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Development and application of non-rejectable skin substitute to improve wound healing Forouzandeh, Farshad 2009

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DEVELOPMENT AND APPLICATION OF NON REJECTABLE SKIN SUBSTITUTE TO IMPROVE WOUND HEALING by  FARSHAD FOROUZANDEH M.D., TEHRAN UNIVERSITY OF MEDICAL SCIENCES, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June2009  © Farshad Forouzandeh, 2009  ABSTRACT Skin substitutes consist of dermal and epidermal components are very beneficial to improve the current strategies of wound healing. However, these substitutes are not yet a routine treatment for burns or non-healing wounds mainly due to the difficulty of timely obtaining autologous skin cells. The aim of this study is to investigate the immuno protective role of IDO in bilayer skin substitutes made out of non-autologous cells. To address this aim, first we asked the question of whether tryptophan deficient environment caused by IDO expression is safe for the survival of nonimmune cells, mainly primary skin cells. The results of our study showed a significant activation of apoptotic pathway as well as GCN2 kinase pathway in T cells, but not in skin cells, in response to the tryptophan deficient environment mediated by IDO expression. We then studied whether there is any differences between the main T cell populations in response to IDO mediated low tryptophan environment. Our results showed a marked immunosuppressive effect of IDO expression on human T cells with more suppressive effect on proliferation of CD8compared to that of CD4 T cells which is, at least in part, due to differences in the level of GCN2 kinase pathway activation between these two sets of immune cells. Thereafter, we developed a bilayer skin substitute equipped with IDO expression ability. We found that IDO expressed by treated fibroblasts of this skin substitute suppress the proliferation of bystander lymphocytes in vitro considerably. Further, in our in vivo experiments, we found that the expression of IDO by cells of skin substitute significantly improved the wound healing rate, reduce the number of infiltrating T cells, and induced more revascularization in the wounds received IDO expressing skin substitutes compared to the non-IDO expressing ones. In conclusion, the results of this thesis research revealed that IDO expression can improve the efficacy of non-autologous bilayer skin substitutes  11  to be used not oniy as a wound coverage, but also as a source of wound healing process improvement. A better revascularization seen in IDO grafted skin substitute further improved the graft take and survival.  111  TABLE OF CONTENTS Abstract Table of Contents List of Tables List of Figures Acknowledgements Dedication Co-Authorship Statement CHAPTER 1: Introduction 1.1 Introduction 1.2 Wound Healing Process 1.3 Wound Healing and the Role of Dermal-Epidermal Interaction 1.4 Skin Substitutes and Wound Healing 1.5 Wound Healing and the Immune System 1.6 Immune System and Skin Graft Rejection 1.7 Indoleamine 2, 3-Dioxygenase as a Local Immunosuppressive Factor 1.8 Thesis Theme 1.9 References CHAPTER 2: Skin cells, but not T cells, are resistant to indoleamine 2,3dioxygenase (IDO) expressed by allogeneic fibroblasts 2.1 Introduction 2.2 Materials and Methods 2.3 Results 2.4 Discussion 2.5 References ChAPTER 3: Differential immunosuppressive effect of indoleamine 2,3dioxygenase (IDO) on primary human CD4 and CD8 T cells 3.1 Introduction 3.2 Materials and Methods 3.3 Results 3.4 Discussion 3.5 References CHAPTER 4: Indoleamine 2, 3-dioxygenase (IDO) expression improves the engraftment of non-autologous engineered skin substitute 4.1 Introduction 4.2 Materials and Methods 4.3 Results 4.4 Discussion 4.5 References CHAPTER 5: Conclusion 5.1 Conclusion 5.2 Significance and Applications 5.3 Limitations and Suggestions 5.4 References  137 143 144 146  APPENDICES A.1 Reprints of Published Papers A.2 UBC Research Ethics Board Certificates  150 166  ii iv v vi vii viii ix  .  1 2 8 10 13 13 15 28 37  60 62 68 78 81  86 87 90 96 98  101 104 112 124 129  iv  LIST OF TABLES Table 1.1 The main types of the skin substitutes  12  V  LIST OF FIGURES Figure 1.1 The time line of wound healing process  3  Figure 1.2 The proposed mechanisms for immuno-modulatory effects of IDO  17  Figure 2.1 IDO expression in fibroblasts  70  Figure 2.2 Proliferation rates of human T cells stimulated with allogeneic pieces of either epidermis or full thickness skin in the presence or absence of IDO expressing cells 71 Figure 2.3 The effect of kynurenine on the survival rate of human skin cells and T cells  72  Figure 2.4 Effect of IDO expression on cell survival rate of human skin cells and T cells  74  Figure 2.5 Role of GCN2 kinase pathway in selective apoptotic effect of IDO expression on human T cells vs. skin cells 77 Figure 3.1 Fluorescence-activated cell sorting of human PBMC for T cells  92  Figure 3.2 The effect of IDO expression on resting and stimulated human CD4 and CD8 T cells 93 Figure 3.3 Effect of IDO-induced low tryptophan and high kynurenine environment on proliferation of T cell subsets 94 Figure 3.4 GCN2 kinase pathway is involved in suppressive effect of 1DO expression on human T cell subsets 95 Figure 4.1 Histological structure of the Engineered Skin Substitute (ESS)  113  Figure 4.2 IDO expression and activity in ESSs  116  Figure 4.3 Closure of 6mm circular wounds treated with different ESSs  119  Figure 4.4 Detection of CD3-infiltrated lymphocytes in wound sections after engraftment  121  Figure 4.5 Detection of vessel-like structures in wound sections after treatment with ESSs 123  vi  ACKNOWLEDGEMENTS I offer my enduring gratitude to the faculty, staff and my fellow students at the University of British Columbia, especially all of my colleagues in Burn and Wound Healing Professional Laboratory at Jack Bell Research Centre. I am also very grateful to the members of my advisory committee, Dr Aziz Ghahary, Dr Vincent Duronio, Dr Nicholas Carr, and Dr Kevin McElwee from whom I received many thoughtful comments as well as very kind encouragements and supports during the course of my research project. Also, I gratefully acknowledge the Canadian Institute of Health Research (CIHR) to grant my research project and the funders of following scholarships and awards that supported me during the course of my PhD: -  -  -  -  -  -  -  -  -  -  -  University of British Columbia Graduate Fellowship British Columbia Industrial Innovation Council Scholarship Pacific Century Graduate Scholarship Anne and John Brown Fellowship in Diabetes and Obesity Related Research CIHR’MSFHR Training Program in Transplantation Mary Norman Graduate Award in Medical Research Vancouver Coastal Health Research Institute Rising Star Award l2 and  th 14  Annual Chung Research Day Lectureship Awards  American Society for Cell Biology Travel Award Centre for Human Islet Transplantation and 13-cell Regeneration Travel Grant World Union of Wound Healing Societies Travel Scholarship  vii  DEDICATION  To all burn victims andpatients sufferingfrom non-healing wounds whose pain drove me to seek them out a remedy,  To my great mentor, Professor Aziz Ghahary, who taught me not only the science but more how to contribute to it,  To my parents who have dedicated me their lives and taught me how to dedicate mine to others, &  To my forever beloved wife andfriend, Aneseh, whose love has prompted me to enjoy every moment of my lfe...  viii  CO-AUTHORSHIP STATEMENT I gratefully appreciate all the valuable contributions that I received from all the co authors of the manuscripts published or submitted based on this thesis. This is to confirm that I, Farshad Forouzandeh, as the first author of these manuscripts, as presented in chapter 2 to 4, had the main role in the following areas:  -  -  -  -  identification and design of research program performing the research data analyses manuscript preparation  ix  CHAPTER 1 Introduction Skin is the largest organ in the body and it protects our body against many hazardous toxins and microorganisms in the environment and also serves to prevent dehydration of all non-aquatic animals. In addition, there are many other critical functions for the skin such as sensory detection, immune surveillance, and self-healing. Loss of skin integrity because of injury or illness may result acutely in substantial physiologic imbalance and ultimately in significant disability or even death (1). Therefore, the ability to generate or repair injured skin tissue is essential to the continuity of human life. As in all other organs, wound healing in the skin is a dynamic process involving tissue response to different types of insults. This process involves a continuous sequence of signals and responses in which platelets, fibroblasts, epithelial, endothelial and immune cells come together outside of their usual domain in order to orchestrate a very complex event which results in tissue repair. These signals, which are mainly growth factors (GFs) and cytokines, orchestrate the initiation, continuation and termination of wound healing (2,3). An imbalance in the synthesis and release of these cytokines and GFs at the wound site may result in either difficult-to-heal wounds, as seen in the diabetic and elderly population, or over-healing wounds as seen in fibroproliferative disorders, a frequent complication following surgical incision, traumatic wounds and severe electrical and thermal injury (4-6). Although the promotion of healing in patients with non-healing wounds is desirable, the over healing process such as post-burn hypertrophic scarring is not only disfiguring, but also limits the range of motion, mobility and dexterity, affecting return to occupational and lifestyle pursuits (7,8). These two extreme wound-healing conditions are pervasive medical  1  problems with far reaching clinical and economic implications. In this chapter, the wound healing process and different types of skin substitutes which are among the very promising ways to address the difficult-to-heal wounds will be reviewed. Thereafter, our novel approach in this project in addressing some of the short coming of these skin substitutes will be discussed in details.  Wound Healing Process Over the past three decades, our understanding of the cellular and molecular aspects of the wound healing has been improved substantially (1,9). Wound healing is a multi-step process that can be generally divided into 3 overlapping phases of inflammatory, proliferation, and remodeling phases (figure 1.1). The inflammatory phase involves vascular responses characterized by blood coagulation and hemostasis as well as cellular events, including infiltration of leukocytes with varied functions in antimicrobial and cytokine release. Indeed, the factors released by the cells during the inflammatory phase will initiate the proliferation phase (10). During the proliferative phase, there is formation of the epithelium to cover the wound surface, reepithelialization, along with simultaneous growth of granulation tissue to fill the wound gap. Granulation tissue formation involves proliferation of fibroblasts, loosely deposition of collagens and other extracellular matrices, and development of angiogenesis. After the formation of the new tissue, the remodeling phase begins to restore tissue structural integrity and functional abilities (10).  2  Phase I: Inflammation Hemostasis Inflammation  Phase II: Proliferation Reepithelialization Angiogenesis Reconstitution of the dermis Fibroplasia  Phase Ill: Remodeling Wound contraction Scar maturation Matrix turn over Changes In collagen type/tensile strength  Minutes  Hours  Days  Weeks  Months-Years  Figure 1.1 The time line of wound healing process. The 3 phases of wound repair are not simple linear events but rather overlapping in time  Acute wounds refer to those wounds, such as burns, other traumatic injuries, and surgically created wounds, that heal in a timely fashion such as a clean and uninfected surgical incisional wound approximated by surgical sutures. Although the desirable final result of coordinated healing would be the formation of tissue with a similar structure and comparable functions as with intact skin, regeneration is unusual (with extraordinary exceptions such as early fetal healing); however healing results in a structurally and functionally acceptable but not identical result. Alterations that disrupt controlled timely healing processes would extend tissue damage and prolong repair (10,11). The main phases of wound healing are as follows:  3  -  Phase I: Inflammation  The first consequence of any injury to our body is the inflammation process as the first innate initial reaction which most of the time leads to tissue repair and restoration of the lost function. During the early steps of the wounding process, local vasodilatation, blood and fluid extravasation into the extravascular space, and blocking of lymphatic drainage can produce the four cardinal signs of inflammation including heat, pain, redness, and swelling (calor, dolor, rubor, and tumor). This acute inflammatory response usually lasts between 1 to 2 days and may persist for up to 2 weeks. As a result of tissue injury blood vessels will disrupted and bleeding will occur. Platelets adhere, aggregate, and release many mediators to facilitate coagulation. Although hemostasis is the major function of blood coagulation, a secondary but equally important function of platelets is to initiate the healing cascade via release of chemoattractants and growth factors. At the same time, the clot provides a matrix scaffold which facilates the recruitment of cells to the injured area. Then, the chemokines released during the vascular step of the inflammation will recruit leukocytes, including neutrophils and macrophages to the wound site. These infiltrated cells to the wounded area will assist in cleaning and removing damaged tissue debris and foreign particles. Once in the wound site, activated macrophages release several important growth factors and cytokines, initiating granulation tissue formation. Indeed, the mediators and signals triggering the proliferation phase will be released early on during the inflammation phase of wound healing. Therefore, inflammation phase is absolutely beneficial to the wound healing process. However, too much or too long inflammatory phase can significantly can interfere with the normal wound healing process. These chronic wounds usually are sealed by necrotic tissue which is  4  contaminated with pathogens, or contains foreign material that cannot be phagocytized or solubilized during the acute inflammatory phase (10). Indeed, in experimental models of repair, too long inflammatory phase has been shown to delay healing and to result in increased scarring. Furthermore, chronic inflammation, a hallmark of the non-healing wound, predisposes tissue to cancer development (12).  -  Phase II: Proliferation  In the proliferation phase of wound healing cellular activity predominates. The major events during this phase are reepithelialization, angiogenesis, and reinforcement of the injured dermal tissue (i.e., fibroplasia).  Reepithelialization  Reepithelialization is the process of restoring the epidermis after skin injury. It generally involves several processes, including the migration of epidermal keratinocytes from the wound edges into the wound and their proliferation, the differentiation of the neoepithelium into a stratified epidermis, and the restoration of an intact basement membrane zone that connects the epidermis and the underlying dermis. Angiogenesis  Angiogenesis refers to new vessel growth by the budding of preexisting vessels adjacent to the wound. In response to the injury, microvascular endothelial cells initiate an angiogenic process consisting of activation of endothelial cells, local degradation of their basement membrane, budding into the wound clot, cell proliferation, tubulur structure  5  formation, reconstruction of the basement membrane, and, eventually, regression and involution of the newly formed vasculature as tissue remodeling (13,14). Newly formed vessels participate in granulation tissue development and provide nutrition and oxygen to emerging tissues. During angiogenesis, endothelial cells also produce and secrete growth factors and cytokines useful for the wound healing process.  Reconstitution of the dermis and fibroplasia  Dermal reconstitution which begins approximately 3 to 4 days after injury, is characterized clinically by granulation tissue formation, which includes angiogenesis, and the accumulation of fibroblasts and ground matrices, named fibroplasia. The interim extracellular matrix that is formed in part by the fibrin clot, which is rich in fibronectin, promotes granulation tissue formation by providing scaffolding and contact guidance for cells to migrate into wound gaps and for angiogenesis and fibroplasia to occur in an effort to replace the wounded dermal tissue. Fibroplasia is described as a process of fibroblast proliferation, migration into wound fibrin clot, and production of new collagen and other matrix proteins, which contribute to the formation of granulation tissue (10).  