in colorectal cancer patients.

2 Basic Immunological Principles of Tumor and Host Interactions

In the context of tumor immunology, the survival of a malignant cell, after its generation from the benign precursor due to genetic alteration or transformation, strongly depends on the interaction of this cell with various components of the immune system. Generally, three basic mechanisms govern how the immune system interacts with tumor cells. A malignant cell can be eliminated either by cytotoxic T-cells or natural killer cells (NK cells) because of the recognition of tumor associated antigens (TAA). Second, a tumor can be resistant to immune attack due to the loss of major histocompatibility complex (MHC) class I molecules or the production of anti-inflammatory cytokines like transforming growth factor-β (TGF-β). Or finally, some tumors are able to induce T-cell anergy, ultimately leading to tumor antigen-specific tolerance. The tumor microenvironment, which is composed of malignant cells, immune cells, stromal cells and the extracellular matrix, is the main scene of action during the neoplastic process. Crosstalk between the players in this field, which are normal and tumor cells, is increasingly recognized to influence various stages of carcinogenesis [1]. During the initial stages of tumor formation, stromal cells can provide signals that determine tumor-cell growth and differentiation, whereas later in tumor progression, stromal-cell-derived cues can modulate cancer-cell invasion, metastasis or propagate conditions that favor tumor immune tolerance leading malignant cells to escape from tumor immunity [2]. One variable of particular importance for tumor-host interaction is the mixture of cytokines produced within this microenvironment; among these cytokines, interferons have been identified as one of the key players [3, 4].

Human cancer development is a slow process that, like chronic infection, can occur over several years. In addition, tumorigenesis and malignant disease expose patients to a high risk for opportunistic infections and the development of sepsis and multiple organ failure [5]. Impaired immune responsiveness, in particular of effector T-cells, is considered a crucial factor for the development of an immunocompro-mised status in these patients. T-cell anergy appears to be mostly due to a deficient production of forward-regulatory cytokines such as interleukin-2 and IFN-γ [6]. During the initiation of cellular anti-tumor immune activation IFN-γ increases T-cell activity, supporting the development of a Th1-type immune response.

2.1 Immunological Mechanisms Facilitating Tumor Immune Escape

Recent findings, however, have challenged this hypothesis since some tumors develop strategies to bypass IFN-γ-mediated anti-tumor responses. However, there are a number of such different mechanisms by which tumors may actively evade or silence immune responses and thereby create a state of immunological tolerance towards their own antigens [2]. Tumor antigen-specific immune tolerance is initiated by a constitutive interaction between tumors and the patient’s immune system and controlled by various modifications to the immune response present in the tumor environment [7]. The expression of apoptosis-inducing Fas ligand causes deletion of tumor-reactive T-cells and immune evasion [8]. Secretion of immunosuppressive cytokines like IL-10 or TGF-β is important for direct tolerization [9, 10]. Another mechanism responsible for the shutdown of T-cell responses against tumors might be the presence of regulatory CD4+CD25+ T-regulatory cells [11]. Some tumors are localized so that they are not accessible to circulating T-cells, which therefore are unable to detect the presence of such tumors [12]. And finally, cross-presentation of tumor antigens by APCs is also a major feature inducing T-cell tolerance [13]. Despite the various ways in which tumors can evade or subvert immune responses, the exact mechanisms, by which such unresponsiveness to malignant cells is generated or maintained, are not fully understood.

Recently an additional mechanism during tumor immunosurveillance has been proposed for IFN-γ via induction of the immunomodulatory enyzme indoleamine 2,3-dioxygenase (IDO). IDO is the extrahepatic rate-limiting enzyme in the degradation of the essential amino acid tryptophan via the kynurenine pathway to form N-formyl kynurenine, which – depending on the cell type and species [38] – is subsequently converted to niacin. IDO is widely distributed in mammals and is induced in various cell types particularly by IFN-γ [14]. For many years IDO has been considered as an innate defence mechanism limiting growth of viruses, bacteria, intracellular pathogens and malignant cells by withdrawing tryptophan from the microenvironment [15]. Recently, it has been shown that activation of IDO is also critically involved in the regulation of immune responses [16], in establishing immune tolerance in pregnant mice upon their fetuses, and in inducing T-cell unresponsiveness [17, 18]. Proliferation of alloreactive T-cells is thereby arrested via local tryptophan deprivation and possibly also by the accumulation of toxic, pro-apoptotic catabolites such as kynurenine and quinolinic acid [19].

