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Posts Tagged ‘T cell’


Immune System in Perspective

Curator: Larry H. Bernstein, MD, FCAP

LPBI

How regulatory T cells work
Vignali DAA, Collison LW & Workman CJ
Nature Reviews Immunology 8, 523-532 (July 2008) |   doi:10.1038/nri2343
http://www.nature.com/nri/journal/v8/n7/full/nri2343.html

Regulatory T (TReg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting antitumour immunity. Given that TReg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we describe the basic mechanisms used by TReg cells to mediate suppression and discuss whether one or many of these mechanisms are likely to be crucial for TReg-cell function. In addition, we propose the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate TReg-cell function.

How regulatory T cells work.

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Basic mechanisms used by Treg cells

This schematic depicts the various regulatory T (Treg)-cell mechanisms arranged into four groups centred around four basic modes of action. ‘Inhibitory cytokines’ include interleukin-10 (IL-10), interleukin-35 (IL-35) and transforming growth factor-β (TGF-β). ‘Cytolysis’ includes granzyme-A- and granzyme-B-dependent and perforin-dependent killing mechanisms. ‘Metabolic disruption’ includes high affinity IL-2 receptor α (CD25)-dependent cytokine-deprivation-mediated apoptosis, cyclic AMP (cAMP)-mediated inhibition, and CD39- and/or CD73-generated, adenosine–purinergic adenosine receptor (A2A)-mediated immunosuppression. ‘Targeting dendritic cells’ includes mechanisms that modulate DC maturation and/or function such as lymphocyte activation gene-3 (LAG3; also known as CD223)–MHC-class-II-mediated suppression of DC maturation, and cytotoxic T lymphocyte antigen-4 (CTLA4)–CD80/CD86-mediated induction of indoleamine 2,3-dioxygenase (IDO), which is an immunosuppressive molecule, by DCs.

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Model for how effector T cells might boost Treg-cell function

This occurs in three stages. (a) Initial regulatory T (Treg)-cell activation induces production of regulatory factors such as interleukin-35 (IL-35). (b) Treg cells ‘sense’ the presence of recently activated effector T cells through a receptor–ligand interaction (cell surface or soluble). (c) This in turn boosts or potentiates Treg-cell function resulting in the enhanced production of regulatory mediators, such as IL-35, and perhaps the induction of new mediators.

 

Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity. Given that Treg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we discuss the basic mechanisms used by Treg cells to mediate suppression, and discuss whether one or many of these mechanisms are likely to be crucial for Tregcell function. In addition, we present the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate Treg-cell function.

Several sophisticated regulatory mechanisms are used to maintain immune homeostasis, prevent autoimmunity and moderate inflammation induced by pathogens and environmental insults. Chief amongst these are regulatory T (Treg) cells that are now widely regarded as the primary mediators of peripheral tolerance. Although Treg cells play a pivotal role in preventing autoimmune diseases, such as type 1 diabetes1,2, and limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease (IBD)3,4, they also block beneficial responses by preventing sterilizing immunity to certain pathogens5,6 and limiting anti-tumour immunity7. A seminal advance in the analysis of Treg cells came with the identification of a key transcription factor, forkhead box P3 (FOXP3), that is required for their development, maintenance and function8,9. Mice and patients that lack FOXP3 develop a profound autoimmune-like lymphoproliferative disease that graphically emphasizes the importance of Treg cells in maintaining peripheral tolerance10-12 (BOX 1). Although FOXP3 has been proposed as the master regulator of Treg cells that controls the expression of multiple genes that mediate their regulatory activity13,14, this has been recently challenged raising the possibility that other transcriptional events may operate upstream of and/or concurrently with FOXP3 to mediate Treg-cell development15.

While Foxp3 has proven to be an invaluable marker for murine Treg cells, its role in human Treg cells is less straightforward (see BOX 2 for a discussion of Treg-cell markers). Humans that lack FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), a severe autoimmune disease that presents early in infancy. Although FOXP3 appears to be required for human Treg-cell development and function, expression of FOXP3 alone is clearly not sufficient as a significant percentage of human activated T cells express FOXP3 and yet do not possess regulatory activity16-20. Furthermore, induction of FOXP3 in human T cells by transforming growth factor-β (TGFβ) does not confer a regulatory phenotype, in contrast to their murine counterparts20. Consequently, FOXP3 is not a good marker for human Treg cells (BOX 2). Whether this distinction is due to intrinsic differences between mouse and human FOXP3 and/or a requirement for an additional cofactor/ transcription factor is an important question that needs to be resolved.

Significant progress has been made over the last few years in delineating the molecules and mechanisms that Treg cells use to mediate suppression21,22. In this Review, we outline our current understanding of the mechanisms used by Treg cells to mediate suppression, and the challenges that lie ahead in defining their mode of action. We also discuss whether Treg cells are likely to depend on one, a few or many of these mechanisms. In addition, we propose that effector T cells may have a significant role in boosting and/or modulating Treg-cell function. Unless stated, we focus here primarily on the mechanisms that are used by thymus-derived natural CD4+CD25+ FOXP3+ Treg cells.

Basic mechanisms of Treg-cell function Defining the mechanisms of Treg-cell function is clearly of crucial importance. Not only would this provide insight into the control processes of peripheral tolerance but it would probably provide a number of potentially important therapeutic targets. Although this quest has been ongoing since interest in Treg cells was reignited in 199523, there has been significant progress in the last few years. From a functional perspective, the various potential suppression mechanisms of Treg cells can be grouped into four basic ‘modes of action’: suppression by inhibitory cytokines, suppression by cytolysis, suppression by metabolic disruption, and suppression by modulation of dendritic-cell (DC) maturation or function (FIG. 1).

Suppression by inhibitory cytokines Inhibitory cytokines, such as interleukin-10 (IL-10) and TGFβ, have been the focus of considerable attention as a mechanism of Treg-cell-mediated suppression. There has also been significant interest in their ability to generate induced (also known as adaptive) Treg-cell populations, either naturally in vivo or experimentally as a potential therapeutic modality (BOX 3). Although the general importance of IL-10 and TGFβ as suppressive mediators is undisputed, their contribution to the function of thymus-derived, natural Treg cells is still a matter of debate24. This is partly due to the general perception that Treg cells function in a contactdependent manner25,26. Indeed, in vitro studies using neutralizing antibodies or T cells that are unable to produce or respond to IL-10 and TGFβ suggested that these cytokines may not be essential for Treg-cell function25-28. However, this contrasts with data from in vivo studies29,30.

In allergy and asthma models, evidence suggests that both natural and antigen-specific Treg cells control disease in a manner that is, in part, dependent on IL-1029 and in some reports dependent on both IL-10 and TGFβ 31. Adoptive transfer of allergen-specific Treg cells induced significant IL-10 production by CD4+ effector T cells in the lung following allergen challenge and this Treg-cell-mediated control of disease was reversed by treatment with an IL-10- receptor-specific antibody32. However, suppression of allergic inflammation and airway hyper-reactivity, and increased production of IL-10 still occurred following transfer of IL-10- deficient Treg cells, suggesting that Treg cells can suppress the Th2-driven response to allergens in vivo through an IL-10-dependent mechanism, but that the production of IL-10 by Treg cells themselves is not required for the suppression observed. This contrasts with a recent study suggesting that the Treg-cell-specific ablation of IL-10 expression resulted in increased lung allergic inflammation and hyperreactivity33.

This scenario might occur in other disease models. For instance, the effects of IL-10 can only be partially attributed to Treg-cell-derived IL-10 in the immune response to hepatitis B virus34 and in the allograft tolerance response elicited by splenocytes exposed to non-inherited maternal antigens35. Recently, it was also shown that IL-10 is crucial for the control of various infections in which Treg cells have been reported to be involved including Mycobacterium tuberculosis36, Toxoplasma gondii37, Leishmania major38, and Trichinella spiralis39. However, Treg cells were not the source of IL-10 in all of these infection models.

By contrast, several studies have shown that IL-10 production by Treg cells is essential for the prevention of colitis in mouse models of IBD40. Moreover, it appears that the tumour microenvironment promotes the generation of FOXP3+ Treg cells that mediate IL-10- dependent, cell-contact independent, suppression41. Similarly, in UV-radiation-induced carcinogenesis, IL-10 production by Treg cells appears to be important for blocking anti-tumour immunity42. IL-10 produced by Treg cells also appears to be crucial for IL-10-mediated tolerance in a model of hepatitis induced by concanavalin A43 and tolerance to bacterial and viral superantigens44. In addition, recent papers suggest new roles for Treg-cell-derived IL-10 in the induction of feto-maternal tolerance45 and B-cell-enhanced recovery from experimental autoimmune encephalomyelitis46. Collectively, the picture that appears to be emerging is that the relative importance of Treg-cell-derived IL-10 is very dependent on the target organism or disease and on the experimental system. Furthermore, the Treg-cell-specific deletion of IL-10 did not result in the development of spontaneous systemic autoimmunity, but did result in enhanced pathology in the colon of older mice and in the lungs of mice with induced airway hypersensitivity, suggesting that the function of Treg-cell-derived IL-10 may be restricted to controlling inflammatory responses induced by pathogens or environmental insults33.

While some early in vitro studies using neutralizing antibodies to TGFβ or Treg cells lacking TGFβ 25,47 indicated that TGFβ was not required for natural Treg-cell function, other studies, both in vitro and in vivo suggested a critical role for Treg-cell surface bound TGFβ 48,49. Therefore, the importance of TGFβ for natural Treg-cell function has also been a controversial topic. Indeed, there has been considerably more focus recently on the importance of TGFβ in the development of induced Treg cells and perhaps in Treg-cell maintenance in general (BOX 3). However, there are studies that suggest that TGFβ produced by Treg cells may directly participate in effector T-cell suppression. For instance, effector T cells that are resistant to TGFβ-mediated suppression cannot be controlled by Treg cells in an IBD model50. In addition, TGFβ produced by Treg cells has been found to be important in the control of the host immune response to M. tuberculosis36, suppression of allergic responses31 and prevention of colitis in an IBD model51. Interestingly, TGFβ produced by Treg cells has also been implicated in limiting anti-tumour immunity in head and neck squamous-cell carcinoma52 and in follicular lymphoma53 by rendering T cells unresponsive to the tumour. TGFβ also appears to limit the anti-tumour activity of cytokine-induced killer cells54.

Membrane-tethered TGFβ can also mediate suppression by Treg cells in a cell-cell contactdependent manner48. Treg cells can control islet infiltration of CD8+ T cells and delay the progress of diabetes through membrane-tethered TGFβ 49. However, experiments using mice deficient in TGFβ-receptor (TGFβR) signalling in effector T cells or using TGFβ or TGFβR blocking reagents failed to show that membrane-tethered TGFβ is required for natural Treg cell development or function47. More recently, however, interest in membrane-tethered TGFβ has re-surfaced with the description of a previously unappreciated role for it in the tumour microenvironment. TGFβ associated with tumour exosome membranes appears to enhance the suppressive function of Treg cells and skew T cells away from their effector functions and towards regulatory functions55. Furthermore, ovalbumin-induced airway inflammation can be attenuated by heme oxygenase-1 through membrane-tethered TGFβ and IL-10 secretion by Treg cells56, a process that activates the Notch1–HES1 (hairy and enhancer of split 1) axis in target cells57. Thus, in light of the most current data, it now appears that soluble and/or membrane-tethered TGFβ may have a previously unappreciated role in natural Treg-cell function.

Recently, a new inhibitory cytokine, IL-35, has been described that is preferentially expressed by Treg cells and is required for their maximal suppressive activity58. IL-35 is a novel member of the IL-12 heterodimeric cytokine family and is formed by the pairing of Epstein–Barr virus induced gene 3 (Ebi3), which normally pairs with p28 to form IL-27, and p35 (also known as  Il12a), which normally pairs with p40 to form IL-12. Both Ebi3 and Il12a are preferentially expressed by murine Foxp3+ Treg cells58,59, but not resting or active effector T cells, and are significantly upregulated in actively suppressing Treg cells58. As predicted for a heterodimeric cytokine, both Ebi3−/− and Il12a−/− Treg cells had significantly reduced regulatory activity in vitro and failed to control homeostatic proliferation and cure IBD in vivo. This precise phenocopy suggested that IL-35 is required for the maximal suppressive activity of Treg cells. Importantly IL-35 was not only required but sufficient, as ectopic expression of IL-35 conferred regulatory activity on naive T cells and recombinant IL-35 suppressed T cell proliferation in vitro58. Although IL-35 is an exciting addition to the Treg-cell portfolio, there is clearly much that remains to be defined about this cytokine and its contribution to Treg-cell function. For instance, it remains to be determined if IL-35 suppresses the development and/or function of other cell types such as DCs and macrophages.

It is now clear that three inhibitory cytokines, IL-10, IL-35 and TGFβ, are key mediators of Treg-cell function. Although they are all inhibitory, the extent to which they are utilized in distinct pathogenic/homeostatic settings differs suggesting a non-overlapping function, which needs further refinement.

……….

How many mechanisms do Treg cells need? Although efforts to define the suppressive mechanisms used by Treg cells continue, an important question looms large. Is it likely that all these molecules and mechanisms will be crucial for Treg-cell function? There are three broad possibilities.

One, a single, overriding suppressive mechanism is required by all Treg cells Until the entire mechanistic panoply of Treg cells is defined, one cannot completely rule out this possibility. However, this possibility would seem unlikely as none of the molecules and/ or mechanisms that have been defined to date, when blocked or deleted, result in the complete absence of regulatory activity — a consequence that one might predict would result in a ‘Scurfy-like’ phenotype (BOX 1). So, although Treg cells that lack a single molecule, for instance IL-10, IL-35 or granzyme B, exhibit significantly reduced suppressor function, a scurfy phenotype does not ensue. Given that none of the current Treg-cell mechanisms can exclusively claim this distinction, it seems unlikely that any ‘unknown’ molecules or mechanisms could do so either.

Two, multiple, non-redundant mechanisms are required for maximal Treg-cell function In the studies conducted to date, Treg cells that lack various suppressive molecules have been shown to be functionally defective. This favours a scenario where there are multiple mechanisms that can be used by Treg cells but they are non-redundant, with each molecule contributing to the mechanistic whole. At present, this possibility would seem plausible. Indeed, this is supported by the recent analysis of mice possessing a Treg-cell-specific ablation of IL-10 expression, in which enhanced pathology was observed following environmental insult33. One would predict that at some point we should be able to generate knockout mice that lack a particular set of genes which results in a complete loss of Treg-cell activity. For this to be truly non-redundant, this list would probably be restricted and small (2–4 genes).

Three, multiple, redundant mechanisms are required for maximal Treg-cell function With the plethora of regulatory mechanisms described to date and the possibility of more yet to be identified, it is conceivable that there are multiple mechanisms that function redundantly. Such a redundant system would help to mitigate against effector T-cell escape from regulatory control. Also, given the very small size of the Treg-cell population, a sizable arsenal may be required at the height of an effector T-cell attack. Of course, it is possible that a semi-redundant scenario exists.

These possibilities have been discussed from the perspective of there being a single homogeneous Treg-cell population. However, as for helper T cell subsets it remains possible that a few or even many different Treg-cell subsets exist24. Each of these may rely on one or multiple regulatory mechanisms. Several recent studies have provided support for both phenotypic and functional heterogeneity amongst Treg cells. For instance, it has recently been shown that a small sub-population of Treg cells express the chemokine receptor CCR6, which is associated with T cells possessing an effector-memory phenotype102. CCR6+ Treg cells appeared to accumulate in the central nervous systems of mice with experimental autoimmune encephalomyelitis (EAE) suggesting that they may have a prevalent role in controlling responses in inflamed tissues. Heterogeneous expression of HLA-DR has also been suggested to mark different subpopulations of functionally distinct human Treg cells103. Indeed, HLADR positive Treg cells were found to be more suppressive than their DR negative counterparts. One might speculate that their enhanced inhibitory activity is due to DR-mediated ligation of the inhibitory molecule LAG3 expressed by activated effector T cells95,96.

So, if multiple suppressor mechanisms exist, how might these be integrated and used productively by Treg cells in vivo? We would propose the following possible models21. First, a ‘hierarchical’ model in which Treg cells possess many mechanisms that could be used but only one or two that are really crucial and consistently important in a variety of regulatory settings. Second, a ‘contextual’ model where different mechanisms become more or less important depending on the background or context in which the Treg cells reside and the type of target cell that they have to repress. For example, some cell types may be inhibited primarily by cytokines, whereas others are most effectively suppressed through lysis by Treg cells. Alternatively, different mechanisms may be more effective in different tissue compartments or in different disease settings. This notion is supported by the recent analysis of mice in which IL-10 expression was specifically ablated in Treg cells33. Whereas Treg-cell-derived IL-10 was not required for the systemic control of autoimmunity, it did seem to be required from the control of inflammatory events at mucosal interfaces such as the lungs and colon. As a clear picture of the available Treg-cell weaponry emerges, an important challenge will be to determine their relative importance and contribution to Treg-cell function in different disease models.

A hypothesis: effector T cells potentiate Treg-cell function? Most cellular interactions within the immune system are bidirectional, with molecular signals moving in both directions even though the interaction has broader unidirectional intentions (for example, CD4+ T-cell help). However, to date the general perception is that Treg cells suppress and effector T cells capitulate. We hypothesize that this is in fact an incomplete picture and that effector T cells have a very active role in their own functional demise. Three recent observations support this view. First, we have recently examined the molecular signature of activated Treg cells in the presence and absence of effector T cells and were surprised to find that it was strikingly different, with hundreds of genes differentially modulated as a consequence of the presence of effector T cells (C.J.W. and D.A.A.V., unpublished observations). Second, we have shown that Ebi3 and Il12a mRNA are markedly upregulated in Treg cells that were co-cultured with effector T cells, supporting the idea that effector T cells may provide signals which boost IL-35 production in trans58. Third, we found that Treg cells were able to mediate suppression of effector T cells across a permeable membrane when placed in direct contact with effector T cells in the upper chamber of a Transwell™ plate (L.W.C. and D.A.A.V., unpublished observations). Interestingly, this suppression was IL-35 dependent, as Ebi3−/− Treg cells were unable to mediate this ‘long-distance’ suppression. Collectively, these data suggest that it is the ‘induction’, rather than the ‘function’, of Treg-cell suppression that is contact-dependent and that effector T cells have an active role in potentiating Treg-cellmediated suppression. Therefore, we hypothesize that receptor–ligand interactions between the co-cultured CD4+ effector T cells and Treg cells initiate a signalling pathway that leads to enhanced IL-35 secretion and regulatory activity (FIG. 2). While the molecule that mediates this enhanced Treg-cell suppression is unknown, it is possible that IL-2 may serve this function104. Given the contrasting genetic profiles of activated Treg cells in the presence and absence of effector T cells, it seems possible that this interaction may boost the expression of other regulatory proteins. It may well be that effector T cells unwittingly perform the ultimate act of altruism.

Concluding remarks Although significant progress has been made over the last few years in defining the mechanisms that Treg cells use to mediate their suppressive function, there is clearly much that remains to be elucidated and many questions persist. First, are there more undiscovered mechanisms and/ or molecules that mediate Treg-cell suppression? What is clear is that the transcriptional landscape of Treg cells is very different from naive or activated effector T cells. There are literally thousands of genes that are upregulated (or downregulated) in Treg cells compared with effector T cells. Although it seems unlikely that all or many of these will be crucial for Treg-cell function, it is quite possible that a few undiscovered genes might be important. It should be noted that although we are discussing mechanisms here, it is clear that some of these molecules may perform key Treg-cell functions, such as Treg-cell homing and homeostasis, which are likely to indirectly influence their suppressive capacity in vivo but don’t directly contribute to their inhibitory activity. It is also possible that some of these unknown molecules may represent more specific markers for the characterization and isolation of Treg cells, a particularly important issue for the analysis and use of human Treg cells (BOX 2).

Second, which mechanisms are most important? An important but potentially complex challenge will be to determine if a few mechanisms are important in many Treg-cell settings or whether different mechanisms are required in different cellular scenarios. At present it is difficult to assess this objectively as these mechanisms have predominantly been elucidated in different labs using distinct experimental systems and thus none have really been compared in side-by-side experiments. Furthermore, only recently have conditional mutant mice been examined that have a regulatory component specifically deleted in Treg cells33.

It almost goes without saying that although defining the Treg-cell mode of action is of great academic importance, it is also essential in order to develop effective approaches for the clinical manipulation of Treg cells. Given the capacity of Treg cells to control inflammation and autoimmunity, and their implication in blocking effective anti-tumour immunity and preventing sterilizing immunity, it seems probable that a clear understanding of how Treg cells work will present definitive opportunities for therapeutic intervention.

Box 1 Scurfy mice: misplaced mechanistic expectations?

Mice that carry a spontaneous loss-of-function mutation (known as Scurfy mice) or a deletion of Foxp3 develop a fatal autoimmune-like disease with hyperresponsive CD4+ T cells9,12. More recently Foxp3:diptheria toxin receptor (DTR) knockin mice have allowed for the selective depletion of Treg cells following DT treatment105. These mice have been invaluable for dissecting the role of Foxp3 in Treg-cell function. Given the profound phenotype in these mice, there is a general expectation that genetic disruption of any key Treg-cell inhibitory molecule or mechanism would probably result in a Scurfy-like phenotype. Of course, it is also possible that deletion of a key Treg-cell gene may be more synonymous with DT-mediated Treg-cell depletion where Foxp3 may still serve to prevent expression of proinflammatory cytokines105. Nonetheless, this has lead to the notion that if mutant mice don’t have a Scurfy-like or a Treg-cell-depleted phenotype, then the disrupted gene probably isn’t important for Treg-cell function. This may not necessarily be correct. Indeed, it is possible that no mouse lacking a Treg-cell inhibitory effector molecule will ever be generated that develops a profound, spontaneous autoimmune disease21. It should be noted that mutant mice that are Helicobacter spp. and/or Citrobacter rodentium positive may have an exacerbated phenotype, as several studies have shown that opportunistic enteric bacteria can significant exacerbate gut pathology4. Ultimately, the occurrence of disease in knockout mice will depend on whether Treg cells rely on a single or multiple suppressive mechanisms. Given the number of genes induced or modulated by FOXP3, it is probable that a programme of intrinsic and extrinsic regulation is induced that involves multiple proteins9,13. Therefore, it would not be surprising if deletion of a single molecule does not provoke the profound Scurfy-like phenotype seen in mice that lack Foxp3.

Box 2. Treg-cell markers

Identifying discriminatory cell surface markers for the characterization and isolation of Treg cells has always been a critical goal. Although excellent markers exist for murine Treg cells, this goal has remained elusive for human Treg cells. Traditionally, murine and human Treg cells have been characterized as CD4+CD25+ (also known as interleukin-2 receptor α (IL-2Rα)). Indeed, murine Treg cells can be effectively isolated based on staining for CD4+CD25+CD45RBlow expression. However, the purity of isolated human Treg cells has always been an issue because T cells up-regulate CD25 upon activation106. Indeed, during the influenza or allergy season a substantial proportion of human CD4+ T cells can express CD25. Although the identification of forkhead box P3 (Foxp3) as a key regulator of Treg-cell development and function has facilitated their identification in the mouse8, many activated (non-regulatory) human T cells express FOXP3, precluding it as a useful marker for human Treg cells16-20. Consequently, the search for Treg-cell-specific cellsurface markers, particularly in humans, has continued in earnest with a growing number of candidates proposed (reviewed by Zhao and colleagues107). For instance, it was shown that the expression of CD127 (also known as IL-7R) is down-regulated on Treg cells and that this could be used to increase the purity of human Treg-cell isolation. Indeed, there is a 90% correlation between CD4+CD25+CD127low T cells and FOXP3 expression108, 109. In addition, it was recently found that Treg cells expressed a higher level of folate receptor 4 (FR4) compared with activated effector T cells110. It is also important to recognize that Treg cells, like their T helper cell counterparts, may be heterogeneous and thus a collection of cell surface markers could facilitate their isolation and functional characterization. Indeed, such heterogeneity has recently been described based on differential expression of HLA-DR or CCR6102,103. However, the general use of both markers remains to be fully established so it is quite probable that the search for better Treg-cell markers will continue for some time.

Box 3 Induced or adaptive Treg cells: development and mode of action

Naturally occurring FOXP3+CD4+CD25+ Treg cells develop in the thymus and display a diverse T-cell receptor (TCR) repertoire that is specific for self-antigens111,112. However, Treg cells can also be ‘induced’, ‘adapted’ or ‘converted’ from effector T cells during inflammatory processes in peripheral tissues, or experimentally generated as a possible therapeutic29,113,114. For instance, T regulatory 1 cells (Tr1) and T helper 3 cells (Th3) can be generated experimentally by, and mediate their suppressive activity through interleukin-10 (IL-10) and transforming growth factor-β (TGFβ), respectively114,115. Typically, these regulatory populations do not express FOXP3. In vivo, it has recently been suggested that stimulation of mouse effector T cells by CD103+ dendritic cells (DCs) in the presence of TGFβ and retinoic acid induces the generation of Foxp3+ T cells in the gutassociated lymphoid tissue (GALT)116-121. Furthermore, Treg cells can be preferentially induced in the periphery by exposure to αVβ8-integrin-expressing DCs122 or suppressor of cytokine signalling 3 (Socs3) −/− DCs123. Interestingly, independent of its role in generating induced Treg cells, TGFβ may also have an important role in helping to maintain Foxp3 expression in natural Treg cells124, a process that can be blocked by IL-4 or interferon-γ (IFNγ) 125. In contrast to mouse T cells, FOXP3 induction by TCR stimulation in the presence of TGFβ in human T cells does not confer a regulatory phenotype20. The mechanism of action of adaptive Treg cells may not necessarily be restricted to suppressive cytokines. Indeed, human adaptive Treg cells (CD4+CD45RA+ T cells stimulated with CD3- and CD46-specific antibodies) have also been shown to express granzyme B and killing target cells in a perforin-dependent manner126. Treg cells often have a restricted specificity for particular cell types, tumours or foreign antigens127. Therefore, induced Treg cells may be ideally suited to respond to infectious agents. This may also be of particular importance in the GALT and in the tumour microenvironment where TGFβ drives the conversion of induced Treg cells118,128. A significant challenge in deciphering data from in vivo experiments is to assess the contribution of natural Treg cells versus induced Treg cells, and to determine whether inhibitory molecules, such as IL-10 or TGFβ, were derived from the former or the latter (or elsewhere).

 

 

Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers.

Keisuke Kataoka, Yuichi Shiraishi, Yohei Takeda, Seiji Sakata, et al.
Nature may 23,2016; http://dx.doi.org:/10.1038/nature18294

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development1, 2, 3, 4, 5, 6. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma6, 7, 8, 9, 10. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3′ region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3′-untranslated region (UTR). Disruption of the Pd-l1 3′-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3′-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

 

Viruses are a dominant driver of protein adaptation in mammals.

David Enard, Le Cai, Carina Gwennap and Dmitri A Petrov.
eLife May 16, 2016; 5:e12469. http://dx.doi.org/10.7554/eLife.12469

Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes.

 

Purdue scientists use adaptors to advance CAR-T therapy

by Oliver Worsley | May 25, 2016

http://www.fiercebiotech.com/research/purdue-scientists-use-adaptors-to-advance-car-t-therapy

Chimeric antigen receptor (CAR) T cells, developed in the 1990s, are a genetically engineered type of T cell that can target a specific cancer. Now, scientists at Purdue University say they’ve made improvements in this strategy–overcoming the several limitations of traditional CAR-T therapy.

Purdue professor of chemistry Philip Low and his team presented their findings at the American Association for Cancer Research meeting in New Orleans last month.

T cells are a type of immune cell that recognizes and clears the body of invading cells or pathogens, like cancer. They are fine-tuned by the immune system in order to specifically target and kill these foreign invaders–but cancer cells may respond by jumping these safety barriers.

CAR-T therapy was therefore proposed and has been recently used for cancer treatment. It has been hailed for its promising remission rates after early stage clinical trials for acute lymphoblastic leukemia.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said in a university news release. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

They did this by teaming up with Endocyte ($ECYT) scientist Haiyan Chu and designing CAR T cells that require activation by a small molecule adaptor before proceeding. In this way, they can carefully control the amount of active CAR T cells in the circulation.

So far, they have only tried the novel therapy in animal models, but when they tested it in mice they observed antitumor activity only when both the CAR T cells and the correct adaptor molecules were present.

Low believes it will allow clinicians to target multiple cancer subtypes at once. “Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said in the release. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

– here’s the release

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Read More: CAR-T   Cancer

 

Purdue research may expand engineered T-cell cancer treatment

PURDUE UNIVERSITY

http://www.eurekalert.org/multimedia/pub/116141.php

A graphic depicting the activation and inactivation of CAR T cells through a small molecule adaptor is shown. Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery, and graduate student Yong Gu Lee led a team that designed new engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The system has the potential to control the engineered cells to overcome existing limitations in CAR T-cell therapy. CREDIT Purdue University image courtesy of Yong Gu Lee

Purdue University researchers may have figured out a way to call off a cancer cell assassin that sometimes goes rogue and assign it a larger tumor-specific “hit list.”

T cells are the immune system’s natural defense against cancer and other harmful entities in the human body. However, the cells must be activated and taught by the immune system to recognize cancer cells in order to seek out and destroy them. Unfortunately, many types of cancer manage to thwart this process.

 

In the 1990s scientists found a way to genetically engineer T cells to recognize a specific cancer. These engineered T cells, called chimeric antigen receptor, or CAR, T cells, have been recently used as treatment for cancer, said Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery who led the work.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

Low and Purdue graduate student Yong Gu Lee collaborated with Endocyte Inc. scientist Haiyan Chu to design genetically engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The technology has been tested in animal models but no human trials have been performed. A poster presentation describing the work was presented Tuesday (April 19, 2016) at the American Association for Cancer Research annual meeting in New Orleans.

“While the traditional CAR T cells could remain and replicate in the human body for many years, the adaptors we have created are expected to be excreted fairly quickly,” Lee said. “By controlling the level of adaptors in the system, we can control the numbers and potencies of active CAR T cells. Those that aren’t stimulated by an adaptor molecule are blind and do not recognize or target any cells. Eventually, if they remain inactive for a while, they should die and be eliminated from the body.”

A study in mice showed the anti-tumor activity was induced only when both the engineered CAR T cell and the correct adaptor molecules were present.

The system also offers the potential to treat multiple cancer subtypes at once, Low said.

“Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

The adaptor molecule serves as a bridge between the CAR T-cell and the cancer cell. It is made with a yellow dye called fluorescein isothiocyanate on one end, to which the engineered CAR T cells have been designed to bind, and a cancer-targeting molecule on the other.

Low’s research has focused on the design and synthesis of technologies for targeted delivery of therapeutic and imaging agents to treat cancer, inflammatory and autoimmune diseases, and infectious diseases.

He has developed molecules that target folate-receptors and prostate-specific membrane antigen on the surfaces of cancer cells. Approximately 85 percent of ovarian cancers; 80 percent of endometrial and lung cancers; and 50 percent of breast, kidney and colon cancers express folate receptors on their cellular surfaces. Prostate-specific membrane antigen receptors are found on nearly 90 percent of all prostate cancers. Other tumor-specific ligands developed by Low’s lab can target each of the other major human cancers, he said.

Each CAR T cell has thousands of receptors on its surface to which an adaptor molecule can bind. One CAR T cell could have a variety of adaptor molecules bound to its surface and the cancer cell it targets will depend on which of those adaptors first encounters a targeted cancer cell. Once the CAR T cell binds to a cancer cell, it begins the process of destroying it. When that process is complete, the CAR T cell is released and can bind to a new cancer cell, he said.

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

In addition to Low, Chu and Lee, members of the research group include Purdue postdoctoral research associates at the time of the study Srinivasarao Tenneti and Ananda Kumar Kanduluru.

Drug discovery is one of the priorities within Purdue Moves, an initiative designed to broaden the university’s global impact and enhance educational opportunities for its students. All of the moves fall into four broad categories: science, technology, engineering and math (STEM) leadership; world-changing research; transformative education; and affordability and accessibility.

The Purdue University Center for Drug Discovery supports more than 100 faculty in six colleges with research focused on several major disease categories: cancer; diabetes, obesity and cardiovascular; immune and infectious disease; and neurological disorders and trauma.

The center and drug discovery initiative builds upon Purdue’s strengths along all points of the drug discovery pipeline, including 14 core units to provide shared resources for analysis, screening, synthesis and testing of potential therapeutic compounds.

With more than 44 Purdue-developed compounds at various stages of preclinical development, and 16 in human clinical trials, Purdue is among the most productive universities in the world of drug discovery.

The center also is aligned with the university’s recently announced $250 million investment in the life sciences.

Endocyte Inc., a Purdue Research Park-based company that develops receptor-targeted therapeutics for the treatment of cancer and autoimmune diseases, funded the study, holds exclusive rights to the technology and assisted Purdue researchers in the development of the technology. Low is a founder and chief science officer of Endocyte Inc. and serves on the Endocyte board of directors.

AACR press release: http://www.aacr.org/Newsroom/Pages/News-Release-Detail.aspx?ItemID=874#.VxZFs2N8V0c

Endocyte press release: http://investor.endocyte.com/releasedetail.cfm?ReleaseID=965753

ABSTRACT

A Universal Remedy for CAR T Cell Limitations

Yong Gu Lee, Haiyan Chu, Srinivasarao Tenneti, Ananda Kumar Kanduluru, Philip S. Low

Chimeric antigen receptor (CAR) T cells show significant potential for treating cancer due to their tumor-specific activation and ability to focus their killing activity on cells that express a tumor antigen. Unfortunately, this promising therapeutic technology is still limited by: (1) an inability to control the rate of cytokine release and tumor lysis; (2) the absence of an “off switch” that can terminate cytotoxic activity when tumor eradication is complete; (3) a failure to eliminate tumor cells that do not express the targeted antigen; and (4) a requirement to generate a different CAR T cell for each unique tumor antigen. In order to address these limitations, we have exploited a low molecular weight bi-specific adaptor molecule that must bridge between the CAR T cell and its targeted tumor cell by simultaneously binding to the chimeric antigen receptor on the CAR T cell and the unique antigen on the tumor. Using this bispecific adaptor, one can control CAR T cell cytotoxicity by adjusting the concentration and rate of administration of the adaptor. Because the half life of the adapter is <20 minutes in vivo, termination of CAR T cell killing can be accomplished by cessation of adapter administration. Moreover, when heterogeneous tumors containing cells that express orthogonal antigens must be treated, the same CAR T cell can be targeted to multiple antigens by attachment of the same CAR ligand to the appropriate selection of tumor-specific ligands. Finally, when the targeted tumor antigen is also expressed at low levels on normal cells, tumor specificity can be achieved by adjusting the affinity of the tumor-specific ligand to enable CAR T cell engagement only when a highly multivalent interaction is possible. To experimentally demonstrate the aforementioned benefits of using low molecular weight bispecific adaptors, CAR T cells were constructed by fusing an anti-fluorescein isothiocyanate (FITC) scFv to a CD3 zeta chain containing the intracellular domain of CD137 (i.e. CAR4-1BBZ T cells). Then, to enable their tumor-specific cytotoxicity, a bispecific adaptor molecule comprised of fluorescein linked to a small organic ligand with high affinity and specificity for a tumor-specific antigen (FITC-SMC) was synthesized. For these studies, the tumor-specific ligands were: i) folate for recognition of the folate receptor that is over-expressed on ~1/3 of human cancers, ii) DUPA for binding to prostate specific membrane antigen that is over-expressed on prostate cancers, and iii) NK-1R ligand that is over-expressed on neuroendocrine tumors. The ability of the same clone of CAR4-1BBZ T cells to eliminate tumors expressing each of the above antigens was then demonstrated by administration of the desired FITC-SMC to mice injected with the CAR4-1BBZ T cells. Our data show that anti-tumor activity: i) is only induced when both CAR4-1BBZ T cells and the correct antigen-specific FITC-SMC are present, ii) anti-tumor activity and toxicity can be sensitively controlled by adjusting the dosing of FITC-SMC, and iii) treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired FITC-SMCs. Taken together, these data show that many of the limitations of CAR T cell technology can be addressed by use of a bi-specific adaptor molecule to mediate tumor cell recognition and killing.