Wound contraction  Fibroblasts are also modulated into phenotypes of myofibroblasts and participate in wound contraction. Contraction of the wound begins soon after wounding and peaks at 2 weeks and it will continue till the very end stages of the remodeling phase. The degree of wound contraction depends mostly to the depth of the wound. For full-thickness wounds, contraction can cause up to 40% decrease in the size of the wound (15). There is  6  increased expression of smooth muscle differentiation markers of a—smooth muscle actin, smooth muscle myosin, and desmin starting on day 6 in myofibroblasts which reaches a maximum on day 15 and then regress progressively (16).  -  Phase III: Remodeling  Remodeling consists of the continuation of wound contraction, deposition of the matrix and its subsequent changes over time. It occurs throughout the entire wound repair process as fibrin clot formed in the early inflammatory phase is replaced by the granulation tissue that is rich in type III collagen and blood vessels during the proliferative phase and subsequently replaced by a collagenous scar predominantly of type I collagen predominant with much less mature blood vessels (17,18). In healthy adults, type I collagen accounts for approximately 80% of collagens and type III collagen constitutes 10% of collagens in the dermis. During early wound healing, however, similar to the case in the fetal dermis, type III collagen is the major collagen synthesized by fibroblasts in granulation tissue. Type 111 collagen first appears after 48 to 72 hours and is maximally secreted between 5 and 7 days. The total amount of collagen increases early in repair, reaching a maximum between 2 and 3 weeks after injury. Over the period of I year or longer, the dermis gradually returns to the stable preinjury phenotype, consisting largely of type I collagen. Tensile strength, a functional assessment of collagen, increases to 40% of strength before the injury at 1 month and may continue to increase for 1 year, reaching up to 70% of its preinjury strength (10).  The regulation of collagen synthesis is controlled by several growth factors, including transforming growth factor- f3 (TGF-13) and fibroblast growth factor (FGF). TGF—13  7  stimulates types I and III collagen production. Surplus TGF-131 has been found in the dermis of chronic venous ulcers and may play a role in fibrosis. Also, matrix metalloproteinases (MMPs) play an important role in wound remodeling. Unbalanced expression of MMPs and tissue inhibitors of metalloproteinases (IMPs) may also contribute to delayed healing or excessive fibrosis (10). In summary, while acute wounds go through the linear but overlapping phases of the 3 wound healing, these phases do not progress normally in difficult-to-heal wounds. Indeed, in chronic wounds some areas of the wound are found in different phases, having lost the ideal synchrony of events that leads to normal (rapid) healing. Therefore, it is critical to understand the normal repair process to better understand the mechanisms of delayed and/or non-healing wounds or, alternatively, excessive fibrosis.  Wound Healing and the Role of Dermal-Epidermal Interaction As discussed earlier, the process of wound healing involves at least three major overlapping phases, the lag phase (homeostasis, inflammation, cytokine release), proliferative phase (fibroblast number increases, myofibroblasts appear, matrix deposited, epithelial cell migration, vascularization) and the remodeling phase (cell number decreases, collagen fiber organization) (19,20). The initial phase of wound reepithelialization involves activation of basal and suprabasal keratinocytes located at the wound edge that migrates underneath the dried out portion of the wound edges to re epithelialize the wound (21). When an injury occurs that is too large for the wound edges to be approximated, epithelial cells from sub-epidermal appendages, like hair follicles and  8  sweat glands, migrate toward the wound surface and rapidly proliferate to form a new epidermal layer. Thus, surviving a large body surface area injury to the skin depends heavily on the efficiency of an epithelial lining to be restored over the wound site (22). Keratinocytes, which are the main cell type in the epidermis, are the major source of different cytokines and GFs and therefore their role in dermal wound healing is critical (19,23). Keratinocyte-fibroblast interactions has been the focus of several studies. It has been demonstrated that when a cultured keratinocyte sheet is used as a temporary wound coverage, it promotes wound healing and increases wound epithelialization (24). Further studies revealed that lysates of cultured keratinocytes contain mitogenic activity for keratinocytes, endothelial cells and fibroblasts (25,26). Epidermal cell-derived factors seem to control wound healing through stimulation of migration and proliferation of keratinocytes from sweat glands, hair follicles and wound edges. These findings collectively suggest that keratinocyte-derived factors are likely to be a mixture of several previously identified and unrecognized factors with overlapping biological activities important in the healing process (27). Thus, local cytokines and GFs, released from both keratinocytes and fibroblasts functioning in a paracrine and/or autocrine fashion are considered to be the main regulators of activation, migration, proliferation and differentiation of keratinocytes during the dynamic process of wound healing (3,19). Therefore, considering these very important interactions of the epidermal and dermal layers in wound healing process, using a wound healing promoting system that has these both layers, such as a bilayer skin substitute composing alive keratinocytes and fibroblasts, seems to be very beneficial approach to address the problem of difficult-to heal wounds.  9  Skin Substitutes and Wound Healing  Burn and wound management has been changed enormously in recent decades (28). It has been clearly verified that early wound closure leads to lessen the chance of post-burn hypermetabolic state in the acute phases of burn injury and improved epidermal-dermal interactions with rapid wound closure. This itself will result in reduction of the frequency of developing fibrotic conditions, such as scar hypertrophy and contractures (23). Autologous skin grafts, the main desired source for early wound closure, however have limited availability and are associated with additional scarring at the donor sites. Severe burn patients invariably lack sufficient adequate skin donor sites. Additionally, skin grafting creates additional donor site wounds equivalent to second degree burns thus further increasing the total body surface area that is affected directly or indirectly by the injury (29). These difficulties have encouraged the development and use of skin substitutes and replacement materials of natural and biotechnological origin. Ideally, skin substitutes for wound healing should replace all the structures and functions of native skin including the epidermal and dermal layers. Unfortunately, there are currently no engineered skin substitutes that can completely duplicate the complexity of human skin. Available skin substitutes can act as temporary wound coverage or permanent skin replacements, depending upon their design. Temporarily, they cover the wound and preventing dehydration and keep the wound bed moist and prepare it for skin grafting. This will give time to the donor site to heal and become ready for reharvesting. Permanent skin substitutes tend to replace one or both layers of skin facilitating wound healing in several different clinical settings. In addition, some skin substitutes can release cytokines and GFs to the wound site as well as stimulating the host to produce a variety  10  of cytokines and GFs that promote wound healing (30,3 1). There are many different characteristics of the patients and the reconstruction method itself that must be considered before choosing any specific therapy including an appropriate skin substitute to be used. These investigations should be very comprehensive and considering the normal skin anatomy, the patient’s condition, patient’s comorbidities, wound type, the type of the tissue that is missing, the level of contamination, visibility of the area, contour abnormalities, adjacent tissue laxity, vascularity of the wound bed, ability to immobilize the patient postoperatively, and aesthetics. Within this framework, a reconstructive plan may be formulated with the goals of wound closure, prevention of infection, and stablerobust coverage, which maximizes function while minimizing donor defects (30).  As mentioned before, despite of many previous and ongoing efforts, there is still no perfect or ideal skin substitute available that can replace all the functions of intact human skin (30,32). However, there are many different skin substitutes available that can be used with some success to improve the wound healing process and prevent some of the disastrous consequent of skin injury. A list of some of the main types of the skin substitutes is presented in Table. 1.1 (1,30-32).  11  Table 1.1- The main types of the skin substitutes XENOGRAFTS  Ranafilrn Permacol OASIS  -  -  Frog skin, the first recorded one was made 1500 b.C. Processed porcine acelliilar dermis Processed acdllular extracellular matrix from porcine jejamum  ALLOGRAFTS  Epidermal Laser skin Dermal Cadaveric skin Alloderm TransCyte Dermagraft -  -  Cultured allogeneic keratinocytes  -  Icx-sKI4 Composite Apligraf  Processed allogeneic acellular dermis Processed allogeneic acellular dermis Processed allogeneic neonatal foreskin fibroblasts seeded on nylon mesh Cryopreserved allogeneic neonatal foreskin fibroblasts seeded on bioabsorbabale polyglactin mesh scaffold Cultured allogeneic fibroblasts seeded on human-based extracellular matrix -  -  -  -  -  -  OrCel  Cultured allogeneic neonatal keratinocytes and fibroblasts seeded in bovine collagen gel Cultured allogeneic neonatal keratinocytes and fibroblasts seeded in bovine collagen sponge -  -  AUTOGRAFTS  Epidermal Epicel  -  Epidex  TranCell  -Composite css  Cultured autologous keratinocytes from patient skin biopsy, transplanted as epidermal sheet using petrolatum gauze support Cultured autologous keratinocytes isolated from patient’s outer root sheath of scalp hair follicles, transplanted as epidermal sheet discs with a silicone membrane support Cultured autologous keratinocytes from patient skin biopsy, grown on acrylic acid polymer-coated surface, transplanted as epidermal sheets -  -  -  Cultured Skin Substitute, Bilayered autologous keratinocytes and fibroblasts cultured from patients’ skin biopsy, combined with degradable bovine collagen matrix -  SYNTHETIC Suprathel Biobrane Integra  Monolayer epidermal substitute composed of DL-Lactatide Bilayer skin substitute composed of very thin semipermeable silicone membrane bonded to nylon fabric and collagen Bilayer skin substitute composed of biodegradable dermal layer made of porous bovine collagen-chondroitin-6-sulfate matrix and a temporary epidermal layer made of synthetic silicone polymer -  -  -  12  Wound Healing and the Immune System Considering the crucial roles of the immune cells in the process of wound healing, it is worthwhile to review some aspects of the roles of immune system in details. In fact, cells of the immune system are among the most active participants in the repair of injured skin. Immune cells can greatly impact the repair process at each of the three main phases of wound healing. Immediately after dermal injury, platelets enter the wounded area. They aggregate and release lots of mediators that initiate the coagulation cascade, as well as GFs and cytokines involved in the recruitment of inflammatory and immune cells (9,11). Resident mast cells degranulate after injury and also release immune mediators and histamine which causes more vasodilation. Neutrophils recruited to the wound site will destroy any invading microorganisms. Thereafter, macrophages will enter the area to clear the wound from debris and foreign particles. Both neutrophils and macrophages release a series of GFs and cytokines. Lymphocytes play a distinct and regulatory role in normal wound healing through the secretion of lymphokines (33). In addition to performing their protective functions, immune cells and their releasable mediators are also believed to be important for later stages of healing mainly the proliferative phase, reepithelialization, angiogenesis, and the remodeling (9). Based on all of theses evidence, cells of the immune system are regarded as absolute necessities for proper wound healing (II).  Immune System and Skin Graft Rejection The immune system comprises a nonlinear network of pathways that orchestrate the balance of survival of an individual in its environment (34). After allogeneic cell  13  transplantation, a state of immune activation driven by recognition of major or minor histocompatibility antigens, invariably will emerge in the recipient, even in human leukocyte antigen (HLA)-matched donors. In addition, even the surgery process itself causes some tissue damage that can stimulate the immune reaction. This state of the immune activation will include the secretion of pro-inflammatory cytokines including interferon-y (IFN-y) by antigen presenting cells (APCs) or activated T cells (34). Like many other organs, rejection of allogeneic grafted skin is a major obstacle in dermal transplantation (35,36). As skin is not a primary vascularized organ, hyperacute rejection involving antibody responses to allogeneic protein does not occur (37). In fact skin rejection is believed to be an acute rejection which is mostly T cell dependent (38,39). Indeed, there are different cell types that contribute in the acute graft rejection process, however, only T lymphocytes appear to be absolutely required for acute rejection (35,40,4 1). For this reason, the most effective immuno-suppressive agents to prevent this type of rejection are those that function as anti-T cell proliferatives such as cyclosporin and FK506. Although the use of immunosuppressive drugs has significantly improved graft take, the lack of specificity on T cell proliferation, systemic immuno-suppressive effects on patient’s immune system and their side-effects are still a major concern in their administration (3 7,42). Although the immunogenicity of the cultured skin substitutes either from xenogeneic or allogeneic primary cells are less substantial compared to normal human skin, still the immune rejection process can threaten the survival of these skin substitutes at the grafted sites and a use of appropriate immunosuppressive agent or tolerance induction strategy is needed to at least postpone their rejection (35,43-45).  14  Indoleamine 2, 3-Dioxygenase as a Local Immunosuppressive Factor The immune system continuously modulates the balance between responsiveness to pathogens and tolerance to non-harmful antigens. Although the mechanisms that mediate tolerance are not well understood, recent findings have implicated tryptophan catabolism through the kynurenine metabolic pathway as at least one of the main mechanisms involved in immune tolerance (46). Tryptophan is essential for protein synthesis in mammals and many other important molecules and it is the least abundant of all essential amino acids in the human body. The major catabolic route of tryptophan in mammals is the kynurenine pathway that can ultimately lead to the biosynthesis of nicotinamide adenine dinucleotide (NAD) (46). The initial and rate-limiting reaction of the kynurenine pathway is the oxidation of tryptophan to N-formyl-l-kynurenine, catalysed by hepatic tryptophan 2,3-dioxygenase (TDO) or the ubiquitous, extra-hepatic, indoleamine 2,3dioxygenase (IDO or INDO) (46). Approximately 99% of the dietary tryptophan that is not used in protein synthesis is metabolized by IDO (47). IDO has recently been proposed to have profound immunoregulatory activity and the concept that cells expressing IDO can suppress T cell responses and promote tolerance is a relatively new paradigm in immunology (figure 1.2). Considerable evidence now supports this hypothesis, including studies of mammalian pregnancy, tumor resistance, chronic infections and autoimmune diseases (34,48-50). IFN-y but not IFN-u and IFN-13 which is a cytokine mainly produced by activated T cells (51), is a potent IDO inducer in a variety of cell types. The immuno-modulatory and antiproliferative effects of IFN-y both in vitro and in vivo seem to be due, at least in part, to IFN- y inducing cellular proteins such as TDO (52) There is compelling evidence to .  