Insufficient availability of tryptophan seems to be of particular relevance for the development of immune deficiency and infection because of its multiple important roles in regulating monocytes, macrophages, and T-cells, in response to inflammation and infection (Fig. 27.2). Insufficient T-cell responses together with inadequate production of IFN-γ in cancer patients would result in insufficient killing of pathogens. As a consequence, bacterial overgrowth and sepsis develop, which increases the risk of death in these patients and has substantial implications on long term outcome.

Fig. 27.2
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Interaction between tumor tissue and immunocompetent cells leads to activation of T-cells and macrophages (MΦ). Among various regulatory molecules, Th1-type cytokine interferon-γ (IFN-γ) stimulates macrophages to a number of tumoricidal activities, which include the formation of reactive oxygen species (ROS) mainly via NADPH oxidase, the expression of indoleamine 2,3-dioxygenase (IDO), of nitric oxides synthase (iNOS) and of GTP-cyclohydrolase I (GCH). IDO degrades tryptophan and thus causes deprivation of this essential amino acid as well as the production of toxic tryptophan catabolites. iNOS produces nitric oxide and together with reactive oxygen species, cytotoxic peroxynitrite (ONOO-) is formed. Nitric oxide formation is also involved in the withdrawal of iron from the tumor cells. GCH gives rise for the production of tetrahydrobiopterin, the necessary cofactor of iNOS. However, due to distinct repertoire of subsequent enzymes, human macrophages produce neopterin at the expense of biopterin derivatives. Neopterin contributes to amplification of ROS effects, it super-induces the production of pro-inflammatory cytokine as well as apoptosis in tumor cells

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As discussed above, IFN-γ is an important mediator of innate and adaptive immune responses that play many critical roles in promoting both protective immune responses and immunopathologic processes and influences a remarkable range of distinct cellular programs [20]. In addition to its forward-regulatory role in T-cell activation, within Th1-type immune response IFN-γ initiates several antimicrobial and antitumoral biochemical pathways in certain target cells. For example, in various cells, but especially in monocyte-derived macrophages, IFN-γ, together with tumor necrosis factor-α (TNF-α), induces enzymes like nitric oxide synthase (iNOS), GTP cyclohydrolase I (GCH) and IDO and triggers the formation of reactive oxygen species (ROS) [21]. iNOS produces nitric oxide which, upon reaction with superoxide anion, produces highly toxic peroxynitrite. IDO converts the essential amino acid tryptophan into kynurenine and subsequent degradation products in various cells [22]. Thereby, growth of microbes and tumor cells is affected, because deprivation of essential amino acid tryptophan limits protein biosynthesis. Increased IDO activity is indicated by an elevated ratio of kynurenine to tryptophan concentrations (kyn/trp) [23]. Activation of GCH in human macrophages and dendritic cells (DC) leads to the increased formation of neopterin, which is a sensitive marker of immune activation in humans [24]. Both enzymatic pathways are induced in parallel in peripheral blood mononuclear cells (PBMC) upon stimulation with mitogens (Fig. 27.3).

Fig. 27.3
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When human peripheral blood mononuclear cells are stimulated with mitogens such as phytohemagglutinin (PHA) and concanavalin A (con A), T-cells release Th1-type cytokine interferon-γ (IFN-γ), which in turn stimulates tryptophan degradation by indoleamine 2,3-dioxygenase (IDO). Due to IDO activation, significant decline of tryptophan concentrations in culture supernatants is observe, at the same time kynurenine concentrations increase. Increased IDO activity is reflected by an increased kynurenine to tryptophan ratio in stimulated cells (note: log-scale) and goes in parallel to enhanced neopterin formation in stimulated cells. IFN-γ also induces enzyme GTP-cyclohydrolase I and thus, the degradation of tryptophan is paralleled by increased neopterin formation

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Like IDO activity, several other IFN-γ-mediated biochemical effects aim to affect growth of cells carrying non-self-surface structures after infection with viruses, parasites or mycobacteria or after malignant transformation. Such data have given rise to the generally accepted concept that IFN-γ is critically involved and plays a physiologically relevant role in promoting host resistance to tumor development and microbial infection.