 

 

CTLA-4 found in dendritic cells suggests New cancer treatment possibilities

Matthew Halpert, et al. Dendritic Cell Secreted CTLA-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Stem Cells and Development, 2016; http://dx.doi.org:/10.1089/scd.2016.0009

Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the “general” leading T cells “soldiers” to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.

  Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Halpert MM1, Konduri V1, Liang D1, Chen Y1, Wing JB2, Paust S3,4, Levitt JM1,5, Decker WK1,6.  Stem Cells Dev. 2016 May 15;25(10):774-87. doi: 10.1089/scd.2016.0009. Epub 2016 May 2.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

 

Non-invasive strategy to guide personalized cancer immunotherapy

Cancer immunotherapy is the rising hope to offer ultimate solutions for cancer. Neoantigens, derived from products of mutated genes in tumor cells, are found to be closely related to the efficacy of cancer immunotherapies. A non-invasive approach to identify unique, patient-specific neoantigens has been advanced by Dr. Steven Rosenberg’s group. A recent article published in Nature Medicine reported that a small population of circulating CD8+PD-1+ tumor-reactive T lymphocytes can be used to identify neoantigens, in addition to tumor-infiltrating T cells. The study paves the way for designing personalized cancer immunotherapy with a novel non-invasive approach.

Gros, A. et al.
Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.
Nat. Med. (2016)   http://dx. doi.org:/10.1038/nm.4501

Detection of lymphocytes that target tumor-specific mutant neoantigens-derived from products encoded by mutated genes in the tumor-is mostly limited to tumor-resident lymphocytes, but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(-), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8+ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8+ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8+lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8+ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8+PD-1+ compared with CD8+PD-1 TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8+ and the CD8+PD-1+ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8+ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

Cancer immunotherapy has experienced major progress in the last decade. Adoptive transfer of ex vivo–expanded tumor-infiltrating lymphocytes (TILs) can cause substantial regression of metastatic melanoma (1, 2). Blockade of the interaction of cytotoxic T lymphocyte antigen 4 (CTLA-4; also known as CD152) or programmed cell death 1 receptor (PD-1; also known as CD279) with their ligands using blocking antibodies alone or in combination have been shown to unleash an otherwise-ineffective immune response against melanoma (37), renal cell carcinoma (3), and non–small cell lung cancer (3). The antitumor responses observed in these clinical trials support the presence of naturally occurring tumor-reactive CD8+ T cells and their immunotherapeutic potential. In the particular case of TIL therapy, persistence of transferred tumor-specific T cell clones is associated with tumor regression (8). Moreover, retrospective clinical studies have shown an association of autologous tumor recognition by TILs and clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. However, the identification of the diverse repertoire of tumor-reactive cells limits the ability to study these cells, enhance clinical efficacy, and extend this therapy to other malignancies.

Melanoma TILs represent a heterogeneous population that can target a variety of antigens, including melanocyte differentiation antigens, cancer germline antigens, self-antigens overexpressed by the tumor, and mutated tumor neoantigens (11). The latter appear to be of critical importance for the antitumor responses observed after transfer of TILs, given the substantial regression of metastatic melanoma in up to 72% of patients in phase 2 clinical trials, in the absence of any autoimmune side effects in the great majority of patients (2). This contrasts with the modest antitumor activity but high prevalence of severe autoimmune manifestations observed after transfer of peripheral blood gene-engineered T cells expressing TCRs targeting shared melanocyte differentiation antigens MART1 and gp100 (12,13). Furthermore, T cells targeting mutated neoepitopes are not subject to negative selection in the thymus and may constitute the predominant naturally occurring tumor-reactive population in cancer patients. In support of this notion, a recent study reported the frequent detection and dominance of T cell populations targeting mutated epitopes in melanoma-derived TILs (14). Conversely, T cells targeting shared melanocyte differentiation antigens and cancer germline antigens in bulk melanoma TILs were represented at a strikingly low frequency (15). These findings have shifted our interest from the more accessible and commonly studied T cells targeting melanocyte differentiation antigens to T cells targeting unique patient-specific mutations. However, the often rare availability of autologous tumor cell lines necessary to study these reactivities, and the hurdles associated with the identification of the unique mutations targeted, have thus far hindered immunobiological studies of these T cell populations in the tumor.

Naturally occurring tumor-reactive cells are exposed to their antigen at the tumor site. Thus, the immunobiological characterization of T cells infiltrating tumors represents a unique opportunity to study their function and to identify the patient-specific repertoire of tumor-reactive cells. TCR stimulation triggers simultaneous upregulation of both costimulatory and coinhibitory receptors, which can either promote or inhibit T cell activation and function. Expression of the inhibitory receptors PD-1, CTLA-4, lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) is regulated in response to activation and throughout differentiation (16, 17). Chronic antigen stimulation has been shown to induce coexpression of inhibitory receptors and is associated with T cell hyporesponsiveness, termed exhaustion (18). Exhaustion in response to persistent exposure to antigen was first delineated in a murine model of chronic lymphocytic choriomeningitis virus (19), but has been observed in multiple human chronic viral infections (2022) as well as in tumor-reactive MART1-specific TILs (23, 24), and has provided the rationale for restoring immune function using immune checkpoint blockade. Conversely, 4-1BB (also known as CD137) is a costimulatory member of the TNF receptor family that has emerged as an important mediator of survival and proliferation, particularly in CD8+ T cells (2527). 4-1BB is transiently expressed upon TCR stimulation, and its expression has been used to enrich for antigen-specific T cells in response to acute antigen stimulation (28). However, expression of this marker has not been extensively explored in CD8+ lymphocytes infiltrating human tumors. In addition to changes in the expression of cosignaling receptors on the surface of T cells, antigen-specific stimulation typically results in clonal expansion. TCR sequence immunoprofiling can be used to monitor T cell responses to a given immune challenge even without a priori knowledge of the specific epitope targeted, through determination of the abundance of specific clonotypes (29, 30). However, there is limited knowledge regarding the TCR repertoire and the frequency of tumor-reactive clonotypes infiltrating human tumors.

We hypothesized that the assessment of unique phenotypic traits expressed by CD8+ TILs and TCR β chain (TCRβ; encoded by TRB) clonotypic immunoprofiling of lymphocytes infiltrating the tumor could provide a powerful platform to study antitumor T cell responses and evaluated their usefulness in identifying the diverse repertoire of tumor-reactive cells. Despite the accepted negative regulatory role of PD-1 in T cells, our findings establish that expression of PD-1 on CD8+ melanoma TILs accurately identifies the repertoire of clonally expanded tumor-reactive, mutation-specific lymphocytes and suggest that cells derived from this population play a critical role in tumor regression after TIL administration.

PD-1 was initially described to be expressed on a T cell hybridoma undergoing cell death (37). Its negative effect on T cell responses was first delineated in PD-1 knockout mice (38, 39). Since then, PD-1 expression and coexpression of other inhibitory receptors such as CTLA-4, TIM-3, BTLA, CD160, LAG-3, and 2B4 have become a hallmark of chronically stimulated T cells during chronic infection or in the tumor microenvironment. This altered phenotype, and the interaction of these receptors with their corresponding ligands on target cells, is associated with impaired proliferation and effector function frequently referred to as exhaustion (18, 24, 40). Expression of PD-1 in patients with chronic viral infections correlates with disease progression (22, 41). Additionally, CD8+ lymphocytes targeting melanoma differentiation antigens in the tumor express PD-1, CTLA-4, TIM-3, and LAG-3 and exhibit impaired IFN-γ and IL-2 secretion (23, 24), supporting a negative regulatory role of PD-1 and inhibitory receptors in naturally occurring T cell responses to cancer and providing a rationale for the treatment of cancer with immune checkpoint inhibitors.

In the present study, we found that expression of PD-1 on CD8+ melanoma TILs captured the diverse repertoire of clonally expanded tumor-reactive lymphocytes. TCRβ sequencing revealed that tumor-reactive and mutation-specific clonotypes were highly expanded in the CD8+ population and preferentially expanded in the PD-1+ population. This is consistent with the TCR stimulation-driven expression of this receptor on T cells (42). The inhibitory receptors TIM-3 and LAG-3 and the costimulatory receptor 4-1BB were also expressed on CD8+PD-1+ TILs and could also be used to enrich for tumor-reactive cells. PD-1 was consistently expressed at a higher frequency and was found to be more comprehensive at identifying the diverse repertoire of tumor-reactive cells infiltrating melanoma tumors, although the less frequent PD-1/TIM-3+ and PD-1/LAG-3+ subpopulations could also represent tumor-reactive cells (Supplemental Figure 4 and Supplemental Table 6). Additionally, previous studies from our laboratory showing coexpression of PD-1 and CTLA-4 (23), and our preliminary data supporting coexpression of PD-1 and ICOS (Supplemental Figure 5), suggest that other receptors may also be used to distinguish tumor-reactive cells. Our present results further support immunotherapeutic intervention using immune checkpoint blockade using PD-1, TIM-3, and LAG-3 blocking antibodies or 4-1BB agonistic antibody to restore the function of tumor-reactive lymphocytes, which is currently being actively pursued in the clinic (3, 4, 6, 7, 43). The potential cooperative mechanisms of inhibition of these receptors when engaged with their ligands (44, 45) suggests that the combined targeting of different inhibitory receptors can further enhance antitumor efficacy, as already shown with the combination of anti–PD-1 and anti–CTLA-4 (5). Our present results demonstrate that PD-1 identifies the clonally expanded CD8+ tumor-reactive population and suggest that expression of PD-1 on CD8+TILs could function as a potential predictive biomarker of antitumor efficacy using immune checkpoint inhibitors.

Naturally occurring tumor-reactive cells play a pivotal role in mediating antitumor responses after TIL transfer. Currently, expansion of TILs for patient treatment involves nonspecific growth of TILs from tumor fragments in IL-2, and the diversity and frequency of antitumor T cells present in the final T cell product used for treatment remains largely uncharacterized. Prospective clinical studies have reported that in vitro recognition of autologous tumor by TILs is associated with a higher probability of clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. This is consistent with the idea that both tumor-reactive and non–tumor-reactive cells may compete for cytokines in vivo, especially in the absence of vaccination. However, the isolation of the patient-specific repertoire of tumor-reactive cells is not possible with current technologies (14, 28, 4650). Our findings established that expression of PD-1, TIM-3, LAG-3, and 4-1BB in CD8+ TILs can be used to enrich for tumor-reactive cells, regardless of the specific antigen targeted. One potential concern with isolating T cells expressing inhibitory receptors for therapy is that these cells may be exhausted or functionally impaired (23, 24, 44, 51, 52). However, we found that PD-1+, TIM-3+, and LAG-3+ CD8+ cells expanded in IL-2 were capable of secreting IFN-γ and lyse tumor in vitro. This supports the notion that immune dysfunction associated with coexpression of inhibitory receptors on CD8+ TILs can be reversed (21, 41, 51, 53), and may enable the reproducible enrichment of tumor-reactive cells for patient treatment. Notably, in a preliminary experiment (n= 8 nonresponders; 14 responders), there was no association between the frequency of expression of any of the markers studied in the CD8+ TILs in the fresh tumor and the clinical response to TILs derived from these tumor samples. However, the fresh tumors included in this study belonged to patients treated in several TIL protocols over the course of 10 years, and TILs were generated from these tumors using different methods, which makes these data difficult to interpret. In addition, the frequency of cells initially expressing PD-1 in the tumor may not reflect the frequency of the PD-1 derived cells in the infusion bag. For example, a low frequency of PD-1+ cells may be highly enriched during the process of TIL culture as a result of the presence of tumor cells. Although in vivo antitumor activity of tumor-isolated TILs based on PD-1 expression requires testing in a clinical trial, the observation that the overwhelming majority of tumor-reactive cells were derived from cells expressing PD-1 suggests that cells expressing PD-1 and inhibitory receptors in the tumor play a critical role in tumor regression after TIL administration.

The functional implications of selecting PD-1–, LAG-3–, TIM-3–, or 4-1BB–expressing T cells to enrich for tumor-reactive cells for patient treatment remain unclear. Although previous studies have reported differential expression of PD-1, LAG-3, and TIM-3 throughout differentiation (17), or preferential expression of TIM-3 in IFN-γ–secreting cells (54), our preliminary results have failed to show consistent phenotypic or functional differences between PD-1+, LAG-3+, TIM-3+, and 4-1BB+ selected TILs, including cytokine secretion, proliferation, and susceptibility to apoptosis (data not shown). We found that PD-1 expression was almost completely lost in the PD-1+ derived populations upon in vitro culture in IL-2. Conversely, TIM-3 and LAG-3 expression increased in the TIM-3 and LAG-3 populations after expansion. Overall, there were no differences in the expression of PD-1, TIM-3, or LAG-3 between any the populations after expansion. Thus, in agreement with previous reports (55, 56), we conclude that expansion in IL-2 alters the expression of these markers and compromises the potential use of inhibitory receptors to select for tumor-reactive cells after in vitro expansion. Recent work in animal models suggests that chronic antigen stimulation (5759) or a tolerizing microenvironment (60) may lead to permanent epigenetic changes in T cells, raising the possibility that the restoration of function observed in previously exhausted or tolerized cells in presence of cytokines may only be transient. These results have not yet been corroborated in human tumor-specific cells. However, given that the overwhelming majority of tumor-reactive cells appear to derive from cells expressing PD-1 in the tumor, studying permanent versus transient reversion of exhaustion may have important implications for adoptive cell transfer of TILs.

Tumor-reactive cells can also be found infiltrating other tumor malignancies, such as renal cell carcinoma (61) or ovarian (62), cervical (63), or gastrointestinal tract cancers (64), albeit at lower frequencies. Our findings provide alternatives to enrich and study tumor-reactive CD8+ TILs through selection of cells expressing the cell surface receptors PD-1, LAG-3, TIM-3, and 4-1BB, a hypothesis that we are actively investigating. Additionally, our present findings showed that the frequency of a specific clonotype in the CD8+ and PD-1+ populations can be used to predict its ability to recognize tumor and isolate tumor-specific TCRs, thus providing means to overcome potential irreversible functional impairments of TILs (52).

2 reports with opposing results have generated controversy regarding which may be the optimal marker for the identification of the tumor-reactive repertoire, PD-1 or 4-1BB. In one report studying PD-1 expression in the tumor, the authors showed promising although inconsistent ability to enrich for shared melanoma-reactive cells (55). In a more recent article studying the role of 4-1BB in fresh ovarian TILs, Ye et al. concluded that expression of 4-1BB, but not PD-1, on lymphocytes defines the population of tumor-reactive cells in the tumor (65). The results of Ye et al. appear to contradict our present findings, showing that expression of PD-1 rather than 4-1BB more comprehensively identifies the repertoire of tumor-reactive cells in the tumor. However, these inconsistencies can be explained by different experimental approaches undertaken to study the immunobiology of TILs. First, Ye et al. found that expression of 4-1BB in fresh ovarian TILs and tumor-associated lymphocytes was low, and thus exposed the tumor to IL-7 and IL-15 (65). In the 1 patient sample in which the authors enriched for tumor-reactive cells from fresh ovarian TILs or tumor-associated lymphocytes exposed to IL-7 and IL-15, expression of 4-1BB was dependent on in vitro activation, but no longer represented the natural expression of 4-1BB in the fresh tumor. Second, with the exception of the 1 experiment described above, the enrichment experiments reported were carried out with melanoma or ovarian TIL lines expanded in IL-2 and cocultured with tumor cell lines in vitro. It is well known that IL-2 can change the activation status and also the expression of inhibitory receptors on T cells (data not shown and ref. 56). Thus, the experiment comparing expression of PD-1 and 4-1BB performed by Ye et al. (65) addressed the significance of these receptors after in vitro coculture of a highly activated melanoma TIL line with a tumor cell line, rather than the role of PD-1 and 4-1BB expression in CD8+ lymphocytes in the fresh tumor. Finally, both Inozume et al. and Ye et al. used matched HLA-A2 cell lines to assess tumor reactivity (55, 65). However, the use of HLA-matched tumor cell lines does not enable the assessment of reactivities against unique mutations that are present only in the autologous tumor cell line. In our current study, we used fresh melanoma tumors for all our experiments, and these were rested in the absence of cytokines to preserve the phenotype of TILs. Moreover, we used autologous tumor cell lines to assess tumor recognition. We believe that our experimental approach overcomes the limitations described above, enabling us to conclude that tumor-reactive cells can be detected in both the PD-1+/4-1BB+ and PD-1+/4-1BB CD8+ TIL populations.

In summary, expression of PD-1 in CD8+ TILs in the fresh tumor identified and selected for the diverse patient-specific repertoire of tumor-reactive cells, including mutation-specific cells. In addition, analysis of the CD8+ TIL TCRβ repertoire in 2 melanomas showed that the frequency of a specific TCRβ clonotype in the CD8+ and PD-1+ populations could be used to predict its ability to recognize the autologous tumor. The use of inhibitory receptors and the frequency of individual TCRs to prospectively identify and select the diverse repertoire of tumor-reactive cells holds promise for the personalized treatment of cancer with T cell therapies, but may also facilitate the dissection and understanding of the immune response in human cancer patients.

Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?

Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.

Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:

  • LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
  • TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
  • VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice

In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets

  • OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
  • 4-1BB – Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
  • Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.

Sharma P and Allison JP.
Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential.
Cell. April 2015;161:205-214

 

Targeting single immune-checkpoint proteins has proven to be clinically effective at treating specific tumor types; can targeting two different proteins synergize effects?

Despite the success of targeting immune-checkpoint proteins, such as CTLA-4, PD-1, LAG-3, TIM-3 among others, percentages of patient response vary and rarely exceed 50%. It is highly tempting to speculate a strategy of dual-targeting of these checkpoint proteins. A recent presentation at the Keystone Symposium for Tumor Immunology: Multidisciplinary Science Driving Combination Therapy detailed findings of dual-targeting two immune-checkpoint proteins in mouse tumor models. Their key findings are summarized below:

  • Dual-targeting PD-1 and LAG-3 demonstrates superior efficacy over blocking either target alone
  • In addition to previous reported data on superior dual-targeting efficacy against fibrosarcoma (Sa1N) and colorectal adenosarcoma (MC38) tumor types1, anti-tumor activity against myeloma (SC J558L) and B-cell lymphoma (A20) hematological tumor types were also reported to be effacious.2

These exciting pre-clinical findings may result in further exploration of dual-targeting antibodies in the clinic, either as combination of existing antibody therapies, or as a new bi-specific antibody therapeutic.

Camelid single domain antibodies are a novel bi-specific antibody platform that may be used to develop a new generation of dual-targeting antibodies against multiple immune-checkpoint proteins.

1Woo SR et al.
Immune Inhibitory Molecules Lag-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape.
Cancer Res. Feb 2012. 15(4):917-927.

2Lewis KE et al.
Dual Targeting of PD-1 and LAG-3 demonstrates Superior Efficacy to Blocking Either PD-1 or LAG-3 Alone in Pre-Clinical Solid and Hematological Tumor Models.
Abstract J7 2033. Keystone Symposia: Tumor Immunology: Multidisciplinary Science Driving Combination Therapy. February 8-13, 2015. Banff, Alberta, Canada.

 

New insight behind the success of fighting cancer by targeting immune checkpoint proteins

Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.

In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.

In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.

Gubin MM. et al.
Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.
Nature. Nov 2014. 515:577-584

 

 

Myeloid-derived-suppressor cells as regulators of the immune system
Dmitry I. Gabrilovich and Srinivas Nagaraj  Nat Rev Immunol. 2009 March ; 9(3): 162–174. http://dx.doi.org:/10.1038/nri2506

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expands during cancer, inflammation and infection, and that has a remarkable ability to suppress T-cell responses. These cells constitute a unique component of the immune system that regulates immune responses in healthy individuals and in the context of various diseases. In this Review, we discuss the origin, mechanisms of expansion and suppressive functions of MDSCs, as well as the potential to target these cells for therapeutic benefit.

The first observations of suppressive myeloid cells were described more than 20 years ago in patients with cancer1-3. However, the functional importance of these cells in the immune system has only recently been appreciated due to accumulating evidence that has demonstrated their contribution to the negative regulation of immune responses during cancer and other diseases. It is now becoming increasingly clear that this activity is contained within a population known as myeloid-derived suppressor cells (MDSCs). Features common to all MDSCs are their myeloid origin, immature state and a remarkable ability to suppress T-cell responses (Box 1). In addition to their suppressive effects on adaptive immune responses, MDSCs have also been reported to regulate innate immune responses by modulating the cytokine production of macrophages4. Non-immunological functions of MDSC have also been described, such as the promotion of tumour angiogenesis, tumour-cell invasion and metastasis. However, as a discussion of these aspects of MDSC biology is beyond the scope of this article, the reader is referred to another recent Review on this topic5.

MDSCs represent an intrinsic part of the myeloid-cell lineage and are a heterogeneous population that is comprised of myeloid-cell progenitors and precursors of myeloid cells. In healthy individuals, immature myeloid cells (IMCs) generated in bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). In pathological conditions such as cancer, various infectious diseases, sepsis, trauma, bone marrow transplantation or some autoimmune disorders, a partial block in the differentiation of IMCs into mature myeloid cells results in an expansion of this population. Importantly, the activation of these cells in a pathological context results in the upregulated expression of immune suppressive factors such as arginase (encoded by ARG1) and inducible nitric oxide synthase (iNOS; also known as NOS2) and an increase in the production of NO (nitric oxide) and reactive oxygen species (ROS). Together, this results in the expansion of an IMC population that has immune suppressive activity; these cells are now collectively known as MDSCs. In this

Origin and subsets of MDSCs It is important to note that MDSCs that are expanded in pathological conditions (see later) are not a defined subset of myeloid cells but rather a heterogeneous population of activated IMCs that have been prevented from fully differentiating into mature cells. MDSCs lack the expression of cell-surface markers that are specific for monocytes, macrophages or DCs and are comprised of a mixture of myeloid cells with granulocytic and monocytic morphology6. Early studies showed that 1–5% of MDSCs are able to form myeloid-cell colonies7-9 and that about one third of this population can differentiate into mature macrophages and DCs in the presence of appropriate cytokines in vitro and in vivo7-9. In mice, MDSCs are characterized by the co-expression of the myeloid lineage differentiation antigen Gr1 (also known as Ly6G) and CD11b (also known as αM-integrin)10. Normal bone marrow contains 20–30% of cells with this phenotype, but these cells make up only a small proportion (2–4%) of spleen cells and are absent from the lymph nodes in mice (Fig. 1). In humans, MDSCs are most commonly defined as CD14-CD11b+ cells or, more narrowly, as cells that express the common myeloid marker CD33 but lack the expression of markers of mature myeloid and lymphoid cells and the MHC-class-II molecule HLA-DR11, 12. MDSCs have also been identified within a CD15+ population in human peripheral blood13. In healthy individuals, immature myeloid cells with described above phenotype comprise ∼0.5% of peripheral blood mononuclear cells.
Recently, the morphological heterogeneity of these cells has been defined more precisely in part based on their expression of Gr1. Notably, Gr1-specific antibodies bind to both Ly6G and Ly6C,  which are encoded by separate genes. However, these epitopes are recognized by different antibodies specific for each individual epitopes: anti-Ly6C and anti-Ly6G. Granulocytic MDSCs have a CD11b+Ly6G+Ly6Clow phenotype, whereas MDSCs with monocytic morphology are CD11b+Ly6G-Ly6Chigh 6,14. Importantly, evidence indicates that these two subpopulations may have different functions in cancer and infectious and autoimmune diseases15-17. During the analysis of ten different experimental tumour models, we found that both of these subsets of MDSCs were expanded. In most cases, however, the expansion of the granulocytic MDSC population was much greater than that of the monocytic subset6 and, interestingly, the two subpopulations used different mechanisms to suppress Tcell function (see later). In addition, the ability to differentiate into mature DCs and macrophages in vitro has been shown to be restricted to monocytic MDSCs6.
In recent years, several other surface molecules have been used to identify additional subsets of suppressive MDSCs, including CD80 (also known as B7.1)18, CD115 (the macrophage colony-stimulating factor receptor)19, 20 and CD124 (the IL-4 receptor α-chain)20. In our own studies, we observed that many MDSCs in tumour-bearing mice co-express CD115 and CD1246; however, direct comparison of MDSCs from tumour-bearing mice and Gr1+CD11b+ cells from naive mice showed that they expressed similar levels of CD115 and CD124. In addition, sorted CD115+ or CD124+ MDSCs from EL-4 tumour-bearing mice had the same ability to suppress T-cell proliferation on a per cell basis as did CD115- or CD124-MDSCs. This suggests that, although these molecules are associated with MDSCs, they might not be involved in the immunosuppressive function of these cells in all tumour models.

Overall, current data suggest that MDSCs are not a defined subset of cells but rather a group of phenotypically heterogeneous myeloid cells that have common biological activity.

MDSCs in pathological conditions MDSCs were first characterized in tumour-bearing mice or in patients with cancer. Inoculation of mice with transplantable tumour cells, or the spontaneous development of tumours in transgenic mice with tissue-restricted oncogene expression, results in a marked systemic expansion of these cells (Fig. 1 and Table 1). In addition, up to a tenfold increase in MDSC numbers was detected in the blood of patients with different types of cancer11, 12, 21, 22. In many mouse tumour models, as many as 20–40% of nucleated splenocytes are represented by MDSCs (in contrast to the 2-4% seen in normal mice). In addition, these cells are found in tumour tissues and in the lymph nodes of tumour-bearing mice.
Although initial observations and most of the current information regarding the role of MDSCs in immune responses has come from studies in the cancer field, accumulating evidence has shown that MDSCs also regulate immune responses in bacterial and parasitic infections, acute and chronic inflammation, traumatic stress, surgical sepsis and transplantation. A systemic expansion of both the granulocytic and monocytic subset of MDSCs was observed in mice primed with Mycobacterium tuberculosis as part of complete Freund’s adjuvant (CFA). Acute Trypanosoma cruzi infection, which induces T-cell activation and increased production of interferon-γ (IFNγ), also leads to the expansion of MDSCs23, 24. A similar expansion of MDSCs has been reported during acute toxoplasmosis25, polymicrobial sepsis26, acute infection with Listeria monocytogenes or chronic infection with Leishmania major27 and infection with helminths28,29, 30, Candida albicans31 or Porphyromonas gingivalis32.

MDSC expansion is also associated with autoimmunity and inflammation. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, an increase in CD11b+Ly6ChiLy6G− MDSCs was observed in the spleen and blood and these cells were found to enter the central nervous system during the inflammatory phase of the disease16. A significant increase in the number of MDSCs was also detected in experimental autoimmune uveoretinitis, an animal model of human intraocular inflammatory disease33, in the skin and spleens of mice that were repeatedly treated with a contact sensitizer to induce an inflammatory response34 and in inflammatory bowel diseases35. MDSCs were also found to infiltrate the spleen and suppress T-cell function in a model of traumatic stress36. Finally, a significant transient increase in MDSC numbers was also demonstrated in normal mice following immunization with different antigens such as ovalbumin or peptide together with CFA, a recombinant vaccinia virus expressing interleukin-2 (IL-2) or staphylococcal enterotoxin A 8, 37, 38. Therefore, current information clearly indicates that the expansion of an immunosuppressive MDSC population is frequently observed in many pathological conditions.

Expansion and activation of MDSCs Studies have demonstrated that the MDSC population is influenced by several different factors (Table 1), which can be divided into two main groups. The first group includes factors that are produced mainly by tumour cells and promote the expansion of MDSC through stimulation of myelopoiesis and inhibiting of the differentiation of mature myeloid cells. The second group of factors is produced mainly by activated T cells and tumour stroma, and is involved in directly activating MDSCs.
Mechanisms of MDSC expansion—Factors that induce MDSC expansion can include cyclooxygenase-2 (COX2), prostaglandins 39-41, stem-cell factor (SCF)39, macrophage colony-stimulating factor (M-CSF), IL-642, granulocyte/macrophage colony-stimulating factor (GM-CSF)41 and vascular endothelial growth factor (VEGF) 43 (Table 1). The signalling pathways in MDSCs that are triggered by most of these factors converge on Janus kinase (JAK) protein family members and signal transducer and activator of transcription 3 (STAT3) (Fig. 2), which are signalling molecules that are involved in cell survival, proliferation, differentiation and apoptosis44. STAT3 is arguably the main transcription factor that regulates the expansion of MDSCs. MDSCs from tumour-bearing mice have markedly increased levels of phosphorylated STAT3 compared with IMCs from naive mice45. Exposure of haematopoietic progenitor cells to tumour-cell-conditioned medium resulted in the activation of JAK2 and STAT3 and was associated with an expansion of MDSCs in vitro, whereas inhibition of STAT3 expression in haematopoietic progenitor cells abrogated the effect of tumour-derived factors on MDSC expansion46. Ablation of STAT3 expression in conditional knockout mice or selective STAT3 inhibitors markedly reduced the expansion of MDSCs and increased T-cell responses in tumour-bearing mice45, 47. STAT3 activation is associated with increased survival and proliferation of myeloid progenitor cells, probably through upregulated expression of STAT3 target genes including B-cell lymphoma XL, (BCL-XL), cyclin D1, MYC and survivin. So, abnormal and persistent activation of STAT3 in myeloid progenitors prevents their differentiation into mature myeloid cells and thereby promotes MDSC expansion.

Recent findings suggest that STAT3 also regulates MDSC expansion through inducing the expression of S100A8 and S100A9 proteins. In addition, it has been shown that MDSCs also express receptors for these proteins on their cell surface. S100A8 and S100A9 belong to the family of S100 calcium-binding proteins that have been reported to have an important role in inflammation48. STAT3-dependent upregulation of S100A8 and S100A9 expression by myeloid progenitor cells prevented their differentiation and resulted in the expansion of MDSCs in the spleens of tumor-bearing and naive S100A9-transgenic mice. By contrast, MDSCs did not expand in the peripheral blood and spleens of mice deficient for S100A9 following challenge with tumour cells or CFA49. In a different study, S100A8 and S100A9 proteins were shown to promote MDSC migration to the tumour site through binding to carboxylated N-glycan receptors expressed on the surface of these cells 50. Blocking the binding of S100A8 and S100A9 to their receptors on MDSCs in vivo with a carboxylated glycan-specific antibody reduced MDSC levels in the blood and secondary lymphoid organs of tumour-bearing mice50. In human colon tumour tissue, and in a mouse model of colon cancer, myeloid progenitor cells expressing S100A8 and S100A9 have been shown to infiltrate regions of dysplasia and adenoma. Furthermore, administration of a carboxylated glycan-specific monoclonal antibody (mAbGB3.1) was found to markedly reduced chronic inflammation and tumorigenesis51. Although the mechanisms involved require further study, these studies suggest that S100A9 and/or S100A8 proteins have a crucial role in regulating MDSC expansion, and may provide a link between inflammation and immune suppression in cancer.

Mechanisms of MDSC activation—Recently, it has become clear that the suppressive activity of MDSCs requires not only factors that promote their expansion but those that induce their activation. The expression of these factors, which are produced mainly by activated T cells and tumour stromal cells, is induced by different bacterial or viral products or as a result of tumour cell death 26. These factors, which include IFNγ, ligands for Toll-like receptors (TLRs), IL-13, IL-4 and transforming growth factor-β (TGFβ), activate several different signalling pathways in MDSCs that involve STAT6, STAT1, and nuclear factor-κB (NF-κB) (Fig. 2).

Blockade of IFNγ, which is produced by activated T cells, abolishes MDSC-mediated T-cell suppression17, 52. STAT1 is the major transcription factor activated by IFNγ-mediated signalling and, in the tumour microenvironment, the upregulation of ARG1 and iNOS expression in MDSCs involved a STAT1-dependent mechanism. Indeed, MDSCs from Stat1-/- mice failed to up regulate ARG1 and iNOS expression and therefore did not inhibit Tcell responses53. Consistent with other findings, IFNγ produced by activated T cells and by MDSCs triggered iNOS expression and synergized with IL-4Rα and ARG1 pathways that have been implicated in the suppressive function of MDSCs20.
An important role for the signalling pathway that involves IL-4 receptor α-chain (IL-4Rα) and STAT6 (which is activated by the binding of either IL-4 or IL-13 to IL-4Rα) in MDSC activation has been demonstrated in several studies. It has been shown that ARG1 expression is induced by culturing freshly isolated MDSCs or cloned MDSC lines with IL-454. In addition, IL-4 and IL-13 upregulate arginase activity, which increases the suppressive function of MDSCs55. In line with these observations, other experiments have shown that STAT6 deficiency prevents signalling downstream of the IL-4Rα and thereby blocks the production of ARG1 by MDSCs56. In addition, the IL-4Rα–STAT6 pathway was also found to be involved in IL-13-induced TGFβ1 production by MDSCs in mice with sarcoma, which resulted in decreased tumour immunosurveillance57. This could be regulated by neutralizing both TGFβ and IL-1357. However, in breast tumor model IL-4Rα knockout mice retain high levels of MDSC after surgery56. In a different study that evaluated the separate role of TGFβ (not involving study of IL-4Rα) TGFβ-specific blocking antibody failed to reverse T-cell anergy in B-cell lymphoma in vitro58. It is possible that, the IL4Rα–STAT6 pathway might not be involved in promoting tumour immunosuppression in all tumour models.

TLRs have a central role in the activation of innate immune responses. Polymicrobial sepsis induced by the ligation and puncture of the caecum, which releases microbial products into the peritoneum and systemic circulation, was shown to result in an expansion of the MDSC population in the spleen that was dependent on the TLR adaptor molecule myeloid differentiation primary-response gene 88 (MyD88)26. However, wild-type mice and mice lacking a functional TLR4 protein had comparable expansion of the MDSC during polymicrobial sepsis, which suggests that signalling through TLR4 is not required for MDSC expansion and that MyD88-dependent signalling pathways that are triggered by other TLRs probably contribute to the expansion of MDSCs in sepsis26. This indicates that the activation of MDSCs is a fundamental outcome of the host innate immune response to pathogens that express TLR ligands.

It is important to note that an increase in the production and/or recruitment of IMCs in the context of acute infectious diseases or following vaccination does not necessarily represent an expansion of an immunosuppressive MDSC population. It is likely that under pathological conditions, the expansion of a suppressive MDSC population is regulated by two different groups of factors that have partially overlapping activity: those that induce MDSC expansion and those that induce their activation (which leads to increased levels of ROS, arginase, and/ or NO). This two-tiered system may allow for flexibility in the regulation of these cells under physiological and pathological conditions.
Mechanisms of MDSC suppressive activity Most studies have shown that the immunosuppressive functions of MDSCs require direct cell– cell contact, which suggests that they act either through cell-surface receptors and/or through the release of short-lived soluble mediators. The following sections describe the several mechanisms that have been implicated in MDSC-mediated suppression of T-cell function.

Arginase and iNOS—Historically, the suppressive activity of MDSCs has been associated with the metabolism of L-arginine. L-arginine serves as a substrate for two enzymes: iNOS, which generates NO, and arginase, which converts L-arginine into urea and L-ornithine. MDSCs express high levels of both arginase and iNOS, and a direct role for both of these enzymes in the inhibition of T-cell function is well established; this has been reviewed recently59, 60. Recent data suggest that there is a close correlation between the availability of arginine and the regulation of T-cell proliferation11, 61. The increased activity of arginase in MDSCs leads to enhanced L-arginine catabolism, which depletes this non-essential amino acid from the microenvironment. The shortage of L-arginine inhibits T-cell proliferation through several different mechanisms, including decreasing their CD3ζ expression62 and preventing their upregulation of the expression of the cell cycle regulators cyclin D3 and cyclin-dependent kinase 4 (CDK4)63. NO suppresses T-cell function through a variety of different mechanisms that involve the inhibition of JAK3 and STAT5 in T cells64, the inhibition of MHC class II expression 65 and the induction of T-cell apoptosis66.

ROS—Another important factor that contributes to the suppressive activity of MDSCs is ROS. Increased production of ROS has emerged as one of the main characteristics of MDSCs in both tumour-bearing mice and patients with cancer6, 10, 13, 53, 67-70. Inhibition of ROS production by MDSCs isolated from mice and patients with cancer completely abrogated the suppressive effect of these cells in vitro10, 13, 67. Interestingly, ligation of integrins expressed on the surface of MDSCs was shown to contribute to increased ROS production following the interaction of MDSCs with T cells10. In addition, several known tumour-derived factors, such as TGFβ, IL-10, IL-6, IL-3, platelet-derived growth factor (PDGF) and GM-CSF, can induce the production of ROS by MDSCs (for review see Ref 71).