15  suggest that IDO may in fact function as the cellular defense mechanism through which intracellular tryptophan is deprived and thereby functions as an anti-proliferative factor for intracellular pathogens (53-56). IDO possesses an anti-proliferative effect on T cells both in vitro and in vivo (48,50,57). Moreover, IDO expression by macrophage arrests the activated T cells in the G1-S phase through a marked reduction in tryptophan concentration and an increase in tryptophan metabolite, kynurenine. This arrest was restored by the addition of tryptophan or IDO inhibitor, 1 -methyl-tryptophan (1 -MT) (58). Furthermore, Munn et al. (49,59) reported that antigen-presenting cells (APC5) can regulate T cell activation through tryptophan catabolism and speculated that the expression of IDO by certain APCs in vivo allows them to suppress unwanted T cell responses. Moreover, Munn et a!. (48) demonstrated that the expression of IDO during murine pregnancy is required to prevent rejection of semi-allogeneic fetus by maternal T cells. This finding is consistent with the fact that systemic tryptophan in normal pregnancy is low and IDO produced by trophoblasts is localized to the zone of contact between fetal-derived tissues and the maternal immune system (48). Based on these findings, in this project we suggest that induction of IDO by IDO genetically modified skin substitute at the wound site suppresses the infiltrated T cells and thereby delays or prevents skin graft rejection without affecting the patient’s general immune system. Therefore, the role of IDO as a local immunosuppressive factor in a cultured skin substitute model is the main focus of this project and as such herein we will discuss the characteristics of IDO in more details.  16  IDO  Tryptophan metabolites  1-MT Uncharged tRNA  Jr  t  GCN2 activation  Immunosuppression: -r cell anergy Apoptosis Cell cycle arrest -  -  Figure 1.2- The proposed mechanisms for immuno-modulatory effects of IDO. Both tryptophan deficiency and increased in the level of tryptophan metabolites, i.e. kynurenine, cause immunosuppression through different ways.  -  Biochemical Characteristics of IDO  Mature IDO is a 42-45 KDa monomeric protein containing heme as its only prosthetic group. The tertiary structure of recombinant human IDO was recently defined using X ray crystallography (60). Overall, IDO is folded into two distinct alpha-helical domains, one small and one large, with the heme prosthetic group positioned between them. Once synthesized, the IDO holoenzyme catalyzes the oxidative cleavage of the pyrrole ring of L-tryptophan to generate an unstable metabolite, N-formylkynurenine, which is metabolized to formic acid and the stable-end product, kynurenine. IDO has high affinity for L-tryptophan (Km—0.02 mM) and therefore can rapidly catabolize it to create a local tissue microenvironment devoid of this essential amino acid (46).  17  -  IDO Expression and its Regulation  IDO is expressed constitutiyely as an intracellular protein and also can be induced in different cell and tissue types. Other than in the male epididymis, with unclear significance, IDO is constitutively expressed only in the lower gastrointestinal tract (61). IFN-y is a potent inducer of IDO expression in placenta (62), macrophages (63), dendritic cells, cultured fibroblasts (64), and many cancer cell lines (65). IDO also has been detected in other cells which may be important to allergic inflammation including eosinophils (66), endothelial cells (67,68), and lung epithelial cells (69). IDO in mice and humans is encoded by a single gene, termed Indo, with 10 exons spread over ‘-1 .5 kbp of DNA located on the short arm of chromosome 8 (8p1 2—8pl 1) (70) and, as shown in murine and human dendritic cells, it was found to be co-regulated by a limited number of genes (71). IDO gene transcription, in general, occurs in response to inflammatory mediators, most prominently IFN-’y, or toll-like receptor (TLR) ligation (e.g. through Iipopolysaccharide) (72). In fact IDO gene is regulated by a promoter that contains a single IFN-y-activated site specific for IFN- ‘y, as well as two nonspecific IFN stimulated response elements, which can respond to IFN-a and IFN-f3 as well as IFN-’y. Based on the cell type being cultured, IFN-y has been described as being up to TOO times more effective in inducing IDO expression than 1FN-o or IFN-f3 (64). In contrast, T helper 2 (Th2) cytokines such as IL-4 and IL-13 inhibit the expression of IDO (73,74). Intracellular signaling following ligation of IDO inducers occurs along the JAK-STAT pathway and nuclear factor iB (NFKB) (72,75) to finally result in expression of the monomeric, cytosolic, 45 KDa IDO glycoprotein (76). Furthermore, some of the IDO dependent effects are initiated by CpG-rich oligodeoxynucleotides and depend on cell  18  signaling through TLR9, (77,78) whose activation typically results in type I IFN release by plasmacytoid DCs (79). In the immune system, certain types or subsets of APCs seem to be preferentially willing to express functional IDO when challenged with proinflammatory stimuli or exposed to signals from activated T cells. In mice, these “IDO-competent” APCs include a subset of plasmacytoid DCs (80), CD8o+ splenic DCs (or a subset there of) (71,8 1), and doubtless other subsets of DCs and macrophages as well (82). On the other hand, even in APCs that are IDO competent, the actual presence or absence of functional IDO enzymatic activity is tightly regulated by specific maturation and activation signals (83-85). Theoretically, this ability to upregulate or downregulate IDO in response to external stimuli seems reasonable, as APCs are required to sometimes present antigens in an activating fashion and sometimes in a tolerating fashion to serve their immune roles in different situations (86). -  IDO and its Immuno-modulatory Roles  As mentioned earlier, IDO has recently emerged as an important immuno-modulator of T cell function and inducer of tolerance. Expression of IDO plays critical roles in regulation of T cell-mediated immune responses. IDO dependent T cell suppression by dendritic cells suggests that biochemical changes due to tryptophan catabolism have substantial effects on T cell proliferation, differentiation, effector functions and viability (72). In addition to the potential role of IDO in promoting tolerance in pregnancy, transplantation, and autoimmunity, its role in modulating allergic responses has recently been investigated, raising the possibility that IDO and its metabolites may be novel targets for immuno-modulation in allergy and asthma (87). IDO has been implicated as a possible immunosuppressive effector mechanism of regulatory T cells (Tregs).  19  Grohmann et a!. (88) showed that Tregs could trigger high levels of functional IDO expression in mouse DCs in vitro. This occurred through binding of CTLA4 on Tregs to B7-1 and B7-2 on DCs, which transduced a signal in the DCs that upregulated IDO protein expression and functional enzymatic activity (88,89). A similar ability of CTLA4B7-1 and/or B7-2 interactions to induce IDO has been shown in human monocyte derived DCs (71,90). Therefore, IDO might function as a downstream mechanism through which CTLA4+ Tregs mediate immuno-modulation (91). Another biologic function of IDO is a counter regulatory mechanism to suppress excessive immune activation. Such counter regulatory pathways are vital in the immune system because uncontrolled immune responses can cause unacceptable damage to the host. At sites of inflammation, IDO expression is not limited to DCs and macrophages but can be found in epithelial cells (92,93), eosinophils (66),endothelial cells (68) and possibly other cell types as well. Therefore, IDO is an important endogenous counterregulatory mechanism that helps protect the host. -  IDO and its Role in Pregnancy  Fetus has paternally derived genes that encode antigens that are immunologically considered to be foreign for the mother immune system. Indeed, fetus is semi-allogeneic graft for the pregnant woman. However, most of the time, this semi-allogeneic graft can survive the gestational period with no immunorejection. This fascinating paradox was first raised by the Nobel Laureate Sir Peter Medawar in 1953. Interestingly, endogenous IDO expressed by the trophoblasts has been implicated as one the main mechanism that maintains this maternal immunotolerance and the mice treated early in pregnancy with 1methyl-tryptophan (1-MT), which is an inhibitor of IDO, underwent immune mediated  20  rejection of their fetus (48,94,95). In brief, these findings suggest that IDO expressing trophoblastic cells provide an immunosuppressive barrier protecting semi-allogeneic fetus tissue from maternal T cell immunity. -  IDO-Mediated Immune Suppression and GCN2 Kinase Pathway  It appears that some of the biological effects of IDO can be mediated via local depletion of tryptophan, whereas others are mediated via immuno-modulatory tryptophan metabolites. As mentioned earlier, IDO initiates the degradation of tryptophan along the kynurenine pathway. IDO and the downstream enzymes in this pathway produce a series of immunosuppressive tryptophan metabolites. Some of these metabolites suppress T cell proliferation in vitro or cause T cell apoptosis, and some can affect NK cell function (9699). The molecular mechanism by which tryptophan metabolites exert their immunologic effects is not known, but at least one recent report has described an orphan G proteincoupled receptor, GPR35 receptor, which binds to a specific metabolite of tryptophan (kynurenic acid) (100). In addition to the immunosuppressive effects of tryptophan metabolites, the cellular stress imposed by local depletion of tryptophan also mediate some of the immunosuppressive effects of IDO. This was first suggested when some effects of IDO on T cells can be reversed by the addition of excess tryptophan in vitro (80,101,102). Recently, the stress-responsive kinase general control nonderepressible 2 (GCN2) has been recognized as a signaling molecule that enables T cells to sense and respond to stress conditions created by IDO (103,104) As mentioned earlier, one .  possible mechanism for immunoregulatory effect of IDO is through depleting the essential amino acid tryptophan in the microenvironment to the level that T cells are unable to proliferate (72). In this way, IDO might affect pathways known to respond to  21  amino acid metabolism. There are two known amino-acid-responsive signal-transduction pathways through which T cells might sense decreased levels of free tryptophan. One of these pathways is the essential amino acid deficiency antagonizing signaling through the mammalian target of rapamycin (mTOR) kinase pathway (72,105,106). mTOR signalling is required for normal beginning of ribosomal translation. This pathway is important for growth-factor signaling. T cells are particularly sensitive to inhibitors of mTOR such as the immunosuppressant drug, rapamycin (.1 07,108). However, it has been found that inhibitors of mTOR such as rapamycin did not recapitulate the profound proliferative arrest seen with IDO-mediated suppression (109). In yeast, there is a second amino acidsensitive pathway mediated by the kinase GCN2 (110). GCN2 contains a regulatory domain that binds the uncharged form of transfer RNA (tRNA). Amino acid insufficiency causes a rise in uncharged tRNA, which activates the GCN2 kinase domain and initiates downstream signaling (111). Recently, the mammalian homolog of GCN2 has been recognized and shown to have similar signaling properties (112,113). GCN2 is one of a family of four related kinases (GCN2, PERK, HRI, and PKR), which share as their only known substrate the alpha subunit of translation eukaryotic Initiation Factor 2 (eTF2cL). Indeed, GCN2 was originally identified as a regulator of translation control in response to starvation for one of many different amino acids (114). Uncharged tRNA that accumulates during amino-acid depletion binds to a GCN2 regulatory domain homologous to histidyl—tRNA synthetase enzyme, triggering eIF2ci kinase activity (113, 115). Indeed, GCN2 participates in nutritional stress management that guides food selection for survival. Animals faced with a diet lacking in essential amino acids quickly reject the imbalanced food and forage for complete or complementary sources of protein  22  (116,117). Activation of GCN2 kinase pathway, which has been termed the integrated stress response (ISR), can trigger cell-cycle arrest, differentiation, compensatory adaptation, or apoptosis, depending on the cell type and the initiating stress (118-12 1). It has been found that expression of IDO by APCs activates the GCN2 kinase pathway in responding T cells, generating an intracellular signal that mediates key biologic effects of IDO. Specifically, it has been shown GCN2 is required for CD8 T cells to sense and respond to conditions created by IDO (57). T cells lacking GCN2 proliferated normally in the presence of IDO+ APCs both in vitro and in vivo and were not susceptible to IDO induced anergy (103). Exactly how IDO creates the stress that activates GCN2 in T cells is not yet known. In the published literature, the only well-characterized and proven stimulus for GCN2 activation is a rise in uncharged tRNA, as would occur if IDO depleted the T cells of tryptophan. The fact that IDO-mediated suppression was reversed by addition of excessive amount of tryptophan might also be consistent with a mechanism of tryptophan depletion. However, we cannot exclude the alternative possibility that a downstream metabolite produced by IDO might interfere with the acylation reaction by which tryptophan is ligated to its tRNA. Although an inhibitory metabolite would also cause a rise in uncharged tRNATRP (by inhibiting the charging reaction), its effect might be overcome by high tryptophan concentration. GCN2 is broadly expressed, and other cell types (including the APC itself) might respond to IDO via their own endogenous GCN2 pathway. It has been shown that IDO, itself, can mediate some events that modify the biology of the IDO-expressing cells (122-124). Thus, the IDO/GCN2 pathway might influence the biology of the APC itself, and perhaps even other bystander cells as well. In brief, studies have identified GCN2 as a  23  downstream mediator for several key effects of IDO. This constitutes the first clarification of a specific molecular target for the immunoregulatory action of IDO in T cells. -  Other Clinical Roles of IDO  IDO also controls the severity of a variety of experimental autoimmune disorders (125128). According to the model suggested by Grohmann eta!. (126), IFN-’y acts on tolerogenic DCs to activate the expression of IDO which leads to the induction of immunosuppressive tryptophan catabolism and to the onset of specific self tolerance. Furthermore, Gurtner et al. (125) showed that IDO plays an important role in the downregulation ofThl responses within the gastrointestinal tract and may play a similar role in human inflammatory bowel disease (IBD). Moreover, pharmacologic inhibition of IDO causes marked exacerbation of inflammation and worsened symptoms of diseases in experimental models of IBD (125), allergic asthma (69), and autoimmune encephalomyelitis (128). Thus, IDO might play a physiological role in regulating immune responses against self antigens. The role of IDO in defense mechanisms against some pathogens is also considerable. This role of IDO is more pronounced against microorganisms which are sensitive to tryptophan deficiency including, but not limited to, Chiamydia pneumoniae, Toxop!asma gondii and certain bacteria, such as group B streptococci and mycobacteria species (63,129,130). Similarly, during viral infection, IDO inhibits the replication of cytomegalovirus (CMV) and herpes simplex virus (HSV) in vitro (131). In all of these examples, the effect of IDO was found to be specifically due to its ability to deplete tryptophan, because adding supra-physiological levels of exogenous tryptophan restored pathogen or viral replication. Although IDO has an effect  24  on pathogen replication in vitro, the biological relevance of IDO in controlling infections in vivo remains unclear. IFN-’y- deficient mice, which fail to induce IDO during infection, are less able to control C. pneumoniae and T. gondii infection in vivo (132); however, it is unknown whether this is due specifically to the lack of IDO expression or to one of the many other effects of IFN-’y. Additional studies are thus required to address the specific contribution of IDO to host defense against infection. This question is particularly relevant, given that one (highly unwanted) outcome of inducing IDO expression by the innate system might be the suppression of T cell responses by the adaptive system (133). One may wonder that slowing pathogen replication by IDO in vitro does not necessarily mean that it would be a useful host defense in vivo (particularly in the case of chronic, slow-growing infections such as those described). Therefore, like the Th2 cell paradox in chronic leishmaniasis (134), it is probable that IDO could benefit the pathogen more than the host. -  The Role of IDO in Transplantation  As discussed earlier, after allogeneic cell transplantation a state of immune activation, even in HLA-matched donors will occur. This state of immune activation will include the secretion of pro-inflammatory cytokines including IFN7 by APCs or activated T cells. Because of the close association of IFN-y and induction of IDO, one can speculate that IDO by its immunoregulatory effects may actively contribute in down-regulating allogeneic immune responses in transplantation (34). In fact, it has been recently suggested that cells expressing IDO might contribute to the underlying mechanism of donor specific tolerance without the use of immunosuppressive drugs. Several experiments, mostly carried out in vitro, corroborated the evidence that IDO activity  25  possesses the potential to down-regulate alloresponses (135,136). In an in vivo study, recipient mice exposed to 1-MT, a specific inhibitor of IDO activity, rejected the murine liver allografts at the time of engrafiment (137). More evidence on immuno-protective role of IDO in cell and tissue allografts comes from a study using adenoviral transfection to increase IDO expression in donor islet cells. The study reported prolonged survival of transplanted allogeneic pancreatic islet cells (138). Our recently published data provide compelling evidence in supporting our working hypothesis that the expression of IDO in bystander fibroblasts through either IDO genetic modification or IFN-y treatment suppresses immune cell proliferation (44,139-142). This hypothesis has been substantiated by the fact that co-culturing IDO genetically modified fibroblasts with different types of immune cells, significantly increased the number of damaged bystander human peripheral blood mononuclear cells (PBMC), T cells, Jurkat cells, THP-1 monocytes, relative to those of controls. This bystander effect proved to be due to IDO induction of a tryptophan deficient cell culture environment. In addition, the finding of this study further demonstrated that, by so far rather unknown mechanism, only immune, but not primary skin cells are sensitive to IDO induced low tryptophan environments (142-145). In another study (122), we have demonstrated a significant down-regulation of cell membrane associated MHC class I antigen in IDO genetically modified keratinocytes relative to that of either nontransfected or empty vector transfected cells. More experiments showed that an addition of tryptophan or IDO inhibitor markedly restored the expression of MHC class I on IDO transfected keratinocytes. Therefore, the findings of this study suggest for the first time that down-regulation of MHC class I expression by IDO might be one of the mechanisms through which IDO mediates local  26  immunosuppression. In another series of studies by Sarkhosh eta!. (140,141,146), we provided compelling evidence that IFN-y induced IDO expression suppresses the proliferation of immune cells co-cultured with IDO-expressing fibroblasts and addition of an IDO-inhibitor (1-MT) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependent fashion. As an alternative method to genetic modification, we have used a temperature-sensitive polymer, conjugated IFN-y as a slow release system in a skin substitute to further prolong the effect of IFN-’ on IDO expression in skin cells. We, therefore, concluded that in an in vitro setting IDO-expressing allogeneic fibroblasts embedded within a collagen gel suppress the proliferation of allogeneic immune cells, while they still remain viable in this IDO-induced tryptophan deficient environment. Given, IDO does have a physiologic role in transplantation, the ultimate understanding of its role and effects will be challenging. Because of the multiple microenvironmental factors regulating its activity, one has to be aware that more of IDO does not necessarily mean more immunoregulatory activity in the direction of tolerance. On the other hand it is this complexity that makes IDO a captivating field of research. An ultimately better understanding of its complex role in regulating allogeneic immune responses will probably contribute to better understand the principle mechanisms of ups and downs in immunoregulation. The elaboration of conditions in which IDO-mediated immunoregulation is optimized towards the induction of antigen-specific tolerance will potentially open new horizons of therapeutic opportunities.  27  Thesis theme -  Rationales for Developing a Non-Rejectable Skin Substitute  Non- and over-healing wounds are considered to be two extremes of wound healing with serious pathologic conditions in which the biomechanical properties of normal tissue are disturbed due to either deficiencies or alteration in ECM composition and organization. As discussed earlier, the current skin substitutes such as the autologous keratinocyte sheets which are used in some burn centers to treat patients with large thermal injury (147,148), have not become a routine procedure because of so many limitations: firstly sheets of keratinocytes prepared from layers of cultured keratinocytes without matrix are very fragile and difficult to cultivate, secondly the rate of graft-take is relatively low (50— 60%), thirdly patients with large injuries do not have enough of uninjured skin to be used for cell culture, and lastly generating another wound increases the risk of infection and development of hypertrophic scarring. In fact, for diabetic patients whose wound healing process is impaired (5) making another wound(s) to obtain cells for culturing cannot be acceptable. Furthermore, preparation of an autologous skin substitute is time consuming; reducing the benefit for those patients whose survival depends on early application of wound coverage. Considering the fact that all of these factors are pervasive medical problems with far-reaching clinical and economic implications, utilizing an allogeneic and readily available skin substitute seems to be logical to overcome these problems. The rationale for development and use of non-rejectable skin substitute for this purpose is based on the fact that the majority of wounds, particularly those seen in diabetic, elderly and immuno-compromised patients heal poorly. More than 90% of diabetic patients are over 50 years of age when most foot problems occur (149). These patients have an  28  increased rate of morbidity and prolonged hospital stay due to vascular complications, infections and delayed wound healing. Delayed wound healing may occur in diabetic patients due to glycosylation of important structural proteins (150), a reduction in neovascularisation, and impairment of leukocyte or macrophage function (151). Despite the fact that many wound healing promoting factors have so far been identified, their exogenous application failed to improve the healing process due to their short half life, cost and multi-factorial requirement. It is now recognized that improved quality of wound healing with acceptable biologic function is achievable only if the skin substitute has both dermal and epidermal layers. Therefore, the ultimate goal of this study is to explore the possible approaches through which the clinical complications of both non-healing and over-healing wounds can be improved. Indeed, there is a need to develop a non-rejeetable and readily available skin substitute to function as: 1) an early wound coverage to prevent or reduce heat and fluid loss and prevent wound infection and 2) a wound coverage through which dermal-epidermal wound healing modulating factors are released locally to facilitate granulation tissue formation, reepithelialization and improve closure of non-healing wounds such as the one seen in diabetic, elderly and immuno-compromised patients. There are potentially two main skin substitute models to address these wounds. First is the use of commercially available ‘living’ skin substitutes. Although these products, i.e. keratinocytes sheets with a synthetic dermal matrix, have been heavily promoted by industry to meet the tremendous need for a reliable skin substitute, trials in burn patients on full thickness wounds or on donor sites in many institutions have demonstrated that each of the main three products, Apligraf , TM TM and Transcyte Dermagraft TM fail to revascularize, adhere to the wound for only short  29  periods of time (10 days or less), and the latter two contain few if any viable cells, rendering them of little utility as permanent skin replacements for the burn wound (30-32). They are simply not accepted by the host tissue. Integra , an artificial acellular dermal matrix is TM useful in the burn wound, but is very expensive, highly susceptible to infection, requires a thin split thickness skin graft for epithelial reconstruction and at least two operative stages 3-4 weeks apart and thus is not a one stage dermal and epidermal reconstruction as proposed  herein. This leaves us with the second option through which a non-rejectable and readily available skin substitute composed of non-autologous cells can be developed. As rejection is a major obstacle in any type of grafting, this proposal seeks a novel approach through which local induction of the immunosuppressive factor, indoleamine 2, 3-dioxygenase (IDO), a tryptophan catabolizing enzyme, generates a tryptophan deficient wound microenvironment in which infiltrated immune T cells, but not skin cells are unable to proliferate and destroy the grafted skin substitute. Therefore, we hypothesize that local induction of IDO by cellular components of a skin substitute will suppress the proliferation of infiltrated T cells at the wound site and thereby delay or prevent the rejection of non-autologous skin substitute engraftment used not only as a wound coverage but also as a rich source of wound healing promoting factors.  -  Our Previous Findings Relevant to This Project  We hypothesized that IDO would function as a local immuno-suppressive factor for the immune cells (CD4 and CD8) involved in rejection of a non-autologous bilayer skin substitute engraftment. Our previous findings provided evidence that: 1) An adenoviral vector bearing IDO gene can be constructed and used to prepare IDO genetically modified skin cells (44), 2) IDO expression at the levels of mRNA, protein production,  30  and enzyme activity can be confirmed (140), 3) Genetically modified IDO expressing dermal cells can generate a tryptophan deficient environment in which the proliferation of different types of immune cells such as CD4Jurkat cells, THP-1 monocytes and human peripheral blood lymphocytes (44), but not primary keratinocytes, endothelial and fibroblasts is suppressed by apoptotic pathway within 3 days of being in this environment, 4) Cell membrane associated MHC class I antigen in IDO genetically modified keratinocytes is significantly down-regulated (122), and finally 5) IFN-y induced IDO expression in dermal cells suppressed the proliferation of immune cells in a co-culture system (141,146). Indeed, before the current project, we have already constructed and employed an adenoviral vector bearing the human IDO gene and a gene marker, green fluorescence protein (GFP) to prepare IDO genetically modified skin cells. The success of IDO expression at the levels of mRNA, protein production and enzyme activity was also confirmed. To confirm the mediating role of IDO in suppression of immune cell proliferation, a series of experiments were carried out using different types of immune cells such as CD4 Jurkat cells, THP-1 monocytes and human peripheral blood lymphocytes (PBL) co-cultured with IDO genetically modified expressing cells for 3 days. FACS analysis of the PT stained (dead cells) Jurkat, THP-1 and PBL were 16, 9 and 6 fold higher than those of their controls, respectively (44). The results of this study suggest that the expression of IDO by dermal fibroblasts mediates immune cell death. In another series of studies by Sarkhosh eta!. (140,141,146), we provided compelling evidence that IFN-y induced IDO expression suppresses the proliferation of immune cells co-cultured with IDO-expressing fibroblasts. This finding was supported by the fact that the addition of an IDO-inhibitor, I -MT, reversed the suppressive effects of IDO on  31  PBMC proliferation in a dose-dependant fashion. We therefore concluded that IDO expressing non-autologous fibroblasts embedded within the matrix of our skin substitute suppress the proliferation of the host immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment. Collectively, these findings signify the potential use of IDO as a local immuno-protective factor to prevent rejection of a non-autologous bilayer skin substitute engraftment. These novel findings set the stage for a new question of whether a non-rejectable skin substitute can be further developed for use as a wound coverage to promote wound healing process. -  Hypothesis and Main Objectives  In this project, first we want to investigate whether there is any differential effect caused by low tryptophan environment induced by IDO expression on skin cells versus immune T cells and if so whether there is any difference between the main T cell subsets, CD4 versus CD8 T cells. If we confirm this differential effect, then it will be our working hypothesis that promoting immunotolerance by IDO expression by genetical modification of fibroblasts populated in collagen matrix can improve the result of engrafiment of a cultured skin substitute from a non-autologous source, consisting of both keratinocytes and treated fibroblasts, applied to the wounds in animal models. Thus, IDO expressing cells will provide a tryptophan deficient environment which infiltrated immune cells are unable to proliferate and so it will improve the wound healing process at the wounds received the IDO expressing skin substitute. We will test these hypotheses through 3 main objectives: 1- To investigate the cell survival rate of the skin and immune cells in tryptophan deficient environment generated by IDO. Through this objective, we tried to further explore the  32  potential mechanism by which the primary dermal, but not immune cells are resistant to IDO generated a tryptophan deficient environment. 2- To further explore which main subsets of T cells (CD4 or CD8 T cells) is more sensitive to tryptophan deficient environment induced by IDO expression. 3. To develop and apply an IDO-genetically-modified skin substitute consisting of keratinocytes, fibroblasts and collagen matrix as well as appropriate control skin substitutes to full thickness wounds in rats and to evaluate and compare the wound healing outcome.  -  Experimental Research Plan  Objective 1: To investigate the cell survival rate ofthe skin cells and immune cells in tryptophan deficient environment generated by IDO. Through this objective, we triedfurther to explore the potential mechanism by which the primary dermal, but not immune cells are resistant to IDO generated a tryptophan deficient environment. Rationale: One of the main concerns regarding the use of IDO as a local immunosuppressive factor is whether the survival of skin cells are also compromised when they are growing in an IDO induced tryptophan deficient environment. A series of experiments were, therefore, conducted to specifically address the issue of resistancy of two main primary skin cells, fibroblasts and keratinocytes co-cultured with IDO expressing fibroblasts. Further, during the course of this study as well as our previous studies, we realized that IDO expressing 293 cells, used as packaging cells to propagate the adenoviral vectors, are resistant to low tryptophan conditioned medium. These cells were firmly attached to the bottom of the culture dish and expressed a high level GFP and IDO. These findings are consistent with those reported for IDO expressing trophoblasts in placenta in a mouse model indicating that bystander infiltrated immune cells, but not trophoblasts, are influenced by an IDO induced tryptophan deficient environment (48). Thus, the cellular response to IDO induced low tryptophan environment is different from one cell type to 33  another. The findings of this objective would help us to further understand the mechanism by which skin, but not immune cells, survive in a low tryptophan environment. This would provide supporting evidence that preparation of a non-rejectable skin substitute is feasible and safe to be used as a wound coverage without compromising skin cell survival. To address this objective, in a co-culture system, IDO expressing cells were co-cultured with bystander IDO sensitive and resistant cells for 72 hrs and the survival and apoptotic pathways in these bystander cells were evaluated. The detail outline, experimental method and the result of this study is presented in chapter 2 of this thesis.  Objective 2: To further explore which main subsets of T cells (CD4 or CD8 T cells) is more sensitive to tryptophan deficient environment induced by IDO expression. Rational: We have previously demonstrated that indoleamine 2,3-dioxygenase (IDO) expression by skin cells generates a tryptophan deficient environment in which THP-1, Jurkat cells as well as human PBMC are unable to survive (142). However, the subsets of primary human T cells that are more sensitive to tryptophan depletion have not been identified. Due to the importance of T cells in immune reaction to non-autologous grafts this finding would help us to have a better understanding of the immuno-modulatory role of IDO in this setting. In this objective, we asked whether the proliferation and viability of bystander CD4 and CD8 T cells are modulated in response to IDO induced tryptophan deficient environment and if so, whether their response is different. To address this objective, we co-cultured IDO-expressing fibroblasts with bystander human CD4 and CD8 T cells for 4 days and then the survival and proliferation rates as well as downstream metabolic pathway of tryptophan degradation in these cells were  34  evaluated. The detail outline, experimental method and the result of this study is presented in chapter 3 of this thesis.  