Taking into account that NO and tryptophan metabolism are non-redundant, alternative systems that control T-cell responses, current data obtained largely in murine models and in vitro suggest that a temporal relay between both pathways could be beneficial in tolerance induction. Recently, Hill et al. demonstrated that the interplay between NO and IDO is an important determinant in the clinical efficacy of the costimulatory blocking molecule cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) to induce immunological tolerance upon fully MHC mismatched murine heart allografts [25]. The proposed mechanism for this potent pro-tolerogenic action was, that both, IDO and iNOS together, are responsible for the impaired capacity of DCs from CTLA4Ig-treated animals to stimulate allogeneic T-cells. These data substantiate the fact that the NO and IDO pathways are cross-regulated, since certain tryptophan catabolites can inhibit IFN-γ-induced iNOS transcription in murine macrophages by inhibition of NF-κB activation and tryptophan starvation also inhibits IFN-γ-induced iNOS expression in these cells [26].

3 Role of IFN-γ During Innate and Adaptive Anti-tumor Immune Responses

Since the introduction of the cancer immunosurveillance concept by Burnet and Thomas in 1957, the idea that the immune system plays a protective role in the development of malignant disease and might be able to control or eliminate cancer cells has been a subject of controversy [27]. In general an immune response towards tumor cells can be broadly divided into innate and adaptive components, with intense crosstalk between the two. Innate immunity, including components such as complement proteins, granulocytes, macrophages and NK cells, serves as the first line of defense against infection orchestrating inflammatory reactions [28]. NK cells thereby do not employ classical antigen receptors to recognize their cellular target, they rather use pattern-recognition receptors and other cell-surface molecules such as NKG2D, Ly-49 and KIRs to directly detect tumor cells [29]. Activation of NK cells subsequently increases their production of IFN-γ and thus helps establish an appropriate cytokine milieu that favours the generation of further protective cell-mediated immune responses towards tumor cells.

The adaptive immune system acts via an indirect pathway, which is mediated through cross-priming by dendritic cells (DCs) to achieve initial recognition of cancer. Malignant cells do not express co-stimulatory molecules like B7 family members that are essential for full T-cell activation and hence are insufficient to prime cellular immune responses [30]. By contrast, DCs, the key antigen-presenting cells (APC) for initiating immune responses, capture tumor antigens and process the material for MHC presentation following increased expression of costimulatory molecules such as B7-1 and B7-2 within the regional lymph nodes. DCs thereby activate both CD4+ and CD8+ T-cells, which represent the major source of IFN-γ during the adaptive phase of immune responses for tumor clearance [31].

Taken together, IFN-γ exerts significant features in regulating tumor development in several animal models. The major issues thereby are activation of the innate and adaptive immune response against tumors (Fig. 27.2), anti-proliferative and pro-apoptotic actions of IFN-γ and its signaling system as well as inhibition of angiogenesis.

In humans there are also several hints for a similar existing role of IFN-γ in cancer immunosurveillance. First, transplant recipients requiring life-long immunosupp-ressive therapy display a higher incidence of malignancies as compared to immunocompetent individuals [32]. Second, cancer patients may spontaneously develop specific adaptive immune responses toward tumor antigens [33] and third, there have been several reports showing that the presence of tumor-infiltrating lymphocytes (TILs) correlates with clinical outcome and patient survival [34].

3.1 Effects of Sustained IFN-γ Production in Cancer Patients

As discussed so far, IFN-γ is probably the most important anti-proliferative cytokine which is released in large amounts during tumor development. It induces several biochemical pathways and mechanisms in order to stop growth of microbes and tumor cells. However, if the immune system is unable to eliminate the malignant tumor cells, immune activation may persist, which causes overproduction of IFN-γ. Because of its outstanding potency it is not surprising to find side-effects, especially during such clinical conditions of continuous immune system stimulation and IFN-γ production which may exert a strong negative impact on host cells [35]. One underlying mechanism for these detrimental IFN-γ effects appears to be the increased tryptophan depletion, which can affect T-cell responses and thus contribute to the development of immunodeficiency in cancer patients. IFN-γ may also provide a key to understand how the complex interplay between tumor and stroma is affected by IDO activity. First evidence for such a tumoral immune resistance mechanism based on tryptophan degradation was provided by Uyttenhove et al. in a murine model, where they showed that IDO reduces anti-tumoral T-cell attack [36]. They observed that expression of IDO by immunogenic mouse tumor cells prevented their rejection in preimmunized mice. This effect was accompanied by a lack of accumulation of T-cells at the tumor site and was partly reverted by systemic treatment of mice with a pharmacological IDO inhibitor.