The involvement of ROS and NO in mechanisms of MDSC suppression are not restricted to neoplastic conditions, as inflammation and microbial products are also known to induce the development of a MDSC population that produces ROS and NO following interactions with activated T cells15. Similar findings were observed in models of EAE16 and acute Toxoplasmosis infection 16. In addition, it has been observed that MDSCs mediated their suppressive function through IFNγ-dependent NO production in an experimental model of Trypanosoma cruzi infection23.

Peroxynitrite—More recently, it has emerged that peroxynitrite (ONOO-) is a crucial mediator of MDSC-mediated suppression of T-cell function. Peroxynitrite is a product of a chemical reaction between NO and superoxide anoion (O2-) and is one of the most powerful oxidants produced in the body. It induces the nitration and nitrosylation of the amino acids cystine, methionine, tryptophan and tyrosine72. Increased levels of peroxynitrite are present at sites of MDSC and inflammatory-cell accumulation, including sites of ongoing immune reactions. In addition, high levels of peroxynitrite are associated with tumour progression in many types of cancer72, 73,74-78, which has been linked with T-cell unresponsiveness. Bronte and colleagues reported that human prostate adenocarcinomas were infiltrated by terminallydifferentiated CD8+ T cells that were in an unresponsive state. High levels of nitrotyrosine were present in the T cells, which suggested the production of peroxynitrites in the tumour environment. Inhibiting the activity of arginase and iNOS, which are expressed in malignant but not in normal prostate tissue and are key enzymes of L-arginine metabolism,, led to decreased tyrosine nitration and restoration of T-cell responsiveness to tumour antigens79. In addition, we have demonstrated that peroxynitrite production by MDSCs during direct contact with T cells results in nitration of the T-cell receptor (TCR) and CD8 molecules, which alters the specific peptide binding of the T cells and renders them unresponsive to antigen-specific stimulation. However, the T cells maintained their responsiveness to nonspecific stimuli80. This phenomenon of MDSC induced antigen-specific T-cell unresponsiveness was also observed in vivo in tumour-bearing mice53.

Subset-specific suppressive mechanisms?—Recent findings indicate that different subsets of MDSC might use different mechanisms by which to suppress T-cell proliferation. As described earlier, two main subsets of MDSCs have been identified: a granulocytic subset and a monocytic subset. The granulocytic subset of MDSC was found to express high levels of ROS and low levels of NO, whereas the monocytic subset expressed low levels of ROS and high levels of NO and both subsets expressed ARG16 (Fig.3). Interestingly, both populations suppressed antigen-specific T-cell proliferation to an equal extent, despite their different mechanisms of action. Consistent with these observations, Movahedi et al. also reported two distinct MDSC subsets in tumour-bearing mice, one that consisted of mononuclear cells that resembled inflammatory monocytes and a second that consisted of polymorphonuclear cells that were similar to immature granulocytes. Again, both populations were found to suppress antigen-specific T-cell responses, although by using distinct effector molecules and signalling pathways. The suppressive activity of the granulocytic subset was ARG1-dependent, in contrast to the STAT1- and iNOS-dependent mechanism of the monocyte fraction17. Finally, the same trend was observed in Trypanosoma cruzii infection. In this case, monocytic MDSCs produced NO and strongly inhibited T-cell proliferation, and granulocytic MDSCs produced low levels of NO and did not inhibit T-cell proliferation, although they did produce superoxide15. The biological significance of such functional dichotomy of these two MDSC subsets remains to be elucidated.
Induction of TReg cells—Recently, the ability of MDSCs to promote the de novo development of FOXP3+ regulatory T (TReg) cells in vivo has been described18, 19. The induction of TReg cells by MDSCs was found to require the activation of tumour-specific Tcells and the presence of IFNγ and IL-10 but was independent of NO19. In mice bearing 1D8 ovarian tumours, the induction of TReg cells by MDSCs required the expression of cytotoxic lymphocyte antigen 4 (CTLA-4; also known as CD152) by MDSCs18. In a mouse model of lymphoma, MDSCs were shown to induce TReg-cell expansion through a mechanism that required arginase and the capture, processing and presentation of tumour-associated antigens by MDSCs, but not TGFβ58. By contrast, Movahedi et al. found that the percentage of TReg cells was invariably high throughout tumour growth and did not relate to the kinetics of expansion of the MDSC population, suggesting that MDSCs were not involved in TReg-cell expansion17. Furthermore, in a rat model of kidney allograft tolerance that was induced with a CD28-specific antibody, MDSCs that were co-expressing CD80 and CD86 were found to have a limited effect on the expansion of the TReg-cell population81. Although further work is required to resolve these discrepancies and to determine the physiological relevance of these studies, it seems possible that MDSCs are involved in TReg-cell differentiation through the production of cytokines or direct cell–cell interactions. Furthermore, MDSCs and TReg cells might be linked in a common immunoregulatory network (see later).
Tissue-specific effects on MDSCs A major unresolved question in this field is whether MDSCs mediate antigen-specific or nonspecific suppression of T-cell responses. Provided that MDSCs and T cells are in close proximity, the factors that mediate MDSC suppressive function (ROS, arginase and NO) can inhibit T-cell proliferation regardless of the antigen specificity of the T cells. Indeed, numerous in vitro studies have demonstrated the antigen nonspecific nature of MDSC-mediated suppression of T cells82 83. However, whether the situation is the same in vivo is not clear, and evidence suggests that MDSC-mediated immunosuppression in peripheral lymphoid organs is mainly antigen-specific. The idea that MDSC-mediated T-cell suppression occurs in an antigen-specific manner is based on findings that antigen-specific interactions between antigen-presenting cells and T cells result in much more stable and more prolonged cell–cell contact than nonspecific interactions82, 84, 85. Such stable contacts are necessary for MDSCderived ROS and peroxynitrite to mediate effects on the molecules on the surface of T cells that render the T cells unresponsive to specific antigen. It should be noted that such modification of cell-surface molecules does not lead to T-cell death nor prevent nonspecific T-cell activation. Other evidence that supports the idea that MDSCs mediate antigen-specific suppression is the finding that that MDSCs can take up soluble antigens, including tumourassociated antigens, and process and present them to T cells17 80; blockade of MDSC–T-cell interactions with a MHC-class-I-specific antibody abrogated MDSC-mediated inhibition of T cell responses in vitro86. The MHC-class-I-restricted nature of MDSC-mediated CD8+ T-cell suppression has also been demonstrated in vivo in tumor models53 and in the model of inflammatory bowel disease 35. This is consistent with the recent observation that large numbers of tumour-induced MDSCs did not inhibit CD8+ T-cell responses specific for unrelated antigens in a model of sporadic cancer87. Notably, it is currently unclear whether similar antigen-specific mechanisms of MDSC-mediated suppression operate on CD4+ T cells, as published studies have only assessed the effects of MDSCs on CD8+ T cells. Addressing this question is complicated by the fact that only a small proportion of MDSCs in many tumour models expresses MHC class II molecules.

The theory that MDSCs suppress T-cell responses in an antigen-specific manner helps to explain the finding that T cells in the peripheral lymphoid organs of tumour-bearing mice and in the peripheral blood of cancer patients can still respond to stimuli other than tumourassociated antigens, including viruses, lectins, co-stimulatory molecules, IL-2 and CD3- and CD28-specific antibodies21, 80, 88-90. Furthermore, even patients with advanced stage cancer do not have systemic immunodeficiency except in cases in which the patient has received high doses of chemotherapy or is at a terminal stage of the disease.

Evidence suggets that the nature of MDSC-mediated suppression at the tumour site is quite different to that which occurs in the periphery. MDSCs actively migrate into the tumour site10, where they upregulate the expression of ARG1 and iNOS, downregulate the production of ROS and/or rapidly differentiate into tumour-associated macrophages (TAMs) 52. The levels of NO and arginase produced by tumour-associated MDSCs and TAMs are much higher than those of MDSCs found in peripheral lymphoid organs of the same animals. In addition, TAMs produce several cytokines (reviewed in REFs91, 92) that suppress T-cell responses in a nonspecific manner (Fig. 4). The mechanisms by which MDSC functions are regulated within the tumour microenvironment, and how they differ from those that operate at peripheral sites, remain unclear. It is possible that tumour stroma, hypoxia and/or the acidophilic environment have a role.
Therapeutic targeting of MDSCs The recognition that immune suppression has a crucial role in promoting tumour progression and contributes to the frequent failure of cancer vaccines to elicit an immune response has resulted in a paradigm shift with respect to approaches for cancer immunotherapy. Indeed, it has become increasingly clear that successful cancer immunotherapy will be possible only with a strategy that involves the elimination of suppressive factors from the body. As MDSCs are one of the main immunosuppressive factors in cancer and other pathological conditions, several different therapeutic strategies that target these cells are currently being explored (Table 2). Although the studies described below were carried out in tumor-bearing hosts, it is likely that the same strategies will be useful in other pathological conditions in which inhibition or elimination of MDSCs is a therapeutic aim.

Promoting myeloid-cell differentiation—One of the most promising approaches by which to target MDSCs for therapy is to promote their differentiation into mature myeloid cells that do not have suppressive abilities. Vitamin A has been identified as a compound that can mediate this effect: vitamin A metabolites such as retinoic acid have been found to stimulate the differentiation of myeloid progenitors into DCs and macrophages 86, 93. Mice that are deficient in vitamin A94 or that have been treated with a pan-retinoic-acid-receptor antagonist95, show an expansion of MDSCs in the bone marrow and spleen. Conversely, therapeutic concentrations of all-trans retinoic acid (ATRA) results in substantial decrease in the presence of MDSCs in cancer patients and tumour-bearing mice. ATRA induced MDSCs to differentiate into DCs and macrophages in vitro and in vivo 12, 86, 96. It is probable that ATRA preferentially induces the differentiation of the monocytic subset of MDSCs, whereas it causes apoptosis of the granulocytic subset. The main mechanism of ATRA-mediated differentiation involved an upregulation of glutathione synthesis and a reduction in ROS levels in MDSCs 97. Decreasing the number of MDSCs in tumour-bearing mice resulted in increased tumour-specific T-cell responses, and the combination of ATRA and two different types of cancer vaccine prolonged the anti-tumour effect of the vaccine treatment in two different tumour models 96. Moreover, administration of ATRA to patients with metastatic renal cell carcinoma resulted in a substantial decrease in the number of MDSCs in the peripheral blood and improved antigen-specific response of T cells 21. Further studies will lead to identification of other agents that have a similar effect. So far, evidence suggests that Vitamin D3 may be another agent with the potential to decrease MDSC numbers in patients with cancer, as it is also known to promote myeloid-cell differentiation98.

Inhibition of MDSC expansion—Because MDSC expansion is known to be regulated by tumour-derived factors (Table 1), several studies have focused on neutralizing the effects of these factors. Recently, SCF has been implicated in causing MDSC expansion in tumourbearing mice39. Inhibition of SCF-mediated signalling by blocking its interaction with its receptor, c-kit, decreased MDSC expansion and tumor angiogenesis39. VEGF, another tumourderived factor that is involved in promoting MDSC expansion, might also be a useful target by which to manipulate MDSC. However, in a clinical trial of 15 patients with refractory solid tumours, treatment with VEGF–trap (a fusion protein that binds all forms of VEGF-A and placental growth factor) showed no effect on MDSC numbers and did not result in increased T-cell responses99. By contrast, treatment of patients with metastatic renal cell cancer with a VEGF-specific blocking antibody (known as avastin) resulted in a decrease in the size of a CD11b+VEGFR1+ population of MDSCs in the peripheral blood 100. However, whether avastatin treatment resulted in an improvement in antitumour responses in these patients has not been determined. Finally, inhibition of matrix metalloproteinase 9 function in tumorbearing mice decreased the number of MDSCs in the spleen and tumour tissues and resulted in a significant delay in the growth of spontaneous NeuT tumours in transgenic BALB/c mice101. However, the mechanism responsible for this outcome remains to be elucidated.

Inhibition of MDSC function—Another approach by which to inhibit MDSCs is to block the signalling pathways that regulate the production of suppressive factors by these cells. One potential target by which this might be achieved is COX2. COX2 is required for the production of prostaglandin E2, which in 3LL tumour cells61 and mammary carcinoma40 has been shown to induce the upregulation of ARG1 expression by MDSCs, thereby inducing their suppressive function. Accordingly, COX2 inhibitors were found to downregulate the expression of ARG1 by MDSCs, which improved antitumour T-cell responses and enhanced the therapeutic efficacy of immunotherapy102, 103. Similarly, phosphodiesterase-5 inhibitors such as sildenafil were found to downregulate the expression of arginase and iNOS expression by MDSCs, thereby inhibiting their suppressive function in growing tumours104. This resulted in the induction of a measurable anti-tumour immune response and a marked delay of tumour progression in several mouse models 104.
ROS inhibitors have also been shown to be effective for decreasing MDSC-mediated immune suppression in tumour-bearing mice. The coupling of a NO-releasing moiety to a conventional non-steroidal anti-inflammatory drug has proven to be an efficient means by which to inhibit the production of ROS. One such drug, nitroaspirin, was found to limit the activity of ARG1 and iNOS in spleen MDSCs105. In combination with vaccination with endogenous retroviral gp70 antigen, nitroaspirin inhibited MDSCs function and increased the number and function of tumour-antigen-specific T cells105.

Elimination of MDSCs—MDSCs can be directly eliminated in pathological settings by using some chemotherapeutic drugs. Administration of one such drug, gemcitabine, to mice that were bearing large tumours resulted in a dramatic reduction in the number of MDSCs in the spleen and resulted in a marked improvement in the anti-tumour response induced by immunotherapy106, 107. This effect was specific to MDSCs, as a significant decrease in the number of T or B cells was not observed in these animals. Furthermore, in a study of 17 patients with early-stage breast cancer that were treated with doxorubicin–cyclophosphamide chemotherapy, a decrease in the level of MDSCs in the peripheral blood was observed22.

Evidence suggests that there is a broad range of methods that will be effective for targeting of the number and/or function of MDSCs in vivo. These strategies will undoubtedly help to further investigate the biology of these cells as well as expedite clinical applications to treat cancer and other pathological conditions.

MDSCs as regulatory myeloid cells? The wealth of information that has accumulated in recent years regarding the biology of MDSCs suggests that these cells might have evolved as a regulatory component of the immune system. These cells are absent under physiological conditions, as IMCs in naive mice are an intrinsic part of normal haematopoiesis that are not immunosuppressive in an unactivated state. In conditions of acute stress, infection or immunization, there is a transient expansion of this IMC population, which then quickly differentiates into mature myeloid cells. This transient IMC population can mediate the suppressive functions that are characteristic of MDSCs but, because the acute conditions are short-lived, the suppressive functions of this transient population have a minimal impact on the overall immune response. However, these cells probably function as important ‘gatekeepers’ that prevent pathological immune-mediated damage.

The role of the MDSC population in settings of chronic infections and cancer is very different. In these pathological conditions, the prolonged and marked expansion of IMCs and their subsequent activation leads to the expansion of a large population of MDSCs with immunosuppressive abilities. MDSCs accumulate in peripheral lymphoid organs and migrate to tumour sites, where they contribute to immunosuppression. Furthermore, some evidence suggests that MDSCs can also induce expansion of regulatory T cells. Future studies will reveal whether MDSCs can be considered part of a natural immune regulatory network.

Concluding remarks The field of MDSC research has more outstanding questions than answers. The roles of specific MDSC subsets in mediating T-cell suppression, and the molecular mechanisms responsible for inhibition of myeloid-cell differentiation, need to be elucidated. The issue of whether Tcell suppression occurs in an antigen-specific manner remains to be clarified, as do the mechanisms that cause MDSC migration to peripheral lymphoid organs. Some of the main priorities in this field should include a better characterization of human MDSCs and a clear understanding of whether targeting these cells in patients with various pathological conditions will be of clinical significance. Conversely, adoptive cellular therapy with MDSCs may be an attractive opportunity by which to inhibit immune responses in the setting of autoimmune disease or transplantation. The challenge for these approaches will be to devise methods by which to generate these cells ex vivo in clinical-grade conditions such that they are suitable for administration to patients. If the past 5–6 years are an indication of the potential for progress in this area, it is safe to estimate that there will soon be significantly more discoveries that further our understanding about the biology and clinical utility of MDSCs.

Box 1. Definition of myeloid-derived suppressor cells (MDSCs)

• a heterogeneous population of cells of myeloid origin that consist of myeloid progenitors and immature macrophages, immature granulocytes and immature dendritic cells

• present in activated state that is characterized by the increased production of reactive oxygen and nitrogen species, and of arginase

• potent suppressors of various T-cell functions • in mice, their phenotype is CD11b+Gr1+, although functionally distinct subsets within this population have been identified (see main text)

• in humans, their phenotype is Lin-HLA-DR-CD33+ or CD11b+CD14-CD33+.

Human cells do not express a marker homologous to mouse Gr1. MDSC have also been identified within a CD15+ population in human peripheral blood.

• in the steady state, immature myeloid cells lack suppressive activity and are present in the bone marrow, but not in secondary lymphoid organs

• accumulation of MDSCs in lymphoid organs and in tumours in response to various growth factors and cytokines is associated with various pathological conditions (most notably cancer)

• in tumour tissues, MDSCs can be differentiated from tumour-associated macrophages (TAMs) by their high expression of Gr1 (not expressed by TAMs) by their low expression of F4/80 (expressed by TAMs), by the fact that a large proportion of MDSCs have a granulocytic morphology and based the upregulated expression of both arginase and inducible nitric oxide synthase by MDSCs but not TAMs.

References

1. Young MRI, Newby M, Wepsic TH. Hematopoiesis and suppressor bone marrow cells in mice bearing large metastatic Lewis lung carcinoma tumors. Cancer Res 1987;47:100–106. [PubMed: 2947676]
2. Buessow SC, Paul RD, Lopez DM. Influence of mammary tumor progression on phenotype and function of spleen and in situ lymphocytes in mice. J Natl Cancer Inst 1984;73:249–255. [PubMed: 6610791]
3. Seung L, Rowley D, Dubeym P, Schreiber H. Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection. Proc Natl Acad Sci U S A 1995;92:6254–6258. [PubMed: 7603979]
4. Sinha P, Clements VK, Bunt SK, Albelda SM, Ostrand-Rosenberg S. Crosstalk between myeloidderived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. J Immunol 2007;179:977–983. [PubMed: 17617589]
5. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008;8:618–631. [PubMed: 18633355]
6. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumorbearing mice. J Immunol 2008;181:5791–5802. [PubMed: 18832739] Together with reference # 17 this paper described functional differences between subsets of MDSC.
7. Bronte V, et al. Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells. Blood 2000;96:3838. [PubMed: 11090068]
8. Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in cancer: The role of reactive oxygen species. J Leukoc Biol 2003;74:186–196. [PubMed: 12885935]
9. Li Q, Pan PY, Gu P, Xu D, Chen SH. Role of immature myeloid Gr-1+ cells in the development of antitumor immunity. Cancer Res 2004;64:1130–1139. [PubMed: 14871848] …..

 

 

Aurelian Udristioiu commented on your update
“The proto-oncogenic transcription factor Myc is known to promote transcription of genes for the cell cycle as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role to induce the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor HIF1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions. “

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Phosphorylation-dependent interaction between antigenic peptides and MHC class I

Curator: Larry H. Bernstein, MD, FCAP

 

 

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Nat Immunol. 2008 Nov;9(11):1236-43.    http://dx.doi.org:/10.1038/ni.1660.  Epub 2008 Oct 5.
Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of ‘transformed self’. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.
Figure 1
Bioinformatic characterization of the HLA-A2–restricted phosphopeptide repertoire. (a) Distribution of phosphorylated residues among naturally processed (A2 phosphopeptide) and predicted HLA-A2 binding phosphopeptides (Phosphosite, EMBL). The frequency of phosphorylated residues at each position is displayed for naturally processed HLA-A2 associated phosphopeptides, and for peptides in EMBL and Phosphosite datasets that contain phosphorylation sites and are predicted, according to criteria described in Methods, to bind HLA-A2. (b) Representation of positively charged residues (Arg or Lys) at P1 among naturally processed HLA-A2 associated phosphopeptides, phosphopeptides from the EMBL or Phosphosite datasets that are predicted to bind HLA-A2 and contain a p-Ser residue at the P4 position, and datasets of naturally processed non-phosphorylated peptides (B-LCL) and known HLA-A2 binding peptides (Immune Epitope). Selection criteria for the latter two datasets are described in Methods. * = P<0.001, NS= not significant. (c, d) Representation of subdominant residues at the P2 anchor position (c) and the PC (P9) position (d) in naturally processed HLA-A2 associated phosphopeptides and in datasets of naturally processed non-phosphorylated peptides and known HLA-A2 binding peptides.
Changes in protein expression or metabolism due to intracellular infection or cellular transformation modify the repertoire of peptides generated and therefore displayed by class I MHC molecules, resulting in presentation of “altered self” to the immune system. T cell receptor (TCR)-mediated recognition of specific MHC-bound peptides by CD8 T lymphocytes results in cytolytic activity and release of pro-inflammatory cytokines, which are key components of anti-viral and anti-tumor immunity. Evidence suggests that peptides containing post-translational modifications (PTM), including deamidation, cysteinylation, glycosylation, and phosphorylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition2. Indeed, the majority of naturally occurring PTM-bearing peptides defined to date can be discriminated from their unmodified homologs specifically by T cells2-4.  …..
Recent studies have highlighted protein phosphorylation as a process with the capacity to generate unique peptides bound to class I MHC molecules. Significant numbers of different phosphorylated peptides are presented by several HLA-A and HLA-B alleles that are prevalent in humans3,4, demonstrating their widespread potential as antigens. Moreover, CD8+ T lymphocytes recognize these phosphopeptides in a manner that is both peptide sequence-specific and phosphate-dependent3, 4. Thus, phosphopeptides can be immunologically distinguished from their non-phosphorylated counterparts. Consistent with their presentation by class I MHC molecules, most phosphorylated peptides are derived from proteins that function intracellularly, and processing of both model and naturally occurring phosphopeptides is dependent on transport into the endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP)3, 5. Furthermore, rapid degradation by the proteasome, a process that regulates the activity of many transcription factors, cell growth modulators, signal transducers and cell cycle proteins6-8, is frequently dependent on target protein phosphorylation9-11. ….
Phosphopeptide antigens are of significant therapeutic interest because deregulation of protein kinase activity, normally tightly controlled, is one of the hallmarks of malignant transformation and is thought to contribute directly to oncogenic signaling pathways involved in cell growth, differentiation and survival13-15. In addition, mutation-induced deregulation of a limited number of critical kinases can often lead to activation of several signaling cascades and increases in the extent of protein phosphorylation within the cell16-18. These considerations strongly suggest that alterations in protein phosphorylation during malignancy represent a distinctive immunological signature of “transformed self”. Consistent with this notion, the phosphopeptides presented by HLA-A*0201….

Nα-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I–Restricted Nα-Acetylpeptide Presentation

As one of the most common posttranslational modifications (PTMs) of eukaryotic proteins, Nα-terminal acetylation (Nt-acetylation) generates a class of Nα-acetylpeptides that are known to be presented by MHC class I at the cell surface. Although such PTM plays a pivotal role in adjusting proteolysis, the molecular basis for the presentation and T cell recognition of Nα-acetylpeptides remains largely unknown. In this study, we determined a high-resolution crystallographic structure of HLA (HLA)-B*3901 complexed with an Nα-acetylpeptide derived from natural cellular processing, also in comparison with the unmodified-peptide complex. Unlike the α-amino–free P1 residues of unmodified peptide, of which the α-amino group inserts into pocket A of the Ag-binding groove, the Nα-linked acetyl of the acetylated P1-Ser protrudes out of the groove for T cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide but also switches the residues in the α1-helix of HLA-B*3901, which may impact the T cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from different species reveal reduced stability. Our findings provide the insight into the mode of Nα-acetylpeptide–specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.

Produced by Ag processing and proteasomal degradation of intracellular proteins, polypeptides serve as CTL epitopes presented by MHC class I molecules, which play a critical role in cellular immunity (1). Eukaryotic proteins bearing various posttranslational modifications (PTMs) can generate a group of modified Ags, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for TCR-mediated recognition. A modified peptide may become a new Ag because of the distinguished antigenicity compared with its unmodified homolog. A variety of natural peptide Ags containing modification have been observed that can be immunologically discriminated by T cells from their unmodified homologs as “altered self” (2). Thus, the significance of PTMs on epitopes and the application of modified peptides in vaccine development for immunotherapy against cancer and autoimmune diseases have been increasingly appreciated (3, 4).

The molecular bases of the presentation of peptides with several PTMs by MHC class I molecules have been successfully explicated. For instance, the formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of H2-M3 (5); both the glycan and the phosphate moieties of the central region of the glycopeptides (6, 7) and the phosphopeptides (8, 9), respectively, are exposed to enable TCR binding, and the deimination (citrullination) of arginine on a peptide presented by two HLA-B27 subtypes induces distinct peptide conformations (10).

Nα-terminal acetylation (Nt-acetylation) is one of the most common PTMs, occurring on the vast majority of eukaryotic proteins. In humans, >80% of the different varieties of intracellular proteins are irreversibly Nt-acetylated by Nα-acetyltransferases, often after the removal of the initiator methionine. Only a subset of the penultimate residues (Ala, Ser, Thr, Cys, and Val) or the retained initiator methionine can be acetylated at the α-amino (NH2) groups (11). A recent study found that acetylated N-terminal residues of eukaryotic proteins act as specific degradation signals (Ac-N-degrons) that are recognized by specific ubiquitin ligases (12). A subsequent systematic analysis demonstrated that Nt-acetylation can also represent an early determining factor in the cellular sorting for prevention of protein targeting to the secretory pathway (13). These findings suggested that Nt-acetylation–mediated inhibition of secretion could contribute to the retention of proteins in the cytosol where they may subsequently be ubiquitinylated through the specific recognition of their Ac-N-degrons and thereby generating Nt-acetylated proteasomal digestion products (14). Hence, these Nt-acetylated polypeptides in the form of MHC-associated neoantigens stand a good chance to be recognized by T cells. This has indeed been illuminated in an Nt-acetylated MHC class II–restricted peptide derived from myelin basic protein, which stimulates murine T cells to elicit experimental autoimmune encephalomyelitis, whereas the nonacetylated form does not (15). A structural study subsequently suggested that the Nt-acetylation of this peptide is essential for MHC class II binding (16).

For MHC class I, the first Nt-acetylated natural ligand was identified more than a decade ago (17). However, the mode of interaction of this acetylated peptide with class I molecules remained largely enigmatic. To understand this, we determined the crystal structures of a naturally occurring Nt-acetylated self-peptide (NAc-SL9) and two nonmodified variants (SL9 and HL8), respectively, in complex with HLA-B*3901. Taken together with the thermostability analyses of Nα-acetylpeptides complexed with a series of class I molecules of human and murine origin, we elucidated that Nt-acetylation exerts a destabilizing effect on peptide–MHC (pMHC) complex, thereby influencing TCR recognition.

……

Our results here provide the structural and thermodynamic insights into the presentation of Nt-acetylated peptides by MHC class I molecules. The structure of the Nα-acetylpeptide in complex with HLA-B*3901 outlines a molecular interpretation of the reduced stability of MHC class I–bound Nt-acetylated peptides and also highlights a potential influence of Nt-acetylation on antigenic identity and T cell recognition. In addition, the structure elucidation of HLA-B*3901, the predominant B39 subtype, also is valuable in studying immune diseases associated with this MHC allele.

In a previous report, the Nt-formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of the murine MHC class I H2-M3 playing an anchoring role for MHC class I binding (Supplemental Fig. 2A) (5). In our study, the methyl and carbonyl groups of the acetyl are rotated upwards like two arms that push the peptide-binding groove open (Fig. 2G, Supplemental Fig. 2B), thereby altering its immunogenicity at the expense of the pMHC stability. The thermostability we tested from seven human and one murine complexes indicates a general feature of Nα-acetylpeptide in weakening the binding affinity to MHC class I, which could be revealed by the gel-filtration chromatography of pMHC refolding assays as well (Supplemental Fig. 3). Their instability would partially explain why, as yet, such epitopes are rarely found. Within N-terminal residues of eukaryotic proteins, Ser is the most frequently acetylated in vivo (11). The Ala, Thr, Cys, and Val residues can also be Nt-acetylated and have small side chains like Ser. Thus, the rotation of P1 residues observed in the pHLA-B*3901 complex with an acetylated P1-Ser could very well be a general mode in Nα-acetylpeptide binding. In contrast, the long side chain of Met precludes it from being rotated into pocket A, but a certain reorientation is presumed to take place in the acetylated P1-Met based on the thermal instability (Fig. 6H). Besides the accommodation of the acetyl moiety, Nt-acetylation is presumed to decrease the stability of the pHLA-B*3901 complex as a result of the conformational switch of the Arg62. Arg62 in the α1-helix is largely conserved in almost all HLA-B and -C allotypes (Table V). For other HLA class I (Table V, Fig. 8), the long charged side chains of the residues in position 62 (Glu62 of A24 and Gln62 of A11 and so on) also may interact with the acetyl. Hence, the residue in position 62 plays a key role in the interaction between acetyl group and the H chain, which may influence not only the Nα-acetylpeptide binding to HLA molecules but also the TCR docking.

The discoveries that intracellular proteins with Ac-N-degrons are inhibited from being secreted (13) and then are degraded via ubiquitylation (12) raise many questions on the biological significance of acetylation-mediated proteolysis (14). The Nt-acetylated peptides with the size of MHC class I ligands (8–11 aa) as neoepitopes for CD8+ T cells, represent one of the possible roles of the Nt-acetylated digestion products. The vast armory of intracellular proteins that are frequently Nt-acetylated can create a large pool of Nα-acetylpeptides for Ag presentation and T cell surveying. The Nt-acetylation potentially impacts the TCR-MHC interaction in three different aspects: 1) the direct interaction of the solvent-exposed acetyl moiety; 2) the altered conformation of the central region of the peptide main chain; and 3) the conformational switches of the MHC residues. The Nt-acetylation creation of a distinctive pMHC landscape and participation in a potential binding element for TCR engagement described in our results highlights needs for further investigation into the Nα-acetylpeptide–specific TCR repertoires.  ……

see…J Immunol 2014; 192:5509-5519   http://dx.doi.org:/10.4049/jimmunol.1400199   http://www.jimmunol.org/content/192/12/5509

Supplementary http://www.jimmunol.org/content/suppl/2014/05/14/jimmunol.1400199.DCSupplemental.html
References http://www.jimmunol.org/content/192/12/5509.full#ref-list-1

 

The Cellular Redox Environment Alters Antigen Presentation*

Jonathan A. Trujillo,§12Nathan P. Croft,1Nadine L. Dudek,1Rudragouda ChannappanavarAlex TheodossisAndrew I. Webb,…., Jamie Rossjohn,‡‡,§§5Stanley Perlman,§6 and Anthony W. Purcell,7
The Journal of Biological Chemistry 289; 27979-27991.
http://dx.doi.org:/10.1074/jbc.M114.573402

Capsule

Background: Modification of cysteine residues, including glutathionylation, commonly occurs in peptides bound to and presented by MHC molecules.

Results: Glutathionylation of a coronavirus-specific T cell epitope results in diminished CD8 T cell recognition.

Conclusion: Cysteine modification of a T cell epitope negatively impacts the host immune response.

Significance: Cross-talk between virus-induced oxidative stress and the T cell response probably occurs, diminishing host cell recognition of infected cells.

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5–10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.

Antigen Presentation     Antigen Processing     Glutathionylation     Mass Spectrometry (MS)     Oxidation-Reduction (Redox)     Redox Regulation     T-cell     Viral Immunology

Small fragments of proteins (peptides) derived from both intracellular and extracellular sources are displayed on the surface of cells by molecules encoded within the major histocompatibility complex (MHC). These peptides are recognized by T lymphocytes and provide the immune system with a surveillance mechanism for the detection of pathogens and cancer cells. The fidelity with which antigen presentation communicates changes in the intracellular proteome is critical for immune surveillance. Not only do antigens expressed at vastly different abundances need to be represented within the array of peptides selected and presented at the cell surface (collectively termed the immunopeptidome (1, 2)), but changes in their post-translational state also need to be conveyed within this complex mixture of peptides. For example, changes in antigen phosphorylation have been linked to cancer, and the presentation of phosphorylated peptides has been shown to communicate the cancerous state of cells to the immune system (36). Other types of post-translational modification play a central role in the pathogenesis of autoimmune diseases (7), such as arginine citrullination in arthritis (810), deamidation of glutamine residues in wheat proteins in celiac disease (1115), and cysteine oxidation in type 1 diabetes (16, 17). Cysteine is predicted to be present in up to 14% of potential T cell epitopes based on its prevalence in various pathogen and host proteomes (18). However, reports of cysteine-containing epitopes are much less frequent due to technical difficulties associated with synthesis and handling of cysteine-containing peptides and their subsequent avoidance in many epitope mapping studies (19). Cysteine can be modified in numerous ways, including cysteinylation (the disulfide linkage of free cysteine to peptide or protein cysteine residues), oxidation to cysteine sulfenic (oxidation), sulfinic (dioxidation) and sulfonic acids (trioxidation), S-nitrosylation, and S-glutathionylation. Such modifications may occur prior to or during antigen processing; however, the role of cysteine modification in T-cell-mediated immunity has not been systematically addressed.

In addition to constitutive presentation of a subset of oxidatively modified peptides, it is anticipated that changes in the proportion of these ligands will occur upon infection because oxidative stress, triggering of the unfolded protein response, and modulation of host cell synthesis by the virus are hallmarks of this process (2027). For example, host cell stress responses modulate expression, localization, and function of Toll-like receptors, a key event in the initiation of the immune response (28). Oxidative stress would also be predicted to affect protein function through post-translational modification of amino acids, such as cysteine. Indeed, because of the reactive nature of cysteine and the requirements for cells to regulate the redox state of proteins to maintain function, a number of scavenging systems for redox-reactive intermediates exist. The tripeptide glutathione (GSH) is one of the key intracellular antioxidants, acting as a scavenger for reactive oxygen species. Reduced GSH is equilibrated with its oxidized form, GSSG, with normal cytosolic conditions being that of the reduced state in a ratio of ∼50:1 (GSH/GSSG) (29). Modification of proteins and peptides with GSH (termed S-glutathionylation) occurs following reaction of GSSG with the thiol group of cysteine in a reaction catalyzed by the detoxifying enzyme, glutathione S-transferase (GST). A variety of cellular processes and signaling pathways, such as the induction of innate immunity, apoptosis, redox homeostasis, and cytokine production, are modulated by this GST-catalyzed post-translational modification (3032). S-Glutathionylation can eventuate via oxidative stress, whereby the intracellular levels of GSSG increase.

Given that viruses are known to induce oxidative stress (3335), the intracellular environment of viral infection may lead to an increase inS-glutathionylated cellular proteins and viral antigens. For instance, HSV infection induces an early burst of reactive oxygen species, resulting in S-glutathionylation of TRAF family members, which in turn is linked to downstream signaling and interferon production (36). The potential for modification of viral antigens subsequent to reactive oxygen species production is highlighted by S-glutathionylation of several retroviral proteases, leading to host modulation of protease function (37). Indeed large scale changes in protein S-glutathionylation are observed in HIV-infected T cell blasts (38), suggesting that functional modulation of both host and viral proteins occurs via this mechanism. Whether these S-glutathionylated proteins inhibit or enhance immune responses to the unmodified epitope or generate novel T-cell epitopes that are subsequently recognized by the adaptive immune system is unclear.

Here, we investigate the frequency of modification of cysteine-containing MHC-bound peptides by interrogating a large database of naturally processed self-peptides derived from B-lymphoblastoid cells, murine tissues, and cytokine-treated cells. In addition, the functional consequences of Cys modification of T cell epitopes was investigated using an established model of infection that involves an immunodominant cysteine-containing epitope derived from a neurotropic strain of mouse hepatitis virus, strain JHM (JHMV)8(3941). We describe S-glutathionylation of this viral T cell epitope and the functional and structural implications of redox-modulated antigen presentation. Collectively our studies suggest that S-glutathionylation plays a key, previously unappreciated role in adaptive immune recognition.