Objective 3: To develop and apply an JDO-genetically-modfled skin substitute consisting ofkeratinocytes, Jlbroblasts and collagen matrix as well as appropriate control skin substitutes to full thickness wounds in rats and to evaluate and compare the wound healing process in these wounds. Rational: Like many other organs, rejection of non-autologous grafted skin is a major  problem in dermal transplantation (36). As stated before, there is compelling evidence that an immunologic response to non-autologous skin graft requires T cell activation and proliferation to generate a sufficient number of infiltrating immune cells at the graft site (39). Thus, to prevent acute rejection, suppression of infiltrated immune cells at early time points of engraftment is necessary. The rationale for development and use of non-rejectable skin substitute for this purpose is based on the fact that the majority of wounds, particularly those seen in diabetic, elderly and immuno-compromised patients heal poorly. These wounds represent a severe physiologic, immunologic and metabolic derangement for many patients and caregivers. Despite the fact that many wound healing promoting factors have so far been identified, their exogenous application failed to improve the healing process due to their short half-life, cost and multi-factorial requirement. Here, we propose to develop and apply a non-rejectable skin substitute prepared from non-autologous dermal (fibroblasts embedded in collagen-GAG matrix) and epidermal (multi-layers of keratinocytes) layers which have been modified to express IDO at the wound site. Thus, a natural cocktail of wound healing modulating factors is then released by dermal and epidermal cells at the wound site where they influence many wound healing related events such as ECM modulation and tissue remodeling. Previously, in an in vivo rat model, we showed that an  35  IDO expressing fibroblast-populated collagen-GAG gel placed on wounds accelerated  healing and provided protection of non-autologous fibroblasts (44). However, we did not show whether including an epidermal layer of non-autologous keratinocytes which are more antigenic than fibroblasts can also be immuno-protected by local expression of IDO. 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(129) Gupta SL, Carlin JM, Pyati P, Dai W, et a!. Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts. Infect Immun.1994 Jun;62(6):2277-84.  (130) Pfefferkorn ER. Interferon gamma Blocks the Growth of Toxoplasma gondii in Human Fibroblasts by Inducing the Host Cells to Degrade Tryptophan. PNAS 1 984;8 1 (3):908-9 12.  (131) Bodaghi B, Goureau 0, Zipeto D, Laurent L, Virelizier JL, Michelson S. Role of IFN-gamma-induced indoleamine 2,3 dioxygenase and inducible nitric oxide synthase in the replication of human cytomegalovirus in retinal pigment epithelial cells. J.Immunol. I 999;l 62(2):957-964.  (132) Fujigaki S, Saito K, Takemura M, Maekawa N, Yamada Y, Wada H, et al. L tryptophan-L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferon-gene-deficient mice: cross-regulation between inducible nitric oxide synthase and indoleamine-2,3-dioxygenase. Infect.Immun. 2002 ;70(2):779-786.  (133) Widner B, Weiss G, Fuchs D. Tryptophan degradation to control T-cell responsiveness. Immunol.Today 2000;2 1(5) :250.  55  (134) Rogers KA, DeKrey GK, Mbow ML, Gillespie RD, Brodskyn Cl, Titus RG. Type 1 and type 2 responses to Leishmania major. FEMS Microbiol.Lett. 2002 ;209(1):1-7.  (135) Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. Indoleamine 2,3dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J.Immunol. 2000; 1 64(7):3 596-3599.  (136) Bertera S, Alexander AM, Crawford ML, Papworth G, Watkins SC, Robbins PD, et al. Gene combination transfer to block autoimmune damage in transplanted islets of Langerhans. Exp.Diabesity.Res. 2004 ;5(3):20 1-210.  (137) Miki T, Sun H, Lee Y, Tandin A, Kovscek AM, Subbotin V, et al. Blockade of tryptophan catabolism prevents spontaneous tolerogenicity of liver allografts. Transplant.Proc. 2001;33(1-2):129-130.  (138) Alexander AM, Crawford M, Bertera 5, Rudert WA, Takikawa 0, Robbins PD, et al. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Diabetes 2002;5 1(2):356365.  (139) Jalili RB, Rayat GR, Rajotte RV, Ghahary A. Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts. J.Cel I .Physiol. 2007;213(0021-9541; l):137-143.  (140) Sarkhosh K, Tredget EE, Li Y, Kilani RT, Uludag H, Ghahary A. Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2,3-dioxygenase  56  expression of interferon-gamma-treated skin cells in a co-culture system. Wound Repair Regen. 2003;1 1(5):337-345.  (141) Sarkhosh K, Tredget EE, Karami A, Uludag H, Twashina T, Kilani RT, et a!. Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG). J.Cell.Biochem. 2003;90(1):206-217.  (142) Ghahary A, Li Y, Tredget EE, Kilani RT, Iwashina T, Karami A, et a!. Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions as a local immunosuppressive factor. J.Invest.Dermatol. 2004; 122(4) :953-964.  (143) Forouzandeh F, Jalili RB, Germain M, Duronio V. Ghahary A. Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts. Wound Repair Regen. 2008; 1 6(3):379-3 87.  (144) Frolova LY, Grigorieva AY, Sudomoina MA, Kisselev LL. The human gene encoding tryptophanyl-tRNA synthetase: interferon-response elements and exon-intron organization. Gene 1993; 1 28(2):237-245.  (145) Fleckner J, Martensen PM, Tolstrup AB, Kjeldgaard NO, Justesen J. Differential regulation of the human, interferon inducible tryptophanyl-tRNA synthetase by various cytokines in cell lines. Cytokine 1995;7(1):70-77.  (146) Sarkhosh K, Tredget EE, Uludag H, Kilani RT, Karami A, Li Y, et al. Temperature-sensitive polymer-conjugated IFN-gamma induces the expression of IDO  57  mRNA and activity by fibroblasts populated in collagen gel (FPCG). J.CeIl.Physiol. 2004 ;201(1):146-154.  (147) Morhenn VB, Benike CJ, Charron DJ, Cox A, Mahrle 0, Wood GS, et al. Use of the fluorescence-activated cell sorter to quantitate and enrich for subpopulations of human skin cells. J.Invest.Dermatol. 1982;79(5):277-282.  (148) Thivolet CH, Chatelain P, Nicoloso H, Durand A, Bertrand J. Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures. Exp.Cell Res. 1985; 1 59(2):3 13-322.  (149) Kratz 0, Jansson K, Gidlund M, Haegerstrand A. Keratinocyte conditioned medium stimulates type IV collagenase synthesis in cultured human keratinocytes and fibroblasts. Br.J.Dermatol. 1995; 1 33(6):842-846.  (150) Harding KG, Morris HL, Patel OK. Science, medicine and the future: healing chronic wounds. BMJ 2002;324(7330): 160-163.  (151) Goulet F, Poitras A, Rouabhia M, Cusson D, Germain L, Auger FA. Stimulation of human keratinocyte proliferation through growth factor exchanges with dermal fibroblasts in vitro. Burns 1996;22(2):107-1 12.  58  CHAPTER 2’ Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts  A version of this chapter has been published. With kind permission from Wiley Blackwell: Wound Repair and Regeneration, Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts, 2008 May Jun;16(3):379-387, Forouzandeh F, Jalili RB, Germain M, Duronio V, Ghahary A.  59  Introduction Endoleamine 2, 3-dioxygenase (IDO), a heme-containing rate-limiting enzyme, catalyzes conversion of tryptophan to kynurenine as the main tryptophan metabolite (1). The initial and rate-limiting reaction of the kynurenine pathway is the oxidation of tryptophan to N-formyl-L-kynurenine, catalyzed by hepatic tryptophan 2,3-dioxygenase (TDO) or the ubiquitous, extra-hepatic, IDO (2). IDO has been found in nonhepatic cells mainly in trophoblasts, dendritic cells, monocytes, and macrophages (3-5). The expression of IDO has immuno-modulatory effects on T cells that are related to the pericellular degradation of tryptophan (6-8). Tryptophan is the least available essential amino acid and is required by all forms of life for protein synthesis and other important metabolic functions. Based on this information, we hypothesize that an allogeneic skin substitute whose cellular components ectopically express IDO would be non-rejectable. As IDO degrades the essential amino acid tryptophan, it might affect pathways known to respond to amino acid metabolism. One possibility is the activation of nutrient-sensitive mammalian target of rapamycin (mTOR) kinase pathway. However, it has been shown that inhibitors of mTOR such as rapamycin do not recapitulate the profound proliferative arrest in immune cells seen with IDO-mediated suppression (9). A second amino acid sensitive pathway is mediated by the general control nonderepressing-2 (GCN2) kinase. GCN2 contains a regulatory domain that binds the uncharged form of tRNA. Amino acid insufficiency causes a rise in uncharged tRNA, which activates the GCN2 kinase domain and initiates downstream signaling. Recently, it has been shown that the GCN2 kinase pathway is involved in the immunosuppressive effect of IDO expression (9). In an attempt to use IDO expression as a local immunosuppressive factor to immunoprotect an  60  allogeneic skin substitute made of an epidermal portion of primary cultured keratinocytes and a dermal portion of dermal fibroblasts populated in a collagen-GAG gel, a series of experiments were conducted; previously, we showed that different types of immune cells such as CD4Jurkat cells, THP-1 monocytes, and human peripheral blood lymphocytes (PBL) become apoptotic within 3 days when co-cultured with IDO expressing cells (10). Moreover, under a similar experimental condition, a significant down-regulation of cell membrane associated MHC class I antigen in IDO genetically modified keratinocytes relative to that of either noninfected or infected cells with adenovirus without IDO was shown (6,10). In an in vivo study, IDO-expressing human fibrob lasts embedded within bovine collagen placed on rat wounds accelerated wound healing by promoting revascularization during the early stages of healing and by providing protection of xenogeneic fibroblasts (11). These findings collectively suggest that IDO expression may function as a local immunosuppressive factor to protect an allogeneic skin substitute in which keratinocytes and fibroblasts serve as the cellular components of this wound coverage. However, what is not known is why primary skin cells, but not immune cells, are resistant to IDO induced low tryptophan environment. Here, by conducting a series of experiments we provided evidence that: (1) small pieces of human skin are highly antigenic for primary human T cells and can be suppressed by bystander IDO expressing fibroblasts, (2) Caspase-3 and CHOP levels as two indicators of activation of apoptotic and GCN2 kinase pathways, respectively, either slightly increased or remained undetectable in fibroblasts and keratinocytes relative to those of T cells grown in an environment generated by co-cultured IDO expressing fibroblasts.  61  Materials and methods Adenoviral vector construction The procedure of construction of IDO expressing adenoviral vectors has been described before (10). Briefly, to construct the adenovirus encoding human IDO, we cloned the polymerase chain reaction product encoding the full length protein into a shuttle vector, which coexpresses GFP as a reporter gene following the manufacturer’s instructions  (Q  Biognene, Carlsbad, CA). The recombinant adenoviral plasmids were generated by electroporation ofBJ5l 83 Escherichia coli using the shuttle vector either with or without IDO. Recombinant adenoviral plasmids were then purified and transfected to 293 cells using Fugene-6 transfection reagent (Roche Applied Science, Laval, QC, Canada). Infected cells were monitored for GFP expression and after three cycles of freezing in ethanol/dry ice bath and rapid thawing at 37 °C, the cell lysate were used to amplify viral particles in large scale to prepare adenoviral stock. Then, viral titration was done using 293-cell line as described previously (10).  Fibroblasts and keratinocytes culture Neonatal foreskin pieces were used as source of fibroblasts and keratinocytes and the procedure was done based on the approval of Ethics Committee of the University of British Columbia (UBC). Cultures of human foreskin fibroblasts were established as described previously (8). In brief, punch biopsy samples were prepared from human foreskins. The tissue was collected in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY), minced into small pieces of< 0.5mm in any dimension, washed with sterile medium six times, and distributed into 60x 15mm Petri culture dishes (Corning Inc., Corning, NY), four pieces per dish. A  62  sterile glass coverslip was attached to the dish with a drop of sterile silicone grease to immobilize the tissue fragment. DMEM  +  antibiotics (penicillin 6 sodium 100 U/mL,  streptomycin sulfate 100 mg/mL, and amphotericin B 0.25 mg/mL, GIBCO) with 10% FBS was added to each dish and incubated at 37 °C in a water-jacked humidified incubator in an atmosphere of 5% C02. The medium was replaced twice weekly. After 4 weeks of incubation, cells were released from dishes by brief (5 minute) treatment with 0.1% trypsin (Life technologies Inc., Gaithersburg, MD) and 0.02% EDTA (Sigma, St. Louis, MO) in phosphate-buffered saline solution (PBS; pH 7.4) and transferred to 75 2 culture flasks (Corning Inc.). Thereafter, once visual confluence was reached, the cm cells were subcultured 1: 6 by trypsinization. Fibroblasts from passages 4—7 were used for this study. In order to culture human foreskin keratinocytes, keratinocyte serum-free medium (KSFM, GIBCO) supplemented with bovine pituitary extract (25 mg/mL) and epidermal growth factor (0.5 nglmL) were used. Primary cultured keratinocytes at passages 3—5 were used for this study. Infection of fibroblasts with IDO adenoviral vector Recombinant adenoviruses were used to infect fibroblasts at the multiplicity of infection of 100 (MOI: 100). Free viral particles were removed from culture medium 30 hours after infection. The success of infection was determined by flow cytometry measuring the GFP protein expression and fluorescent microscopy using a Motic inverted microscope equipped with an fluorescein isothiocyanate (FITC) filter to view GFP (Motic Instruments, Richmond, BC, Canada). Images were captured using a digital camera. Moreover, the efficacy of infection and expression of functional IDO were assessed by  63  immunohistochemistry for IDO expression, and by western blot using anti-human IDO Ab, and measuring the levels of kynurenine. Co-culture systems of IDO expressing fibroblasts and bystander cells Using 30mm Millicell Sterilized Culture Plate Inserts (Millipore, Bedford, MA), we set up a co-culture system in which IDO-expressing fibroblasts were grown on the upper chamber of a six-well plate, while bystander either keratinocytes, fibroblasts or T cells were cultured on the lower chamber. Therefore, there was no direct contact between IDO expressing fibroblasts and bystander cells. It should also be mentioned that adenovirus or IFN-’y treated fibroblasts were washed with PBS before co-culturing them with other cells, to remove any excess adenovirus and IFN-y, respectively. Immunohistochemical staining IDO expression was detected by intracellular staining. Fibroblasts remained untreated, treated with IFN-y or infected with Ad-IDO-GFP for 3 days and fixed by paraformaldehyde 4%. Cells were then incubated with polyclonal anti-human IDO antibody, raised in rabbits by Washington Biotechnology Inc. (Baltimore, MD), at final concentration of 1: 1,000 at 4 °C overnight. The procedure was followed by incubating cells with biotinylated-goat anti rabbit immunoglobulin (Vector Laboratories, Burlingame, CA). The signal detection was carried out using 3, 30-diaminobenzidineenhanced liquid substrate system (Sigma). The slides were counterstained with hematoxylin for 5 seconds, and then sections were dehydrated, mounted, and examined under microscope.  V  64  Fluorescence activated cell sorting of human peripheral blood mononuclear cells (PBMC) for T cells  Total PBMC were isolated by density gradient sedimentation on I-Iistopaque-1077 (Sigma) according to the manufacturer’s protocol. Briefly, whole blood was layered on an equal volume of Histopaque and centrifuged at 2,000 r.p.m. for 20 minutes at room temperature and stopped without any brake. PBMC were isolated and resuspended in RPMI 1640+10% FBS and pelleted by centrifugation at 2,000 r.p.m. for 10 minutes and were further washed twice in PBS+1% FBS. Using PE-conjugated mouse anti-human CD3 mAb (BD, Oakville, ON), FITC-conjugated mouse anti-human CD4 mAb (BD), and allophycocyanin (APC)-conjugated mouse anti-human CD8 mAb (BD) at the concentration of 20 jiL/10 6 cells, we stained the PBMCs. The cells were then incubated at room temperature for 30 minutes. Thereafter, cells were washed twice and resuspended at i0 cells/mL in PBS+1% FBS for fluorescent activated cell sorting (FACS). For preparation of a pure population of CD3CD4 and CD3CD8 T cells, we gated on CD3CD4 and CD3CD8 T cells after excluding the dead cells and cell debris based on FSC and SSC parameters and sorted these two cell populations into separate tubes. T cell culture  After being sorted from blood, I cells were propagated in RPMI 1,640 (Hyclone, UT) supplemented with 10% FBS, 0.1U penicillin/mL, and 0.1 mg streptomycin/mL at 37 °C in a humidified 5% CO2 atmosphere to be used for further treatment. Cell survival evaluation  The survival of two different sensitive (CD4and CD8 T cells) and two different resistant (dermal fibroblasts and keratinocytes) cell types in an IDO-induced tryptophan  65  deficient environment was compared by 7-AAD staining. 