4 IFN-γ Induces IDO-Mediated Tryptophan Catabolism

The release of IFN-γ is the main stimulus for activating IDO in monocyte-derived macrophages, DCs, fibroblasts and various other cell types [37]. However, other cytokines like IFN-α, IFN-β, TNF-α or lipopolysaccharides are also able to induce IDO, although to a much lesser extent [38]. IDO, a 42kDA hem- containing enzyme, degrades the essential amino acid L-tryptophan to form N-formyl kynurenine which sets free a 1-carbon unit and kynurenine is produced. Thus, also the production of formaldehyde and formate during, e.g. malignant disease [39] may relate to IDO activity. Kynurenine, depending on cell type and enzymatic repertoires, is subsequently converted to finally form niacin [40]. This process involves the cleavage of the five-membered indole ring. Once cleaved, the indole ring cannot be resynthesized by human metabolism. L-tryptophan is therefore an essential amino acid which is required for the biosynthesis of proteins and is the precursor for several biologically important compounds such as 5-hydroxytryptamine (serotonin), formed by tryptophan (5)-hydroxylase following decarboxylation, or melatonin. Raised IFN-γ concentrations during immune responses have been shown to lead to robust and sustained tryptophan depletion [41]. In recent years, a clear association has been made between tryptophan catabolism and inflammatory reactions in a wide array of different diseases with much of the focus centering on the kynurenine pathway of tryptophan degradation occurring in the immune system [42].

IFN-γ-mediated tryptophan breakdown acts to reduce substrate availability for certain intracellular pathogens, bacteria or cancer cells and contributes substantially to its antimicrobial and antitumor response [43]. Consequently, tryptophan depletion is regarded as a natural defense mechanism of immunocompetent cells, which is induced by IFN-γ during immune response. Furthermore, local tryptophan depletion has recently been hypothesized to be a mechanism for suppressing T-cell responses. Activation of IDO inhibits responsiveness of T-cells to mitogenic stimulation in vitro and in vivo [44]. This is especially true when the enzyme is induced by IFN-γ in macrophages and DCs. It appears that not only tryptophan deprivation is important to arrest T-cells within the G1 phase of the cell cycle, but also the pro-apoptotic effect of certain tryptophan catabolites like kynurenine is of relevance [45]. In addition, tryptophan depletion is also critically involved in DC functions. DCs are known as APCs that prime T helper cells and are also involved in tolerance induction towards self and non-pathogenic foreign antigens [46]. Dependent on the state of DC maturation, these cells can activate lymphocytes to respond to a certain antigen or to induce tolerance to the presented antigen (Fig. 27.4). IFN-γ is one of the primary cytokines that cause DC maturation and hence induction of effector T-cell responses. Recently it has been shown that CTLA4-Ig is able to upregulate IDO expression in DCs via an IFN-γ-dependent mechanism [47]. CTLA4 is a T-cell surface receptor that plays an important role in mediating immune tolerance. Thus, these data indicate that IDO-mediated tryptophan depletion is involved in the development and maintenance of tolerance. IDO+ DCs have also been identified in breast tumor tissue and tumor-draining lymph nodes of patients with melanoma and patients with colon, lung and pancreatic cancers, and it is conceivable that such DCs contribute to tumor-mediated immunosuppression or immune evasion. [48]. All these observations together support the view that activation of IDO together with other biochemical pathways induced by IFN-γ is an important anti-proliferative mechanism of monocyte-derived macrophages and DCs which however can also decrease the responsiveness of stimulated T-cells and thus contribute to the development of immunodeficiency. In general IFN-γ-mediated metabolic activation of IDO during prolonged disease stages can be regarded as harmful to the host by slowing down T-cell responses.