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CD-4 Therapy for Solid Tumors

Curator: Larry H. Bernstein, MD, FCAP

 

CD4 T-cell Immunotherapy Shows Activity in Solid Tumors

Alexander M. Castellino, PhD

http://www.medscape.com/viewarticle/862095

For the first time, treatment with genetically engineered T-cells has used CD4 T-cells instead of the CD8 T-cells, which are used in the chimeric antigen receptor (CAR) T-cell approach. Early data suggest that this CD4 T-cell approach has activity against solid tumors, whereas the CAR T-cell approach so far has achieved dramatic success in hematologic malignancies.

In the new approach, CD4 T-cells were genetically engineered to target MAGE-A3, a protein found on many tumor cells. The treatment was found to be safe in patients with metastatic cancers, according to data from a phase 1 clinical study presented here at the American Association for Cancer Research (AACR) 2016 Annual Meeting.

“This is the first trial testing an immunotherapy using genetically engineered CD4 T-cells,” senior author Steven A. Rosenberg, MD, PhD, chief of the Surgery Branch at the National Cancer Institute (NCI), told Medscape Medical News.

Most approaches use CD8 T-cells. Although CD8 T-cells are known be cytotoxic and CD4 T-cells are normally considered helper cells, CD4 T-cells can induce tumor regression, he said.

Louis M. Weiner, MD, director of the Lombardi Comprehensive Cancer Center at Georgetown University, in Washington, DC, indicated that in contrast with CAR T-cells, these CD4 T-cells target proteins on solid tumors. “CAR T-cells are not tumor specific and do not target solid tumors,” he said.

Engineering CD4 Cells

Immunotherapy with engineered CD4 T-cells was personalized for each patient whose tumors had not responded to or had recurred following treatment with least one standard therapy. The immunotherapy was specific for patients in whom a specific human leukocyte antigen (HLA) — HLA-DPB1*0401 — was found to be expressed on their cells and whose tumors expressed MAGE-A3.

MAGE-A3 belongs to a class of proteins expressed during fetal development. The expression is lost in normal adult tissue but is reexpressed on tumor cells, explained presenter Yong-Chen William Lu, PhD, a research fellow in the Surgery Branch of the NCI.

Targeting MAGE-A3 is relevant, because it is frequently expressed in a variety of cancers, such as melanoma and urothelial, esophageal, and cervical cancers, he pointed out.

 Researchers purified CD4 T-cells from the peripheral blood of patients. Next, the CD4 T-cells were genetically engineered with a retrovirus carrying the T-cell receptor (TCR) gene that recognizes MAGE-A3. The modified cells were grown ex vivo and were transferred back into the patient.

Clinical Results

Dr Lu presented data for 14 patients enrolled into the study: eight patients received cell doses from 10 million to 30 billion cells, and six patients received up to 100 billion cells.

This was similar to a phase 1 dose-finding study, except the researchers were seeking to determine the maximum number of genetically engineered CD4 T-cells that a patient could safely receive.

One patient with metastatic cervical cancer, another with metastatic esophageal cancer, and a third with metastatic urothelial cancer experienced partial objective responses. At 15 months, the response is ongoing in the patient with cervical cancer; after 7 months of treatment, the response was durable in the patient with urothelial cancer; and a response lasting 4 months was reported for the patient with esophageal cancer.

Dr Lu said that a phase 2 trial has been initiated to study the clinical responses of this T-cell receptor therapy in different types of metastatic cancers.

In his discussion of the paper, Michel Sadelain, MD, of the Memorial Sloan Kettering Cancer Center, New York City, said, “Although therapy with CD4 cells has been evaluated using endogenous receptor, this is the first study using genetically engineered CD4 T-cells.”

Although the study showed that therapy with genetically engineered T-cells is safe and efficacious at least in three patients, the mechanism of cytotoxicity remains unclear, Dr Sadelain indicated.

Comparison With CAR T-cells

CAR T-cells act in much the same way. CARs are chimeric antigen receptors that have an antigen-recognition domain of an antibody (the V region) and a “business end,” which activates T-cells. In this case, CD8 T-cells from the patients are used to genetically engineer T-cells ex vivo. In the majority of cases, dramatic responses have been seen in hematologic malignancies.

CARs, directed against self-proteins, result in on-target, off-tumor effects, Gregory L. Beatty, MD, PhD, assistant professor of medicine at the University of Pennsylvania, in Philadelphia, indicated when he reported the first success story of CAR T-cells in a solid pancreatic cancer tumor.

Side effects of therapy with CD4 T-cells targeting MAGE-A3 were different and similar to side effects of chemotherapy, because patients received a lymphodepleting regimen of cyclophosphamide and fludabarine. Toxicities included high fever, which was experienced by the majority of patients (12/14). The fever lasted 1 to 2 weeks and was easily manageable.

High levels of the cytokine interleukin-6 (IL-6) were detected in the serum of all patients after treatment. However, the elevation in IL-6 levels was not considered to be a cytokine release syndrome, because no side effects occurred that correlated with the syndrome, Dr Liu indicated.

He also indicated that future studies are planned that will employ genetically engineered CD4 T-cells in combination with programmed cell death protein 1–blocking antibodies.

This study was funded by Intramural Research Program of the National Institutes of Health. The NCI’s research and development of T-cell receptor therapy targeting MAGE-A3 are supported in part under a cooperative research and development agreement between the NCI and Kite Pharma, Inc. Kite has an exclusive, worldwide license with the NIH for intellectual property relating to retrovirally transduced HLA-DPB1*0401 and HLA A1 T-cell receptor therapy targeting MAGE-A3 antigen. Dr Lu and Dr Rosenberg have disclosed no relevant financial relationships.

American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract CT003, presented April 17, 2016.

 

Searches Related to immunotherapy using genetically engineered CD4 T-cells

 

Genetic engineering of T cells for adoptive immunotherapy

To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
Adoptive T cell therapy is a form of transfusion therapy involving the infusion of large numbers of T cells with the aim of eliminating, or at least controlling, malignancies or infectious diseases. Successful applications of this technique include the infusion of CMV-or EBVspecific CTLs to protect immunosuppressed patients from these transplantation-associated diseases [1,2]. Furthermore, donor lymphocyte infusions of ex vivo-expanded allogeneic T cells have been used to successfully treat hematological malignancies in patients with relapsed disease following allogeneic hematopoietic stem cell transplant [3]. However, in many other malignancies and chronic viral infections such as HIV-1, adoptive T cell therapy has achieved inconsistent and/or marginal successes. Nevertheless, there are compelling reasons for optimism on this strategy. For example, the existence of HIV-positive elite non-progressors [4], as well as the correlation between the presence of intratumoral T cells and a favorable prognosis in malignancies such as ovarian [5,6] and colon carcinoma [7,8], provides in vivo evidence for the critical role of the immune system in controlling both HIV and cancer.
The key to successful adoptive immunotherapy strategies appears to consist of (1) using the “right” T cell type(s) and (2) obtaining therapeutically effective numbers of these cells without compromising their effector functions or their ability to engraft within the host. This article is focused on strategies employed in our laboratory to generate the “right” cell through genetic engineering approaches, with an emphasis on redirecting the antigen specificity of CD8 T cells, and rendering CD4 T cells resistant to HIV-1 infection. The article by Paulos et al. describes the evolving process of how to best obtain therapeutically effective numbers of the “right” cells by optimizing ex vivo cell expansion strategies.
Our laboratory’s overall strategy and flow plan for development and evaluation of engineered T cells is depicted in Fig. 1. We work almost exclusively with primary human T cells; little or no work is performed with conventional established cell lines. Thus, we benefit substantially from our close association with the UPenn Human Immunology Core. The Core performs leukaphereses on healthy donors 2–3 times a week, and provides purified peripheral blood mononuclear cell subsets, ensuring a constant influx of fresh human T cells into our laboratory. We have extensive experience in developing both bead- and cell-based artificial antigen presenting cells (aAPCs), as described in detail in the article by Paulos et al. The ability to genetically modify T cells at high efficiency is critical for virtually every project within the laboratory. We have adapted the lentiviral vector system described by Dull [15] for most, but not all, of the engineering applications in our laboratory.
CD4 T cells are the primary target of HIV-1, and decreasing CD4 T cell numbers is a hallmark of advancing HIV-1 disease [34]. Thus, strategies that protect CD4 T cells from HIV-1 infection in vivo would conceivably provide sufficient immunological help to control HIV-1 infection. Our early observations that CD3/CD28 costimulation resulted in improved ex vivo expansion of CD4 T cells from both healthy and HIV-infected donors, as well as enhanced resistance to HIV-1 infection [35,36], ultimately led to the first-in-human trial of lentiviral vector-modified CD4 T cells [37]. In this trial, CD4 T cells from HIV-positive subjects who had failed antiretroviral therapy were transduced with a lentiviral vector encoding an antisense RNA that targeted a 937 bp region in the HIV-1 envelope gene. Preclinical studies demonstrated that this antisense region, directed against the HIV-1NL4-3 envelope, provided robust protection from a broad range of both R5-and X4-tropic HIV-1 isolates [38]. One year after administration of a single dose of the gene-modified cells, four of the five enrolled patients had increased peripheral blood CD4 T cell counts, and in one subject, a 1.7 log decrease in viral load was observed. Finally, in two of the five patients, persistence of the gene-modified cells was detected one year post-infusion.
Since its identification as the primary co-receptor involved in HIV transmission, CCR5 has attracted considerable attention as a target for HIV therapy [42,43]. Indeed, “experiments of nature” have shown that individuals with a homozygous CCR5 Δ32 deletion are highly resistant to HIV-1 infection. Thus, we hypothesized that knocking out the CCR5 locus would generate CD4 T cells permanently resistant to infection by R5 isolates of HIV-1. To test this hypothesis we took advantage of zinc-finger nuclease (ZFN) technology [44]. ZFNs introduce sequencespecific double-strand DNA breakage, which is imperfectly repaired by non-homologous endjoining. This results in the permanent disruption of the genomic target, a process termed genome editing (Fig. 3).
Genetic modification of T cells to redirect antigen specificity is an attractive strategy compared to the lengthy process of growing T cell lines or CTL clones for adoptive transfer. Genetically modified, adoptively transferred T cells are capable of long-term persistence in humans [37, 46,47], demonstrating the feasibility of this approach. When compared to the months it can take to generate an infusion dose of antigen-specific CTL lines or clones from a patient, a homogeneous population of redirected antigen-specific cells can be expanded to therapeutically relevant numbers in about two weeks [3]. Several strategies are being explored to bypass the need to expand antigen-specific T cells for adoptive T cell therapy. The approaches currently studied in our laboratory involve the genetic transfer of chimeric antigen receptors and supraphysiologic T cell receptors.
Chimeric antigen receptors (CARs or T-bodies) are artificial T cell receptors that combine the extracellular single-chain variable fragment (scFv) of an antibody with intracellular signaling domains, such as CD3ζ or Fc(ε)RIγ [48–50]. When expressed on T cells, the receptor bypasses the need for antigen presentation on MHC since the scFv binds directly to cell surface antigens. This is an important feature, since many tumors and virus-infected cells downregulate MHCI, rendering them invisible to the adaptive immune system. The high-affinity nature of the scFv domain makes these engineered T cells highly sensitive to low antigen densities. In addition, new chimeric antigen receptors are relatively easy to produce from hybridomas. The key to this approach is the identification of antigens with high surface expression on tumor cells, but reduced or absent expression on normal tissues.  Since one can redirect both CD4 and CD8 T cells, the T-body approach to immunotherapy represents a near universal “off the shelf” method to generate large numbers of antigen-specific helper and cytotoxic T cells.
Many T-bodies targeting diverse tumors have been developed [51], and four have been evaluated clinically [52–55]. Three of the four studies were characterized by poor transgene expression and limited T-body engraftment. However, in a study of metastatic renal cell carcinoma using a T-body directed against carbonic anhydrase IX [55], T-body-expressing cells were detectable in the peripheral blood for nearly 2 months post-administration.
The major goals in the T-body field currently are to optimize their engraftment and maximize their effector functions. Our laboratory is addressing both problems simultaneously through an in-depth study of the requirements for T-body activation. We hypothesize that their limited persistence is due to incomplete cell activation due to the lack of costimulation. While naïve T cells depend on costimulation through CD28 ligation to avoid anergy and undergo full activation in response to antigen, it is recognized that effector cells also require costimulation to properly proliferate and produce cytokines [56]. Previous studies have shown that providing CD28 costimulation is crucial for the antitumoral function of adoptively transferred T cells and T-bodies [57–59]. Unlike conventional T cell activation, which requires two discrete signals, T-bodies can be engineered to provide both costimulation and CD3 signaling through one binding event.
A different approach for redirecting specificity to T cells for adoptive immunotherapy involves the genetic transfer of full-length TCR genes. A T cell’s specificity for its cognate antigen is solely determined by its TCR. Genes encoding the α and β chains of a T cell receptor (TCR) can be isolated from a T cell specific for the antigen of interest and restricted to a defined HLA allele, inserted into a vector, and then introduced into large numbers of T cells of individual patients that share the restricting HLA allele as well as the targeted antigen. In 1999, Clay and colleagues from Rosenberg’s group at the National Cancer Institute were the first to report the transfer of TCR genes via a retroviral vector into human lymphocytes and to show that T cells gained stable reactivity to MART-1 [67]. To date, many others have shown that the same approach can be used to transfer specificity for multiple viral and tumor associated antigens in mice and human systems. These T cells gain effector functions against the transferred TCR’s cognate antigen, as defined by proliferation, cytokine production, lysis of targets presenting the antigen, trafficking to tumor sites in vivo, and clearance of tumors and viral infection.
In 2006, Rosenberg’s group redirected patients’ PBLs with the naturally occurring, MART-1- specific TCR reported in 1999 by Clay. In the first clinical trial to test TCR-transfer immunotherapy, these modified T cells were infused into melanoma patients [68]. While the transduced T cells persisted in vivo, only two of the 17 patients had an objective response to this therapy. One issue revealed by the study was the poor expression of the transgenic TCRs by the transferred T cells. Nonetheless, the results from this trial showed the potential of TCR transfer immunotherapy as a safe form of therapy for cancer and highlighted the need to optimize such therapy to attain maximum potency.
The adoptive immunotherapy field is advancing by a tried-and-true method: learning from disappointments and moving forward. Our ability to fully realize the therapeutic potential of adoptive T cell therapy is tied to a more complete understanding of how human T cells receive signals, kill targets, and modulate effective immune responses. Our goal is to perform labbased experiments that provide insight into how primary T cells function in a manner that will facilitate and enable adoptive T cell therapy clinical trials. Our ability to efficiently modify (and expand) T cells ex vivo provides the opportunity to deliver sufficient immune firepower where it has heretofore been lacking. Sustained transgene expression, coupled with enhanced in vivo engraftment capability, will move adoptive immunotherapy into a realm where longterm therapeutic benefits are the norm rather than the exception.
Genetic Modification of T Lymphocytes for Adoptive Immunotherapy

Claudia Rossig1 and Malcolm K. Brenner2
Molecular Therapy (2004) 10, 5–18;   http://dx.doi.org:/10.1016/j.ymthe.2004.04.014      http://www.nature.com/mt/journal/v10/n1/full/mt20041193a.html

Adoptive transfer of T lymphocytes is a promising therapy for malignancies—particularly of the hemopoietic system—and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.

That gene transfer could be used to improve the effectiveness of T lymphocytes was apparent from the beginning of clinical studies in the field. T cells were the very first targets for genetic modification in human gene transfer experiments. Rosenberg’s group marked tumor-infiltrating lymphocytes ex vivo with a Moloney retroviral vector encoding neomycin phosphotransferase before reinfusing them and attempting to demonstrate selective accumulation at tumor sites. Shortly thereafter, Blaese and Anderson led a group that infused corrected T cells into two children with severe combined immunodeficiency due to ADA deficiency. While neither study was completely successful in terms of outcome, both showed the feasibility of ex vivo gene transfer into human cells and set the stage for many of the studies that followed. More recently, a second wave of interest in adoptive T cell therapies has developed, based on their success in the prevention and treatment of viral infections such as EBV and cytomegalovirus (CMV) and on their apparent ability to eradicate hematologic and perhaps solid malignancies1,2,3,4,5,6. There has been a corresponding increase in studies directed toward enhancing the antineoplastic and antiviral properties of the T cells. In this article we will review how gene transfer may be used to produce the desired improvements focusing on vectors and genes that have had clinical application.

Currently available viral and nonviral vector systems lack a pattern of biodistribution that would favor T cell transduction in vivo—as occurs, for example, with adenovectors and the liver or liposomal vectors and the lung. This lack of favorable biodistribution cannot yet be compensated for by the introduction of specific T-cell-targeting ligands into vectors. Hence, all T cell gene transfer studies conducted to date have used ex vivo transduction followed by adoptive transfer of gene-modified cells. This approach is inherently less attractive for commercial development than directin vivo gene transfer and has probably restricted interest in developing clinical applications using these cells. On the other hand, ex vivo transduction may be more readily controlled, characterized, and standardized than in vivo efforts and may ultimately produce a better defined final product (the transduced cell).

The gene products of suicide and coexpressed resistance genes are highly immunogenic and may induce immune-mediated rejection of the transduced cells. In one study, the persistence of adoptively transferred autologous CD8+ HIV-specific CTL clones modified to express the hygromycin phosphotransferase (Hy) gene and the herpesvirus thymidine kinase gene as a fusion gene was limited by the induction of a potent CD8+ class I MHC-restricted CTL response specific for epitopes derived from the Hy-tk protein126. Less immunogenic suicide and selection marker genes, preferably of human origin, may reduce the immunological inactivation of genetically modified donor lymphocytes. Human-derived prodrug-activating systems include the human folylpolyglutamate synthetase/methotrexate127, the deoxycytidine/cytosine arabinoside128, or the carboxylesterase/irinotecan129 systems. These systems do not activate nontoxic prodrugs but are based on enhancement of already potent chemotherapeutic agents. The administration of methotrexate to treat severe GVHD may not only kill transduced donor lymphocytes but may also have additional inhibitory activity on nontransduced but activated T cells.

Finally, endogenous proapoptotic molecules have been proposed as nonimmunogenic suicide genes. A chimeric protein that contains the FK506-binding protein FKBP12 linked to the intracellular domain of human Fas130 was recently introduced. Addition of the dimerizing prodrug induces Fas crosslinking with subsequent triggering of an apoptotic death signal.

Genetic engineering of T lymphocytes should help deliver on the promise of immunotherapies for cancer, infection, and autoimmune disease. Improvements in transduction, selection, and expansion techniques and the development of new viral vectors incapable of insertional mutagenesis will reduce the risks and further enhance the integration of T cell and gene therapies. Nonetheless, successful application of the proposed modifications to the clinical setting still requires many iterative studies to allow investigators to optimize the individual components of the approach.

Genetically modified T cells in cancer therapy: opportunities and challenges
Michaela Sharpe, Natalie Mount

 

The feasibility of T-cell adoptive transfer was first reported nearly 20 years ago (Walter et al., 1995) and the field of T-cell therapies is now poised for significant clinical advances. Recent clinical trial successes have been achieved through multiple small advances, improved understanding of immunology and emerging technologies. As the key challenges of T-cell avidity, persistence and ability to exert the desired anti-tumour effects as well as the identification of new target antigens are addressed, a broader clinical application of these therapies could be achieved. As the clinical data emerges, the challenge of making these therapies available to patients shifts to implementing robust, scalable and cost-effective manufacture and to the further evolution of the regulatory requirements to ensure an appropriate but proportionate system that is adapted to the characteristics of these innovative new medicines.

 

 

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Obesity Issues

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

The Changing Face of Obesity

Science tells us obesity is a chronic disease. Why does the outmoded and injurious notion that it is a problem of willpower persist?

By Joseph Proietto | November 1, 2015   http://www.the-scientist.com//?articles.view/articleNo/44288/title/The-Changing-Face-of-Obesity/

In Dante Alighieri’s Divine Comedy the narrator meets a man named Ciacco who had been sent to Hell for the “Damning sin of Gluttony.” According to Catholic theology, in order to end up in Hell one must willfully commit a serious sin. So Dante believed that fat people chose to be fat. This antiquated view of the cause of obesity is still widespread, even among medical professionals. The consequences of this misconception are significant, because it forms the basis for the discrimination suffered by the obese; for the wasting of scarce resources in attempts to change lifestyle habits by public education; and for the limited availability of subsidized obesity treatments.

http://www.the-scientist.com/November2015/critic1.jpg

While obesity is often labeled a lifestyle disease, poor lifestyle choices alone account for only a 6 to 8 kg weight gain. The body has a powerful negative feedback system to prevent excessive weight gain. The strongest inhibitor of hunger, the hormone leptin, is made by fat cells. A period of increased energy intake will result in fat deposition, which will increase leptin production. Leptin suppresses hunger and increases energy expenditure. This slows down weight gain. To become obese, it may be necessary to harbor a genetic difference that makes the individual resistant to the action of leptin.

Evidence from twin and adoption studies suggests that obesity has a genetic basis, and over the past two decades a number of genes associated with obesity have been described. The most common genetic defect in European populations leading to severe obesity is due to mutations in the gene coding for the melanocortin 4 receptor (MCR4). Still, this defect can explain severe obesity in only approximately 6 percent to 7 percent of cases (J Clin Invest, 106:271-79, 2000). Other genes have been discovered that can cause milder increases in weight; for example, variants of just one gene (FTO) can explain up to 3 kg of weight variation between individuals (Science, 316:889-94, 2007).

Genes do not directly cause weight gain. Rather, genes influence the desire for food and the feeling of satiety. In an environment with either poor access to food or access to only low-calorie food, obesity may not develop even in persons with a genetic predisposition. When there is an abundance of food and a sedentary lifestyle, however, an obesity-prone person will experience greater hunger and reduced satiety, increasing caloric intake and weight gain.

Since the 1980s, there has been a rapid rise in the prevalence of obesity worldwide, a trend that likely results from a variety of complex causes. There is increasing evidence, for example, that the development of obesity on individual or familial levels may be influenced by environmental experiences that occur in early life. For example, if a mother is malnourished during early pregnancy, this results in epigenetic changes to genes involved in the set points for hunger and satiety in the developing child. These changes may then become fixed, resulting in a tendency towards obesity in the offspring.

The biological basis of obesity is further highlighted by the vigorous defense of weight following weight loss. There are at least 10 circulating hormones that modulate hunger. Of these, only one has been confirmed as a hunger-inducing hormone (ghrelin), and it is made and released by the stomach. In contrast, nine hormones suppress hunger, including CCK, PYY, GLP-1, oxyntomodulin, and uroguanylin from the small bowel; leptin from fat cells; and insulin, amylin, and pancreatic polypeptide from the pancreas.

 

After weight loss, regardless of the diet employed, there are changes in circulating hormones involved in the regulation of body weight. Ghrelin levels tend to increase and levels of multiple appetite-suppressing hormones decrease. There is also a subjective increase in appetite. Researchers have shown that even after three years, these hormonal changes persist (NEJM, 365:1597-604, 2011; Lancet Diabetes and Endocrinology, 2:954-62, 2014). This explains why there is a high rate of weight regain after diet-induced weight loss.

Given that the physiological responses to weight loss predispose people to regain that weight, obesity must be considered a chronic disease. Data show that those who successfully maintain their weight after weight loss do so by remaining vigilant and constantly applying techniques to oppose weight regain. These techniques may involve strict diet and exercise practices and/or pharmacotherapy.

It is imperative for society to move away from a view that obesity is simply a lifestyle issue and to accept that it is a chronic disease. Such a change would not only relieve the stigma of obesity but would also empower politicians, scientists and clinicians to tackle the problem more effectively.

Joseph Proietto was the inaugural Sir Edward Dunlop Medical Research Foundation Professor of Medicine in the Department of Medicine, Austin Health at the University of Melbourne in Australia. He is a researcher and clinician investigating and treating obesity and type 2 diabetes.

 

 

A Weighty Anomaly

Why do some obese people actually experience health benefits?

By Jyoti Madhusoodanan | November 1, 2015     http://www.the-scientist.com//?articles.view/articleNo/44304/title/A-Weighty-Anomaly/

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THE ENDOCRINE THEORY: Some researchers have posited that fat cells may secrete molecules that affect glucose homeostasis in muscle or liver tissue.COURTESY OF MITCHELL LAZAR

In the early 19th century, Belgian mathematician Adolphe Quetelet was obsessed with a shape: the bell curve. While helping with a population census, Quetelet proposed that the spread of human traits such as height and weight followed this trend, also known as a Gaussian or normal distribution. On a quest to define a “normal man,” he showed that human height and weight data fell along his beloved bell curves, and in 1823 devised the “Quetelet Index”—more familiar to us today as the BMI, or body mass index, a ratio of weight to height.

Nearly two centuries later, clinicians, researchers, and fitness instructors continue to rely on this metric to pigeonhole people into categories: underweight, healthy, overweight, or obese. But Quetelet never intended the metric to serve as a way to define obesity. And now, a growing body of evidence suggests these categories fail to accurately reflect the health risks—or benefits—of being overweight.

Although there is considerable debate surrounding the prevalence of metabolically healthy obesity, when obesity is defined in terms of BMI (a BMI of 30 or higher), estimates suggest that about 10 percent of adults in the U.S. are obese yet metabolically healthy, while as many as 80 percent of those with a normal BMI may be metabolically unhealthy, with signs of insulin resistance and poor circulating lipid levels, even if they suffer no obvious ill effects. “If all we know about a person is that they have a certain body weight at a certain height, that’s not enough information to know their health risks from obesity,” says health-science researcher Paul McAuley of Winston-Salem State University. “We need better indicators of metabolic health.”

The dangers of being overweight, such as a higher risk of heart disease, type 2 diabetes, and other complications, are well known. But some obese individuals—dubbed the “fat fit”—appear to fare better on many measures of health when they’re heavier. Studies have found lower mortality rates, better response to hemodialysis in chronic kidney disease, and lower incidence of dementia in such people. Mortality, it’s been found, correlates with obesity in a U-shaped curve (J Sports Sci, 29:773-82, 2011). So does extra heft help or hurt?

To answer that question, researchers are trying to elucidate the metabolic reasons for this obesity paradox.

In a recent study, Harvard University epidemiologist Goodarz Danaei and his colleagues analyzed data from nine studies involving a total of more than 58,000 participants to tease apart how obesity and other well-known metabolic risk factors influence the risk of coronary heart disease. Controlling these other risk factors, such as hypertension or high cholesterol, with medication is simpler than curbing obesity itself, Danaei explains. “If you control a person’s obesity you get rid of some health risks, but if you control hypertension or diabetes, that also reduces health risks, and you can do the latter much more easily right now.”

Danaei’s team assessed BMI and metabolic markers such as systolic blood pressure, total serum cholesterol, and fasting blood glucose. The three metabolic markers only explained half of the increased risk of heart disease across all study participants. In obese individuals, the other half appeared to be mediated by fat itself, perhaps via inflammatory markers or other indirect mechanisms (Epidemiology, 26:153-62, 2015). While Danaei’s study was aimed at understanding how obesity hurts health, the results also uncovered unknown mechanisms by which excess adipose tissue might exert its effects. This particular study revealed obesity’s negative effects, but might these unknown mechanisms hold clues that explain the obesity paradox?

Other researchers have suggested additional possibilities—for example, that inflammatory markers such as TNF-α help combat conditions such as chronic kidney disease, or that obesity makes a body more capable of making changes to, and tolerating changes in, blood flow depending on systemic needs (Am J Clin Nutr, 81:543-54, 2005).

According to endocrinologist Mitchell Lazar at the University of Pennsylvania, the key to explaining the obesity paradox may be two nonexclusive ways fat tissue is hypothesized to function. One mechanism, termed the endocrine theory, suggests that fat cells secrete, or don’t secrete enough of, certain molecules that influence glucose homeostasis in other tissues, such as muscle or liver. The first such hormone to be discovered was leptin; later studies reported several other adipocyte-secreted factors, including adiponectin, resistin, and various cytokines.

The other hypothesis, dubbed the spillover theory, suggests that storing lipids in fat cells has some pluses. Adipose tissue might sequester fat-soluble endotoxins, and produce lipoproteins that can bind to and clear harmful lipids from circulation. When fat cells fill up, however, these endotoxins are stashed in the liver, pancreas, or other organs—and that’s when trouble begins. In “fat fit” people, problems typically linked to obesity such as high cholesterol or diabetes may be avoided simply because their adipocytes mop up more endotoxins.

“In this model, one could imagine that if you could store even more fat in fat cells, you could be even more obese, but you might be protected from problems [associated with] obesity because you’re protecting the other tissues from filling up with lipids that cause problems,” says Lazar. “This may be the most popular current model to explain the fat fit.”

Although obesity greatly increases the risk of type 2 diabetes—up to 93-fold in postmenopausal women, for example—not all obese people suffer from the condition. Similarly, a certain subtype of individuals with “normal” BMIs are at greater risk of developing insulin resistance and type 2 diabetes than others with BMIs in the same range. Precisely what distinguishes these two cohorts is still unclear. “Just as important as explaining why some obese people don’t get diabetes is to explain why other subgroups—normal-weight people or those with lipodystrophy—sometimes get it,” Lazar says. “If there are multiple subtypes of obesity and diabetes, can we figure out genetic aspects or biomarkers that cause one of these phenotypes and not the other?”

To Lazar, McAuley, and other researchers, it’s increasingly evident that BMI may not be that metric. Finding better ways to assess a healthy weight, however, has proven challenging. Researchers have tested measures, such as the body shape index (ABSI) or the waist-hip ratio, which attempt to gauge visceral fat—considered to be more metabolically harmful than fat in other body locations. However, these metrics have yet to be implemented widely in clinics, and few are as simple to understand as the BMI (Science, 341:856-58, 2013).

Independent of metrics, however, the health message regarding weight is still unanimous: exercise and healthy dietary choices benefit everyone. “At a certain point, despite all the so-called fit-fat people, the demographics say that there’s a huge risk of diabetes and heart disease at very high BMI,” notes Lazar. “We can’t assume we’ll be one of the lucky ones who will have a BMI in the obese category but will still be protected from heart disease.”

Correction (November 2): The original version of this article misattributed the pull quote above. The attribution for this quote has been corrected, and The Scientist regrets the error.

 

 

THE HEALTH RISK OF OBESITY—BETTER METRICS IMPERATIVE

 Science 23 Aug 2013;  341(6148): 856858     DOI: http://dx.doi.org:/10.1126/science.1241244
Obesity paradoxes.
In this review, we examine the original obesity paradox phenomenon (i.e. in cardiovascular disease populations, obese patients survive better), as well as three other related paradoxes (pre-obesity, “fat but fit” theory, and “healthy” obesity). An obesity paradox has been reported in a range of cardiovascular and non-cardiovascular conditions. Pre-obesity (defined as a body mass index of 25.0-29.9 kg · m⁻²) presents another paradox. Whereas “overweight” implies increased risk, it is in fact associated with decreased mortality risk compared with normal weight. Another paradox concerns the observation than when fitness is taken into account, the mortality risk associated with obesity is offset. The final paradox under consideration is the presence of a sizeable subset of obese individuals who are otherwise healthy. Consequently, a large segment of the overweight and obese population is not at increased risk for premature death. It appears therefore that low cardiorespiratory fitness and inactivity are a greater health threat than obesity, suggesting that more emphasis should be placed on increasing leisure time physical activity and cardiorespiratory fitness as the main strategy for reducing mortality risk in the broad population of overweight and obese adults.
Obesity, insulin resistance, and cardiovascular disease.
Recent Prog Horm Res. 2004;59:207-23.
The ability of insulin to stimulate glucose disposal varies more than six-fold in apparently healthy individuals. The one third of the population that is most insulin resistant is at greatly increased risk to develop cardiovascular disease (CVD), type 2 diabetes, hypertension, stroke, nonalcoholic fatty liver disease, polycystic ovary disease, and certain forms of cancer. Between 25-35% of the variability in insulin action is related to being overweight. The importance of the adverse effects of excess adiposity is apparent in light of the evidence that more than half of the adult population in the United States is classified as being overweight/obese, as defined by a body mass index greater than 25.0 kg/m(2). The current epidemic of overweight/obesity is most-likely related to a combination of increased caloric intake and decreased energy expenditure. In either instance, the fact that CVD risk is increased as individuals gain weight emphasizes the gravity of the health care dilemma posed by the explosive increase in the prevalence of overweight/obesity in the population at large. Given the enormity of the problem, it is necessary to differentiate between the CVD risk related to obesity per se, as distinct from the fact that the prevalence of insulin resistance and compensatory hyperinsulinemia are increased in overweight/obese individuals. Although the majority of individuals in the general population that can be considered insulin resistant are also overweight/obese, not all overweight/obese persons are insulin resistant. Furthermore, the cluster of abnormalities associated with insulin resistance – namely, glucose intolerance, hyperinsulinemia, dyslipidemia, and elevated plasma C-reactive protein concentrations — is limited to the subset of overweight/obese individuals that are also insulin resistant. Of greater clinical relevance is the fact that significant improvement in these metabolic abnormalities following weight loss is seen only in the subset of overweight/obese individuals that are also insulin resistant. In view of the large number of overweight/obese subjects at potential risk to be insulin resistant/hyperinsulinemic (and at increased CVD risk), and the difficulty in achieving weight loss, it seems essential to identify those overweight/obese individuals who are also insulin resistant and will benefit the most from weight loss, then target this population for the most-intensive efforts to bring about weight loss.
Long-Term Persistence of Hormonal Adaptations to Weight Loss

Priya Sumithran, Luke A. Prendergast, Elizabeth Delbridge, Katrina Purcell, Arthur Shulkes, Adamandia Kriketos, and Joseph Proietto

N Engl J Med 2011; 365:1597-1604   October 27, 2011http://dx.doi.org:/10.1056/NEJMoa1105816

After weight loss, changes in the circulating levels of several peripheral hormones involved in the homeostatic regulation of body weight occur. Whether these changes are transient or persist over time may be important for an understanding of the reasons behind the high rate of weight regain after diet-induced weight loss.

Weight loss (mean [±SE], 13.5±0.5 kg) led to significant reductions in levels of leptin, peptide YY, cholecystokinin, insulin (P<0.001 for all comparisons), and amylin (P=0.002) and to increases in levels of ghrelin (P<0.001), gastric inhibitory polypeptide (P=0.004), and pancreatic polypeptide (P=0.008). There was also a significant increase in subjective appetite (P<0.001). One year after the initial weight loss, there were still significant differences from baseline in the mean levels of leptin (P<0.001), peptide YY (P<0.001), cholecystokinin (P=0.04), insulin (P=0.01), ghrelin (P<0.001), gastric inhibitory polypeptide (P<0.001), and pancreatic polypeptide (P=0.002), as well as hunger (P<0.001).

What’s new in endocrinology and diabetes mellitus

Large genome wide association studies have demonstrated that variants in the FTO gene have the strongest association with obesity risk in the general population, but the mechanism of the association has been unclear. However, a nonocoding causal variant in FTO has now been identified that changes the function of adipocytes from energy utilization (beige fat) to energy storage (white fat) with a fivefold decrease in mitochondrial thermogenesis [17]. When the effect of the variant was blocked in genetically engineered mice, thermogenesis increased and weight gain did not occur, despite eating a high-fat diet. Blocking the gene’s effect in human adipocytes also increased energy utilization. This observation has important implications for potential new anti-obesity drugs. (See “Pathogenesis of obesity”, section on ‘FTO variants’.)

Liraglutide for the treatment of obesity (July 2015)

Along with diet, exercise, and behavior modification, drug therapy may be a helpful component of treatment for select patients who are overweight or obese. Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist, used for the treatment of type 2 diabetes, and can promote weight loss in patients with diabetes, as well as those without diabetes.