7-AAD intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. After each treatment, cells were harvested, washed in PBS, stained for 7-AAD and then examined using FACS analysis, as per the manufacturer’s protocol (BD). In vitro proliferation assay for T cells T cells were pulsed with [3H]-thymidine (1 jiCi/mL, Perkin Elmer, Boston, MA) on day 3 and harvested on day 4 for measurement of cell lysate-associated tritium by b scintillation counting. All proliferation experiments were performed in triplicate and reported as total of cell count per minute (c.p.m.). Western blot analysis Total proteins from cell lysate (20 ig) were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The blots were probed for CHOP using anti-GADDI53/ CHOP-b Ab (1: 250 dilution, Sigma) and for active caspase-3 with anti-active caspase-3 Ab (1: 1,000 dilution, BD). Blots were stripped and reprobed for 3-actin as equal loading control. For detection of IDO expression, fibroblasts were harvested after 48 hours of IFN-y treatment or 72 hours post viral infection and washed with PBS. Cells were then lyzed in lysis buffer (50mM Tris—HCI, pH 7.4; 10mM EDTA; 5mM EGTA; 0.5% NP4O; 1% Triton X- 100, and protease inhibitor cocktail, Sigma). Equal amounts of total protein from each individual fibroblast culture were separated by 10% SDS-PAGE. Proteins were then transferred to a PVDF membrane (Millipore) and immunoblotted with polyclonal anti-human IDO antibody raised in rabbits by Washington Biotechnology Inc. at final  66  dilution of 1: 5,000. Enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Little Chalfont, UK) was used in all blots to detect the secondary Ab. Kynurenine measurement in the conditioned medium  The biological activity of IDO was evaluated by measuring the levels of tryptophan degraded product, L-kynurenine, present in the conditioned medium derived from IDO and control vector-infected cells. The amount of L-kynurenine was measured by a previously established method (12). Briefly, proteins in the conditioned medium were precipitated by trichloroacetic acid, and after centrifugation, 0.5mL of supernatant was incubated with an equal volume of Ehrich’s reagent (Sigma) for 10 minutes at room temperature. Absorption of the resultant solution was measured at 490nm by a spectrophotometer within 2 hours. The values of kynurenine in the conditioned medium were calculated by a standard curve with the defined kynurenine (0—100mM) concentration. In this study, we have used this assay as an evidence of IDO expression wherever we have induced the fibroblasts to express IDO either by Ad-IDO-GFP infection or by IFN-’ treatment. Statistical analysis  Data were expressed as Mean+SD and analyzed with one-way ANOVA among different groups of each cell type where indicated, p-values <0.05 are considered statistically significant in this study.  67  Results IDO expression inhibits T cell proliferation induced by co-cultured allogeneic pieces of epidermis or full intact skin Before evaluating the immunosuppressive effect of IDO expression on immune cells in response to allogeneic pieces of epidermis or full thickness skin, the expression of IDO was validated in fibroblasts infected with an Ad-IDO-GFP vector (MOl: 100) or treated with IFN-y (1,000 U/mL), a potent inducer of IDO expression (8,10,13,14). Indeed, the efficacy of IDO expression by these fibroblasts was tested by different methods. The results of immunohistochemical experiments showed a strong IDO protein staining in fibroblasts either infected with Ad-IDO-GFP vector or treated with IFN-y (figure 1A). This finding was confirmed under fluorescence microscopy for GFP expression as well (figure 1 B). To further confirm this finding and demonstrate that the immunostaining of IDO expressing cells is not due to nonspecific staining, cells were then harvested and the total proteins were subjected to western blot analysis. As shown in figure Ic, the results showed an intense IDO protein band in those fibroblasts that were either infected with Ad-IDO-GFP vector or treated with IFN-y. Under a similar experimental condition, cells treated with vector without IDO gene or untreated cells showed little or no IDO expression. This difference was not due to variation in protein loading as the intensity of 13-actin protein, a loading control, was relatively the same in all samples (Figrue IC). As our ultimate goal is to make use of the IDO immunosuppressive effect to develop a non rejectable bilayered skin substitute, we were interested in looking at the effect of IDO expression on human T cells stimulated with the intact pieces of either allogeneic epidermis or full thickness skin in an ex vivo co-culture models. For this purpose, human  68  T cells were co-cultured with allogeneic pieces of either epidermis or full thickness skin in the presence of fibroblasts infected with Ad-IDO-GFP vector or control fibroblasts. Photomicrographs were taken from this coculture system after 72 hours. As shown in figure 2A, human T cells proliferated in response to stimulation by allogeneic epidermis or full-thickness skin when co-cultured with either nontreated fibroblasts (first row) or Ad-GFP treated fibroblasts (second row). However, there was a remarkable reduction in the number of lymphocytes when co-cultured with allogeneic epidermis or full thickness skin in the presence of genetically modified IDO-expressing fibroblasts (third row). The suppressive effect of IDO was markedly reduced in the presence of IDO inhibitor, 1-MT (fourth row). To confirm and quantif’ these findings, proliferation of lymphocytes was measured by [3H]-thymidine incorporation assay. The results showed an almost six fold reduction in the proliferation rates of T cells when co-cultured with allogeneic skin in the presence of IDO-expressing fibroblasts compared with those of lymphocytes co-cultured with allogeneic skin and non—IDO-expressing fibroblasts in genetically modified preparations (p  <  0.00 1, figure 2B). The suppression of immune cell proliferation was  specific to 1D0 expression, because this effect was reversible upon addition of 1 -MT, an IDO inhibitor. Further, control human T cells co-cultured with allogeneic fibroblasts alone showed no significant proliferation (figure 2A and B).  69  A  B  Ad-OFP infected  Ad-IDO-GEP infected  IFNV treated  Ad..GFP infected  C IDO (42 KD  Non-infected  Non-infected  Ad.IDOGFP infected  Ad-GFP  Ad.IDO-GFP  IFNy  —  i. actin—.  Figure 2.1. IDO expression in fibroblasts. IDO expression in fibroblasts either infected with Ad-IDOGFP or treated with IFN-’y IDO expression in fibroblasts either infected with Ad-IDO-GFP (MOl: 100) or treated with IFN-y (1,000 U/mL) was shown by immunohistochemistry of these cells for IDO protein after 3 days of culturing them in four chamber slides and compared with the control groups (panel A). Also, the GFP expression of Ad-GFP or Ad-IDO-GFP infected fibroblasts viewed under fluorescent microscope is also shown in panel B. Moreover, the confirmation of IDO expression by the same cells has been shown by western blot analysis for IDO protein by using IDO polyclonal Ab at concentration of 1:5,000 (panel C). IDO, indoleamine 2, 3-dioxygenase.  70  A Fib +T cells  Fib +T cells +Ep.  Fib +T cells +3km Figure 2.2. Proliferation rates of human T cells stimulated with allogeneic pieces of either epidermis or full thickness skin in the presence or absence of IDO expressing cells. Human fibroblasts (F) were infected with Ad IDO-GFP (MOl: 100) and then washed with PBS, irradiated, and co cultured with human T cells (T) and allogeneic pieces of either epidermis (E) or full thickness skin (5). To one preparation of each experimental group IDO inhibitor, 1-MT, was also added at the final concentration of 800 jiM. Co-cultures of fibroblasts with T cells alone were also included as another controls (panel A).  Non-treated  AdGFP  Ad-lDOGFP  Ad-IDO-GFP + IMT  B 0 C)  0  Fib+T cells  Fib+T cells+Ep.  Fib+T cefls+S kin  After 3 days, T cells were then subjected to [3H]-thymidine incorporation assay (panel B). Data represent [3H]- thymidine incorporation (count per minute, c.p.m.) into DNA of bystander T cells co cultured with either untreated fibroblasts (solid bars), Ad-GFP (Mock-Ad) infected fibroblasts (open bars), Ad-IDO- GFP infected fibroblasts (hatched bars) or IDO-expressing fibroblasts in the presence of 1-MT (checked board bars). The significant (p value <0.001) differences have been indicated by asterisks (*). 1-MT. 1methyl-D-tryptophan; IDO, Indoleamine 2, 3-dioxygenase.  71  Kynurenine causes loss of viability in T cells, but not in fibroblasts and keratinocytes In order to evaluate the possible toxic effect of kynurenine, the main IDO-induced tryptophan degradative product, on human T cells, fibroblasts and keratinocytes, we treated each of these cell types with various concentrations of kynurenine. The concentrations of kynurenine were chosen based on probable concentrations that we usually can get from our IDO-expressing fibroblasts and also our previous study (1). Cells were then harvested after 4 days and evaluated for their survival rate based on 7AAD staining examined by flow cytometry. The results showed that increasing concentration of kynurenine causes a significant reduction (from 98+1.7% to 81 + 1.3%, p <0.001) in T cell viability at concentration as low as 128 jiM of kynurenine used. However, under the same experimental conditions, the viability of fibroblasts and keratinocytes remained unchanged even at higher concentration of kynurenine (figure 3). This finding indicates that T cells, but not skin cells, are sensitive to kynurenine. 125 100 75 *  * *  25  0  64  128  256  512  1024  Kynurenine Concentration (pM)  Figure 2.3. The effect of kynurenine on the survival rate of human skin cells and T cells. Three different cell types including fibroblasts (squares), keratinocytes (circles), and T cells (triangles) were cultured in the presence of increasing concentration of kynurenine for 4 days. At day 4 the cells were harvested to evaluate their survival rate based on 7-AAD staining with flow cytometry. The significant (p value <0.001) differences have been indicated by asterisks (*).  72  The survival rate of T cells, but not libroblasts and keratinocytes, was reduced by co culturing them with IDO expressing cells As the viability of the skin cells is crucial to make any allogeneic skin substitute to be used as wound coverage with IDO-expressing cells, it is important to demonstrate whether IDO induced environment differentially influences the viability of skin and immune cells. For this reason, fibroblasts were infected with Ad-IDO-GFP or treated with IFN-y and co cultured with CD4 T cells, CD8 T cells, keratinocytes, or another strain of fibroblasts for 4 days. In these experiments, cells cultured either alone, co cultured with noninfected cells or co-cultured with Ad-GFP infected fibroblasts were included as negative controls. At day 4, the bystander cells were harvested and evaluated for their survival rates based on 7-AAD staining with flow cytometry. As demonstrated in figure 4A, the control groups including cells co-cultured with nontreated fibroblasts and co-cultured with Ad-GFP treated fibroblasts showed no significant differences in survival rate compared with non—co-cultured cells, whereas, as shown in figure 4B, there were significant reductions in survival rates of both types of T cells (p  <  0.00 1) cocultured with  Ad-IDO-GFP treated fibroblasts. In CD4 T cells, the survival rate decreased by about 14% and in CD8 T cells 19%. In addition, as demonstrated in figure 4C, in IFN-y treated groups the survival rate reductions of T cells were even greater (p  <  0.001) In CD4 T  cells, the reduction was 27% and in CD8 T cells, it was 26%. These inhibitory effects were restored markedly by adding the IDO inhibitor, 1-MT, to the preparations. On the other hand, as shown in figure 4A—C, no significant decrease in the survival rates of fibroblasts and keratinocytes in any of the treatment groups was observed compared with their control cells, which were cultured alone.  73  A 110 100 0 90  ‘  c z  80 70 60  U)  NL Fib.  Ad-GFP  B 110 100 90 ü 80 70 60 50  M-GFP-IOO  M-GFP400  +  IMT  C  IFN  IFN÷IMT  Figure 2.4. Effect of IDO expression on cell survival rate of human skin cells and T cells. Fibroblasts were either left untreated or treated with various adenovirus (MOl: 100) or IFN-’ (1,000 U/mL). Cells were then washed and co-cultured with the indicated cell type in two chamber co-culture systems for 4 days, in the absence or presence of the IDO inhibitor I-MT (800 M). Cell viability was then analyzed by FACS using 7-AAD (panels A—C). Bar charts corresponding to the survival rate of each bystander cell type in each treatment group was compared to that of the normal non—co-cultured cells that constitutes 100% survival rate for each cell type. The results of this comparison are shown for fibroblasts (solid bars), keratinocytes (open bars), purified CD4 T cells (hatched bars), and purified CD8 T cells (checked board bars). The significant (p value < 0.00 1) differences have been indicated by asterisks (*). IDO, indoleamine 2, 3dioxygenase; FACS, fluorescent activated cell sorting.  74  Caspase-3 activation in bystander T cells, but not fibroblasts and keratinocytes, co cultured with IDO expressing cells The results presented so far indicate that IDO expression causes a loss of viability specifically in T cells. To determine whether this was the consequence of increased apoptosis, we analyzed these cells for caspase-3 activation. Caspase-3 is an effector caspase ubiquitously activated through proteolytic cleavage following induction of apoptosis. As shown in figure 5A, there was a significant activation in caspase-3 in human T cells co cultured with IFN-y treated fibroblasts, as indicated by the appearance of the 17 and 12 KDa subunit of active caspase-3. Activation of caspase-3 was significantly inhibited by an addition of 1-MT. Moreover, no caspase-3 activation was seen in any of the fibroblasts or keratinocytes in different preparations. To further confirm this finding, in another set of experiments, Ad-IDO-GFP infected fibroblasts, as a clean source of IDO expression, was used and the levels of caspase-3 in bystander cells were evaluated (figure 5B). As anticipated, the result was consistent with that obtained from the IFN-y induced IDO expression. These findings collectively confirmed that regardless of the strategies used to induce IDO, the level of caspase-3 in bystander T cells, but not fibroblasts and keratinocytes, co-cultured with IDO expressing cells will be increased. GCN2 kinase pathway is involved in selective apoptotic effect of IDO on human T cells vs. skin cells It has been suggested that activation of GCN2 kinase in T cells by uncharged tRNAs mediates proliferative arrest in T cells and anergy induction (9). Herein, we propose that the selective apoptotic effect of IDO expression on human T cells vs. skin fibroblasts and  75  keratinocytes can be due to their different responses to tryptophan deficiency via the GCN2 kinase pathway. To examine this point, immune and nonimmune cells co-cultured for 3 days with IFN-y treated fibroblasts were harvested and evaluated for the expression of a downstream effector of GCN2, CHOP (9). As shown in figure 5A, there was a significant increase in CHOP expression in T cell subsets and this was restored by adding 1-MT in cultures. Moreover, the expression of CHOP was not seen in any of the examined fibroblasts or keratinocytes co-cultured with IDO expressing cells. Similarly, when Ad-IDO-GFP infected fibroblasts were used as a clean source of IDO expression, the result of CHOP induction in bystander T cells was the same as that found in IFN-y experiments (figure 5B). As shown for caspase-3, regardless of strategies used to induce IDO, the level of CHOP in bystander T cells, but not fibroblasts and keratinocytes, co cultured with IDO expressing cells will be increased.  76  A  IFN  t&iib. CD4CDS CHOP  (29KD)  (171(D) Cleaved Caspase 3 (12K0)  a  IFN+1MT  NLF1b.  C04CD8  Cb4 COB  F  HN F  K  IFN÷IMT K  F  K  :  .  actrn (431(D)  B  100  Mock-Ad  ‘F  F  K  ‘F  F  K  I  DO÷1-MT F K  CHOP (29 1(D)  Cleaved  (171(D)  Caspase-3 (12 1(0)  -actn (43 ((0)  Figure 2.5. Role of GCN2 kinase pathway in selective apoptotic effect of IDO expression on human T cells vs. skin cells. Fibroblasts were left either untreated or treated with IFN-y (1,000 U/mL). Cells were then washed and co-cultured with purified CD4 T cells (CD4), purified CD8 T cells (CD8), keratinocytes (K), or a different strain of fibroblasts (F) for 3 days in a two chamber co-culture system, in the absence or presence of media supplemented with 1-MT (800 IIM). Cell lysates were prepared and analyzed for the expression of CHOP and cleaved caspase-3 by western blot. Blots were stripped and reprobed for 13-actin as equal loading control. A representative experiment is shown (panel A). Moreover, fibroblasts either infected with Ad-GFP (Mock-Ad) or Ad IDO-GFP (IDO) were co cultured with human T cells (T), fibroblasts (F) or keratinocytes (K) for 3 days in a two chamber co-culture system, in the absence or presence of specific IDO inhibitor, 1-MT (800 i.tM). Bystander cells were harvested and cell lysates were prepared and analyzed for the expression of CHOP and cleaved caspase-3 by western blot. Blots were stripped and reprobed for 13-actin as equal loading control. A representative experiment is shown (panel B). GCN-2, general control nonderepressing 2; IDO, indoleamine 2, 3-dioxygenase; 1-MT, 1-methyl-D-tryptophan.  