Fig. 27.4
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Monocyte-derived macrophages (MΦ) and dendritic cells (DC) stimulated via pro-inflammatory cytokines or via Toll like receptor (TLR) share several biochemical pathways including the expression of indoleamine 2,3-dioxygenase (IDO), of nitric oxides synthase (iNOS) or of GTP-cyclohydrolase I (GCH). In stimulated MΦ, these enzymatic pathways appear to preferentially contribute to the killing repertoire of cells, whereas in DC their main task is immunoregulation

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5 IDO-Mediated Tryptophan Catabolism in Patients with Malignant Disease – A Predictive Marker

In patients with malignant tumors, significantly accelerated degradation of tryptophan with lowered serum concentrations of tryptophan and increased kynurenine as well as an increased kyn/trp was recognized earlier by our group and others [49, 50]. Recently, significantly lower tryptophan levels and higher kyn/trp as compared to healthy volunteers were observed in colorectal cancer patients, and the lower tryptophan levels were associated with reduced quality of life and poor outcome [51]. A concomitant increase in kynurenine suggested that the observed tryptophan deficiency is related to IDO-mediated tryptophan degradation. Such a phenomenon can be best explained by IFN-γ-mediated IDO expression within the tumors. This conclusion is also supported by a correlation found between neopterin concentrations, an established marker of cellular immune responses, and kyn/trp, which suggests that under these conditions endogenous production of IFN-γ is increased, and by the finding of an increased expression of IFN-γ in cancer patients reported earlier [52].

In several other malignant diseases, including various solid tumors as well as hematological neoplasias, accelerated tryptophan catabolism has been described as well [53, 54]. Lower tryptophan concentrations and increased kyn/trp are associated with more advanced stages of the disease, and in patients with adult T-cell leukemia [55] or with colorectal carcinoma [51], lower tryptophan concentrations are predictive for shorter survival. In addition, in patients with colon carcinoma enhanced IDO expression and increased tryptophan degradation were suggested to be an intrinsic immunoescape mechanism of tumor cells [36].

In a very recent study we tested the expression of IDO protein in vivo in human tumor samples by means of semiquantitative immunostaining [55]. We detected colorectal tumor cells expressing IDO in all 143 cases analyzed. These results are in line with previous studies indicating that human tumors frequently express IDO [36]. However, 39.2% of tumor specimens revealed IDO-high expression, whereas in 60.8% of cases the staining was scored as IDO-low expression, indicating certain colon cancer subsets that differ in their ability to express IDO in vivo. Apart from malignant cells, we identified a number of IDO positive cells within the tumor stroma morphologically classified as antigen-presenting cells. IDO-expressing cells have been deemed to create a state of immunological unresponsiveness towards tumor-derived antigens [56]. However, which cells in particular, either tumor cells themselves or host APCs expressing IDO, are responsible for tolerance induction is still unclear. The observation of IDO expression in tumor cells of colorectal cancer patients with distinct expression patterns provides for the first time also a link to the clinical outcome, since IDO expression significantly correlated with the frequency of metastases and overall survival in this study, which implicates the importance of the tumor itself as the primary tolerizing agent. Similar conclusions can be deduced from recent findings in patients with malignant melanoma, which presented with significantly accelerated tryptophan degradation, and the lowering of plasma tryptophan concentrations turned out to be strongest predictor of poor survival [57].

Conceptually, tumor cells in the early stage may be recognized by the host’s immune system which is accompanied by formation of IFN-γ. As a consequence, IDO is activated and tryptophan deprivation and formation of pro-apoptotic, downstream catabolites restrict T-cell proliferation and T-cell numbers decline. This may cause a selective survival benefit of IDO-high-expressing malignant cells. In addition, the suppressive effects of IDO might be mediated by tryptophan depletion within the tumor microenvironment resulting in a local milieu that is rendered immunosuppressive. We hypothesize that IDO-high-expressing tumor cells enable certain cancer subsets to initially avoid immune attack and then reduce T-cell priming and defeat the invasion of effector T-cells via local tryptophan depletion and the production of proapoptotic tryptophan catabolites. Almost 20 years ago it has been already shown that IDO can be drastically induced in most but not all human cells and tumor cell lines by IFN-γ, whereas the same cells, with the exception of one T24, a bladder carcinoma cell lien, did not spontaneously degrade tryptophan to a relevant extent [38]. Once induced, it is unclear for how long IDO activity can be detected in these cells. One may speculate that IDO may continue to degrade tryptophan for long time, even if the initial stimulus has already disappeared. These in vitro findings are well in line with more recent studies indicating that the therapeutic efficacy of IFN-γ in tumor models critically depends on the ability of the tumor cells themselves to respond to IFN-γ [58]. However, correlation found between kyn/trp and neopterin concentrations favours the view that IDO activation in monocyte-derived macrophages, DCs and/or tumor cells is due to enhanced endogenous formation of IFN-γ during the host’s response against the tumor and may suppress T-cell proliferation, thus acting immunosuppressive. Reduced immunoresponsiveness may thereby develop as a consequence of the antiproliferative mechanism induced by this particular cytokine [59]. This gives rise to the conclusion that in patients with malignant disease experiencing chronic states of immune activation, systemically increased IFN-γ is no longer antigen-specific and is associated with the development of immunodeficiency.