In a randomized trial in nondiabetic patients who had a body mass index (BMI) of ≥30 kg/m2 or ≥27 kg/m2 with dyslipidemia and/or hypertension, liraglutide 3 mg once daily, compared with placebo, resulted in greater mean weight loss (-8.0 versus -2.6 kg with placebo) [18]. In addition, cardiometabolic risk factors, glycated hemoglobin (A1C), and quality of life improved modestly. Gastrointestinal side effects transiently affected at least 40 percent of the liraglutide group and were the most common reason for withdrawal (6.4 percent). Liraglutide is an option for select overweight or obese patients, although gastrointestinal side effects (nausea, vomiting) and the need for a daily injection may limit the use of this drug. (See “Obesity in adults: Drug therapy”, section on ‘Liraglutide’.)

In a trial designed specifically to evaluate the effect of liraglutide on weight loss in overweight or obese patients with type 2 diabetes (mean weight 106 kg), liraglutide, compared with placebo, resulted in greater mean weight loss (-6.4 kg and -5.0 kg for liraglutide 3 mg and 1.8 mg, respectively, versus -2.2 kg for placebo) [19]. Treatment with liraglutide was associated with better glycemic control, a reduction in the use of oral hypoglycemic agents, and a reduction in systolic blood pressure. Although liraglutide is not considered as initial therapy for the majority of patients with type 2 diabetes, it is an option for select overweight or obese patients with type 2 diabetes who fail initial therapy with lifestyle intervention and metformin.  (See “Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes mellitus”, section on ‘Weight loss’.)

The Skinny on Fat Cells

Bruce Spiegelman has spent his career at the forefront of adipocyte differentiation and metabolism.

By Anna Azvolinsky | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44312/title/The-Skinny-on-Fat-Cells/

Bruce Spiegelman
Stanley J. Korsmeyer Professor of Cell Biology
and Medicine
Harvard Medical School
Director, Center for Energy Metabolism
and Chronic
Disease, Dana-Farber Cancer Institute, Boston

It’s hard to know whether you have the right stuff to be a scientist, but I had a passion for the research,” says Bruce Spiegelman, professor of cell biology at Harvard Medical School and the Dana-Farber Cancer Institute. After receiving his PhD in biochemistry from Princeton University in 1978, Spiegelman sent an application to do postdoctoral research to just one lab. “I wasn’t thinking I should apply to five different labs. I just marched forward more or less in a straight line,” he says. Spiegelman did know that he had no financial backup and depended on research fellowships throughout the early phase of his science career. “I thought it was fantastic, and still think so, that a PhD in science is supported by the government. I certainly appreciated that, because many of my friends in the humanities had to support themselves by cobbling together fellowships and teaching every semester, whereas we didn’t face similar challenges in the sciences.”

Since his graduate student days, Spiegelman has realized his potential, pioneering the study of adipose tissue biology and metabolism. He was introduced to the field in Howard Green’s laboratory, then at MIT, where Spiegelman began his one and only postdoc in 1978. Green had recently developed a system for culturing adipose cells and asked Spiegelman if he wanted to study fat cell differentiation. “I knew nothing about adipose tissue, but I was really interested in any model of how one cell switches to another. Whether skin or fat didn’t matter too much to me, because I was not coming at this from the perspective of physiology but from the perspective of how do these switches work at a molecular level?”

Spiegelman has stuck with studying the biology and differentiation of fat cells for more than 30 years. While looking for the master transcriptional regulator of fat development—which his laboratory found in 1994—Spiegelman’s group also discovered one of the first examples of a nuclear oncogene that functions as a transcription factor, and, more recently, the team found that brown fat and white fat come from completely different origins and that brown and beige fat are distinct cell types. Spiegelman was also the first to provide evidence for the connection between inflammation, insulin resistance, and fat tissue.

Here, Spiegelman talks about his strong affinity for the East Coast, his laboratory’s search for molecules that can crank up brown fat production and activity, and the culture of his laboratory’s weekly meeting.

Spiegelman Sets Out

First publication. Spiegelman grew up in Massapequa, New York, a town on Long Island. “Birds, insects, fish, and animals were fascinating to me. As a kid, I imagined I would be a wildlife ranger,” he says. Spiegelman and his brother were the first in their family to attend college; Spiegelman entered the College of William and Mary in 1970 thinking he would major in psychology. But before taking his first psychology course, he had to take a biology course, really loved it, and switched his major. For his senior thesis, he chose one of the few labs that did biochemistry-related research. He studied cultures of the filamentous fungus Aspergillus ornatus in which he induced the upregulation of a metabolic enzyme. Spiegelman applied a calculus transformation that related the age of the culture to the age of individual cells, something that had not been previously done. The work earned him his first first-author publication in 1975. “It was not a great breakthrough, but I think it showed that I was maybe applying myself more than the typical undergraduate.”

Full steam ahead. “My interest in laboratory research was intense. Even though it was not particularly inspired work, the first-author publication in a college where not many of the professors published a lot gave me a lot of confidence. It was probably out of proportion to the quality of the actual work.” That confidence and Spiegelman’s interest in the chemistry of living things led him to pursue a PhD in biochemistry at Princeton University. “Very early on, I felt that I couldn’t understand biology if it didn’t go to the molecular level. To me, just describing how an animal lived without understanding how it worked was very unsatisfying. I think it was one of the best decisions that I made in my life, to do a PhD in biochemistry,” he says, “because if you really want to understand living systems, you are very limited in how you can understand them without having a strong background in biochemistry because these are, essentially, chemical systems.”

Embracing molecular biology. Spiegelman initially joined Arthur Pardee’s laboratory, but switched when Pardee left Princeton for Harvard University in 1975. Because he was already collaborating with Marc Kirschner, a cell biologist and biochemist who studies the regulation of the cell cycle and how the cytoskeleton works, it was an easy transition to transfer to the new laboratory. In Kirschner’s group, Spiegelman became the cell biologist among many protein biochemists working on microtubule assembly in vitro. Rather than understanding how the proteins fit together to form the filamentous structures, Spiegelman wanted to understand what controlled their assembly inside cells. Working in mammalian cells, Spiegelman published three consecutive Cell papers on how microtubule assembly occurs in vivo. The firstpaper, from 1977, demonstrated that a nucleotide functions to stabilize the tubulin molecule rather than to regulate tubulin assembly in vivo.

Spiegelman Simmers

A new tool. For his next move, Spiegelman wanted to marry his background in biochemistry and molecular biology with a good cellular model system. He became interested in differentiation at the end of his PhD, while studying how the cytoskeleton is reorganized during neural differentiation, and settled on Green’s MIT laboratory for his postdoc. Green had developed a way to study both skin and fat cell differentiation. Again, Spiegelman was the odd man out, working on the molecular biology of fat cell differentiation while most of the graduate students and postdocs focused on the cellular biology of skin cell differentiation. While there, Spiegelman learned how to clone cDNA—a new method that some researchers thought was just another new fad, he says. “I thought it was pretty obvious that this was a tool that would be a game changer. I could see how I could clone some of the cDNAs and genes that were regulated in the fat cell lineage and then try to understand the regulation of these genes.”

Setting the stage. Spiegelman demonstrated that cAMP regulates the synthesis of certain enzymes in fat cells during differentiation. But while this was the most influential paper from his postdoc, says Spiegelman, it was his demonstration of cloning mRNAs from adipocytes, published in 1983, that set the stage for cloning fat-selective genes. The work, mostly done when Spiegelman was already a new faculty member at the Dana-Farber Cancer Institute, stemmed from his learning molecular cloning in Phillip Sharp’s lab at MIT and Bryan Roberts’s lab at Harvard. “This was the raw material from which we eventually cloned PPARγ and showed it to be the master regulator of fat [cell] development.”

Roots. Spiegelman became an assistant professor at the Harvard Medical School in 1982, when he was not yet 30. Although he had entertained the idea of moving to the West Coast with his wife, whom he had met at Princeton where she obtained a PhD in French literature, Spiegelman says he is really an East Coaster at heart. “My wife and I came to love Boston and were very comfortable there. Our families were both in New York, which was close, but not too close, and we really enjoyed the culture and pace of Boston; it was more ‘us.’ We really liked to visit California but didn’t particularly want to move there. We’re both real Northeastern people.”

Relating to Sisyphus. The transition from doing a postdoc to setting up his own laboratory was “very exciting and terribly stressful,” says Spiegelman. “When I think back, I always tried to be professional with my laboratory, but I was so stressed at suddenly being on my own with no management training.” The people resources he had encountered in his graduate and postdoctoral training labs were also not there yet, and he says his first publication as a principal investigator was like pushing a rock up a hill. But eventually, Spiegelman’s lab built a reputation and reached a critical mass of talented people who advanced the science. Again in 1983, Spiegelman produced a publication showing that morphological manipulation can affect gene expression and adipose differentiation.

End goal. Spiegelman’s goal was to find a master molecule that  orchestrates the conversion of adipocyte precursor cells into bona fide fat cells. Piece by piece, his lab identified the enhancers, promoters, and other regulatory elements involved in adipocyte differentiation. In 1994, graduate student Peter Tontonoz finallyfound that the PPARγ gene, inserted via a retroviral vector into fibroblasts, could induce the cells to become adipose cells. “It took 10 years,” Spiegelman says. Along the way, the laboratory found that c-fos, the product of a famous nuclear oncogene, bound to the promoters of fat-specific genes and worked as a transcription factor. “It was not really known how nuclear oncogenes worked. This was one of the first papers showing that these oncogenes bound to gene promoters and were transcription factors.”

A wider scope. In 1993, graduate student Gökhan Hotamisligil found that tumor necrosis factor-alpha(TNF-α), is induced in the fat tissue of rodent models of obesity and diabetes. The paper sparked the formation of the field of immunometabolism and resulted in the expansion of Spiegelman’s lab into the physiology arena, partly thanks to the guidance of C. Ronald Kahn and Jeff Flier, who both study metabolism and diabetes. But the work initially encountered pushback, says Spiegelman, partly because it was the merging of two fields.

Spiegelman Scales Up

Fat color palette. Brown fat tissue, abundant in infants but scarce in adults, is a metabolically active form of fat that is chock full of mitochondria and is found in pockets in the body distinct from white fat tissue.Pere Puigserver, then a postdoc in Spiegelman’s lab, found that the coactivator PCG-1, binding to PPARγ and other nuclear receptors, could stimulate mitochondrial biogenesis. The PCG-1 gene is turned on by stimuli such as exercise or a cold environment. Later, postdoc Patrick Seale, Spiegelman, and their colleagues showed brown fat cells derive from the same lineage that gives rise to skeletal muscle. “This was a big surprise, maybe the biggest surprise we ever uncovered in the lab,” says Spiegelman.

A paler shade of brown. More recently, in 2012, Spiegelman’s laboratory showed that within adult white adipose tissue, there are pockets of a yet another type of fat tissue that he called beige fat. “I think the evidence is very good from rodents that if you activate brown and beige fat, you get metabolic benefit both in obesity and diabetes. So the question now is: Can that be done in humans in a way that’s beneficial and not toxic?”  The lab is now looking to identify molecules that can either ramp up the activity of brown and beige fat or increase the production of both cell types as possible therapeutics for metabolic disorders or even cancer-associated cachexia. “Anyone who says that either approach will work better is being foolish. We just don’t know enough to go after just one or the other.”

On the irisin controversy. After reporting in 2012 that a muscle-related hormone called irisin could switch white fat to metabolically active brown fat, Spiegelman became embroiled in a media-covered debate about whether the molecule really exists; he was also the victim of a potential fraud plot. Most recently, Spiegelman provided thorough evidence that irisin does in fact exist. On the controversy, he says it’s a fine line between defending his scientific integrity and not adding more fuel to the fire or engaging with his harassers. “We have a long track record of doing credible and reproducible science and it was not that complicated to address the paper that claimed irisin was ‘a myth.’ That study used very outmoded scientific approaches.”

Raw talent. Many of Spiegelman’s trainees have gone on to become very successful scientists, including Tontonoz, Hotamisligil, Evan Rosen, and Randy Johnson. “It’s a quantum change in the experience of doing science when you get people who have their own visions. I would have thought that interacting with smart people would mainly help me get my scientific vision accomplished. And that was partly true, but also it changed my vision. When you have people challenging you on a day-to-day basis, you learn from them through the questions they ask and the way they challenge you in a constructive way. They made me a much better scientist.”

Rigorous mentorship.  “I feel very passionately that a major part of my job is to prepare the next generation of scientists. Everyone who comes through my lab will tell you that I take that very seriously. We make sure my students give a lot of talks and get critical assessments of their presentations to our lab group. I am very hands-on both scientifically and in developing the way students project their vision. I had a very good mentor, Marc Kirschner, and I’d like to think that I learned how to be a mentor from him. I want to make sure that when people walk out of my lab they are prepared to run independent research programs.”

Greatest Hits

  • Identified the master regulator of adipogenesis, the nuclear receptor PPARγ
  • Was the first to show that a nuclear oncogene, c-fos, codes for a transcription factor that binds to the promoters of genes
  • Demonstrated that adipose tissue synthesizes tumor necrosis factor-alpha (TNF-α), providing the first direct link between obesity, inflammation, insulin resistance, and fat tissue.
  • Showed that brown fat cells are not developmentally related to white fat
  • Identified beige fat as a distinct cell type, different from either white or brown fat

 

Fanning the Flames

Obesity triggers a fatty acid synthesis pathway, which in turn helps drive T cell differentiation and inflammation.

By Kate Yandell | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44306/title/Fanning-the-Flames/

EDITOR’S CHOICE IN IMMUNOLOGY

The paper
Y. Endo et al., “Obesity drives Th17 cell differentiation by inducing the lipid metabolic kinase, ACC1,” Cell Reports, 12:1042-55, 2015.

Cell Rep. 2015 Aug 11;12(6):1042-55.   http://dx.doi.org:/10.1016/j.celrep.2015.07.014. Epub 2015 Jul 30.
Obesity Drives Th17 Cell Differentiation by Inducing the Lipid Metabolic Kinase, ACC1.
  • A high-fat diet augments Th17 cell development and the expression of Acaca
  • ACC1 controls Th17 cell development in vitro and Th17 cell pathogenicity in vivo
  • ACC1 modulates RORγt function in developing Th17 cells
  • Obesity in humans induces ACACA and IL-17A expression in CD4 T cells

Chronic inflammation due to obesity contributes to the development of metabolic diseases, autoimmune diseases, and cancer. Reciprocal interactions between metabolic systems and immune cells have pivotal roles in the pathogenesis of obesity-associated diseases, although the mechanisms regulating obesity-associated inflammatory diseases are still unclear. In the present study, we performed transcriptional profiling of memory phenotype CD4 T cells in high-fat-fed mice and identified acetyl-CoA carboxylase 1 (ACC1, the gene product of Acaca) as an essential regulator of Th17 cell differentiation in vitro and of the pathogenicity of Th17 cells in vivo. ACC1 modulates the DNA binding of RORγt to target genes in differentiating Th17 cells. In addition, we found a strong correlation between IL-17A-producing CD45RO(+)CD4 T cells and the expression of ACACA in obese subjects. Thus, ACC1 confers the appropriate function of RORγt through fatty acid synthesis and regulates the obesity-related pathology of Th17 cells.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2035221719/2050630604/fx1.jpg

 

 

http://www.the-scientist.com/November2015/NovMediLit_310px.jpg

FEEDING INFLAMMATION: When mice eat a diet high in fat, their CD4 T cells show increased expression of the fatty acid biosynthesis gene Acaca, which encodes the enzyme ACC1 (1). Products of the ACC1 fatty acid synthesis pathway encourage the transcription factor RORγt to bind near the gene encoding the cytokine IL-17A (2). There, RORγt recruits an enzyme called p300 to modify the genome epigenetically and turn on IL-17A. The memory T cells then differentiate into inflammatory T helper 17 cells.
See full infographic: PDF
© STEVE GRAEPEL

Obesity often comes with a side of chronic inflammation, causing inflammatory chemicals and immune cells to flood adipose tissue, the hypothalamus, the liver, and other areas of the body. Inflammation is a big part of what makes obesity such an unhealthy condition, contributing to Type 2 diabetes, heart disease, cancers, autoimmune disorders, and possibly even neurodegenerative diseases.

To better understand the relationship between obesity and inflammation, Toshinori Nakayama, Yusuke Endo, and their colleagues at Chiba University in Japan started with what often leads to obesity: a high-fat diet. They fed mice rich meals for a couple of months and looked at how gene expression in the animals’ T cells compared to gene expression in the T cells of mice fed a normal diet. Most notably, they found increased expression ofAcaca, a gene that codes for a fatty acid synthesis enzyme called acetyl coA carboxylase 1 (ACC1). They went on to show that the resulting increase in fatty acid levels pushed CD4 T cells to differentiate into inflammatory T helper 17 (Th17) cells.

Th17 cells help fight off invading fungi and some bacteria. But these immune cells can also spin out of control in autoimmune diseases such as multiple sclerosis. Nakayama’s team showed that either blocking ACC1 activity with a drug called TOFA or deleting a key portion of Acaca in mouse CD4 T cells reduced the generation of pathologic Th17 cells. Overexpressing Acaca increased Th17-cell generation.

The researchers also demonstrated that mice fed a high-fat diet had elevated susceptibility to a multiple sclerosis–like disease, and that TOFA reduced the symptoms.

“This is a very intriguing finding, suggesting not only that obesity can directly induce Th17 differentiation but also indicating that pharmacologic targeting of fatty acid synthesis may help to interfere with obesity-associated inflammation,” Tim Sparwasser of the Twincore Center for Experimental and Clinical Infection Research in Hannover, Germany, says in an email. Sparwasser and his colleagues had previously shown that ACC1 is required for the differentiation of Th17 cells in mice and humans.

Nakayama explains that CD4 T cells must undergo profound metabolic changes as they mature and differentiate. “The intracellular metabolites, including fatty acids, are essential for cell proliferation and cell growth,” he says in an email. When fatty acid levels in T cells increase, the cells are activated and begin to proliferate.

“It’s a nice illustration of how, really, immune response is so highly connected to the metabolic state of the cell,” says Gökhan S. Hotamisligil of Harvard University’s T.H. Chan School of Public Health who was not involved in the study. “The immune system launches its responses commensurate with the sources of nutrients and energy from the environment,” he adds in an email.

There are still missing pieces in the path from high-fat diet to increased Acaca expression to ACC1’s influence on T-cell differentiation. It also remains to be seen how this plays out in obese humans, although Nakayama and colleagues did show that inhibiting ACC1 reduced pathologic Th17 generation in human immune cell cultures, and that the T cells of obese humans contain elevated levels of ACC1 and show signs of increased differentiation into Th17 cells.

 

The prevalence of obesity has been increasing worldwide, and obesity is now a major public health problem in most developed countries (Gregor and Hotamisligil, 2011, Ng et al., 2014). Obesity-induced inflammation contributes to the development of various chronic diseases, such as autoimmune diseases, metabolic diseases, and cancer (Kanneganti and Dixit, 2012, Kim et al., 2014,Osborn and Olefsky, 2012, Winer et al., 2009a). A number of studies have pointed out the importance of reciprocal interactions between metabolic systems and immune cells in the pathogenesis of obesity-associated diseases (Kaminski and Randall, 2010, Kanneganti and Dixit, 2012, Kim et al., 2014, Mauer et al., 2014, Stienstra et al., 2012, Winer et al., 2011).

Elucidating the molecular mechanisms by which naive CD4 T cells differentiate into effector T cells is crucial for understanding helper T (Th) cell-mediated immune pathogenicity. After antigen stimulation, naive CD4 T cells differentiate into at least four distinct Th cell subsets: Th1, Th2, Th17, and inducible regulatory T (iTreg) cells (O’Shea and Paul, 2010, Reiner, 2007). Several specific master transcription factors that regulate Th1/Th2/Th17/iTreg cell differentiation have been identified, including T-bet for Th1 (Szabo et al., 2000), GATA3 (Yamashita et al., 2004, Zheng and Flavell, 1997) for Th2, retinoic-acid-receptor-related orphan receptor γt (RORγt) for Th17 (Ivanov et al., 2006), and forkhead box protein 3 (Foxp3) for iTreg (Sakaguchi et al., 2008). The appropriate expression and function of these transcription factors is essential for proper immune regulation by each Th cell subset.

Among these Th cell subsets, Th17 cells contribute to the host defense against fungi and extracellular bacteria (Milner et al., 2008). However, the pathogenicity of IL-17-producing T cells has been recognized in various autoimmune diseases, including multiple sclerosis, psoriasis, inflammatory bowel diseases, and steroid-resistant asthma (Bettelli et al., 2006, Coccia et al., 2012, Ivanov et al., 2006,Leonardi et al., 2012, McGeachy and Cua, 2008, Nylander and Hafler, 2012,Stockinger et al., 2007, Sundrud et al., 2009).

An HFD Promotes Th17 Cell Differentiation and Affects the Expression of Fatty Acid Enzymes in Memory CD4 T Cells In Vivo

Inhibition of ACC1 Function Results in Decreased Th17 Cell Differentiation and Ameliorates the Development of Autoimmune Disease

ACC1 Controls the Differentiation of Th17 Cells Both In Vitro and In Vivo

ACC1 Controls the Function, but Not Expression, of RORγt in Differentiating Th17 Cells

Extrinsic Fatty Acid Supplementation Restored Acaca−/− Th17 Cell Differentiation through the Functional Improvement of RORγt

Obese Subjects Show Upregulation of ACACA and Increased Th17 Cells in CD45RO+ Memory CD4 T Cells

We herein identified a critical role that ACC1 plays in Th17 cell differentiation and the pathogenicity of Th17 cells through the control of the RORγt function under obese circumstances. High-fat-induced obesity augments Th17 cell differentiation and the expression of enzymes involved in fatty acid metabolism, including ACC1. Pharmacological inhibition or genetic deletion of ACC1 resulted in impaired Th17 cell differentiation in both mice and humans. In contrast, overexpression of Acaca induced Th17 cells in vivo, leaving the expression ofIfng and Il4 largely unchanged. ACC1 modulated the binding of RORγt to theIl17a gene and the subsequent p300 recruitment in differentiating Th17 cells. Memory CD4 T cells from peripheral blood mononuclear cells (PBMCs) of obese subjects showed increased IL-17A production and ACACA expression. Furthermore, a strong correlation was detected between the proportion of IL-17A-producing cells and the expression level of ACACA in memory CD4 T cells in obese subjects. Thus, our findings provide evidence of a mechanism wherein obesity can exacerbate IL-17-mediated pathology via the induction of ACC1.

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Abstract:

The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review, the  properties of IDO such as basic molecular genetics, biochemistry and genesis will be discussed.

IDO belongs to globin gene family to carry oxygen and heme.  The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn.

The well understood functional genomics and mechanisms is important to translate basic science for clinical interventions of human health needs. In conclusion, overall purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

The first part of the review concerns the basic science information gained overall several years that lay the foundation where translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

Table of Contents:

  • Abstract

1         Introduction: IDO gene encodes a heme enzyme

2        Location, location, location

3        Molecular genetics

4        Types of IDO:

4.1       IDO1,

4.2       IDO2,

4.3       IDO-like proteins

5        Working mechanisms of IDO

6        Infection Diseases and IDO

7. Conclusion

  1. 1.     Indoleamine 2, 3-dioxygenase (IDO) gene encodes a heme enzyme

IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC=1.13.11.52) that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin.

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.

After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

  1. 2.     Location, Location and Location

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).

IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).

 

 3.     Molecular Genetics of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.
(reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).  Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).

Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).

The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

4.     There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).

There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

4.1.  IDO1:

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).  The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms. Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

4.2. IDO2:

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).   Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46).

Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

4.3. IDO-like proteins: The Origin of IDO:

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.

Bacterial IDO has two types of IDOs that are group I and group II IDO (48).  These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).   Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.

Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins are destined to evolve from a common ancestor, which  is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52) IDO like globin widely distributed among gastropodic mollusks (53; 51).  The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).

The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53) Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.

No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

Mechanisms of IDO:

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

 IDO mechanism for immune response

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63).

Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

 Indoleamine-Pyrrole 2,3,-Dioxygenase; IDO dioxygenase; Indeolamine-2,3

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response. When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).

Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68). SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).

This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91).

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103).

Utilizing specific TLR stimulation to link between innate and acquired responses can be possible through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105).  Double stranded (ds) RNAs through TLR3 (106; 107), Lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

IDO action

Then, the specificity is established by correct pairing of a TLR with its proinflammatory cytokines, so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, if the DCs are stimulated with IL-6, DCs relieve the suppression of effector T cells by regulatory T cells (113).

The modification of IDO+ monocytes manage towards specific subset of T cell activation with specific TLRs are significantly important (94).

The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) and CMV was possible, but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys since single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These data show that TLRs has ligand specificity, signal transduction pathways, expression profiles and cellular localization so design of experiments should follow these rules.

Conclusion:

Overall our purpose of this information is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.  This first part of the review concerns the basic science information gained overall several years that lay the foundation that translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

References

1. Biochemistry of tryptophan in health and disease. BenderDA. 1983, Mol Aspects Med , pp. 6:101–197.

2. Molecular insights into substrate recognition and catalysis by indolamine 2,3-dioxygenase. Forouhar, F., Anderson, R., Mowat, C.F, et al. 2006, PNAS, pp. vol. 104, no:2, 473-478.

3. Importance of the Two Interferon-stimulated Response Element. Konan KV, Taylor, MW. 1996, J. Biol. Chem.-, pp. 19140-5.

4. induction of indolamine 2,3 dioxygenase: A mechanism of the anti-tumor activity of interferon gamma. Ozaki, Y., Edelstein, M.P., Duch, D.S. 1998, PNAS USA., pp. vol:85, 1242-1246.

5. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8. . Burkin, D. J., Kimbro, K. S., Barr, B. L., Jones, C., Taylor, M. W., Gupta, S. L. 1993, Genomics , pp. 17: 262-263.

6. Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-p11 by fluorescent in situ hybridization. Najfeld, V., Menninger, J., Muhleman, D., Comings, D. E., Gupta, S. L. 1993, Cytogenet. Cell Genet. , pp. 64: 231-232.

7. Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA. . Dai, W., Gupta, S. L. 1990, Biochem. Biophys. Res. Commun. , pp. 168: 1-8.

8. Gene structure of human indoleamine 2,3-dioxygenase. Kadoya, A., Tone, S., Maeda, H., Minatogawa, Y., Kido, R. 1992, Biochem. Biophys. Res. Commun. , pp. 189: 530-536.

9. A gene atlas of th emouse and human protein-encoding transcriptomes. Andrew I. Su, Tim Wiltshire, Serge Batalov , Hilmar Lapp , Keith A. Ching , David Block, Jie Zhang , Richard Soden , Mimi Hayakawa , Gabriel Kreiman , Michael P. Cooke , John R. Walker , and John B. Hogenesch. 2004, PNAS, pp. vol. 101, no. 166062-6067 (10.1073/pnas.0400782101).

10. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. 2000, J. Immunol, pp. 164:3596–3599.

11. Inhibition of T cell proliferation by acrophage tryptophan catabolism. Munn, D.H. et al. 1999, J. Exp. Med., p. 189:1363.

12. HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity. Logan, G. J., Smyth, C. M. F., Earl, J. W., Zaikina, I., Rowe, P. B., Smythe, J. A., Alexander, I. E. 2002, Immunology, pp. 105:478-487.

13. Indoleamine 2,3-Dioxygenase – Is It an Immun Suppressor? Soliman H, Mediaville-Varela M, Antonia S. 2010, Cancer J. , pp. 16:354-359.

14. Targeting the immunoregulatory indoleamine 2,3-dioxygenase pathway in immunotherapy. Johnson BA, III, Baban B, Mellor AL. 2009, Immunotherapy. , pp. 645–661.

15. Indoleamine 2,3-dioxygenase and regulation of T cell immunity. AL., Mellor. 2005, Biochem Biophys Res Commun. , pp. 338(1):20–24.

16. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C.Modulation of tryptophan catabolism by regulatory T cells. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C., Alegre, M.-L., Puccetti, P. 2003, Nature Immun., pp. 4: 1206-1212.

17. CTLA-4-Ig regulates tryptophan catabolism in vivo. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni, A., Candeloro, P., Belladonna, M. L., Bianchi, R., Fioretti, M. C., Puccetti, P. 2002, Nature Immun. , pp. 3: 1097-1101.

18. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Grohmann, U., Volpi, C., Fallarino, F., Bozza, S., Bianchi, R., Vacca, C., Orabona, C., Belladonna, M. L., Ayroldi, E., Nocentini, G., Boon, L., Bistoni, F., Fioretti, M. C., Romani, L., Riccardi, C., Puccetti, P. 2007, Nature Med., pp. 13:579-586.

19. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. Mellor, A. L., Keskin, D. B., Johnson, T., Chandler, P., Munn, D. H. 2002, J. Immun. , pp. 168: 3771-3776.

20. Chon, SY, Hassanain, HH, Piine, R., and Gupta, SL. 1995, J. Interferon Cytokine Res. , pp. 15, 517-526.

21. Levy, ED, KEsler, DS, Pine, R., Reich, N, and Darnell, JE.Jr et al. 1988, Genes Dev, pp. 2,383-393.

22. Benoist, C. and Manthis, D. 1990, Annu. Rev of Immunol., pp. 8, 681-715.

23. Dorn, A, Durand, B., Marling, C., Meur, M.L., Beoist, C., and Mathis, D. 1987, PNAS USA, pp. 34, 6249-6253.

24. Konan, K.V. Ph.D. Thesis. Transcriptional Regulation of the Indolamine 2,3-oxygenase Gene. s.l. : Indiana University, Bloominigton, 1995.

25. Tryptophan pyrrolase of rabbit intestine: D- and L–tryptophan cleaving enzyme or enzymes. Yamamoto, S., and Hayashi, O. 1967, J Biol Chem, pp. 242: 5260-5266.

26. Prevention of allogeneic fetal rejection by tryptophan catabolism. Munn, DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. 1998, Science, pp. 281:1191–3.

27. Evidence for a tumoral immune resistance mechanismbased on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove, C. et al. 2003, Nature Med. 9,, pp. 1269–1274 .

28. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Raghupathy, R. 2001., Seminars in Immunology, pp. Volume 13, Issue 4, Pages 219–227.

29. Why is the fetal allograft not rejected? Davies, C. J. March 2007 , J ANIM SCI , pp. vol. 85 no. 13 suppl E32-E35 .

30. Exploring the mechanism of tryptoophan 2,3-dioxygenase. Thackray, S., Mowat, C.G., Chapman, K. 2008, Biochem. Society Transaction., pp. 36, 1120-1123.

31. The new life of a centenarian: signalling functions of NAD(P). Berger F, Ramírez-Hernández MH, Ziegler M. 2004, Trends Biochem Sci , pp. 29:111–118 .

32. Biochemistry of tryptophan in health and disease. DA, Bender. 1983, Mol Aspects Med, pp. 6:101–197.

33. Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain. Heyes MP, Saito K, Jacobowitz D, Markey SP, Takikawa O, Vickers JH. 1992, FASEB J., pp. 6:2977–2989.

34. Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH 1998Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. . Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH. 1998, Am J Pathol, pp. 152:611–619.

35. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. . Yoshida R, Hayaishi O. 1978, Proc Natl Acad Sci USA , pp. 75:3998–4000.

36. Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection. . Yoshida R, Urade Y, Tokuda M, Hayaishi O. 1979, Proc Natl Acad Sci USA , pp. 76:4084–4086.

37. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. Yoshida R, Hayaishi. 1978, PNAS USA, pp. 3998-4000.

38. Sequence of human 2,3-dioxygenase (TDO2): presence of a glucorticoid response-like element composed of a GTT repeat and intronic CCCCT repeat. Comings DE, Muhleman D, Dietz G, Sherman M, Forest. 1995, Genomics, pp. 29:390-396165.

39. Studies on the biosynthesis of Nicotinamide adenine inucleotide. II.Arole of picolinic carboxylase in the Biosynthesisofnicotinamideadeninedinucleotidefromtryptophan in mammals. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, HayaishiO. 1965, J. Biol. Chem. , pp. 240: 1395-1401.

40. The Secret Life of NAD+: An Old Metabolite Controlling New Metabolic Signaling Pathways. Houtkooper R.H., Carles Cantó C. , Wanders, R.J. and Auwerx, J. 2010, Endocrine Reviews , pp. vol. 31 no. 2 194-223, doi: 10.1210/er.2009-0026.

41. Stimulation of Nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. Sasaki Y, Araki T, Milbrandt J. 2006, J Neurosci , pp. 26: 8484–8491.

42. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Gale EA, Bingley PJ, Emmett CL, CollierT. 2004, Lancet., pp. 363:925–931.

43. Safety of high-dose nicotinamide: a review. Knip M, Douek IF, Moore WP, Gillmor HA, McLean AE, Bingley PJ, Gale EA. 2000, Diabetologia, pp. 43:1337–1345.

44. Large supplements of nicotinic acid and nicotinamide increase tissue NAD and poly(ADP-ribose) levels but do not affect diethylnitrosamine-induced altered hepatic foci in Fischer-344 rats. JacksonTM, Rawling JM, Roebuck BD, Kirkland JB. 1995, J Nutr , p. 125:1455.

45. Characterization and evolution of vertebrate indelamine 2,3-dihydrogenases IDOs from monotremes and marsupials. Yuasa, HJ, Ball, HJ, Ho, YF, Austin, CJ, et al. 2009, Comp. Biochem. Physiol. B. Biochem.. Mol. Biol., pp. 153 (2): 137-144.

46. Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indolamine 2,3-dihydrogenase inhibitor compound D-1 methyl-tryptophan. Metz, R., Duhadaway, JB, Kamasani, U, Laury-Kleintop, L., Muller, AJ, Prendergast, GC. 2007, Cancer Res., pp. 67 (15): 7082-7087.

47. Total synthesis of exiguamines A and B inspired by catechollamine chemistry. Sofiyev, V, Lumb, JP, Volgraf, M., Trauner, D. 2012, Chemistry., pp. 18 (16): 4999-5005.

48. Molecular evolution of bacterial indolamine 2,3-dioxygenase. Yuasa, H J, Ushigoe, A, Ball, HJ. 2011, Gene., pp. 484 (1) : 22-31.

49. Infectious tolerance and the long-term acceptance of transplant tissue. Waldman, H., Adams, E., Fairchild, P., and Cobbold, S. 2006, J. Immunol., pp. 212:301-313.

50. Molecular evolution and characterizationof fungal indolamine 2,3-dioxygenases. Yuasa, HJ and Ball, HJ. 2012, J. Mol. Eval., pp. 72 (2): 160-168.

51. convergent evolution. The gene structure of Sulculus 41 kDa myoglobin is homologous with tht of human indolamine dioxygenase. Suzuki, T, Imai, K. 1996, Biochim. Biophys. Acta., pp. 1308(1):41-48.

52. Evolutionof myoglobin. Suzuki, T., Imai, K. 1998, Cell Mol Life Sci, pp. 54(9):979-1004.

53. A myoglobin evolved from indolamine 2,3-dioxygenase, trtptophan-degrading enzyme. Suzuki, T., Kawamichi, H., Imai, K. 1998, Comp Biochem Phisiol. Mol. Biol., pp. 121(2):117-128.

54. Do molluscs possess indolamine 2,3-dioxygenase? Yuasa, HJ and Suzuki, T. 2005, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. , pp. (3) 445-454.

55. Comparison studies of the indolamine dioxygenase-like myoglobin from the abalone Sulculus diversicolor. Suzuki, T., Imai, K. 1997, Comp. Biohem. Phsiol B Biochem Mol Biol, pp. 117 (4)599-604.

56. Orchestration of the immune response by dendritic cells. Buckwalter MR, Albert ML. 2009, Curr Biol., pp. 19(9):355–361.

57. Dendritic cells and the control of immunity. Banchereau J, Steinman RM. 1998, Nature., pp. 245–52.

58. IDO expression by dendritic cells: tolerance and tryptophan catabolism. . Munn DH, Mellor AL. 2004, Nat Rev Immunol. , pp. 762–74.

59. Monocyte and Macrophage. Gordon, S. and Taylor, P.R. 2005, NATURE REVIEWS | IMMUNOLOGY , pp. vol:5, 953-964.

60. Blood monocytes consist of two principal subsets with distinct migratory properties. Geissmann F, Jung S, Littman DR. 2003, Immunity. , pp. 19:71–82.

61. Identification of a novel cell type in peripheral lymphoid organs of mice. I Morphology, quantitation, tissue distribution. . Steinman RM, Cohn ZA. 1973, J Exp Med., pp. 137(5):1142–1162.