77  Discussion Several recent studies demonstrated that IDO might have a role in immuno-modulation (7,9,15-19). Our group has already shown that IDO expression functions as a local immunosuppressive factor to protect allo- or xenogeneic skin cells (II). However, the mechanism(s) by which IDO affects T cells is poorly defined. Two mechanisms have been suggested for the immunosuppressive effect of IDO: first, the production of toxic metabolites of tryptophan (i.e., kynurenine and 3-hydroxyanthralinic acid) results in T cell death (20) and second, the local tryptophan depletion causes a decrease in T cell proliferation and apoptosis (18). In this study, several experimental strategies were used to examine these two possibilities. In the current study, we found that IDO expression has a selective effect on different primary human cell types. Indeed, 7-AAD staining, a very sensitive way to detect the loss of membrane integrity during apoptosis and caspase-3 activation assay, indicated that bystander T cells, but not fibroblasts and keratinocytes, will undergo apoptosis when they are co-cultured with IDO expressing cells (2 1,22). Moreover, we also showed that an increase in proliferation of human T cells stimulated by a high immunogenic effect of pieces of both epidermal and full skin can be suppressed in the presence of IDO expressing cells. This finding may pave the way to practically use IDO expression as a local immunosuppressive factor to prevent the rejection of allogeneic grafts, such as allogeneic skin substitute to be used not only as a wound coverage but also as source of wound healing promoting factors. Munn et al. (9) have recently shown that GCN2 serves as a molecular sensor in murine T cells that allows them to detect and respond to the immunoregulatory signal generated by IDO, and it has also been shown that overexpression of CHOP causes apoptosis in keratinocytes in  78  culture (23). Based on these data, to elaborate the mechanism by which immune cells, but not skin cells, are sensitive to the IDO mediated environment, we studied the activation of proapoptotic factor CHOP as a downstream signal for the GCN2 kinase pathway (24) in these different cell types. The findings showed a significant increase in CHOP expression in stimulated human T cells in response to co-culturing with IDO expressing cells; however, there was no detectable expression of CHOP in fibroblasts and keratinocytes under similar conditions. In other words, we were able to demonstrate that there is a difference in the activation of downstream pathways in response to IDO between primary human T cells and skin cells. In summary, our findings indicate that an IDO-mediated environment differentially influences the biological functions of human T cells compared with either fibroblasts or keratinocytes. This differential effect seems to be, at least in part, due to difference in activation of the GCN2 kinase pathway. The findings of this study, therefore, suggest the selective effect of IDO on some human immune cell subsets vs. some nonimmune cell subsets and well support the proposal of using IDO as a local immunosuppressive factor for engraftment of an allogeneic graft, in our case skin substitute, without compromising the viability of nonimmune cells.  Acknowledgments The Canadian Institutes of Health Research (CIHR) and British Columbia Professional Fire Fighters’ Burn Fund supported this study. Farshad Forouzandeh and Reza B. Jalili are holding University Graduate Fellowships from the University of British Columbia. Marc Germain is the recipient of a Postdoctoral Fellowship from the Michael Smith  79  Foundation for Health Research. The authors are grateful to Dr. J.M. Carlin (Department of Microbiology, Miami University, Oxford, OH) for his gift of IDO cDNA.  Conflict of interest: The authors state no conflict of interest.  80  References  (1) Temess P, Bauer TM, Rose L, Dufter C, Watzlik A, Simon H, et a!. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J.Exp.Med. 2002 08; 1 96(4):447457.  (2) Muller AJ, Prendergast GC. Indoleamine 2,3-dioxygenase in immune suppression and cancer. Curr.Cancer Drug Targets. 2007 02;7(l):31-40.  (3) Allen JB, Wong HL, Costa GL, Bienkowski MJ, et al. Suppression of monocyte function and differential regulation of IL-i and IL-ira by IL-4 contribute to resolution of experimental arthritis. J Immunol.1993 Oct 15;151(8):4344-51.  (4) Ignotz RA, Massague J. Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol. Chem.1986 Mar 25;261(9):4337-45.  (5) Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B, et al. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol.1987 Sep;105(3):1039-45.  (6) Li Y, Tredget EE, Ghahary A. Cell surface expression of MHC class I antigen is suppressed in indoleamine 2,3-dioxygenase genetically modified keratinocytes: implications in allogeneic skin substitute engraftment. Hum.Immunol. 2004 02;65(2):i 14-123.  81  (7) Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J.Exp.Med. 1999 05/03;189(9):1363-1372.  (8) Sarkhosh K, Tredget EE, Li Y, Kilani RT, Uludag H, Ghahary A. Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2,3-dioxygenase expression of interferon-gamma-treated skin cells in a co-culture system. Wound Repair Regen. 2003 09;11(5):337-345.  (9) Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, et al. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 2005 05 ;22(5):633-642.  (10) Ghahary A, Li Y, Tredget EE, Kilani RT, Iwashina T, Karami A, et al. Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions as a local immunosuppressive factor. J.Invest.Dermatol. 2004 04;122(4):953-964.  (11) Li Y, Tredget EE, Ghaffari A, Lin X, Kilani RT, Ghahary A. Local expression of indoleamine 2,3-dioxygenase protects engraftment of xenogeneic skin substitute. J.Invest.Dermatol. 2006 01 ;126(1):128-136.  (12) Ghahary A, Tredget EE, Shen  Q, Kilani RT, Scott PG, Houle Y. Mannose-6-  phosphate/IGF-1I receptors mediate the effects of IGF-1-induced latent transforming growth factor beta I on expression of type I collagen and collagenase in dermal fibroblasts. Growth Factors 2000; 17(3): 167-176.  82  (13) Sarkhosh K, Tredget EE, Karami A, Uludag H, Iwashina T, Kilani RT, et al. Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG). J.Cell.Biochem. 2003 09/01 ;90( I ):206-2 17.  (14) Sarkhosh K, Tredget EE, Uludag H, Kilani RT, Karami A, Li Y, et al. Temperaturesensitive polymer-conjugated IFN-gamma induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG). J.Cell.Physiol. 2004 10;201(1):146-154.  (15) Friberg M, Jennings R, Alsarraj M, Dessureault S, Cantor A, Extermann M, et al.  Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection. lnt.J.Cancer 2002 09/10;101(2): 151-155.  (16) Hayashi T, Beck L, Rossetto C, Gong X, Takikawa 0, Takabayashi K, et al. Inhibition of experimental asthma by indoleamine 2,3-dioxygenase. J.Clin.Invest. 2004 07;1 14(2):270-279.  (17) Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998 08/21;281(5380):1 191-1193.  (18) Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, et al. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J.Clin.Invest. 2004 07; 11 4(2):280-290.  83  (19) Uyttenhove C, Pilotte L, Theate I, Stroobant V. Colau D, Parmentier N, et a!. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat.Med. 2003 10;9(10):1269-1274.  (20) Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, Spreca A, et al. T cell apoptosis by tryptophan catabolism. Cell Death & Differentiation 2002 10;9(10):1069.  (21) Schmid I, Uittenbogaart CH, Giorgi JV. Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry. 1994 -  Jan 1;15(1):12-20.  (22) Schmid 1, Uittenbogaart CH, Keld B, Giorgi JV, et a!. A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J Immunol Methods. 1994 Apr 1 5;170(2): 145-57.  (23) Anand S, Chakrabarti E, Kawamura H, Taylor CR, et al. Ultraviolet light (UVB and UVA) induces the damage-responsive transcription factor CHOP. J Invest Dermatol.2005 Aug; 125(2):323-33.  (24) Wek RC, Jiang HY, Anthony TG. Coping with stress: eIF2 kinases and translational control. Biochem.Soc.Trans. 2006 02/01 ;34:7- 11.  84  CHAPTER  31  Differential immunosuppressive effect of indoleamine 2,3-dioxygenase (IDO) on primary human CD4 and CD8 T cells  A version of this chapter has been published. With Kind permission from Springer Science + Buisiness Media: Mol. Cell. Biochem, Differential immunosuppressive effect of indoleamine 2,3-dioxygenase (IDO) on primary human CD4 and CD8 T cells, 2008 Feb;309(1-2):1-7, Forouzandeh F, Jalili RB, Germain M, Duronio V. Ghahary A. The original publication is available at www.springerlink.com through the following link: http://www.springerlink.com/content/w4805374q7681272/  85  Introduction Indoleamine 2,3-dioxygenase (IDO) is a heme-containing rate-limiting enzyme, which catalyzes the conversion of tryptophan to kynurenine, the main tryptophan metabolite (1). IDO has been found in non-hepatic cells mainly in trophoblasts, dendritic cells, monocytes, and macrophages (2-5). The expression of IDO has immuno-modulatory effects on T cells that are related to the pericellular degradation of tryptophan (6). Tryptophan is the least available essential amino acid and is required by all forms of life for protein synthesis and other important metabolic functions. IDO-generated low tryptophan environment may cause the activation of stress pathways such as nutrientsensitive mammalian target of rapamycin (mTOR) kinase pathway in bystander cell. However, it has been shown that inhibitors of mTOR such as rapamycin do not recapitulate the profound proliferative arrest seen with IDO expression (6). A second amino acid-sensitive pathway is mediated by the GCN2 kinase. GCN2 contains a regulatory domain that binds the uncharged form of tRNAs. Amino acid insufficiency causes a rise in uncharged tRNAs, which activates the GCN2 kinase domain and initiates downstream signaling. Recently, it has been shown that GCN2 kinase pathway is involved in the immunosuppressive effect of IDO expression on murine immune cells (6). As the immuno-modulatory effect of IDO is being investigated for its usage as a local immunosuppressive strategy for allogeneic grafts, it is important to precisely demonstrate its effects on different subsets of immune cells important in graft rejection. Here, we, therefore, asked whether the proliferation and viability of bystander CD4 and CD8 T cells are modulated in response to IDO-induced tryptophan deficient environment and if so, whether their response is different. In this study, for the first time, we provided  86  compelling evidence that both subsets of human T cells are sensitive to IDO induced low tryptophan environment and it is more so for CD8 relative to that of CD4 cells. Furthermore, we found that this differential response is due, at least in part, to the difference in the level of GCN2 kinase activation between these two subsets of T cells.  Materials and methods Adenoviral vector construction The procedure of construction of IDO expressing adenoviral vectors has been described before (7). Briefly, to construct the adenovirus encoding human IDO, we cloned the PCR product encoding the full-length protein into a shuttle vector, which co-expresses GFP as a reporter gene following the manufacturer’s instructions (Q-Biognene, Carlsbad, CA). The recombinant adenoviral plasmids were generated by electroporation of BJ5 183 E. coli using the shuttle vector either with or without IDO. Recombinant adenoviral plasmids were then purified and transfected to 293 cells using Fugene-6 transfection reagent (Roche Applied Science, Laval, QC, Canada). Transfected cells were monitored for GFP expression and after three cycles of freezing in ethanol/dry ice bath and rapid thawing at 37°C, the cell lysate were used to amplifi viral particles in large scale to prepare Adenoviral stock. Then viral titration was done using 293- cell line as described previously (7). Transfection of fibroblasts with IDO adenoviral vector Recombinant adenoviruses were used to infect fibroblasts at a Multiplicity of Infection of 100 (MOl: 100) as described previously (8). Free viral particles were removed from culture medium 30 h after transfection. The success of transfection was determined by  87  flow cytometry measuring the GFP protein expression and fluorescent microscopy using a Nikon inverted microscope equipped with an FITC filter to view GFP. Images were captured using a digital camera. Moreover, the efficacy of transfection and expression of functional IDO were confirmed by detection of IDO protein by western blot analysis and kynurenine level in the conditioned medium was measured according to a procedure described before (8). Fluorescence-activated cell sorting (FACS) of human PBMC for T cells Total PBMC were isolated by density gradient sedimentation on Histopaque-1077 (Sigma) according to the manufacturer’s protocol. Briefly, whole blood was layered on an equal volume of Histopaque and centrifuged at 2,000 rpm for 20 mm at room temperature and stopped without any brake. PBMC were isolated and added to RPMI 1640+10% FBS and pelleted by centrifugation at 2,000 rpm for 10 mm and were further washed twice in PBS+1% FBS. We used 20 1.11/106 cells of PE-conjugated mouse anti human CD3 mAb (BD, Oakville, ON), FITC-conjugated mouse anti-human CD4 mAb (BD), and allophycocyanin (APC)-conjugated mouse anti-human CD8 mAb (BD). The cells were incubated at room temperature for 30 mm. After staining, cells were washed twice and resuspended at iü cells/mL in PBS+1% FBS for FACS. For preparation of a pure population of CD3 + CD4 + and CD3 + CD8 + T cells, we gated on CD3 + CD4 + and CD3CD8 T cells after excluding the dead cells and cell debris based on FSC and SSC parameters and sorted these two-cell population into separate tubes. T cell culture and survival evaluation After being sorted from blood, T cells were propagated in RPMI 1640 (Hyclone, Utah, UT) supplemented with 10% FBS, 0.1 U penicillin/mL, and 0.1 mg streptomycin/mL at  88  37°C in a humidified 5% C02 atmosphere to be used for further treatment. The survival of two different sensitive (CD4 and CD8 T cells) and two different resistant (dermal fibroblasts and keratinocytes) cell types in an IDO-induced tryptophan deficient environment was compared by 7-AAD staining. 7-AAD intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. After each treatment, cells were harvested, washed in PBS, stained for 7-AAD and then examined using FACS analysis, as per the manufacturer’s protocol (BD). T cell stimulation and in vitro proliferation assay  T cells were stimulated with anti-CD3 (1 Ig/mL; BD). The cells were pulsed with [3H] thymidine (1 iiCi/mL, Perkin Elmer, Boston, MA) on day 3 and harvested on day 4 for measurement of cell lysate-associated tritium by f3-scintillation counting. All proliferation experiments were performed in triplicate and reported as Count Per Minute (C.P.M). Western blot analysis  Total proteins from cell lysate (20 .tg) were separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA). The blots were probed for CHOP using anti GADD153/CHOP-10 Ab produced in rabbit (1:250 dilution, Sigma). We have used goat anti-rabbit Ab (1:2500 dilution, Bio-Rad) as the secondary Ab. Enhanced chemiluminescence detection system (ECL; Amersham Biosciences, UK) was used in all blots to detect the related secondary Ab. Blots were then stripped and reprobed for 3actin and used as a control of protein loading. Preparing 1-MT solution  1-MT (Sigma) was prepared as a 20 mM stock solution in 0.1 N NaOH and adjusted to pH 7.4. The reagent was stored at 4°C and protected from light.  89  Statistical analysis Data were expressed as mean ± SD and analyzed with one-way ANOVA among different groups of each cell type where indicated. P-values <0.05 are considered statistically significant in this study.  