6 Enhanced Tryptophan Depletion Due to IDO Results in Immunodefiency

Immunodeficiency with reduced cell-mediated immune response (CMI) occurs in a number of chronic infectious diseases, including tuberculosis, leishmaniasis and human immunodeficiency virus (HIV) infection [60]. More recently, it has become increasingly apparent that CMI is also suppressed in virtually all malignant diseases, including melanoma, colorectal and prostate cancer and this becomes even more evident as the disease progresses [61]. However, the mechanisms underlying the immune defects noticed in cancer patients have not been fully elucidated. In cancer patients, analogous to patients with HIV infection, higher neopterin concentrations and kyn/trp, reflecting increased IFN-γ levels (Fig. 27.3), predict a more rapid disease progression and shorter survival time, and both are associated with the development of immunodeficiency. Enhanced degradation of tryptophan has been demonstrated earlier in several diseases which go hand-in-hand with or are even characterized by acquired immunodeficiency. This is especially true for patients with HIV infection, but also for various other mostly chronic diseases like autoimmune disorders, in which immunodeficiency develops as disease progresses and is a sign of poor prognosis [62]. Acquired immunodeficiency is the hallmark of progressing HIV infection. In parallel, activation of several immune compartments has been observed including activation of B-cells, T-cells and macrophages. Several parameters of immune activation were found to predict disease development in patients with HIV infection, and data show that immune activation coexists with immune deficiency in such patients [63]. Activation of IDO by IFN-γ could be involved in diminishing T-cell responsiveness in patients with HIV infection and reduced T-cell proliferative response to soluble antigens in vitro has been found to be associated with the immune activation status in these patients [64]. Similar relationships also appear to exist in cancer patients and result in reduced CMI.

Decreased blood tryptophan in cancer patients indicates significant depletion of tryptophan in the microenvironment of activated cells, and the enhanced IDO activity is most likely driven by endogenously formed cytokines like IFN-γ. Because tryptophan is essential for the production and function of rapidly proliferating cells and tissues in general, tryptophan degradation may also suppress T-cell proliferation. Expression of IDO by antigen-stimulated macrophages inhibits proliferation of T cells co-cultured with macrophages. Such a nutrient-depleting role of competing cells was recently extended to include novel regulatory pathways for IDO in immunosuppression. The restriction of available tryptophan in microenvironment such as within the tumor stroma could be crucial for a sufficient immune response to tumor-associated antigens and contribute to immune evasion by influencing the quality and quantity of local T-cell responses. T-lymphocytes and natural killer cells (NK-cells), in contrast to almost all other cell types, in particular tumor cells, stop proliferation under conditions of tryptophan deprivation because of a specific tryptophan-sensitive checkpoint which arrests their cell cycle in the G1 phase, whereas at the same tryptophan concentrations malignant cell growth is not affected [65]. In addition, cancer patients have significantly lower absolute numbers of both B and T lymphocytes (CD4+ and CD8+ subsets) in peripheral blood than do healthy subjects, which can seriously affect immune functions [66].