62. T cell apoptosis by tryptophan catabolism. Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, Spreca A, Fioretti MC, Puccetti P. 2002, Cell Death Differ , pp. 9:1069–1077.

63. Kynurenine is a novel endothelium derived relaxing factor produced during inflammation. Wang, et al. 2010, Nat. Med., pp. 16(3): 279-285.

64. Activation of the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

65. B cells inhibit induction of T cell-dependent tumor immunity. Qin, Z., Richter, G., Schuler, T., Ibe, S., Cao, X, Blakenstein, T. 1998, Nat. Med, p. 4:627.

66. Different partners, Opposite Outcmes: A new perspective of immunobiology of Indolamine 2,3 dioxygenase. Orabona, C., Pallotta, M.T., Grohman, U. 2012, Molecular Medicine., pp. 18:834-842.

67. Indolamine 2,3-dioxygenase: From catalyst to signaling function. Fallarino, F., Grohman, U., and Puccetti, P. 2012, Eurepean J. of Immunol. , pp. 42:1932-1937.

68. IDO: more than an enzyme. Chen, W. 2011, Nature Immonology, pp. 809-811.

69. Indolamine2,3-dehydrogenase in lung dendritic cells promotes Th2 responses and allergic inflammation. Xu, H., Oriss, T.B., Fei, M., Henry, A.C., Melgert, B.N., Chen, L., Mellor, A.L. 2008, PNAS USA, pp. 105: 6690-6695.

70. The immunoregulatory enzyme IDO paradoxically drives B-cellmediated autoimmunity. Scott, G.N., DuHadaway, J., Pigott, E., Ridge, N., Prendergast, G.C., Muller, A.J., Mandik-Nayak, L. 2009, J. Immunol., pp. 182:7509-7517.

71. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. 2002, Immunology , pp. 107:452–460.

72. Enzymology of NAD+ homeostasis in man. . Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S. 2004, Cell Mol Life Sci , pp. 61:19–34.

73. Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO. . Belladonna ML, Grohmann U, Guidetti P, Volpi C, Bianchi R, Fioretti MC, Schwarcz R, Fallarino F, Puccetti P. 2006, J Immunol. , pp. ;177:130–7.

74. An indogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Opitz, et. al. 2011, pp. doi: 10.1038/nature10491,.

75. Inhibition of indoleamine 2,3-dioxygenase, animmunoregulatorytarget of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Muller, A. J. et al. 2005, Nature Med. , pp. 11, 312–319 .

76. TGF-b; a master of all T cell trades. Li, M.O., Fravell, R.A. 2008, Cell. , pp. 134: 392-404.

77. Palotta, M.T. et al. 2011, Nat. Immunol., pp. 12:870-878.

78. Chen, W. et al. 2003, J. Exp. Immunol., p. 198: 1875.

79. Smads: transcriptional activators of TGF-beta responses. . Derynck R, Zhang Y, Feng XH. 1998, Cell , pp. 95 (6): 737–40. doi:10.1016/S0092-8674(00)81696-7.PMID 9865691. .

80. Smad transcription factors. Massagué J, Seoane J, Wotton D. 2005, Genes Dev, pp. 19 (23): 2783–810. doi:10.1101/gad.1350705. PMID .

81. A structural basis for mutational inactivation of the tumour suppressor Smad4. Shi Y, Hata A, Lo RS, Massagué J, Pavletich NP. 1997, Nature., pp. 388 (6637): 87–93.doi:10.1038/40431. PMID 9214508.

82. Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. Itoh F, Asao H, Sugamura K, Heldin CH, ten Dijke P, Itoh S. 2001, EMBO J., pp. 20 (15): 4132– doi:10.1093/emboj/20.15.4132. PMC 149146. PMID 11483516.

83. SMAD_Signaling_Network. http://www.sabiosciences.com. [Online] 2013. http://www.sabiosciences.com/pathway.php?sn=SMAD_Signaling_Network.

84. Immune inhibitory receptors. Revetch, J.V., and Lanier, L.L. 2000, Science., pp. 290:84-89.

85. Soc3 drives proteasomal degradation of indolamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Orabona, C., Pallotta, M., Volpi, C., et al. 2008, PNAS USA, pp. 105: 20828-20833.

86. Cutting edge; silencing supressor of cytokine signaling3 expression in dendritic cells turns CD28-Ig from immune adjuvant to supressant. Orabona, C.,, Belladonna, M.L., et all. 2005, J. Immunol., pp. 174: 6582-6586.

87. Molecular signatures of T-cell inhibition in HIV-1 infection. Larsson, M., Shankar. E.M, Che, K.F., Ellegard, R., Barathan, M., Velu, V., and Kamarulzaman, A. 2013, Retrovirology, p. 10:31.

88. TGF-beta and CD4+CD25+ regulatory cells. Huber, S. and Schramn, C. 2006, Front. Bioscie., pp. 11:1014-1023.

89. Immune Escape as a fundemental trait of cancer; focus on IDO. Prendergast, G.C. 2008, Oncogene., pp. 27, 3889-3900.

90. Il-6 inhibits the tolerogenic functionof CD8+ dendritic cells expressing indolamine 2,3-dioxygenase. Grohman, U., Fallarino, F., et al. 2001, J. Immunol., pp. 167:708-714.

91. Avoiding horror autotoxicus: Th eimportance of dentritic cells in peripheral T cell tolerance. Steinman, R.M., and Nussenzweig, M.C. 2002, PNAS, pp. no:1, 351-358.

92. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice . Kaisho, T., Akira, S. 2001, Trends Immunol , pp. 22,78-83.

93. Innate sensing of self and non-self RNAs by Toll-like receptors. Sioud, M. 2006., Trends Mol Med., pp. 12:67–76.

94. Impaired expression of indoleamine 2, 3-dioxygenase in monocyte-derived dendritic cells in response to Toll-like receptor-7/8 ligands. Furset, G., Fløisand, Y. and Sioud, M. 2008, Immunology., pp. 123(2): 263–271, doi: 10.1111/j.1365-2567.2007.02695.x.

95. Toll-;ike receptor 9 mediated induction of the immunorepressor pathway of tryptophan metabolism. Fallarino, F., and Puccetti, P. 2006, Eur. J. of Imm., pp. 36:8-11.

96. Toll-like receptors and host defense against microbial pathogens: bringing specificity to the innate immune system. . Netea MG, der Graaf C, Van der Meer JWM, Kullberg BJ. 2004, J Leukoc Biol. , pp. 75:749–55.

97. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. . Heil F, Hemmi H, Hochrein H, et al. 2004, Science. , pp. 303:1526–9.

98. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. . Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004., Science. , pp. 303:1529–31. .

99. The role of CpG motifs in innate immunity. Krieg, A.M. 2000., Curr Opin Immunol., pp. 12:35–43.

100. Anendogenous tumour-promoting ligand of the human aryl hydrocarbon receptor. Opitz, C.A., Litzenburger, U.M., Sahm, F., Ott,M., Tritschler, I., Trump, S. 2011, Nature, pp. vol 478; 197-203.

101. Impaired impression of Indolamine 2,3-deoxygenase in monocyte derived DCs in response to TLR-7/8. Furset, G., Floisand, Y., Sioud, M. 2007, Immunology, pp. 263-271.

102. Activationof the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

103. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo . de Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., Moser, M. 1996, J. Exp. Med., pp. 184,1413-1424.

104. Subsets of dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens . Kadowaki, N., Ho, S., Antonenko, S., de Waal Malefyt, R., Kastelein, R. A., Bazan, F., Liu, Y-J. 2001, J. Exp. Med., pp. 194,863-869 .

105. TRAF6 is a critical factor for dendritic cell maturation and development . Kobayashi, T., Walsh, P. T., Walsh, M. C., Speirs, K. M., Chiffoleau, E., King, C. G., Hancock, W. W., Caamano, J. H., Hunter, C. A., Scott, P., Turka, L. A., Choi, Y. 2003, Immunity , pp. 19,353-363 .

106. Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA. Sadik CD, Bachmann M, Pfeilschifter J, Mühl H. 2009, Nucleic Acids Res. , pp. 37(15):5041-56. doi: 10.1093/nar/gkp525. Epub 2009 Jun 18.

107. Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts. Karpus ON, Heutinck KM, Wijnker PJ, Tak PP, Hamann J. 2012, PLoS One., p. 7(5):e35606. doi: 10.1371/journal.pone.0035606. Epub 2012 May 10.

108. The structure of the TLR5-flagellin complex: a new mode of pathogen detection, conserved receptor dimerization for signaling. Lu J, Sun PD. 2012, Sci Signal., p. 5(216):pe11. doi: 10.1126/scisignal.2002963. .

109. Flagellin/Toll-like receptor 5 response was specifically attenuated by keratan sulfate disaccharide via decreased EGFR phosphorylation in normal human bronchial epithelial cells. Shirato K, Gao C, Ota F, Angata T, Shogomori H, Ohtsubo K, Yoshida K, Lepenies B, Taniguchi N. 2013, Biochem Biophys Res Commun., pp. doi:pii: S0006-291X(13)00779-1. 10.1016/j.bbrc.2013.05.009. [Epub ahead of print].

110. Differential induction of interleukin-10 and interleukin-12 in dendritic cells by microbial Toll-like receptor activators and skewing of T-cell cytokine profiles Infect. Qi, H., Denning, T. L., Soong, L. 2003, Immun. , pp. 71,3337-3342 .

111. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D.Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10 . Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. 2000, J. Immunol. , pp. 165,3804-3810.

112. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells . Re, F., Strominger, J. L. 2001, J. Biol. Chem. , pp. 276,37692-37699.

113. Pasare, C., Medzhitov, R. (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Pasare, C., Medzhitov, R. 2003, Science , pp. 299,1033-1036 .

 

  

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The immune response

The immune response (Photo credit: Wikipedia)

Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Hemeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP

ABSTRACT:

The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review we will discuss properties of IDO such as basic molecular genetics, biochemistry and genesis. IDO belongs to globin gene family to carry oxygen and heme.

The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity. In addition IDO has a role in vascular tone as well.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn. These mechanisms and their relation with various health and disease will be presented. Overall our purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

Our focus is on cancer prevention with DCvax.  The first study proving the connection between IDO and immune response came from, a very natural event, a protection of pregnancy in human. This led to discover that high IDO expression is a common factor in cancer tumors. Thus, attention promoted investigations on IDO’s role in various disease states, immune disorders, transplantation, inflammation, women health, mood disorders.  Many approaches, vaccines and adjuvants are underway to find new immunotherapies by combining the power of DCs in immune response regulation and specific direction of siRNA.  As a result, with this unique qualities of IDO, DCs and siRNA, we orchestrated a novel intervention for immunomodulation of IDO by inhibiting with small interference RNA, called siRNA-IDO-DCvax.  Proven that our DCvax created a delay and regression of tumor growth without changing the natural structure and characterization of DCs in melanoma and breast cancers in vivo.

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IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC=1.13.11.52) that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin (1; 2; 3; 4).

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.  After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).   IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).

Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).    Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).  The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).  There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

 

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).

 The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms.

Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).

Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46). Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

The Origin of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.
(reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.   Bacterial IDO has two types of IDOs that are group I and group II IDO (48)These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).  

Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.  Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins, destined to evolve from a common ancestor that is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52).  IDO like globin widely distributed among gastropodic mollusks (53; 51).

The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).  The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53).  Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.  No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

The Immune Cells and IDO in DCs:

DCs are the orchestrator of the immune response (56; 57; 58) with list of functions in uptake, processing, and presentation of antigens; activation of effector cells, such as T-cells and NK-cells; and secretion of cytokines and other immune-modulating molecules to direct the immune response. The differential regulation of IDO in distinct DC subsets is widely studied to delineate and correct immune homeostasis during autoimmunity, infection and cancer and the associated immunological outcomes.

Genesis of antigen presenting cells (APCs), eventually the immune system, require migration of monocytes (MOs), which is originated in bone marrow. Then, these MOs move from bloodstream to other tissues to become macrophages and DCs (59; 60). Initiation of immune response requires APCs to link resting helper T-cell with the matching antigen to protect body. DCs are superior to MQs and MOs in their immune action model. When DCs are first described (61) and classified, their role is determined as a highly potent antigen-presenting cell (APC) subset with 100 to 1000-times more effective than macrophages and B-cells in priming T-cells. Both MQs and monocytes phagocytize the pathogen, and their cell structure contains very large nucleus and many internal vesicles. However, there is a nuance between MQ and DCs, since DCs has a wider capacity of stimulation, because MQs activates only memory T cells, yet DCs can activate both naïve and memory T cells.

DCs are potent activators of T cells and they also have well controlled regulatory roles. DC properties determine the regulation regardless of their origin or the subset of the DCs.  DCs react after identification of the signals or influencers for their inhibitory, stimulatory or regulatory roles, before they express a complex repertoire of positive and negative cytokines, transmembrane proteins and other molecules. Thus, “two signal theory” gains support with a defined rule.

The combination of two signals, their interaction with types of cells and time are critical. In short, specificity and time are matter for a proper response.  When IDO mRNA expression is activated with CTL40 ligand and IFNgamma, IDO results inhibition of T cell production (4).  However, if DCs are inhibited by 1MT, an inhibitor of IDO, the response stop but IgG has no affect (10).  In addition, if the stimulation is started by a tryptophan metabolite, which is downstream of IDO, such as 3-hydroxyantranilic or quinolinic acids, it only inhibits Th1 but not Th2 subset of T cells (62). Furthermore, inclusion of signal molecules, such as Fas Ligand, cytochrome c, and pathways also differ in the T cell differentiation mechanisms due to combination, time and specificity of two-signals.  The co-culture experiments are great tool to identify specific stimuli in disease specific microenvironment (63; 12; 64) for discovering the mechanism and interactions between molecules in gene regulation, biochemical mechanism and physiological function during cell differentiation.

As a result, the simplest differential cell development from the early development of DCs impact the outcome of the data. For example, collection of MOs from peripheral blood mononuclear cells (PBMCs) with IL4 and GM-CSF leads to immature DCs (iDCs). On next step, treatment of iDCs with tumor necrosis factor (TNF) or other plausible cytokines (TGFb1, IFNgamma, IFNalpha,  IFNbeta, IL6 etc.) based on the desired outcome differentiate iDCs  into mature DCs (mDCs). DCs live only up to a week but MOs and generated MQs can live up to a month in the given tissue. B cells inhibit T cell dependent immune responses in tumors (65).

Mechanisms of IDO:

IDO mechanism for immune response

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO (expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

 T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63). Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response.

When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).  Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68).  SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

TABLE 3- Kyn induced Genes

Table 2: Kyn induced genes based on the only microarray analysis (based on Opitz et. al 2011 data)
  Upregulators Phenotype Location
 Upregulators MYC Oncogene myc
avian myelocytomatosis viral oncogene homolog
protooncogene homologous to myelocytomatosis virus
INDIRECTLY MANIPULATED TARGET
NfKB complex Inappropriate activation of NF-kappa-B has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. In contrast, complete and persistent inhibition of NF-kappa-B has been linked directly to apoptosis, inappropriate immune cell development, and delayed cell growth. 10q24.32POSSIBLE INDIRECTLY MANIPULATEDTARGET   4q24 
  Downregulators
       

 

ALDH1A3(ALDEHYDE DEHYDROGENASE 1 FAMILY, MEMBER A3) An unique ALDH isozyme in human saliva 15q26.3
ARNT2,ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR 2 Member of a novel transcription factor family consisting of a conserved basic helix-loop-helix (bhlh) structural motif contiguous with a PAS domain. Members of this family include PER, the aryl hydrocarbon receptor,SIM1,and HIF1A. 15q25.1
C2CD2 Myogenesis in C2C12 mouse myoblasts by DUX4 and inhibited zebrafish development past gastrulation or caused severe developmental abnormalities in the surviving embryos. 4q35.2
CDC42EP2,CDC42 EFFECTOR PROTEIN 2 A small RHO gtpase, regulates the formation of F-actin-containing structures through its interaction with several downstream effector proteins.  11q13.1
 CDH1,CADHERIN 1;  Uvomorulin, a specific calcium ion-dependent cell adhesion molecule, expresses its adhesive function during the preimplantation stage of development and in epithelial cells,Endometrial carcinoma, somatic, Ovarian carcinoma, somatic, Gastric cancer, familial diffuse, with or without cleft lip and/or palate, Breast cancer, lobular, Prostate cancer, susceptibility to. 16q22.1
CENPACENTROMERIC PROTEIN A;  Centromeric proteins, see CENPB 2p23.3
CREB3L2cAMP RESPONSE ELEMENT-BINDING PROTEIN 3-LIKE 2; Member of the old astrocyte specifically induced substance (OASIS) DNA binding and basic leucine zipper dimerization (bzip) family of transcription factors, which includes CREB3 and CREB4. 7q33
CYP1B1,CYTOCHROME P450, SUBFAMILY I, POLYPEPTIDE 1
Glaucoma 3A, primary open angle, congenital, juvenile,Or adult onset, Peters anomaly 231300
2p22.2
EGR; Discovered first as a putative G0/G1 switch regulatory gene in human blood lymphocyte cultures and named G0S30 (Forsdyke, 1985). Sequence analysis of the murine gene predicted a protein with 3 DNA-binding zinc fingers POSSIBLE TARGET
EGR1;EARLY GROWTH RESPONSE 1 Displays FOS-like induction kinetics in fibroblasts, epithelial cells, and lymphocyte. EGR1 is also known as KROX24. Or nerve growth factor-induced clone A (NGFIA). 5q31.2(Sukhatme et al., 1988).
EREG;EPIREGULIN Functions as a tumor growth-inhibitory factor inducing morphologic changes and exhibits low affinity for the EGF receptor. Found on hela,on human epidermoid carcinoma A431 cells. Toyoda et al. (1995), Toyoda et al. (1997)
GPR115; G PROTEIN-COUPLED RECEPTOR 115 Expression in pregnant uterus, breast, and genitourinary tract. 6p12.3fredriksson et al. (2002) POSSIBLE target
HK2; HEXOKINASE 2 Hexokinase (EC 2.7.1.1) catalyzes the first step in glucose metabolism, using ATP for the phosphorylation of glucose to glucose-6-phosphate. Four different types of hexokinase, designated HK1, HK2, HK3, and HK4 (encoded by different genes, are present in mammalian tissues. 2p12
HTT; HUNTINGTON DISEASE  Huntington disease (HD) is caused by an expanded trinucleotide repeat (CAG)n, encoding glutamine, in the gene encoding Huntington. An autosomal dominant progressive neurodegenerative disorder with a distinct phenotype characterized by chorea, dystonia, incoordination, cognitive decline, and behavioral difficulties. 4p16.3
IGFBP4; INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 4 Insulin-like growth factor binding proteins (igfbps), such as IGFBP4, are involved in the systemic and local regulation of IGF activity. Igfbps contain 3 structurally distinct domains each comprising approximately one-third of the molecule.). 17q21.2(Kiefer et al., 1992
IL1A; INTERLEUKIN 1-ALPHA IL1A is 1 of 2 structurally distinct forms of IL1, the other being IL1B (147720). The IL1A and IL1B proteins are synthesized by a variety of cell types, including activated macrophages, keratinocytes, stimulated B lymphocytes, and fibroblasts, and are potent mediators of inflammation and immunity 2q13(Lord et al., 1991).
IL1B; INTERLEUKIN 1-BETA {Gastric cancer risk after H. Pylori infection} 2q13
IL6INTERFERON, BETA-2; IFNB2
B-
CELL DIFFERENTIATION FACTOR, B-CELL STIMULATORY FACTOR 2; BSF2,  HEPATOCYTE STIMULATORY FACTOR; HSF,HYBRIDOMA GROWTH FACTOR; HGF 
Crohn disease-associated growth, failure}, {Diabetes, susceptibility to}, {Kaposi sarcoma, susceptibility to}, {Intracranial hemorrhage in brain, Cerebrovascular malformations, susceptibility to}, {Rheumatoid arthritis, systemic juvenile}. 7p15.3 POSSIBLE COSTIMULATION TARGET
IL8SMALL INDUCIBLE CYTOKINE SUBFAMILY B, MEMBER 8; SCYB8, MONOCYTE-DERIVED NEUTROPHIL CHEMOTACTIC FACTOR,
NEUTROPHIL-ACTIVATING PEPTIDE 1;
NAP1
GRANULOCYTE CHEMOTACTIC PROTEIN 1; GCP1
CHEMOKINE, CXC MOTIF, LIGAND 8; CXCL8
 
A member of the CXC chemokine family. These small basic heparin-binding proteins are proinflammatory and primarily mediate the activation and migration of neutrophils into tissue from peripheral blood. 4q13.3 (Hull et al., 2001). POSSIBLE  COSTIMULATION TARGET
ITGAE;INTEGRIN, ALPHA-ECD103 ANTIGEN
HUMAN MUCOSAL LYMPHOCYTE ANTIGEN 1, ALPHA SUBUNIT
Integrins are a family of cell surface adhesion molecules that play a major role in diverse cellular and developmental processes including morphogenesis, hemostasis, leukocyte activation, cellular adhesion, and homing.Immune responses at mucosal sites are mediated by lymphocytes associated with mammary glands and the gastrointestinal, genitourinary, and respiratory tracts. Cerf-Bensussan et al. (1987),Parker et al. (1992)
JUN
kiaa1644;TRIL;  TLR4 INTERACTOR WITH LEUCINE-RICH REPEATS TRIL is a component of the TLR4 complex and is induced in a number of cell types by lipopolysaccharide (LPS) 7p14.3(Carpenter et al., 2009).
LDO C1LLEUCINE ZIPPER, DOWNREGULATED IN CANCER 1; LDOC1 Contains a leucine zipper-like motif in its N-terminal region and a proline-rich region that shares marked similarity to an SH3-binding domain.  Northern blot analysis detected ubiquitous expression of LDOC1 in normal tissues, with high expression in brain and thyroid and low expression in placenta, liver, and leukocytes.  LDOC1 was expressed in 6 of 7 human breast cancer cell lines examined, but, with only 1 exception, was not expressed in any pancreatic or gastric cancer cell lines examined.  Fluorescence microscopy analysis demonstrated that the LDOC1 protein is located predominantly in the nucleus. Xq27.1 Nagasaki et al. (1999) COSTIMULATION TARGET
MID1; MIDLINE 1 Midline 1 ring finger gene
midin
finger on x and y, mouse, homolog of; fxyOpitz gbbb syndrome, type I
 xp22.2
mir-124; MICRO RNA 124-1 Lagos-Quintana et al. (2002) cloned mouse mir124a.Northern blot analysis showed that mir124a was highly expressed in mouse brain, but not in any other mouse tissues examined.Suh et al. (2004) cloned human mirna124a from embryonic stem cells. The mature mirna124a sequence is UUAAGGCACGCGGUGAAUGCCA.Sempere et al. (2004) found that mirna124 was preferentially expressed in brain. Chromosome 8
mir-290 Both of the major editing sites in pri-mir-376 rnas (+4 and +44) are located within the functionally critical 5-prime-proximal ‘seed’ sequences, critical for the hybridization of mirnas to targets, of mir-376, suggesting that edited mature mir-376 rnas may target genes different from those targeted by the unedited mir-376 rnas. Their results suggested that a single A-I base change is sufficient to redirect silencing mirnas to a new set of targets.Editing of mir-376 appears to be one of the mechanisms that ensure tight regulation of uric acid levels in select tissues such as the brain cortex. MICRO RNA 376-B; MIRN376B Kawahara et al. (2007)POSSIBLE COSTIMULATION TARGET
mir548
RB1;RB1 GENE Bladder cancer, somatic, Osteosarcoma, somatic, Retinoblastoma, Retinoblastoma, trilateral, Small cell cancer of the lung, somatic. 13q14.2Dryja et al. (1984)
RELA; V-REL AVIAN RETICULOENDOTHELIOSIS VIRAL ONCOGENE HOMOLOG A NUCLEAR FACTOR KAPPA-B, SUBUNIT 3; NFKB3
TRANSCRIPTION FACTOR NFKB3
NFKB, p65 SUBUNIT
NUCLEAR FACTOR OF KAPPA LIGHT CHAIN
GENE ENHANCER IN B CELLS 3Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine). 
11q13.1   POSSIBLE CO-TIMULATION TARGET
SERPINB2;SERPIN PEPTIDASE INHIBITOR,Clade B (Ovalbumin), Member 2Plasminogen Activator Inhibitor, Type 2; Pai2
Planh2
Monocyte Arginine-Serpin
Monocyte-Derived Plasminogen Activator Inhibitor
Urokinase Inhibitor
The specific inhibitors of plasminogen activators have been classified into 4 immunologically distinct groups: PAI1 type PA inhibitor from endothelial cells; PAI2 type PA inhibitor from placenta, monocytes, and macrophages; urinary inhibitor; and protease-nexin-I.Plasminogen activator inhibitor-2 is also known as monocyte arg-serpin because it belongs to the superfamily of serine proteases in which the target specificity of each is determined by the amino acid residue located at its reactive center; i.e., met or val for elastase, leu for kinase, and arg for thrombin. 18q21.33
SH3RF1; SH3 DOMAIN-CONTAINING RING FINGER PROTEIN 2 Chen et al. (2010) cloned SH3RF2, which they called HEPP1. The deduced 186-amino acid protein contains a PP1-binding motif (KTVRFQ). Northern blot analysis detected 1.24- and 0.68-kb HEPP1 transcripts in heart and testis only. 5q32
STC2;STANNIOCALCIN-RELATED PROTEIN Northern blot analysis revealed that STC2 is expressed as multiple transcripts in several human tissues, with the strongest expression in skeletal muscle and heart. No entry??
TAF9; TAF9 RNA POLYMERASE II, TATA BOX-BINDING PROTEIN-ASSOCIATED FACTOR, 32-KD The tafs are required for activated rather than basal transcription and serve to mediate signals between various activators and the basal transcriptional machinery. 5q13.2
TGFB1; TRANSFORMING GROWTH FACTOR, BETA-1
Camurati-Engelmann disease 131300
{Cystic fibrosis lung disease, modifier of}TGFB is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGFB acts synergistically with TGFA (190170) in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGFB activation and signaling may result in apoptosis. Many cells synthesize TGFB and almost all of them have specific receptors for this peptide. TGFB1, TGFB2 (190220), and TGFB3 (190230) all function through the same receptor signaling systems.
19q13.2
  TIPARP; TCDD-INDUCIBLE POLY(ADP-RIBOSE) POLYMERASE Amplified and upregulated in head and neck squamous cell carcinoma (HNSCC). The N-terminal part of the TPH domain contains a CCCH-type zinc finger. 3q25.31Katoh and Katoh (2003)  Redon et al. (2001)
TOP2A DNA topoisomerase II, resistance to inhibition of, by amsacrine.  DNA topoisomerases (EC 5.99.1.3) are enzymes that control and alter the topologic states of DNA in both prokaryotes and eukaryotes.There are about 100,000 molecules of topoisomerase II per hela cell nucleus, constituting about 0.1% of the nuclear extract. In a human leukemia cell line, HL-60/AMSA, Hinds et al. (1991) found that resistance to inhibition of topoisomerase II by amsacrine and other intercalating agents was dueTo a single base change, AGA (arginine) to AAA (lysine). 17q21.2
TP53; P53
TRANSFORMATION-RELATED PROTEIN 53;
TRP53Osteosarcoma, Choroid plexus papilloma, Breast cancer,Adrenal cortical carcinoma, Colorectal cancer, Hepatocellular carcinoma, Li-Fraumeni syndrome, Nasop haryngeal carcinoma, Pancreatic cancer, {Glioma susceptibility 1}, {Basal cell carcinoma 7} 
The transcription factor p53 responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. In addition, p53 appears to induce apoptosis through nontranscriptional cytoplasmic processes.Activity of p53 is ubiquitously lost in human cancer either by mutation of the p53 gene itself or by loss of cell signaling upstream or downstream 17p13.1 POSSIBLE TARGET FOR CO-STIMULATION
TP73; p53-RELATED PROTEIN p73; p73
TRP73, MOUSE, HOMOLOG OF
The p53 gene (TP53; 191170) is the most frequently mutated tumor suppressor in human cancers. The ability of p53 to inhibit cell growth is due, at least in part, to its ability to bind to specific DNA sequences and activate transcription of target genes, such as that encoding cell cycle inhibitor p21(Waf1/Cip1) 1p36.32
ZIC2; ZIC FAMILY MEMBER 2  Brown et al. (1998) reported that the human ZIC2 gene, a homolog of the Drosophila ‘odd-paired’ (opa) gene, maps to the region of chromosome 13 associated with holoprosencephaly (HPE5; 609637). Have zinc finger domain.Holoprosencephaly-5Holoprosencephaly is the most common structural anomaly of the human brain and is one of the anomalies seen in patients with deletions and duplications of chromosome 13 13q32.3

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which  initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).  This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91)

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103). Specific TLR stimulation links innate and acquired responses through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105), double stranded (ds) RNAs through TLR3 (106; 107), lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

The specificity is established by correct pairing of a TLR with its proinflammatory cytokine(s), so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, IL6 stimulated DCs relieve the suppression of effector T cells by regulatory T cells (113).  The modification of IDO+ monocytes towards specific subset of T cell activation with specific TLRs are significantly important (94).  The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) or CMV was possible but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys.  Single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These futures of TLRs important during design of experiments to target and improve the efficacy.

Double-Edged Sword of IDO: The Good and The Bad for Clinical intervention and Developments

High expression level of IDO has a positive impact during pregnancy (29; 28; 114), transplants (115; 116; 117; 118; 119), infectious diseases (96). On the other hand, high IDO expression leads the system to a tolerance state is negative during autoimmune-disorders (120; 121; 122), tumors of cancer (123; 124; 117; 121; 125; 126; 127) (127), and mood disorders (46).

Prevention of allogeneic fetal rejection is possible by tryptophan metabolism (26) by rejecting at lack of IDO but allocating with abundant IDO  (29; 28; 114). The plasticity of  mammary and uterus during reproduction may hold some more answers to prevent GVHD and tumors of cancer with good understanding of IDO and tryptophan mechanism (129; 130). These studies lead to find “the natural regulation mechanism” for protecting the transplants from graft versus host disease GVHD (128) and getting rid of tumors. After allogeneic bone marrow transplants the risk of solid tumor development increased about 80% among 19,229 patients,  even with a greater risk if patients are under 18 years old (117).  The adaptation of tolerance against host mechanism is connected to the IDO expression (131).   During implantation and early pregnancy IDO has a role by making CD4+CD25+Foxp3+ regulatory T cells (Tregs) and expressing in DCs and  MQs  (114; 132; 133).  Clonal deletion mechanism prevents mother to react with paternal products since female mice accepted the paternal MHC antigen-expressing tumor graft during pregnancy and rejected three weeks after delivery (134). CTLA-4Ig gene therapy alleviates abortion through regulation of apoptosis and inhibition of spleen lymphocytes (135).

AutoImmune Disorders:

The balance of IDO expression becomes necessary to prevent overactive immune response self-destruction, so modulation in tryptophan and NDA metabolisms maybe essential.  When splenic IDO-expressing CD11b (+) DCs from tolerized animals applied, they suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen (136).   The role of Nicotinamide prevention on type 1 diabetes and ameliorates multiple sclerosis in animal model presented with activities of  NDAs stimulating GPCR109a to produce prostaglandins to induce IDO expression, then these PGEs and PGDs converted to the anti-inflammatory prostaglandin, 15d-PGJ(2) (137; 138; 139).  Thus, these events promote endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma. (137).

IDO (indoleamine 2,3-dioxygenase) and IDO2 control a tryptophan catabolism signaling pathway. (a) From tryptophan starvation to LIP activation. By catabolizing the essential amino acid tryptophan, IDO and IDO2 generate kynurenines and other reaction products that can modulate T-cell immunity as well as a local microenvironment that is starved for tryptophan. Little is known as yet of the precise mechanistic effects of the tryptophan catabolites generated. Elaboration of the starvation condition triggers a stress response in local T cells through Gcn2, which responds to amino-acid deprivation by phosphorylating the translation initiation factor eIF2alpha, leading to a blockade of most translation initiation with the exception of certain factors such as LIP involved in mediating responses to the stress. (b) LIP is a dominant negative isoform of the immune regulatory b/ZIP transcription factor NF-IL6, also known as CEBPbeta. LIP is an alternately translated isoform of the transcription factor NF-IL6/CEBPbeta implicated in regulating proliferation and immune response. Starvation responses switch NF-IL6/CEBPbeta expression from LAP isoforms to the LIP isoform through the use of a downstream translation start site in the mRNA. Encoding only a b/ZIP dimerization domain, LIP functions as a 'natural' dominant negative molecule that disrupts NF-IL6/CEBPbeta function by competing with LAP isoforms for binding to target gene promoters. Both IDO and IDO2 can switch on LIP, but subsequent restoration of tryptophan levels will only switch it off in the case of IDO, offering a possible mechanism for distal propagation of immune suppression away from the local tumor microenvironment (Figure 5). NF-IL6/CEBPbeta target genes with relevance to the function of IDO include the immune suppressive cytokines IL-6, IL-10 and TGF-beta, which may be upregulated as a result of LIP induction. (http://www.nature.com/onc/journal/v27/n28/fig_tab/onc200835f3.html)

IDO (indoleamine 2,3-dioxygenase) and IDO2 control a tryptophan catabolism signaling pathway. (a) From tryptophan starvation to LIP activation. By catabolizing the essential amino acid tryptophan, IDO and IDO2 generate kynurenines and other reaction products that can modulate T-cell immunity as well as a local microenvironment that is starved for tryptophan. Little is known as yet of the precise mechanistic effects of the tryptophan catabolites generated. Elaboration of the starvation condition triggers a stress response in local T cells through Gcn2, which responds to amino-acid deprivation by phosphorylating the translation initiation factor eIF2alpha, leading to a blockade of most translation initiation with the exception of certain factors such as LIP involved in mediating responses to the stress. (b) LIP is a dominant negative isoform of the immune regulatory b/ZIP transcription factor NF-IL6, also known as CEBPbeta. LIP is an alternately translated isoform of the transcription factor NF-IL6/CEBPbeta implicated in regulating proliferation and immune response. Starvation responses switch NF-IL6/CEBPbeta expression from LAP isoforms to the LIP isoform through the use of a downstream translation start site in the mRNA. Encoding only a b/ZIP dimerization domain, LIP functions as a ‘natural’ dominant negative molecule that disrupts NF-IL6/CEBPbeta function by competing with LAP isoforms for binding to target gene promoters. Both IDO and IDO2 can switch on LIP, but subsequent restoration of tryptophan levels will only switch it off in the case of IDO, offering a possible mechanism for distal propagation of immune suppression away from the local tumor microenvironment (Figure 5). NF-IL6/CEBPbeta target genes with relevance to the function of IDO include the immune suppressive cytokines IL-6, IL-10 and TGF-beta, which may be upregulated as a result of LIP induction. (http://www.nature.com/onc/journal/v27/n28/fig_tab/onc200835f3.html)

Modulating the immune response at non-canonical at canonocal pathway while keeping the non-canonical Nf-  KB intact may help to mend immune disorders. As a result, the targeted blocking in canonical at associated  kinase IKKβ and leaving non-canonocal Nf-kB pathway intact, DCs tips the balance towards immune supression.  Hence, noncanonical NF-κB pathway for regulatory functions in DCs required effective IDO induction, directly or  indirectly by endogenous ligand Kyn and negative regulation of proinflammatory cytokine production.

As a result, this may help to treat autoimmune diseases such as rheumatoid arthritis, type 1 diabetes,        inflammatory bowel disease, and multiple sclerosis, or allergy or transplant rejection. While the opposite action  needs to be taken during prevention of tumors, that is inhibition of non-canonical pathway.  Inflammation    induces not only relaxation of veins and lowering blood pressure but also stimulate coagulopathies that worsen  the microenvironment and decrease survival rate of patients after radio or chemotherapies .

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors.  (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors. (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Cancer:

Generating tumor vaccines and using adjuvants underway (140).   Comparison of clinical correlation and genetic responses in several studies hopes to diagnose and target the system for cancer therapies (127; 141; 131).  The recent surveys on IDO expression and human cancers show that IDO targeting is a candidate for cancer therapy since IDO expression recruiting Tregs, downregulating MHC class I and creating negative immune microenvironment for protection of development of tumors (125; 27; 142).  Inhibition of IDO expression can make advances in immunotherapy and chemotherapy fields (143; 125; 131; 144).  IDO has a great importance on prevention of cancer development (126).    There are many approaches to create the homeostasis of immune response by Immunotherapy.  However, given the complexity of immune regulations, immunomodulation is a better approach to correct and relieve the system from the disease.  Some of the current IDO targeted immunotherapy or immmunomodulations are with RNA technology for cancer prevention (145; 146; 147; 148; 149; 150) or applied on human or animals  (75; 151; 12; 115; 152; 9; 125) or chemical, (153; 154) or  radiological (155).  The targeted cell type in immune system generally DCs, monocytes (94), T cells (110; 156) and neutrophils (146; 157). On this paper, we will concentrate on DCvax on cancer treatments.

IDO and the downstream enzymes in tryptophan pathway produce a series of immunosuppressive tryptophan     metabolites that may lead into Tregs proliferation or increase in T cell apoptosis (62; 16; 27; 158), and some can   affect NK cell function (159).  The interesting part of the mechanism is, even without presence of IDO itself,    downstream enzymes of IDO in the kynurenine tryptophan degradation still show immunosuppressive outcome   (160; 73) due to not only Kyn but also TGFbeta stimulated long term responses. DC vaccination with IDO is    plausible (161) due to its power in immune response changes and longevity in the bloodstream for reversing  the system for Th17 production (162).

Taking advantage of the DC’s central role and combining with enhancing molecules for induction of immunity may overcome tolerogenic DCs in tumors of cancers (163; 164). The first successful application of DC vaccine used against advanced melanoma after loading DCs with tumor peptides or autologous cell lysate in presence of adjuvants keyhole limpet hematocyanin (KLH) (165).  Previous animal and clinical studies show use of DCs against tumors created success (165; 166; 167) as well as some problems due to heterogeneity of DC populations in one study supporting tumor growth rather than diminishing (168).

DC vaccination applied onto over four thousand clinical trial but none of them used siRNA-IDO DC vaccination method. Clinical trials evaluating DCs, loaded ex vivo with purified TAAs as anticancer immunotherapeutic interventions, also did not include IDO (Table from (169). This data is coming from 30 clinical trials, 3 of which discontinued, evaluating DCs loaded ex vivo with TAAs as an anticancer immunotherapy for 12 types of cancer [(AML(1), Breast cancer (4), glioblastoma (1), glioma (2), hepatocellular carcinoma (1), hematological malignancies (1), melanoma (6), neuroblastoma sarcoma (2), NSCLC (1), ovarian cancer (3), pancreatic cancer (3), prostate cancer (10)] at phase I, II or I/II.

Tipping the balance between Treg and Th17 ratio has a therapeutic advantage for restoring the health.  This is shown in ovarian cancer by DC vaccination with adjuvants (161).  Rebalancing of the immune system towards immunogenicity may restore Treg/Th17 ratio (162; 170) but it is complicated. The stimulation of IL10 and IL12 induce Tregs produce less Th17 while inhibiting CTL activation and its function (76; 171; 172).  When animals were pre-treated with anti-TGFbeta before vaccination, elevation in the plasma levels of IL-15 for tumor specific T cell survival in (173; 174) ovarian cancer studies was observed.   After human papilloma virus infection, the system present an increase of IL12 (175).  Opposing signal mechanism downregulates the TGFbeta to activate CTL and Th1 population with IL12 and IL15 expression (162; 173).  The effects of IL17 on antitumor properties observed by unique subset of CD4+ T cells (176) called also CD8+ T cells secrete even more IL17 (177).

Use of cytokines as adjuvants during vaccination may improve the efficacy of vaccination, since cancer vaccines, unlike infections vaccines, applied after the infection or disease started against the established adoptive immune response.  It is an almost common practice to use adjuvants to improve efficacy in immunotherapy as a combination therapy (178). Enhancing cancer vaccine efficacy via modulation of the microenvironment is another solution, if only know who are the players.  For example, changing intercellular Ca signaling in T cells is necessary to convert them to Tregs.    Several molecules can be used to initiate and lengthen the activity of intervention to stimulate IDO expression without compromising the mechanism (179) because of the positive feedback loops.  The system is complicated so generally induction is completed ex-vivo stimulation of DCs in cell lysates, or in whole tumor lysates, to create the microenvironment and natural stimulatory agents. Introduction of molecules as an adjuvants on genetic regulation on modulation of DCs are critical, because order and time of the signals, specific location/ tissue, and heterogeneity of personal needs (174; 138; 180). These studies demonstrated that IL15 with low TGFb stimulates CTL and Th1, whereas elevated TGFb with IL10 increases Th17 and Tregs in cancer microenvironments.

For example Ret-peptide antitumor vaccine contains an extracellular fragment of Ret protein and Th1 polarized immunoregulator CpG oligonucleotide (1826), with 1MT, a potent inhibitor of IDO, brought a powerful as well as specific cellular and humoral immune responses in mice (152).  The main idea of choosing Ret is to produce vaccine in ret related carcinomas because ret fulfilled two requirements, first choosing patients self-antigens for cancer therapy with a non-mutated gene, and second, there is no evidence of genetic mutations in Ret amino acids 64-269.

Table 2- IDO Clinical Trials

1

Clinical Trials From Clinicaltrials.Gov

Title And Details Of The Study

NCT Number Recruitment Condition Primary Completion Date Sponsor/Collaborator Phase orObservation. Intervention Type
2 IDO Inhibitor Study For Relapsed Or Refractory Solid Tumors NCT00739609

Terminated

Breast Cancer|Lung Cancer|Melanoma|Pancreatic Cancer|Solid Tumors October 2012 NewlLink enetics Corp. 1 CHEM
3  IDO2 Genetic Status Informs The Neoadjuvant Efficacy Of Chloroquine (CQ) In Brain Metastasis Radiotherapy NCT01727531

Recruiting

Brain Metastasis Dec. 2020 Main Line Health NA CHEM
4 Peptide Vaccine And Temozolomide For Metastatic Melanoma Patients NCT01543464

Recruiting

Malignant Melanoma September 2014 Newlink Genetics Corporation 2 CHEM
5 A Phase 1/2 Randomized, Blinded, Placebo Controlled Study Of Ipilimumab In Combination With INCB024360 Or Placebo In Subjects With Unresectable Or Metastatic Melanoma NCT01604889

Recruiting

Metastatic Melanoma August 2014 Incyte Corporation 1/2 BIOLINH+CHEM
6 A Phase 2 Study Of The IDO Inhibitor INCB024360 Versus Tamoxifen For Subjects With Biochemical-Recurrent-Only EOC, PPC Or FTC Following Complete Remission With First-Line Chemotherapy NCT01685255

Recruiting

April 2014 Incyte Corporation 2 CHEM
7   Different Injection Number Of The Same Dose Of Botulinum Toxin A On Overactive Bladder Syndrome NCT01657409

Recruiting

Overactive Bladder March 2014 Buddhist Tzu Chi General Hospital 2 CHEM
8  Study On The Effect Of Intravenous Ascorbic Acid On Intraoperative Blood Loss In Women With Uterine MyomaInterventions: Drug: Ascorbic Acid NCT01715597

Recruiting

Uterine Leiomyoma January 2014 Seoul National University Hospital 3 CHEM
9 1-Methyl-D-Tryptophan In Treating Patients With Metastatic Or Refractory Solid Tumors That Cannot Be Removed By Surgery NCT00567931

Recruiting

Unspecified Adult Solid Tumor, Protocol Specific September 2013 National Cancer Institute (NCI) 1 CHEM
10  Properties Of Mesenchymal Stem Cells In Lung Transplant NCT01668576

Recruiting

Lung Transplantation August 2013 Emory University OBS BIOL
11  The Effects Of Medical Clowns In Children Undergoing Blood Tests NCT01396876

Recruiting

Pain And Anxiety Reduction July 2012 Tel-Aviv Sourasky Medical Center NA BIOL
12   Saline Injection – Assisted Anesthesia In Eyelid Surgery NCT01239498

Recruiting

Blepharoptosis October 2011 Sheba Medical Center 4 CHEMBIOL
13   Effects Of The Consumption Of California Walnuts On Cardiovascular HealthInterventions:            Dietary Supplement: Walnuts NCT01235390

Recruiting

Cardiovascular Disease|Immune Health October 2011 University Of California, Davis|California Walnut Commission 1 FOODALERGY-WALNUT
14  Pomegranate To Reduce Maternal And Fetal Oxidative Stress And Improve Outcome In Pregnancies Complicated With Preterm Premature Rupture Of The Membranes NCT01584323

Recruiting

Preterm Premature Rupture Of Membranes|Pregnant State 2013 Rambam Health Care Campus NA FOODSUP
1 Phase II INCB024360 Study For Patients With Myelodysplastic Syndromes (MDS) NCT01822691

Not Yet Recruiting

Myelodysplastic Syndromes September 2015 H. Lee Moffitt Cancer Center And Research Institute|Incyte Corporation 2 CHEM-BIOL?
2   Title:  Schizophrenia Imaging NCT01655472

Not Yet Recruiting

Foetal Differences Between Healthy And Schizophernic Parents July 2014 Tel-Aviv Sourasky Medical Center IMAGEN
3  C11 AMT Positron Emission Tomography (PET) Imaging In Patients With Metastatic Invasive Breast Cancer NCT01302821

Not Yet Recruiting

Breast Cancer April 2014     H. Lee Moffitt Cancer Center And Research Institute|National Cancer Institute (NCI) NA BIOLDCAV-P53MTRADIA
4   Sonographic Evaluation Of Visceral Fat After Bariatric Surgery NCT01285791

Not Yet Recruiting

Morbid Obesity April 2012 Hadassah Medical Organization OBS BIOL CELL
5 How Our Immune System Can Help Fight Cancer NCT01042847

Not Yet Recruiting

Ovarian Cancer January 2011 Winthrop UniversityHospita EVALNA POLY.BIOL
6  Title: Study Of The Long-Term Effect Of Frequent Anti-VEGF Dosing On Retinal Function In Patients With Neovascular AMD NCT00533689

Not Yet Recruiting

Electrophysiology 2013 NA  Tel-Aviv Sourasky Medical Center NAEYE BIOL
7  Microbial Surveillance In Children Hospitalized For Cardiovascular Surgery NCT00426894

Not Yet Recruiting

Cardiac Surgery|Perioperative Prophylaxis 2013 NA Hadassah Medical Organization OBS
8 Study Of Chemotherapy In Combination With IDO Inhibitor In Metastatic Breast Cancer NCT01792050

Not Recruitinbut  Active  G

Metastatic Breast Cancer January 2015 Newlink Genetics Corporation 2 CHEM
9 A Dose-Escalation Study In Subjects With Advanced Malignancies NCT01195311

Not BUT  Active Recruiting

Advanced Malignancies November 2012 Incyte Corporation 1 CHEM DOSE
SP  Title:   Mesalamine To Reduce T Cell Activation In HIV Infection NCT01090102

Enrolling By Invitation

HIV Infections|Sexually Transmitted Diseases|Immune System Diseases|Lentivirus Infections|Acquired Immunodeficiency Syndrome January 2013 UC, San Francisco|California HIV/AIDS Research Program|Salix Pharmaceuticals 4 CHEM
1 Study Of Indoleamine 2,3-Dioxygenase Activity, Serum Levels Of Cytokines, BDNF, BH4 And NCT00919295

Completed

Fibromyalgia Syndrome October 2011 Mahidol University|University Of Texas|University Of Wuerzburg 2 CHEM.
2 Diagnosis Of Posttraumatic Stress Disorder Following Primary Rhegmatogenous Retinal Detachment NCT01233908

Completed

Stress Disorders, Post-Traumatic|Retinal Detachment September 2010 Sheba Medical Center OBSEYE BIOL
3 Comparison Of DCT, ORA And GAT In Eyes After Penetrating Keratoplasty NCT00834782

Completed

Corneal Transplantation December 2009 Sheba Medical Center 4
4 Disturbances Of Kynurenine Pathway Of Trytophan Metabolism In Schizophrenia: A Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Study NCT00573300

Completed

Schizophrenia May 2009 North Suffolk Mental Health Association OBSV. BIOL
5 Effect Of Biological Therapy On Biomarkers In Patients With Untreated Hepatitis C, Metastatic Melanoma, Or Crohn Disease NCT00897312

Completed

Melanoma August 2008 Vanderbilt-Ingram Cancer Center|National Cancer Institute (NCI) OBSV.  BIOL-CHEM
6 A Prospective Comparative Study Of Induction Of Labor With A Cervical Ripening Double Balloon Vs Foley Catheter NCT00501033

Completed

Induction Of Labor|Cesarean|Endometritis February 2008 Western Galilee Hospital-Nahariya NA DEVICE
7 Tryptophan, Serotonin And Kynurenine In Septic Shock NCT00684736

Completed

Shock, Septic April 2007 Central Hospital, Versailles OBS KYN
8 Imatinib Mesylate In Treating Patients With Metastatic Breast Cancer NCT00045188

Completed

Male Breast Cancer|Recurrent Breast Cancer|Stage IV Breast Cancer July 2004 National Cancer Institute (NCI) 2 CHEM
9 IDO Peptid Vaccination For Stage III-IV Non Small-Cell Lung Cancer Patients. NCT01219348

Completed

NSCLC|Lung Cancer NA IDO Peptide Vaccinantio 1
10  Indoleamine 2,3-Dioxygenase (IDO) Activity In Patients With Chronic Lymphocytic Leukemia (CLL) NCT01397916

Completed

CLL NA Tampere University Hospital NA
11   Tryptophan Metabolism In Kidney Disease NCT00758537

Completed

Chronic Kidney Disease NA Charite University, Berlin, Germany OBS BIOLTRP LEVELS
12 Observational To Investigate The Efficacy Of CRESTOR 5mg In Reaching LDL-C Target Goals In Patients Who Are At High Risk For A Cardiovascular Event NCT00347217

Completed

HypercholesteremiaCardiovascular NA Astrazeneca 4 CHEM
13 The Association Between Delivery Method And Maternal Rehospitalization NCT00501501

Completed

Hospitalization NA Western Galilee Hospital-Nahariya OBS BIOL
14 Uterine Flora During Elective And Urgent Cesarean Sections NCT00500019

Completed

Endometritis NA Western Galilee Hospital-Nahariya OBS BIOL
15   Title: Pilot, Proof-Of-Concept Study Of Sublingual Tizanidine In Children With Chronic Traumatic Brain Injury (TBI) NCT00287157

Completed

 Traumatic Brain Injury NA Teva GTC 1B CHEM

Another example came from demonstration of proliferating hemangiomas, benign endothelial tumors and often referred as hemangiomas of infancy appearing at head or neck, expresses IDO and slowly regressed as a result of immune mediated process. Large scale of genomic analysis shows insulin like growth factor 2 as the key regulator of hematoma growth (Ritter et al. 2003).

We set out to develop new technology with our previous expertise in immunotherapy and immunomodulation (181; 182; 183; 184), correcting Th17/Th1 ratio (185), and siRNA technology (186; 187).  We developed siRNA-IDO-DCvax. Patented two technologies “Immunomodulation using Altered DCs (Patent No: US2006/0165665 A1) and Method of Cancer Treatments using siRNA Silencing (Patent No: US2009/0220582 A1). In melanoma cancer DCs were preconditioned with whole tumor lysate but in breast cancer model pretreatment completed with tumor cell lysate before siRNA-IDO-DCvax applied. Both of these studies presented a success without modifying the autanticity of DCs but decreasing the IDO expression to restore immunegenity by delaying tumor growth in breast cancer (147) and in melanoma (188).  Thus, our DCvax specifically interfere with IDO without disturbing natural structure and content of the DCs in vivo.  Thus, we showed that DCvax can carry on this technology to clinical applications.   Furthermore, our method of intervention is more sophisticated since it has a direct interaction mechanism with ex-vivo DC modulation without creating long term metabolism imbalance in Trp/Kyn metabolite mechanisms with corrective and non-invasive actioins.

There are several reasons for us to combine DCs with siRNA technology for making DCvax.  First, prevention of tumor development studies targeting non-enzymatic pathway initiated by pDCs conditioned with TGFbeta is specific to IDO1 (189). Second, IDO upregulation in antigen presenting cells allowing metastasis show that most human tumors express IDO at high levels (123; 124).  Third, tolerogenic DCs secretes several molecules some of them are transforming growth factor beta (TGFb), interleukin IL10), human leukocyte antigen G (HLA-G), and leukemia inhibitory factor (LIF), and non-secreted program cell death ligand 1 (PD-1 L) and IDO, indolamine 2.3-dioxygenase, which promote tumor tolerance. Thus, we took advantage of DCs properties and IDO specificity to prevent the tolerogenicity with siRNA-IDO DC vaccine in both melanoma and breast cancer.  Fourth, IDO expression in DCs makes them even more potent against tumor antigens and create more T cells against tumors. IDOs are expressed at different levels by both in broad range of tumor cells and many subtypes of DCs including monocyte-derived DCs (10), plasmacytoid DCs (142), CD8a+ DCs (190), IDO compotent DCs (17), IFNgamma-activated DCs used in DC vaccination.  These DCs suppress immune responses through several mechanisms for induction of apoptosis towards activated T cells (156) to mediate antigen-specific T cell anergy in vivo (142) and for enhancement of Treg cells production at sites of vaccination with IDO-positive DCs+ in human patients (142; 191; 192; 168; 193; 194).  If DCs are preconditioned with tumor lysate with 1MT vaccination they increase DCvax effectiveness unlike DCs originated from “normal”, healthy lysate with 1MT in pancreatic cancer (195).  As a result, we concluded that the immunesupressive effect of IDO can be reversed by siRNA because Treg cells enhance DC vaccine-mediated anti-tumor-immunity in cancer patients.

Gene silencing is a promising technology regardless of advantages simplicity for finding gene interaction mechanisms in vitro and disadvantages of the technology is utilizing the system with specificity in vivo yet improved(186; 196).  siRNA technology is one of the newest solution for the treatment of diseases as human genomics is only producing about 25,000 genes by representing 1% of its genome. Thus, utilizing RNA opens the doors for more comprehensive and less invasive effects on interventions. Thus this technology is still improving and using adjuvants.  Silencing of K-Ras inhibit the growth of tumors in human pancreatic cancers (197), silencing of beta-catenin in colon cancers causes tumor regression in mouse models (198), silencing of vascular endothelial growth factor (VGEF) decreased angiogenesis and inhibit tumor growth (199).   Combining siRNA IDO and DCvax from adult stem cell is a novel technology for regression of tumors in melanoma and breast cancers in vivo. Our data showed that IDO-siRNA reduced tumor derived T cell apoptosis and tumor derived inhibition of T cell proliferation.  In addition, silencing IDO made DCs more potent against tumors since treated or pretreated animals showed a delay or decreased the tumor growth (188; 147).

Clinical Trials:

First FDA approved DC-based cancer therapies for treatment of hormone-refractory prostate cancer as autologous cellular immunotherapy (163; 164).  However, there are many probabilities to iron out for a predictive outcome in patients.  Clinical trials report shows 38 total studies specifically IDO related function on cancer (16), eye (3), surgery (2), women health (4), obesity (1), Cardiovascular (2), brain (1), kidney (1), bladder (1), sepsis shock (1), transplant (1),  nervous system and behavioral studies (4), HIV (1).  Among these only 22 of which active, recruiting or not yet started to recruit, and 17 completed and one terminated. Most of these studies concentrated on cancer by the industry, Teva GTC ( Phase I traumatic brain injury), Astra Zeneca (Phase IV on efficacy of CRESTOR 5mg for cardiovascular health concern), Incyte corporation (Phase II ovarian cancer), NewLink Genetics Corporation (Phase I breast/lung/melanoma/pancreatic solid tumors that is terminated; Phase II malignant melanoma recruiting, Phase II active, not recruiting metastatic breast cancer, Phase I/II metastatic melanoma, Phase I advanced malignancies), and Salix Corp-UC, San Francisco and HIV/AIDS Research Programs (for HIV Phase IV enrolling by invitation).  Most of these studies based on chemotherapy but there are few that use biological methods completed study with  IDO vaccine peptide vaccination for Stage III-IV non-small-cell lung cancer patients (NCT01219348), observational study on effect of biological therapy on biomarkers in patients with untreated hepatitis C, metastasis melanoma, or Crohn disease by IFNalpha and chemical (ribavirin, ticilimumab (NCT00897312), polymorphisms of patients after 1MT drug application in treating patients with metastatic or unmovable refractory solid tumors by surgery (NCT00758537), IDO expression analysis on MSCs (NCT01668576), and not yet recruiting intervention with adenovirus-p53 transduced dendric cell vaccine , 1MT , radiation, Carbon C 11 aplha-methyltryptophan (NCT01302821).

Among the registered clinical trials some of them are not interventional but  observational and evaluation studies on Trp/Kyn ratio (NCT01042847), Kyn/Trp ratio (NCT01219348), Kyn levels (NCT00897312, NCT00573300),  RT-PCR analysis for Kyn metabolism (NCT00573300, NCT00684736, NCT00758537), and intrinsic IDO expression of mesenchymal stem cells in lung transplant with percent inhibition of CD4+ and CD8+ T cell proliferation toward donor cells (NCT01668576), determining polymorphisms (NCT00426894). These clinical trials/studies are immensely valuable to understand the mechanism and route of intervention development with the data collected from human populations.

 

Future Actions for Molecular Diagnosis and Targeted Therapies:

Current survival or response rate is around 40 to 50 % range.  By using specific cell type, selected inhibition/activation sequence based on patient’s genomic profile may improve the efficacy of clinical interventions on cancer treatments.

Targeted therapies for specific gene regulation through signal transduction are necessary but there are few studies with genomics based approach.  On the other hand, there are surveys, observational or evaluations (listed in clinical trials section) registered with www.clinicaltrials.gov that will provide a valuable short-list of molecules.  Preventing stimulation of Ido1 as well as Tgfb-1gene expression by modulating receptor mediated phosphorylation between TGFb/SMAD either at Mad-Homology 1 (MH1) or Mad-Homology 1 (MH2) domains is possible (79; 82; 80). Within Smads there is a conserved Mad-Homology 1 (MH1) domain, which is a DNA binding module contains tightly bound Zinc atom. So the zinc can be targeted with a small molecule adjuvant.  Smad MH2 domain is also well conserved as one the most diverse protein-signal interacting molecule during signal transduction due to two important Serine residues located extreme distal C-termini at Ser-Val-Ser in Smad 2 or at pSer-X-PSer in RSmads (80).  Kyn activated orphan G protein–coupled receptor, GPR35 with unknown function with a distinct expression pattern that collides with IDO sites since its expression at high levels of the immune system and the gut (63) (200; 63).

 

The first study to connect IDO with cancer shows that group (75) so direct targeting to regulate IDO expression is another method.  It is best to act through modulating ISREs in its promoter with RNA-peptide combination technology. Indirectly, IDO can be regulated through Bin1 gene expression control over IDO since Bin1 is a negative regulator of IDO and prevents IDO expression.  IDO is under negative genetic control of Bin1, BAR adapter–encoding gene Bin1 (also known as Amphiphysin2). Bin1 functions in cancer suppression, because attenuation of Bin1 observed in many human malignancies (141; 201; 202; 203; 204; 205; 206).  Absence of Bin1, null Bin1-/- mice studies, upregulates IDO through STAT1- and NF-kB-dependent in tumor cells to escape from T cell–dependent antitumor immunity.   Detailed molecular genetics studies showed that alternative spicing of Bin1 creates tumor suppressor affect.  Its activities also depends on these spliced outcome, such as Exon 10, in muscle. On the other hand, alternative spliced Exon12A contributes brain cell differentiation (209; 210).  In turn Exon 13 in mice has importance in role for regulating growth. When Bin1 is deleted or mutated C2C12 myoblasts interrupted due to its missing Myc, cyclinD1, or growth factor inhibiting genes like p21WAF1 (207; 208).  Thus, myc becomes a natural target and biomarker as well.

Myc is a target at the junction between IDO gene interaction and Trp metabolism.  Bin1 interacts with Myc either early-dependent on Myc or late-independent on Myc, meaning Myc is not present. This gene regulation also interfered by the long term signaling mechanism related to moonlighting pathway (73; 74).  Hence, Trp/Kyn, Kyn/Trp, Th1/Th17 ratios are important to be observed in patients peripheral blood. These direct and indirect gene interactions place Bin1 to function in cell differentiation (211; 212; 205).

Moonlighting maintains the tolerance. The key factor is in this pathway is Kyn so by reviewing one of the microarray analysis for Kyn affect is critical. This data showed that there are 25 genes affected by Kyn, two of which are upregulated and 23 of them downregulated (100). The list of genes and additional knowledge based on previous intervention methods are a good place to start creating a diagnostics panel as a biomarker to monitor outcomes of given immunotherapies. The short list of candidates are as an adjuvant or co-stimulation agents are myc, NfKB at IKKA, C2CD2, CREB3L2, GPR115, IL2, IL8, IL6, and IL1B, mir-376 RNA, NFKB3, TGFb, RelA, and SH3RF1. From the preivos studies we can also add LIP, Fox3P, CTLA-4, Bin1 and IMPACT to the list.  In addition, specific use of TLR, conserved sequences of IDO across its homologous structures and ISREs of IDO or glucorticoid response elements of TDO are great direct targets to modulate the mechanism. Furthermore, some of the signaling pathway molecules CCR6, CCR5, RORgammat, Jak, STAT, IRFs, MH1 and MH2 domains of Smads may add a value.

Endothelial cell coagulation activation mechanism and pDC maturation or immigration from lymph nodes to bloodstream should marry to control not only IDO expression but also genesis of preferred DC subsets. Stromal mesenchymal cells are activated by this modulation at vascular system and interferes with metastasis of cancer. First, thrombin (human factor II) is a well regulated protein in coagulation hemostasis has a role in cell differentiation and angiogenesis. Protein kinase activated receptors (PARs), type of GPCRs, moderate the actions. Second, during hematopoietic response endothelial cells produce hematopoietic growth factors (213; 214) to revise the vascular structure.  Third, components of bone marrow stroma cells include monocytes, adipocytes, and mesenchymal stem cells (215), which are addressing occurrence of coagulapathologies, DIC, bleeding, thrombosis, and penalizing patients response rate towards therapies and decreasing survival rates specially in breast, lung, prostate cancers.

Both silencing IDO in DCs and reinstallinig antitumor immunity by inhibiting tumor-derived immunosupressive molecule IDO through RNA inference combined with our specialization in stem cell technology created a novel method with a success in vivo.  This data suggests that IDO siRNA DCvax can provide a clinical intervention to increase survival rate and prevention of cancer.

Personal genomic profiles are powerful tool to improve efficacy in immunotherapies so considering the influence of age (young vs. adult) and state of immune system (innate vs. adopted or acquired immunity) are important as well.

CONCLUSION


IDO has a confined function in immune system through complex interactions to maintain hemostasis of immune responses. The genesis of IDO stem from duplication of bacterial IDO-like genes.  Inhibition of microbial infection and invasion by depleting tryptophan limits and kills the invader but during starvation of tryptophan the host may pass the twilight zone since tryptophan required by host’s T cells.  Thus, the host cells in these small pockets adapt to new microenvironment with depleted tryptophan and oxygen poor conditions. Hence, the cell metabolism differentiates to generate new cellular structures, like nodules and tumors under the protection of constitutively expressed IDO in tumors, DCs to inhibit T cell proliferation. On the other hand, having a dichotomy in IDO function can be a potential limiting factor that means is that IDO’s impact on biological system could be variable at many levels based on target cells, IDO’s capacity, pathologic state of the disease and conditions of the microenvironment.  This complexity requires a very close monitoring to analyze the outcome and to prevent conspiracies over the data since some previous studies generated paradoxical results.  Healthcare cost of current therapies through chemotherapies, radiotherapies is very high and provide low efficacy.  Clinical interventions of immunotherapies require control of more than one system, such as coagulation and vascular biology manipulations for a higher efficacy and survival rate in cancer patients. Our siRNA and DC technologies based on stem cell modulation will provide at least prevention of cancer development and hopefully prevention in cancer.

References

1. Biochemistry of tryptophan in health and disease. BenderDA. 1983, Mol Aspects Med , pp. 6:101–197.

2. Molecular insights into substrate recognition and catalysis by indolamine 2,3-dioxygenase. Forouhar, F., Anderson, R., Mowat, C.F, et al. 2006, PNAS, pp. vol. 104, no:2, 473-478.

3. Importance of the Two Interferon-stimulated Response Element. Konan KV, Taylor, MW. 1996, J. Biol. Chem.-, pp. 19140-5.

4. induction of indolamine 2,3 dioxygenase: A mechanism of the anti-tumor activity of interferon gamma. Ozaki, Y., Edelstein, M.P., Duch, D.S. 1998, PNAS USA., pp. vol:85, 1242-1246.

5. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8. . Burkin, D. J., Kimbro, K. S., Barr, B. L., Jones, C., Taylor, M. W., Gupta, S. L. 1993, Genomics , pp. 17: 262-263.

6. Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-p11 by fluorescent in situ hybridization. Najfeld, V., Menninger, J., Muhleman, D., Comings, D. E., Gupta, S. L. 1993, Cytogenet. Cell Genet. , pp. 64: 231-232.

7. Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA. . Dai, W., Gupta, S. L. 1990, Biochem. Biophys. Res. Commun. , pp. 168: 1-8.

8. Gene structure of human indoleamine 2,3-dioxygenase. Kadoya, A., Tone, S., Maeda, H., Minatogawa, Y., Kido, R. 1992, Biochem. Biophys. Res. Commun. , pp. 189: 530-536.

9. A gene atlas of th emouse and human protein-encoding transcriptomes. Andrew I. Su, Tim Wiltshire, Serge Batalov , Hilmar Lapp , Keith A. Ching , David Block, Jie Zhang , Richard Soden , Mimi Hayakawa , Gabriel Kreiman , Michael P. Cooke , John R. Walker , and John B. Hogenesch. 2004, PNAS, pp. vol. 101, no. 166062-6067 (http://dx.doi.org:/10.1073/pnas.0400782101).

10. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. 2000, J. Immunol, pp. 164:3596–3599.

11. Inhibition of T cell proliferation by acrophage tryptophan catabolism. Munn, D.H. et al. 1999, J. Exp. Med., p. 189:1363.

12. HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity. Logan, G. J., Smyth, C. M. F., Earl, J. W., Zaikina, I., Rowe, P. B., Smythe, J. A., Alexander, I. E. 2002, Immunology, pp. 105:478-487.

13. Indoleamine 2,3-Dioxygenase – Is It an Immun Suppressor? Soliman H, Mediaville-Varela M, Antonia S. 2010, Cancer J. , pp. 16:354-359.

14. Targeting the immunoregulatory indoleamine 2,3-dioxygenase pathway in immunotherapy. Johnson BA, III, Baban B, Mellor AL. 2009, Immunotherapy. , pp. 645–661.

15. Indoleamine 2,3-dioxygenase and regulation of T cell immunity. AL., Mellor. 2005, Biochem Biophys Res Commun. , pp. 338(1):20–24.

16. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C.Modulation of tryptophan catabolism by regulatory T cells. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C., Alegre, M.-L., Puccetti, P. 2003, Nature Immun., pp. 4: 1206-1212.

17. CTLA-4-Ig regulates tryptophan catabolism in vivo. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni, A., Candeloro, P., Belladonna, M. L., Bianchi, R., Fioretti, M. C., Puccetti, P. 2002, Nature Immun. , pp. 3: 1097-1101.

18. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Grohmann, U., Volpi, C., Fallarino, F., Bozza, S., Bianchi, R., Vacca, C., Orabona, C., Belladonna, M. L., Ayroldi, E., Nocentini, G., Boon, L., Bistoni, F., Fioretti, M. C., Romani, L., Riccardi, C., Puccetti, P. 2007, Nature Med., pp. 13:579-586.

19. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. Mellor, A. L., Keskin, D. B., Johnson, T., Chandler, P., Munn, D. H. 2002, J. Immun. , pp. 168: 3771-3776.

20. Chon, SY, Hassanain, HH, Piine, R., and Gupta, SL. 1995, J. Interferon Cytokine Res. , pp. 15, 517-526.

21. Levy, ED, KEsler, DS, Pine, R., Reich, N, and Darnell, JE.Jr et al. 1988, Genes Dev, pp. 2,383-393.

22. Benoist, C. and Manthis, D. 1990, Annu. Rev of Immunol., pp. 8, 681-715.

23. Dorn, A, Durand, B., Marling, C., Meur, M.L., Beoist, C., and Mathis, D. 1987, PNAS USA, pp. 34, 6249-6253.

24. Konan, K.V. Ph.D. Thesis. Transcriptional Regulation of the Indolamine 2,3-oxygenase Gene. s.l. : Indiana University, Bloominigton, 1995.

25. Tryptophan pyrrolase of rabbit intestine: D- and L–tryptophan cleaving enzyme or enzymes. Yamamoto, S., and Hayashi, O. 1967, J Biol Chem, pp. 242: 5260-5266.

26. Prevention of allogeneic fetal rejection by tryptophan catabolism. Munn, DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. 1998, Science, pp. 281:1191–3.

27. Evidence for a tumoral immune resistance mechanismbased on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove, C. et al. 2003, Nature Med. 9,, pp. 1269–1274 .

28. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Raghupathy, R. 2001., Seminars in Immunology, pp. Volume 13, Issue 4, Pages 219–227.

29. Why is the fetal allograft not rejected? Davies, C. J. March 2007 , J ANIM SCI , pp. vol. 85 no. 13 suppl E32-E35 .

30. Exploring the mechanism of tryptoophan 2,3-dioxygenase. Thackray, S., Mowat, C.G., Chapman, K. 2008, Biochem. Society Transaction., pp. 36, 1120-1123.

31. The new life of a centenarian: signalling functions of NAD(P). Berger F, Ramírez-Hernández MH, Ziegler M. 2004, Trends Biochem Sci , pp. 29:111–118 .

32. Biochemistry of tryptophan in health and disease. DA, Bender. 1983, Mol Aspects Med, pp. 6:101–197.

33. Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain. Heyes MP, Saito K, Jacobowitz D, Markey SP, Takikawa O, Vickers JH. 1992, FASEB J., pp. 6:2977–2989.

34. Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH 1998Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. . Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH. 1998, Am J Pathol, pp. 152:611–619.

35. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. . Yoshida R, Hayaishi O. 1978, Proc Natl Acad Sci USA , pp. 75:3998–4000.

36. Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection. . Yoshida R, Urade Y, Tokuda M, Hayaishi O. 1979, Proc Natl Acad Sci USA , pp. 76:4084–4086.

37. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. Yoshida R, Hayaishi. 1978, PNAS USA, pp. 3998-4000.

38. Sequence of human 2,3-dioxygenase (TDO2): presence of a glucorticoid response-like element composed of a GTT repeat and intronic CCCCT repeat. Comings DE, Muhleman D, Dietz G, Sherman M, Forest. 1995, Genomics, pp. 29:390-396165.

39. Studies on the biosynthesis of Nicotinamide adenine inucleotide. II.Arole of picolinic carboxylase in the Biosynthesisofnicotinamideadeninedinucleotidefromtryptophan in mammals. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, HayaishiO. 1965, J. Biol. Chem. , pp. 240: 1395-1401.

40. The Secret Life of NAD+: An Old Metabolite Controlling New Metabolic Signaling Pathways. Houtkooper R.H., Carles Cantó C. , Wanders, R.J. and Auwerx, J. 2010, Endocrine Reviews , pp. vol. 31 no. 2 194-223,  http://dx.doi.org:/10.1210/er.2009-0026.

41. Stimulation of Nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. Sasaki Y, Araki T, Milbrandt J. 2006, J Neurosci , pp. 26: 8484–8491.

42. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Gale EA, Bingley PJ, Emmett CL, CollierT. 2004, Lancet., pp. 363:925–931.

43. Safety of high-dose nicotinamide: a review. Knip M, Douek IF, Moore WP, Gillmor HA, McLean AE, Bingley PJ, Gale EA. 2000, Diabetologia, pp. 43:1337–1345.

44. Large supplements of nicotinic acid and nicotinamide increase tissue NAD and poly(ADP-ribose) levels but do not affect diethylnitrosamine-induced altered hepatic foci in Fischer-344 rats. JacksonTM, Rawling JM, Roebuck BD, Kirkland JB. 1995, J Nutr , p. 125:1455.