Thus, immunodeficiency in cancer patients may result from the enhanced long-term production of IFN-γ in response to malignant transition and tumor formation as part of the immune defense reaction. Antimicrobial and cytocidal biochemical consequences of IFN-γ not only restrict bacterial growth, but may also impair T-cell development and proliferation via a negative feedback loop and contribute to the development of immunodeficiency. This raises the possibility that, as a tumor develops, cancer cells evolve to subvert the CMI response via IFN-γ to increase IDO activity. Consequently, tryptophan deprivation is one of several anti-proliferative events mediated by IFN-γ in tumor patients that might exert detrimental effects in the host. Moreover, the reduced CMI response as seen in colorectal cancer patients is completely reversed following curative surgery, strongly supporting the idea that tumors themselves can suppress the systemic immune response [61]. Notably, paralleling neopterin concentrations, kyn/trp in cancer patients exhibits a steady increase over time and those patients with higher kyn/trp tended to have the greatest impairment in immune function and hence the highest incidence of sepsis and death [67].

7 Sustained IDO Activity Accounts for Several Symptoms Such as Cachexia, Anemia, Organ Failure and Depression of the Terminally ILL Cancer Patient

About 50–80% of all advanced-stage cancer patients experience a wasting syndrome called cachexia, in which the tumor induces metabolic changes in the host leading to loss of adipose tissue, skeletal muscle mass and anemia [68]. These effects are not a local phenomenon of a tumor but are thought to be a type of paraneoplastic syndrome. The process appears to be mediated by circulatory tumor-produced catabolic factors acting either alone or in concert with certain cytokines such as IFN-γ [69]. No effective treatment is currently available for the cancer cachexia syndrome and it must therefore be regarded as a strong independent risk factor. As previously mentioned, tryptophan is essential for many cellular functions, including protein biosynthesis and cell proliferation, and an intracellular tryptophan deficiency alters these cellular functions substantially. Increased neopterin levels and kyn/trp were found to be associated with cachexia and weight loss [53] as well as anemia [70]. Enhanced tryptophan degradation appears to be involved in the development of these symptoms as well (Fig. 27.5). Likewise, in patients with hematological neoplasias, low tryptophan concentrations were found to be associated with low serum albumin concentrations and weight loss [71], this association was apparent at study entry and during patient follow-up. IFN-γ-mediated tryptophan deprivation may be one important underlying mechanism to cause slow-down of protein biosynthesis and in turn accelerate breakdown of muscle proteins.

Fig. 27.5
figure 27_5_212344_1_En

Th1-type immune activation is associated with the induction of numerous biochemical pathways including the expression of indoleamine 2,3-dioxygenase (IDO) in various cells and tissues. Due to IDO activity, tryptophan degradation takes place and reduces the availability of tryptophan. This initially antitumoral and antimicrobial activity of immunocompetent cells can also affect proliferation of T-cells, which increases the risk of immunodeficiency, but also of erythroid progenitor cells, which increases the risk of anemia in patients. Insufficient availability of tryptophan will also reduce protein biosynthesis in the whole organism. Moreover, tryptophan-deprived cells begin to break-down protein to recruit tryptophan for their necessary biochemical functions. Both these consequences of tryptophan degradation are involved in the development of weight-loss and cachexia in patients. Subnormal availability of tryptophan also slows down biosynthesis of serotonin and its subsequent metabolite melatonin. Serotonin deficiency can increase the risk for depression in patients, when exposed to unfavorable experiences

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Cytokines affect the homeostatic loop of body weight regulation in cancer patients either by their involvement in the brain’s serotonergic system or by mimicking leptin, a member of the helical cytokine family and one of the key targets for neuropeptidergic effector molecules that regulate food intake and energy expenditure via the sympathetic nervous system [72]. More direct evidence of cytokine involvement comes from experiments in which specific neutralization of cytokines can relieve cachexia in experimental animal models. Examples are the anti-TNF-α, anti-IL-1 and anti-IFN-γ antibodies, although no single antibody could reverse all of the features seen in cancer cachexia. These studies revealed that cachexia is associated with cytokine activation and other cachectic factors that are orchestrated to induce major metabolic abnormalities [73].