45. Characterization and evolution of vertebrate indelamine 2,3-dihydrogenases IDOs from monotremes and marsupials. Yuasa, HJ, Ball, HJ, Ho, YF, Austin, CJ, et al. 2009, Comp. Biochem. Physiol. B. Biochem.. Mol. Biol., pp. 153 (2): 137-144.

46. Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indolamine 2,3-dihydrogenase inhibitor compound D-1 methyl-tryptophan. Metz, R., Duhadaway, JB, Kamasani, U, Laury-Kleintop, L., Muller, AJ, Prendergast, GC. 2007, Cancer Res., pp. 67 (15): 7082-7087.

47. Total synthesis of exiguamines A and B inspired by catechollamine chemistry. Sofiyev, V, Lumb, JP, Volgraf, M., Trauner, D. 2012, Chemistry., pp. 18 (16): 4999-5005.

48. Molecular evolution of bacterial indolamine 2,3-dioxygenase. Yuasa, H J, Ushigoe, A, Ball, HJ. 2011, Gene., pp. 484 (1) : 22-31.

49. Infectious tolerance and the long-term acceptance of transplant tissue. Waldman, H., Adams, E., Fairchild, P., and Cobbold, S. 2006, J. Immunol., pp. 212:301-313.

50. Molecular evolution and characterizationof fungal indolamine 2,3-dioxygenases. Yuasa, HJ and Ball, HJ. 2012, J. Mol. Eval., pp. 72 (2): 160-168.

51. convergent evolution. The gene structure of Sulculus 41 kDa myoglobin is homologous with tht of human indolamine dioxygenase. Suzuki, T, Imai, K. 1996, Biochim. Biophys. Acta., pp. 1308(1):41-48.

52. Evolutionof myoglobin. Suzuki, T., Imai, K. 1998, Cell Mol Life Sci, pp. 54(9):979-1004.

53. A myoglobin evolved from indolamine 2,3-dioxygenase, trtptophan-degrading enzyme. Suzuki, T., Kawamichi, H., Imai, K. 1998, Comp Biochem Phisiol. Mol. Biol., pp. 121(2):117-128.

54. Do molluscs possess indolamine 2,3-dioxygenase? Yuasa, HJ and Suzuki, T. 2005, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. , pp. (3) 445-454.

55. Comparison studies of the indolamine dioxygenase-like myoglobin from the abalone Sulculus diversicolor. Suzuki, T., Imai, K. 1997, Comp. Biohem. Phsiol B Biochem Mol Biol, pp. 117 (4)599-604.

56. Orchestration of the immune response by dendritic cells. Buckwalter MR, Albert ML. 2009, Curr Biol., pp. 19(9):355–361.

57. Dendritic cells and the control of immunity. Banchereau J, Steinman RM. 1998, Nature., pp. 245–52.

58. IDO expression by dendritic cells: tolerance and tryptophan catabolism. . Munn DH, Mellor AL. 2004, Nat Rev Immunol. , pp. 762–74.

59. Monocyte and Macrophage. Gordon, S. and Taylor, P.R. 2005, NATURE REVIEWS | IMMUNOLOGY , pp. vol:5, 953-964.

60. Blood monocytes consist of two principal subsets with distinct migratory properties. Geissmann F, Jung S, Littman DR. 2003, Immunity. , pp. 19:71–82.

61. Identification of a novel cell type in peripheral lymphoid organs of mice. I Morphology, quantitation, tissue distribution. . Steinman RM, Cohn ZA. 1973, J Exp Med., pp. 137(5):1142–1162.

62. T cell apoptosis by tryptophan catabolism. Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, Spreca A, Fioretti MC, Puccetti P. 2002, Cell Death Differ , pp. 9:1069–1077.

63. Kynurenine is a novel endothelium derived relaxing factor produced during inflammation. Wang, et al. 2010, Nat. Med., pp. 16(3): 279-285.

64. Activation of the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

65. B cells inhibit induction of T cell-dependent tumor immunity. Qin, Z., Richter, G., Schuler, T., Ibe, S., Cao, X, Blakenstein, T. 1998, Nat. Med, p. 4:627.

66. Different partners, Opposite Outcmes: A new perspective of immunobiology of Indolamine 2,3 dioxygenase. Orabona, C., Pallotta, M.T., Grohman, U. 2012, Molecular Medicine., pp. 18:834-842.

67. Indolamine 2,3-dioxygenase: From catalyst to signaling function. Fallarino, F., Grohman, U., and Puccetti, P. 2012, Eurepean J. of Immunol. , pp. 42:1932-1937.

68. IDO: more than an enzyme. Chen, W. 2011, Nature Immonology, pp. 809-811.

69. Indolamine2,3-dehydrogenase in lung dendritic cells promotes Th2 responses and allergic inflammation. Xu, H., Oriss, T.B., Fei, M., Henry, A.C., Melgert, B.N., Chen, L., Mellor, A.L. 2008, PNAS USA, pp. 105: 6690-6695.

70. The immunoregulatory enzyme IDO paradoxically drives B-cellmediated autoimmunity. Scott, G.N., DuHadaway, J., Pigott, E., Ridge, N., Prendergast, G.C., Muller, A.J., Mandik-Nayak, L. 2009, J. Immunol., pp. 182:7509-7517.

71. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. 2002, Immunology , pp. 107:452–460.

72. Enzymology of NAD+ homeostasis in man. . Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S. 2004, Cell Mol Life Sci , pp. 61:19–34.

73. Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO. . Belladonna ML, Grohmann U, Guidetti P, Volpi C, Bianchi R, Fioretti MC, Schwarcz R, Fallarino F, Puccetti P. 2006, J Immunol. , pp. ;177:130–7.

74. An indogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Opitz, et. al. 2011, pp.  http://dx.doi.org:/10.1038/nature10491,.

75. Inhibition of indoleamine 2,3-dioxygenase, animmunoregulatorytarget of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Muller, A. J. et al. 2005, Nature Med. , pp. 11, 312–319 .

76. TGF-b; a master of all T cell trades. Li, M.O., Fravell, R.A. 2008, Cell. , pp. 134: 392-404.

77. Palotta, M.T. et al. 2011, Nat. Immunol., pp. 12:870-878.

78. Chen, W. et al. 2003, J. Exp. Immunol., p. 198: 1875.

79. Smads: transcriptional activators of TGF-beta responses. . Derynck R, Zhang Y, Feng XH. 1998, Cell , pp. 95 (6): 737–40.
http://dx.doi.org:/10.1016/S0092-8674(00)81696-7.PMID 9865691. .

80. Smad transcription factors. Massagué J, Seoane J, Wotton D. 2005, Genes Dev, pp. 19 (23): 2783–810.
http://dx.doi.org:/10.1101/gad.1350705. PMID .

81. A structural basis for mutational inactivation of the tumour suppressor Smad4. Shi Y, Hata A, Lo RS, Massagué J, Pavletich NP. 1997, Nature., pp. 388 (6637): 87–93.  http://dx.doi.org:/10.1038/40431. PMID 9214508.

82. Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. Itoh F, Asao H, Sugamura K, Heldin CH, ten Dijke P, Itoh S. 2001, EMBO J., pp. 20 (15): 4132–   http://dx.doi.org:/10.1093/emboj/20.15.4132. PMC 149146. PMID 11483516.

83. SMAD_Signaling_Network. http://www.sabiosciences.com. [Online] 2013. http://www.sabiosciences.com/pathway.php?sn=SMAD_Signaling_Network.

84. Immune inhibitory receptors. Revetch, J.V., and Lanier, L.L. 2000, Science., pp. 290:84-89.

85. Soc3 drives proteasomal degradation of indolamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Orabona, C., Pallotta, M., Volpi, C., et al. 2008, PNAS USA, pp. 105: 20828-20833.

86. Cutting edge; silencing supressor of cytokine signaling3 expression in dendritic cells turns CD28-Ig from immune adjuvant to supressant. Orabona, C.,, Belladonna, M.L., et all. 2005, J. Immunol., pp. 174: 6582-6586.

87. Molecular signatures of T-cell inhibition in HIV-1 infection. Larsson, M., Shankar. E.M, Che, K.F., Ellegard, R., Barathan, M., Velu, V., and Kamarulzaman, A. 2013, Retrovirology, p. 10:31.

88. TGF-beta and CD4+CD25+ regulatory cells. Huber, S. and Schramn, C. 2006, Front. Bioscie., pp. 11:1014-1023.

89. Immune Escape as a fundemental trait of cancer; focus on IDO. Prendergast, G.C. 2008, Oncogene., pp. 27, 3889-3900.

90. Il-6 inhibits the tolerogenic functionof CD8+ dendritic cells expressing indolamine 2,3-dioxygenase. Grohman, U., Fallarino, F., et al. 2001, J. Immunol., pp. 167:708-714.

91. Avoiding horror autotoxicus: Th eimportance of dentritic cells in peripheral T cell tolerance. Steinman, R.M., and Nussenzweig, M.C. 2002, PNAS, pp. no:1, 351-358.

92. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice . Kaisho, T., Akira, S. 2001, Trends Immunol , pp. 22,78-83.

93. Innate sensing of self and non-self RNAs by Toll-like receptors. Sioud, M. 2006., Trends Mol Med., pp. 12:67–76.

94. Impaired expression of indoleamine 2, 3-dioxygenase in monocyte-derived dendritic cells in response to Toll-like receptor-7/8 ligands. Furset, G., Fløisand, Y. and Sioud, M. 2008, Immunology., pp. 123(2): 263–271, http://dx.doi.org:/10.1111/j.1365-2567.2007.02695.x.

95. Toll-;ike receptor 9 mediated induction of the immunorepressor pathway of tryptophan metabolism. Fallarino, F., and Puccetti, P. 2006, Eur. J. of Imm., pp. 36:8-11.

96. Toll-like receptors and host defense against microbial pathogens: bringing specificity to the innate immune system. . Netea MG, der Graaf C, Van der Meer JWM, Kullberg BJ. 2004, J Leukoc Biol. , pp. 75:749–55.

97. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. . Heil F, Hemmi H, Hochrein H, et al. 2004, Science. , pp. 303:1526–9.

98. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. . Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004., Science. , pp. 303:1529–31. .

99. The role of CpG motifs in innate immunity. Krieg, A.M. 2000., Curr Opin Immunol., pp. 12:35–43.

100. Anendogenous tumour-promoting ligand of the human aryl hydrocarbon receptor. Opitz, C.A., Litzenburger, U.M., Sahm, F., Ott,M., Tritschler, I., Trump, S. 2011, Nature, pp. vol 478; 197-203.

101. Impaired impression of Indolamine 2,3-deoxygenase in monocyte derived DCs in response to TLR-7/8. Furset, G., Floisand, Y., Sioud, M. 2007, Immunology, pp. 263-271.

102. Activationof the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

103. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo . de Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., Moser, M. 1996, J. Exp. Med., pp. 184,1413-1424.

104. Subsets of dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens . Kadowaki, N., Ho, S., Antonenko, S., de Waal Malefyt, R., Kastelein, R. A., Bazan, F., Liu, Y-J. 2001, J. Exp. Med., pp. 194,863-869 .

105. TRAF6 is a critical factor for dendritic cell maturation and development . Kobayashi, T., Walsh, P. T., Walsh, M. C., Speirs, K. M., Chiffoleau, E., King, C. G., Hancock, W. W., Caamano, J. H., Hunter, C. A., Scott, P., Turka, L. A., Choi, Y. 2003, Immunity , pp. 19,353-363 .

106. Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA. Sadik CD, Bachmann M, Pfeilschifter J, Mühl H. 2009, Nucleic Acids Res. , pp. 37(15):5041-56. http://dx.doi.org:/10.1093/nar/gkp525. Epub 2009 Jun 18.

107. Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts. Karpus ON, Heutinck KM, Wijnker PJ, Tak PP, Hamann J. 2012, PLoS One., p. 7(5):e35606.  http://dx.doi.org:/10.1371/journal.pone.0035606. Epub 2012 May 10.

108. The structure of the TLR5-flagellin complex: a new mode of pathogen detection, conserved receptor dimerization for signaling. Lu J, Sun PD. 2012, Sci Signal., p. 5(216):pe11.   http://dx.doi.org:/10.1126/scisignal.2002963. .

109. Flagellin/Toll-like receptor 5 response was specifically attenuated by keratan sulfate disaccharide via decreased EGFR phosphorylation in normal human bronchial epithelial cells. Shirato K, Gao C, Ota F, Angata T, Shogomori H, Ohtsubo K, Yoshida K, Lepenies B, Taniguchi N. 2013, Biochem Biophys Res Commun., pp. doi:pii:S0006-291X(13)00779-1. http://dx.doi.org:/10.1016/j.bbrc.2013.05.009. [Epub ahead of print].

110. Differential induction of interleukin-10 and interleukin-12 in dendritic cells by microbial Toll-like receptor activators and skewing of T-cell cytokine profiles Infect. Qi, H., Denning, T. L., Soong, L. 2003, Immun. , pp. 71,3337-3342 .

111. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D.Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10 . Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. 2000, J. Immunol. , pp. 165,3804-3810.

112. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells . Re, F., Strominger, J. L. 2001, J. Biol. Chem. , pp. 276,37692-37699.

113. Pasare, C., Medzhitov, R. (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Pasare, C., Medzhitov, R. 2003, Science , pp. 299,1033-1036 .

114. What is the role of regulatory T cells in the success of implantation and early pregnancy? Saito, S., Shima, T., Nakashima, A., Shiozaki, A., Ito, M., Sasaki, Y. 2007, J Assist Reprod Genet, pp. 24: 379-386.

115. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. . Liu H, Liu L, Fletcher BS, Visner GA. 2006, FASEB J, pp. 20:2384-2386. .

116. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Alexander AM, Crawford M, Bertera S, et al. 2002, Diabetes. , pp. 51(2):356–365.

117. Solid Cancers after Bone Marrow Transplantatioin. Curtis, R.E., Rowlings, P.A., Deeg, J., Schirer, D.A. et al. 1997, The New England Journal of Medicine., pp. 336, No: 13: 897-904.

118. More ADO about IDO; GVHD (commentary). Curti, A., Trabanelli, S., Lemoli, M. 2008, Blood, p. 2950.

119. Jasperson, et al, . 2008, Blood, p. 3257.

120. Tolerance, DCs and tryptophan: much ado about IDO. Grohmann U, Fallarino F, Puccetti P. 2003, Trends Immunol, pp. 24:242-248.

121. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, et al. 2003, Nat Med , pp. 9:1269–74.

122. Indoleamine 2,3-dioxygenase is a critical regulator of acute graft-versus-host disease lethality. Lisa K. Jasperson, Christoph Bucher, Angela Panoskaltsis-Mortari, Patricia A. Taylor, Andrew L. Mellor, David H. Munn, and Bruce R. Blazar. 2008., Blood., pp. 111:3257-3265.

123. The metabolism of tryptophan. 2. The metabolism of tryptophan in patients suffering from cancer of the bladder. . Boyland, E. & Willliams, D.C. 1956, Biochem. J., pp. 64, 578−582 .

124. Tryptophan metabolism in carcinoma of the breast. . Rose, D. 1967, Lancet , pp. 1, 239−241 .

125. Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees? . Löb S, Königsrainer A, Rammensee HG, Opelz G, Terness P. 2009;, Nat Rev Cancer , pp. 9:445–52.  http://dx.doi.org:/10.1158/1078-0432.CCR-11-1331.

126. The hallmarks of cancer. . Hanahan, D. & Weinberg, R.A. 2000., Cell., pp. 100, 57−70.

127. Indoleamine 2,3-Dioxygenase Expression in Human Cancers: Clinical and Immunologic Perspectives. Godin-Ethier, J., Hanafi,L.A., Piccirillo,C.A. and Lapointe, R. 2011, Clin Cancer Res, pp. 17; 6985,  http://dx.doi.org:/10.1158/1078-0432.CCR-11-1331.

128. Dendritic cell modification as a route to inhibiting corneal graft rejection by the indirect pathway of allorecognition. Khan A, Fu H, Tan LA, Harper JE, Beutelspacher SC, Larkin DF, Lombardi G, McClure MO, George AJ. 2013, Eur J Immunol., pp. 43(3):734-46.  http://dx.doi.org:/10.1002/eji.201242914. Epub 2013 Jan 18.

129. Possible role of the ‘IDO-AhR axis’ in maternal-foetal tolerance. . Hao K, Zhou Q, Chen W, Jia W, Zheng J, Kang J, Wang K, Duan T. 2013, Cell Biol Int., pp. 37(2):105-8. doi: 10.1002/cbin.10023. Epub 2013 Jan 2.

130. Implication of indolamine 2,3 dioxygenase in the tolerance toward fetuses, tumors, and allografts. . Dürr S, Kindler V. 2013, J Leukoc Biol. , pp. 93(5):681-7. doi: 10.1189/jlb.0712347. Epub 2013 Jan 16.

131. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, et al. 2003, Nat Med, pp. 9:1269–74.

132. NAturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Sagaguchi, S. 2004, Annu. Rev. of Immunol., pp. 22: 531-562.

133. Regulatory T cells in transplantation tolerance. Wood, K.J., zZSakaguchi, S.,. 2003, Nat. Rev. Immunol., pp. 3; 199-210.

134. The cell awareness of paternal alloantigens during pregnancy. Tafuri, A., Alferink, J., Hammerling, G.J., Arnold, B. 1995, Science, pp. 270; 630-3.

135. Adenovirus mediated CTLA4Ig transgene therapy alleviates abortion by inhibiting spleen lymphocyte proliferation and regulating apoptosis in the feto-placental unit. Li W, Li B, Li S. 2013, J Reprod Immunol. , pp. 97(2):167-74.

136. A distinct tolerogenic subset of splenic IDO(+)CD11b(+) dendritic cells from orally tolerized mice is responsible for induction of systemic immune tolerance and suppression of collagen-induced arthritis. Park MJ, Park KS, Park HS, Cho ML, Hwang SY, Min SY, Park MK, Park SH, Kim HY. 2012, Cell Immunol. , pp. 278(1-2):45-54. http://dx.doi.org:/10.1016/j.cellimm.2012.06.009. Epub 2012 Jul 10.

137. Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease. Penberthy, W.T. 2007, Curr. Drug Metab., pp. 8:(3):245-266.

138. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Alexander AM, Crawford M, Bertera S, et al. 2002, Diabetes. , pp. 51(2):356–365.

139. Heme oxygenase-1 plays an important protective role in experimental autoimmune encephalomyelitis. . Liu Y, Zhu B, Luo L, Li P, Paty DW, Cynader MS. 2001., NeuroReport. , pp. 12(9):1841–1845. .

140. Tumor vaccines in 2010: need for integration. Koos, D., Josephs, SF, Alexandrescu, DT et al. 2010, Cell Immunol, pp. 263: 138-147.

141. BIN1 is a novel MYC-interacting protein with features of a tumor suppressor. . Sakamuro, D., Elliott, K., Wechsler-Reya, R. & Prendergast, G.C. 1996, Nat. Genet. , pp. 14, 69−77. .

142. Expression of Indolamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor draining nodes. Munn, S.H., Sharma, M.D., Hou, D., Baban, B. et al. 2004, J. Clin. Invest. , pp. 114: 280-290.

143. Indoleamine 2,3-Dioxygenase Expression in Human Cancers: Clinical and Immunologic Perspectives. Jessica Godin-Ethier, Laïla-Aïcha Hanafi, Ciriaco A. Piccirillo, and Réjean Lapointe. 2011 , Clin Cancer Res, pp. 17; 6985,   http://dx.doi.org:/10.1158/1078-0432.CCR-11-1331.

144. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. . Munn, D.H. et al. 2002, Science 297, 1867−1870, pp. 297, 1867−1870 .

145. An HDAC inhibitor enhances cancer therapeutic efficiency of RNA polymerase III promoter-driven IDO shRNA. Yen MC, Weng TY, Chen YL, Lin CC, Chen CY, Wang CY, Chao HL, Chen CS, Lai MD. 2013, Cancer Gene Ther. , p.  http://dx.doi.org:/10.1038/cgt.2013.27. [Epub ahead of print].

146. Systemic delivery of Salmonella typhimurium transformed with IDO shRNA enhances intratumoral vector colonization and suppresses tumor growth. Blache CA, Manuel ER, Kaltcheva TI, Wong AN, Ellenhorn JD, Blazar BR, Diamond DJ. 2012, Cancer Res. , pp. 72(24):6447-56.
http://dx.doi.org:/10.1158/0008-5472.CAN-12-0193. Epub 2012 Oct 22.

147. Silencing IDO in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model. Zheng X, Koropatnick J, Chen D, Velenosi T, Ling H, Zhang X, Jiang N, Navarro B, Ichim TE, Urquhart B, Min W. 2013, Int J Cancer., pp. 132(4):967-77. http://dx.doi.org:/10.1002/ijc.27710. Epub 2012 Jul 20.

148. Immunosuppressive CD14+HLA-DRlow/neg IDO+ myeloid cells in patients following allogeneic hematopoietic stem cell transplantation. Mougiakakos D, Jitschin R, von Bahr L, Poschke I, Gary R, Sundberg B, Gerbitz A, Ljungman P, Le Blanc K. 2013, Leukemia. , pp. 27(2):377-88.
http://dx.doi.org:/10.1038/leu.2012.215. Epub 2012 Jul 25.

149. Upregulated expression of indoleamine 2, 3-dioxygenase in primary breast cancer correlates with increase of infiltrated regulatory T cells in situ and lymph node metastasis. Yu J, Sun J, Wang SE, Li H, Cao S, Cong Y, Liu J, Ren X. 2011, Clin Dev Immunol. , p. 11:469135.
http://dx.doi.org:/10.1155/2011/469135. Epub 2011 Oct 24.

150. Skin delivery of short hairpin RNA of indoleamine 2,3 dioxygenase induces antitumor immunity against orthotopic and metastatic liver cancer. Huang TT, Yen MC, Lin CC, Weng TY, Chen YL, Lin CM, Lai MD. 2011, Cancer Sci. , pp. 102(12):2214-20. http://dx.doi.org:/10.1111/j.1349-7006.2011.02094.x. .

151. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. . Alexander AM, Crawford M, Bertera S, et al. 2002, Diabetes. , pp. 51(2):356–365.

152. Prevention of Spontaneous Tumor Development in a ret Transgenic Mouse Model by Ret Peptide Vaccination with Indoleamine 2,3-Dioxygenase Inhibitor 1-Methyl Tryptophan. Zeng, J., Cai, S., Yi, Y., et al. 2009, Cancer Res., pp. 69: 3963-3970, http://dx.doi.org:/10.1158/0008-5472.CAN-08-2476.

153. Medicinal electronomics bricolage design of hypoxia-targeting antineoplastic drugs and invention of boron tracedrugs as innovative future-architectural drugs. Hori H, Uto Y, Nakata E. 2010, Anticancer Res. , pp. 30(9):3233-42. .

154. Synthesis of 4-cyano and 4-nitrophenyl 1,6-dithio-D-manno-, L-ido- and D-glucoseptanosides possessing antithrombotic activity. Bozó E, Gáti T, Demeter A, Kuszmann J. 2002, Carbohydr Res. , pp. 3;337(15):1351-65.

155. Radiopharmaceuticals XXVII. 18F-labeled 2-deoxy-2-fluoro-d-glucose as a radiopharmaceutical for measuring regional myocardial glucose metabolism in vivo: tissue distribution and imaging studies in animals. Gallagher BM, Ansari A, Atkins H, Casella V, Christman DR, Fowler JS, Ido T, MacGregor RR, Som P, Wan CN, Wolf AP, Kuhl DE, Reivich M. 1977, J Nucl Med. , pp. 18(10):990-6.

156. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. 2002, Immunology, pp. 107:452–460.

157. Induction of indoleamine 2,3-dioxygenase by uropathogenic bacteria attenuates innate responses to epithelial infection. Loughman JA, Hunstad DA. 2012 , J Infect Dis. , pp. 205(12):1830-9. http://dx.doi.org:/10.1093/infdis/jis280.

158. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. . Terness, P., et al. 2002, J. Exp. Med.196:447–457., pp. 196:447–457.

159. The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function. . Chiesa, M.D., et al. 2006, Blood. , pp. 108:4118–4125.38.

160. Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8+ T cells induced by TCR triggering or homeostatic cytokines. Weber, W.P., et al. 2006, Eur. J. Immunol. , pp. 36:296-304.

161. Dendritic cell vaccination against ovarian cancer–tipping the Treg/TH17 balance to therapeutic advantage? Cannon MJ, Goyne H, Stone PJ, Chiriva-Internati M. 2011, Expert Opin Biol Ther. , pp. 11(4):441-5.  http://dx.doi.org:/10.1517/14712598.2011.554812. .

162. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. . Kryczek I, Banerjee M, Cheng P, et al. 2009, Blood., pp. 114:1141–1149. .

163. The use of dendritic cells in cancer immunitherapy. Schuler, G., Schuker-Turner, B., Steinman, RM,. 2003, Curr. Opin. Immunol., pp. 15: 138-147.

164. Clinical applications of dentritic cell vaccines. Morse, MA, Lyerly, HK. 2000, Curr. Opin. Mol Ther., pp. 2:20-28.

165. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nestle, FO, Alijagic, S., Gillet, M. et al. 1998, Nat. Med., pp. 4: 328-332.

166. Dentritic cell based tumor vaccination in prostate and renal cell cancer: a systamatic review. Draube, A., Klein-Gonzales, Matheus, S et al. 2011, Plos One, p. 6:e1881.

167. [Online] http://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapy-Products/ApprovedProducts/ucm210215.htm..

168. Dendritic cell based antitumor vaccination: impact of functional indolamine 2,3-dioxygenase expression. Wobster, m., Voigt, H., Houben, R. et al. 2007, Cancer Immunol Immunother, pp. 56:1017-1024.

169. [Online] oncoimmunology.2012 October1; 1(17):1111-1134, doi: 10.4161/onci.21494.

170. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. 2007 , Nat Immunol. , pp. 8(9):942-9. .

171. IFNgamma promotes generationof Il-10 secreting CD4+ T cells that suppress generationof CD8responses in an antigen-experienced host. Liu, X.S., Leerberg, J., MacDonald, K., Leggatt, G.R., Frazer, I.H. 2009, J. Immunol., pp. 183: 51-58.

172. Antigen, in the presence of TGF-beta, induces up-regulationof FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked supressionof CD8+ T cell responses. . Kapp, J.A., Honjo, K., Kapp, L.M., Goldsmith, K., Bucy, R.P. 2007, J. Immunol., pp. 179: 2105-2114.

173. Opposing effects of TGF-beta and IL-15 cytokines control the number of short lived effecctor CD8+ T cells. Sanjabi, S, Mosaheb, M.M., Flavell, R.A. 2009, Immunity., pp. 31; 131-144.

174. Synergestic enhancement of CD8+ T cell mediated tumor vaccines efficacy by an anti-tumor forming growth factor-beta monoclonal antibody. . Terabe, M., Ambrosino, E., Takaku, S. et al. 2009, Clin. Cancer Res., pp. 15; 6560-9.

175. IL-12 enhances CTL synapse formationand induces self-reactivity. Markinewicz, MA, Wise, EL, Buchwald, ZS et al. 2009, J. Immunol., pp. 182: 1351-1362.

176. Tumor specific Th17-polarized cells eradicate large established melanoma. Muranski, P., Boni, A., Antony, PA, et al. 2008, Blood, pp. 112; 362-373.

177. Type17 CD8+ T cells dispplay enhanced antitumor immunity. Hinrichs, C.S., Kaiser, A., Paulos, C.M., et al. 2008, Blood., pp. 112:362-373.

178. Marying Immunotherapy with Chemotherapy: Why Say IDO? Muller, AJ, and Prendergrast, GC. 2005, Cancer Research, pp. 65: 8065-8068.

179. Enhancing Cancer Vaccine efficacy via Modulationof the Tumor Environment. Disis, ML. 2009, Clin Cancer Res, pp. 15: 6476-6478.

180. Systemic inhibition of transforming growth factor beta 1 in glioma bearing mice improves the therapeutic efficacy of glioma-associated antigen peptide vaccines. Ueda, R., Fujita, M., Zhu, X., et al. 2009, Clin. Cancer res., pp. 15: 6551-9.

181. Immune modulation by silencing IL-12 productionin dendritic cells using smal interfering RNA. Hill, JA, Ichim, TE, Kusznieruk, KP, et al. 2003, J. Immunol, pp. 171:809-813.

182. Immune modulation and tolerance induction by RelB-silenced dentritic cells through RNA interference. Li, M. Zang, X, Zheng, X, et al. 2007, J. Immunol, pp. 178: 5480-7.

183. RNAi mediated CD40-CD54 interruption promotes tolerance in autoimmune arthritis. . Zheng, X., Suzuki, M., Zhang, X., et al. 2010, Arthritis Res. Ther., p. 12:R13.

184. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival. Min, WP. Gorczynki, R., huang, XY et al. 2000, J. Immunol., pp. 164:161-167.

185. LF15-0195 generates tolerogenic dendritic cells by supressionof NF-kappaB signaling through inhibitionof IKK activity. . Yang, J., Bernier, SM, Ichim, TE, et al. 2003, J Leukoc. Biol., pp. 74: 438-447.

186. RNA interfrence: A potent tool for gene specific therapeutics. . Ichim, TE, Li, M., Qian, H., Popov, HI, Rycerz, K., Zheng, X., White, D., Zhong, R., and Min, WP. 2004, Am. J. Transplant, pp. 4:1227-1236.

187. A novel in vivo siRNA delivery system specifically targeting dendritic cells and silencing CD40 genes for immunomodulation. Zheng, X., Vladau, C., Zhang, X. et al. 2009, Blood, pp. 113:2646-2654.

188. Reinstalling Antitumor Immunity by Inhibiting Tumor derived ImmunoSupressive Molecule IDO through RNA interference. Zheng, X et al. 2006, Int. Journal of Immunology., pp. 177:5639-5646.

189. Roles of TGFbeta in metastasis. Padua, D., Massague, J. 2009, Cell Res., pp. 19;89-102.

190. Functional expression of indolamine2,3-dioxygenase by murine CDalpha+dendritic cells. Fallarino, F., Vacca, C, Orabona, C et al. 2002, Int Immunol., pp. 14:65-8.

191. Indolamine2,3-dioxygenase controls conversion of Fox3+ Tregs to TH17-like cells in tumor draining lymph nodes. Sharma, MD, Hou, DY, Liu, Y et al. 2009, Blood, pp. 113: 6102-11.

192. IDO upregulates regulatory T cells via tryptoophan catabolite and supresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis. Yan, Y, Zhang, GX, Gran, B et al. 2010, J Immunol, pp. 185; 5953-61.

193. IDO activates regulatory T cells and blocks their conversion into Th-17-like T cells. Baban, B, Chandler, PR, Sharma, MD et al. 2009, J Immunol, pp. 183; 2475-83.

194. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletionof regulatory T cells. Dannull, J., Farrand, KJ, Mathews, SA, et al. 2005, J Clin Invest, pp. 115: 3623-33.

195. 1-MT enhances potency of tumor cell lysate pulled dentritic cells against pancreatic adenocarcinoma by downregulating percentage of Tregs. Li, Y, Xu, J, Zhou, H. et al. 2010, J Huazhong Univ Sci Technol Med Sci , pp. 30: 344-8.

196. siRNA mediated antitumorigenesis for drug target validation and therapeutics. Lu, PY, Xie, FY and Woodle, MC. 2003, Curr Opin Mol. Ther., pp. 5:225-234.

197. Stable supression of tumorigenicity by virus-mediated RNA interference. Brumellkamp, TR, Bernards, R, Agami, R. 2002, Cancer Cell, pp. 2; 243-247.

198. Small interferring RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Verma, UN, Surabhi, RM, Schmaltieg, A., Becerra, C., Gaynor, RB. 2003, Clin. Cancer. Res., pp. 9:1291-1300.

199. siRNA mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogeneic thromboposdin-1 and slows tumor vascularization and growth. Filleur, S., Courtin, A, Ait-Si-Ali, S., Guglielmi, J., Merel, C., Harel-Bellan, A., CLezardin, P., and Cabon, F. 2003, Cancer Res, pp. 63; 3919-3922.

200. Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35. . Wang, J., et al. 2006, J. Biol.Chem. , pp. 281:22021–22028.

201. Bin1 functionally interacts with Myc in cells and inhibits cell proliferation by multiple mechanisms. Elliott, K. et al. 1999, Oncogene , pp. 18, 3564−3573 .

202. Mechanism for elimination of a tumor suppressor: aberrant splicing of a brain-specific exon causes loss of function of Bin1 in melanoma. . Ge, K. et al. 1999, Proc. Natl. Acad. Sci. USA, pp. 96, 9689−9694. .

203. Losses of the tumor suppressor Bin1 in breast carcinoma are frequent and reflect deficits in a programmed cell death capacity. Ge, K. et al. 2000, Int. J. Cancer , pp. 85, 376−383.

204. Loss of heterozygosity and tumor suppressor activity of Bin1 in prostate carcinoma. Ge, K. et al. 2000, Int. J. Cancer , pp. 86, 155−161.

205. Expression of a MYCN-interacting isoform of the tumor suppressor BIN1 is reduced in neuroblastomas with unfavorable biological features. . Tajiri, T. et al. 2003, Clin. Cancer Res., pp. 9, 3345−3355.

206. Targeted deletion of the suppressor gene Bin1/Amphiphysin2 enhances the malignant character of transformed cells. Muller, A.J., DuHadaway, J.B., Donover, P.S., Sutanto-Ward, E. & Prendergast, G.C. 2004, Cancer Biol. Ther. , p. 3.

207. Interactions of myogenic factors and the retinoblastoma protein mediates muscle commitment and cell differentiation. Gu, WJ., Scheniider,W., Condrolli,G., Kaushal,, S, Mahdavi,V., Nadal-Gnard, B. 1993, Cell, pp. 72; 309-324.

208. Structural analysis of the human BIN1 gene: evidence of tissue-specific transcriptional regualtion and alternate splicing. Wechsler-Reya, R, Sakamuro, J., Zhang, J., DuHadaway, J., and Predengast. 1998, J of Biol Chem.

209. A role for th ePutative Tuimor Supressor Bin1 in Muscle Differentiation. Wechsler-Reya, R., Elliott, KJ, Prendergast, GC. 1998, Molecular and Cellular Biology, p. 18 (1) :566.

210. The putative tumor repressor BIN1 is a short lived nuclear phosphoprotein whose localization is altered in malignant cells. Wechsler-Reya, R., Elliot, K., Herlyn, M., Prendergast, GC. 1997, Cancer Res, pp. 57: 3258-3263.

211. Transformation selective apoptosis by farnesyltransferase inhibitors requires Bin1. DuHadaway, J.B. et al. 2003, Oncogene, pp. 22, 3578−3588 (2003).

212. The c-Myc-interacting adapter protein Bin1 activates a caspase-independent cell death program. Elliott, K., Ge, K., Du, W. & Prendergast, G.C. 2000., Oncogene , pp. 19, 4669−4684.

213. Growth stimulation of human bone marrow cells in agar culture by vascular cells. Knudtzon, S., and Mortensen, BT. 1975, Blood, pp. 46 (6) 937-943.

214. Exogenous endothelial cells as accelerators of hematopoietic reconstitution. Mizer, C., Ichim, TE, Alexandrescu, DT, DAsanu, CA, Ramos, F., Turner, A., Woods, EJ, Bogon, V., Murphy, MP, Koos, D., and Patel, A. 2013, J. Translational Medicine, p. 10: 231.

215. Dissecting the bone marrow microenvironment . Torok-Storb, B. et al. 1999, Annals of New York Academy of Science, pp. 872: 164-170.

217. Yuasa, XX and Ball YY. 2011.

218. Possible role of the ‘IDO-AhR axis’ in maternal-foetal tolerance. Hao K, Zhou Q, Chen W, Jia W, Zheng J, Kang J, Wang K, Duan T. 2013, Cell Biol Int. , pp. 37(2):105-8. doi: 10.1002/cbin.10023. .

219. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Pasare, C., Medzhitov, R. 2003, Science , pp. 299,1033-1036 .

220. Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. 2000, J. Immunol. , pp. 165,3804-3810.

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