Anemia is another frequent complication in cancer, occurring in more than half of all patients with malignancies. However, in a considerable number of patients, no cause other than malignant disease itself can be implicated. Cancer-related anemia is similar to the anemia observed in other chronic diseases, characterized by a hyporegenerative, normocytic, normochromic anemia associated with reduced serum iron and transferrin saturation. Recently, accelerated catabolism of tryptophan was proposed to also be in important in the pathogenesis of anemia in states of chronic inflammation [70]. It is currently well established that pro-inflammatory cytokines IFN-γ and TNF-α suppress growth and differentiation of erythroid progenitor cells, and these cytokines are crucially important in the pathogenesis of anemia. Thus, IFN-γ-induced tryptophan deprivation appears to be involved in hematopoietic suppression in cancer patients in the same way as iron withdrawal [74], and the limitation of tryptophan availability may be a key mechanism in cytokine-mediated inhibition of erythroid progenitor cells.

IFN-γ has been furthermore implicated in the pathogenesis of bone marrow failure. In vitro, bone marrow stromal cells genetically engineered to constitutively express IFN-γ markedly suppressed the proliferative capacity of erythrocyte, granulocyte, and monocyte precursors [75]. Such impaired bone marrow function may also contribute to the development of multiple organ dysfunction syndrome and multiple organ failure in cancer patients.

Other distressing symptoms that debilitate patients with malignant disease and contribute to their profound fatigue are severe mood changes, subtle cognitive changes and depression. Insufficient availability of tryptophan reduces the biosynthesis of neurotransmitter 5-hydroxytryptamine (serotonin), which in turn can increase the susceptibility to develop mood disturbance and depression and which may also impair cognitive function [76]. Furthermore, downstream products of tryptophan – kynurenine metabolism such as 3-hydroxy-anthranilic acid and quninolinic acid can cause neuronal damage and dysfunction [77]. The latter is a potent neurotoxin which interferes with the N-methyl-D-aspartate (NMDA) receptor and may thereby influence the neuroendocrine system in addition to the neuropathologic effects of tryptophan deprivation. Immune-mediated tryptophan degradation by means of IDO may thus elicit neuropsychiatric symptoms when the availability of tryptophan is insufficient for normal serotonin biosynthesis [78]. In patients with major depression decreased serum tryptophan concentrations are found, correlating with increased concentrations of immune activation markers [79]. On the one hand, reduced concentrations of 5-hydroxyindoleacetic acid, the main catabolite of serotonin, are observed and confirm insufficient availability of serotonin. On the other hand, treatment with selective serotonin-reuptake inhibitors (SSRIs) can be very effective in patients with depression. Thus, several indirect evidences are in support of the view that enhanced degradation of tryptophan due to immune stimulation could underlie the increased risk for development of mood disturbances and susceptibility to depression in cancer patients, especially when undergoing prolonged disease. In several types cancer, enhanced degradation of tryptophan was found to coincide with impaired quality of life [51, 80]. Moreover during treatment with IFN-α, a relationship between lower tryptophan levels and increased susceptibility of depression was reported recently in malignant melanoma patients [81].

8 Nutrition and Tryptophan Availability

As an essential amino acid, the tryptophan concentration is influenced by diet. However, dietary changes influence serum/plasma tryptophan levels only to a minor extent, when compared to the dramatic changes, which are induced during an inflammatory response. Notably, all the supplemented tryptophan is degraded within 24–48 h, when cultures of PBMC are stimulated (Fig. 27.3) [82]. In vitro it is observed that antioxidant vitamins possess some anti-inflammatory activity, which includes the suppression of IFN-γ production by stimulated PBMC. As a consequence also neopterin production and tryptophan degradation is diminished by compounds like vitamin C and E, but also aspirin. Thus, a so-called “healthy diet” could indeed have some benefit to slow-down inflammation and thus reduce the cancer risks in the community.

In conclusion, it is apparently becoming clear that increased and sustained immune activation and IFN-γ levels and hence increased IDO activity in the course of malignant diseases are an integral mechanism in initiating long-term, aforementioned side-effects of chronic immune activation and may even aggravate severity of tumor burden in this patient population. IDO expression in certain cancer subtypes has been demonstrated to predict disease progression and overall survival. Recent data revealed a dual role for the immune system in general and for IFN-γ in particular in suppressing and promoting cancer formation. In this context, the interplay of chronic infection, inflammation and cancer immunity helps to determine the outcome of the host response. There is also compelling evidence that the IFN-γ induced IDO pathway is critically involved in the pathogenesis of various diseases composing the cancer-cachexia syndrome, including also the impaired quality of life, which is almost generally observed in the alter course of the disease.