Results Sensitivity of CD4 and CD8 to IDO-induced low tryptophan environment In order to investigate the immunosuppressive effects of IDO expression on human T cells, we set up a co-culture system of either CD4 or CD8 cells with IDO-expressing dermal fibroblasts. Total human PBMC (Fig. 1A) as well as CD4 (Fig. 1B) and CD8 (Fig. 1C) T cells were sorted by FACS and analyzed for purity. The result shown in Fig. I revealed that the purity of both CD4 (1D) and CD8 (1E) sub-population was more than 99%. In parallel, fibroblasts were cultured and infected for 72 h with recombinant adenoviral vectors expressing either GFP (Ad-GFP) or IDO plus GFP (Ad-GFP-IDO), as described in Methods. Based on GFP expression, infection rate was estimated to be around 70—80% in each set of experiments. Moreover, IDO expression was confirmed by western blot analysis (data not shown). We then set up the co-culture system in which IDO-expressing cells were grown in the upper chambers of 6-well plates, while T cells were cultured in the lower chamber as bystander cells (Fig. 2A). Therefore, there was no direct contact between fibroblasts and T cells. It should also be mentioned that Adenovirus or IFN-y treated fibroblasts, as described before (8), were washed with PBS prior to co-culturing them with other cells, to remove excess adenovirus and IFN-y. We used anti-CD3 (1 ig/mL) for activating the resting T cells. Bystander T cells were  90  harvested on day 4 and evaluated for 7-AAD staining by FACS analysis. The histograms of one set of experiment, out of three sets, for CD4 and CD8 T cells are shown in Fig. 2B and c respectively. The results of FACS analysis representing the result of three sets of experiments are shown in Fig. 2D and E. As shown in panel 2D, stimulated CD4 T cells were sensitive to IDO-induced low tryptophan environment as their cell survival rate dropped from 91  ±  1.5% to 77 ± 7.3% and 64  ±  4.1% when co-cultured with non-  treated fibroblasts, Ad-GFP-IDO or IFN7 treated fibroblasts, respectively. However, resting CD4 T cells did not show any significant sensitivity to the same IDO induced environment. On the other hand, both resting and stimulated CD8 T cells were sensitive to IDO induced low tryptophan environment (panel 2E; P<O.OO1). The viability of resting CD8 T cells was reduced from 92 ± 2.9% in co-culture with non-treated fibroblasts to 74 ±  4.5% and 67  ±  3.5% in co-cultured with Ad-GFP-TDO and IFN-y groups, respectively.  These decreases were also evident in stimulated CD8 T cells whose viability of 90 3.6% dropped to 72  ±  ±  2% and 67 ± 5.4%, respectively (P<0.001). Moreover, addition of a  competitive IDO inhibitor, 1-MT (800 jiM) (9), reversed the effect of IDO expression on CD4 and CD8 T cells significantly for both Ad-GFP-IDO and IFN-y treated fibroblasts (Fig. 2D and E).  91  [Total PBMCI  A  4: Cl) 000)4  I)1)0  600)4  104606  202  FSCA  j Total PBMC  B  Total PBMC  C  I  0 1)’ C.) C 4,  ‘0  to’ C,  0 4— 04.  o..  10  4: 10  10  0  10  0  PE-anti C03  D4 t-cells after sorting  D  0  1)’  PE•nti CD3  I  t CD8 T-cells after sorting  E 0)  ‘0  0  0 C.)  0  C.) 4-  0  C ‘I,  0.  10  I’.  to_.. 11  PE -antI C03  1  PE-anF C03  Fig. 3.1 Fluorescence-activated cell Sorting of human PBMC for T cells. Total PBMCs were isolated by density gradient sedimentation on Histopaque 1077 (Sigma). Primary gate based on physical parameters (forward and side light scatter, FSC and SSC) was set to exclude dead cells or debris (A), The two T cell subsets, CD3 CD4 (B) and CD3CD8 (C), were sorted into separate tubes. Following the cell sorting process, the purity of the sorted populations was analyzed on the flow cytometer and found to be more than 99% (D and E).  92  C  B L!iJ  bJ  :Lur  A  ____ ,t  MW)-[21i)  7MD(rL2H  lGFp4oo  ?MD(FLfl)  .1MT  1  TZA  j ,  w  I  1 AD L2  ::r  FN  ;:j4A III,IIOI tI1IFL2If  FN+lMi9  :L—  :r  I  4l  ;j “j  :  lIt l IC  ‘1N+1MT)  I______  C  Fig. 3.2 The effect of IDO expression on resting and stimulated human CD4 and CD8 T cells. Fibroblasts were left either untreated or treated with either adenovirus (MOI:100) or IFN-y (1,000 U/mL). Cells were then washed and co-cultured with purified resting and stimulated primary human CD4 and CD8 T cells in two chamber co-culture system for 4 days (A), in the absence or presence of the IDO inhibitor 1-MT (800 iiM). The viability of CD4 (panels B and D) and CD8 (panels C and E) T cells was determined by FACS analysis using 7-AAD staining. The significant (P-value<0.001) differences have been indicated by asterisks (*) (n 3)  D 110  100  NL Fib.  Ad-GFP  Ad-GF4BO Ad4FP-IDO  IFN  IFN • IMT  E 110  100 ,  90  •I  IL_1L **  80  LFih  M-GP  M.GFP-IDO M.GP4DO + IMT  *  IFN  I  IFN+1rIrr  93  ___  Inhibition of T cell proliferation by IDO-induced low tryptophan environment  In order to clarif’ the immunosuppressive effect of IDO expression on T cell subsets, we looked at cell proliferation of T cells in IDO-induced low tryptophan environment. On the third day of co-culturing stimulated CD4 and CD8 T cells with fibroblasts receiving different treatments, 1 tCi/ mL [3H]-thymidine was added to T cell cultures and after 16 h, incorporation of [3H]-thymidine to cellular DNA was measured. As shown in Fig. 3, there was an almost 5-fold reduction in the proliferation rate of T cells when co-cultured with allogeneic IDO-expressing fibroblasts compared to that of T cells co-cultured with non-IDO-expressing fibroblasts (P<O.OO 1). The suppression of immune cell proliferation was specific to IDO expression, because addition of 1-MT significantly reversed the effect of IDO expression on CD4 and CD8 T cells for both Ad-GFP-IDO and IFN-y treated fibroblasts. Even in this experiment, CD8 T cells showed slightly more sensitivity to IDO compared to CD4 T cells. 45000 40000 35000 30000 25000: 20000  —  4  15000-:  *  *  I  NL Fib.  M4FP  AdGFP-IDO  M-GFP-1E0  IFN  IFN +IMT  Fig. 3.3 Effect of IDO-induced low tryptophan and high kynurenine environment on proliferation of T cell subsets. On the third day of coculture, I iCi/mL [3H]-thymidine was added to stimulated CD4 T cell (solid bars) or stimulated CD8 T cells (open bars) cultures. After 16 h, incorporation of {3H]-thymidine to cellular DNA was measured. The bars show lymphocyte proliferation rates, Count Per Minute (C.P.M.). The significant (P value<O.OO1) differences in lymphocyte proliferation rate compared to that of the control lymphocytes cultured with normal (untreated) fibroblasts have been indicated by asterisks (*)(n=3)  94  GCN2 kinase pathway is involved in the suppressive effect of IDO on human T cells It has been suggested that the activation of GCN2 kinase by uncharged tRNAs in murine T cells mediates T cell proliferative arrest and energy induction (6). To verify this point in primary human T cell subsets, CD4 and CD8 T cells co-cultured with IDO expressing fibroblasts were harvested and evaluated for the expression of a downstream effector of GCN2, CHOP (6). We, therefore, evaluated the level of CHOP expression in human T cell subsets, which were cultured in either tryptophan-free media or in the presence of kynurenine (128 jiM). In another set of experiments, we tested also whether the addition of an excess amount of tryptophan could prevent the effect of IDO expression on T cells. As shown in Fig. 4, there was a significant increase in CHOP expression in CD8, but not in CD4 T cells in IDO-induced low tryptophan environment. This effect was preventable to some extent by adding extra amounts of tryptophan in the environment. Moreover, culturing the human CD8 T cells in the absence of tryptophan or in the presence of kynurenine can induce GCN2 kinase activation in these cells, however, not at the level of IDO induced low tryptophari and high kynurenine environment. Interestingly, CHOP expression remained low in CD4 under all conditions tested (Fig. 4). Non-treated  IFN  IFN+ Trp  Trp free  }cynurenThe  CHOP (291(D)  B.Actin  (4KD)  95  Fig. 3.4 GCN2 kinase pathway is involved in suppressive effect of IDO expression on human T cell subsets. Fibroblasts were left either untreated or treated with JFN-y (1,000 U/mL). Cells were then washed and co-cultured with purified CD4 T cells (CD4), stimulated purified CD8 T cells (CD8) for 4 days in a two chamber co-culture system, in the absence or presence of media supplemented with extra amount of tryptophan. In addition, cells were separately cultured in either tryptophan free media or in the presence of kynurenine. Cell lysates were prepared and analyzed for the expression of CHOP by western blot. Blots were stripped and reprobed for 13-actin as equal loading control. A representative experiment is shown (N = 3)  Discussion In this study, we demonstrated for the first time that the proliferation of CD8 and CD4 T cells is suppressed in response to tryptophan deficient environment caused by IDO expression. This was even more so for CD8 T relative to that of CD4 T cells. This finding is consistent with those reported by Munn et al. using murine T cell populations (6). However, we found a differential sensitivity in cell proliferation between two subsets of CD8 T and CD4T cells when grown in the same IDO-induced low tryptophan deficient environment. Although some reports suggested that T cells are specifically sensitive to IDO expression (10), the mechanism(s) by which IDO affects primary human T cells is poorly defined. Two mechanisms have been suggested for the immunosuppressive effect of IDO: first, the production of toxic metabolites of tryptophan (i.e., kynurenine and 3-hydroxyanthralinic acid) results in T cells death (11). Second, local tryptophan depletion causes a decrease in T cell proliferation and apoptosis (12). In this study, we demonstrated that IDO expression has a marked effect on viability and proliferation of both activated CD4 and CD8 T cells. However, this effect was greater in CD8 cells relative to CD4 T cells. The findings also showed that resting human CD8 lymphocytes are more sensitive to IDO induced low tryptophan environment than CD4 T cells. This difference was also reflected in IDO expression induced cell death of  96  these cells. Munn et a!. has recently shown (2005) that GCN2 serves as a molecular sensor in murine T cells, which allows them to detect and respond to the immunoregulatory signal generated by IDO. We studied the activation of the pro apoptotic factor CHOP as a downstream signal for GCN2 kinase pathway (13). The findings showed a significant increase in CHOP expression in stimulated human CD8 T cells and almost no increase in stimulated human CD4 T cells in response to IDO expression. In summary, our findings indicate that IDO-induced low tryptophan environment differentially influences the biological functions of primary human CD4 and CD8 T cells. This differential effect seems to be due, at least in part, to differences in the activation of GCN2 kinase pathway.  Acknowledgments The Canadian Institutes of Health Research and British Columbia Professional Fire Fighters’ Burn Fund supported this study. Farshad Forouzandeh and Reza B. Jalili are holding University Graduate Fellowships from the University of British Columbia. Marc Germain is the recipient of a Postdoctoral Fellowship from the Michael Smith Foundation for Health Research. The authors are grateful to Dr. JM Carlin (Department of Microbiology, Miami University, Oxford, OH) for his gift of IDO cDNA.  97  References  (1) Terness P, Bauer TM, Rose L, Dufter C, Watzlik A, Simon H, et al. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J.Exp.Med. 2002; 1 96(4):447-45 7.  (2) Allen JB, Wong HL, Costa GL, Bienkowski MJ, et al. Suppression of monocyte function and differential regulation of IL-i and IL-ira by IL-4 contribute to resolution of experimental arthritis. J Immunol.1993 Oct 15;151(8):4344-51.  (3) Ignotz RA, Massague J. Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem. 1986 Mar 25 ;26 I (9):4337-45.  (4) Jalili RB, Forouzandeh F, Bahar MA, Ghahary A. The immunoregulatory function of indoleamine 2, 3 dioxygenase and its application in allotranspiantation. Iran J Allergy Asthma Immunol.2007 Dec;6(4): 167-79.  (5) Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B, et a!. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol.1987 Sep;105(3):1039-45.  (6) Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, et al. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 2005 05 ;22(5):633-642.  98  (7) Ghahary A, Li Y, Tredget EE, Kilani RT, Iwashina T, Karami A, et a!. Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions as a local immunosuppressive factor. J.Invest.Dermatol. 2004 04; 122(4) :953-964.  (8) Jalili RB, Rayat GR, Rajotte RV, Ghahary A. Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts. J.Cell.Physiol. 2007 10;213(0021-9541; 1):137-143.  (9) Li Y, Tredget EE, Ghaffari A, Lin X, Kilani RT, Ghahary A. Local expression of indoleamine 2,3-dioxygenase protects engraftment of xenogeneic skin substitute. J.Invest.Dermatol. 2006 01 ;126(1): 128-136.  (10) Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J.Exp.Med. 1999;1 89(9): 1363-1372.  (11) Fallarino F, Grohmann U, Vacca C, Orabona C, Spreca A, Fioretti MC, et at. T cell apoptosis by kynurenines. Adv.Exp.Med.Biol. 2003 ;527: 183-90.  (12) Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, et al. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J.Clin.Invest. 2004; 11 4(2):280-290.  (13) Wek RC, Jiang HY, Anthony TG. Coping with stress: eIF2 kinases and translational control. Biochem.Soc.Trans. 2006;34:7- 11.  99  CHAPTER  41  Indoleamine 2, 3-dioxygenase (IDO) expression improves the engraftment of non-autologous engineered skin substitute  ‘A version of this chapter has been submitted for publication. Forouzandeh F, Jalili RB, Hartwell RV, Allan SE, Boyce S. Supp D, Ghahary A. Indoleamine 2, 3-dioxygenase (IDO) expression improves the engrafiment of non-autologous engineered skin substitute.  100  Introduction The ability to re-generate or repair injured tissue is essential to the continuity of human life (1). As in all other organs, wound healing in the skin is a dynamic process that involves a variety of responses to different types of insults. This process involves the cooperation of platelets, fibroblasts, epithelial, endothelial and immune cells which together regulate a complex sequence of events which results in tissue repair (2). The skin serves a wide range of protective, sensory and regulatory functions. Importantly, the capacity of the skin to act as a protective barrier is crucial for survival (3). When this barrier is severely disrupted by an injury such as a burn or a diabetic ulcer, healing and/or repair are essential for survival. Despite the fact that many factors that promote wound healing have been identified, the topical application of these agents have a limited value due to their short half lives, high cost, and side effects (4-6). As an alternative, sheets of autologous keratinocytes, first used in the clinic by O’Connor et al. in 1981 (7,8), have been used in some burn centers to treat patients with large thermal injuries. However, this has not been adopted as a routine procedure, as its widespread use has been hampered due to delays in obtaining the grafts, the variable acceptance rate of grafts, the sensitivity of patients to infection and high costs (9) Moreover, sheets of keratinocytes that are .  prepared from layers of engineered keratinocytes in the absence of a matrix are very fragile and are difficult to generate in patients that have a limited area of uninjured tissue as a source of primary cells. In addition, the biopsy wounds generated to obtain the required primary cells can cause complications in patients suffering from impaired wound healing process, such as diabetics (10). Another notably shortcoming of autologous sheets of keratinocytes as skin substitute is missing dermal cells and  101  components in this model. In fact, living fibroblasts play important roles in promoting keratinocytes growth, dermal-epidermal organization, and providing mechanical support to the epidermal layer (7,11,12). Moreover, it has been shown that the presence of both dermal and epidermal layers allows for highly complex ex vivo function of epidermal cells (13). This interaction may be crucial for a better healing quality at a late stage of the wound healing process (14,15). Therefore, the use of a non-autologous and readily available skin substitute consists of both dermal and epidermal cells resembling full thickness skin is among the most promising skin substitute models (16). Although skin was the first tissue-engineered organ to have been successfully developed in the laboratory, immune-mediated rejection of non-autologous skin substitutes limits the use of this strategy for the treatment of wounds similar to the transplantation of other organs (3,17-19). Non-autologous skin rejection is thought to occur mostly as acute rejection and to be mainly T cell dependent (18,20). Accordingly, the most effective immunosuppressive agents for preventing rejection of this tissue are those that act on T cells, such as the anti-proliferative drugs cyclosporine and FK506 (18,20-22). Although the use of immunosuppressive drugs has significantly improved the acceptance of grafts, their lack of specificity, which invariably results in generalized immunosuppression, and their other systemic side effects are among the major concerns of their administration (23). Therefore, the use of a targeted immunosuppressive agent that would suppress the local immune response to a non-autologous graft without inducing unwanted systemic immunosuppression would be an ideal way to approach this problem. To this end, we hypothesized that a functional skin equivalent in which a local immunosuppressive  102  factor, such as indoleamine 2, 3-dioxygenase (IDO), was expressed would improve the engraftment of a non-autologous graft without causing generalized immunosuppression.  IDO is a heme-containing, rate-limiting enzyme that catalyzes tryptophan to N formylkynurenine and then to kynurenine. IDO is an inducible enzyme in non-hepatic cells, mainly in trophoblasts, dendritic cells, monocytes and macrophages, however, the hepatic cells express TDO (tryptophan 2,3- dioxygenase) at low level constitutively for the same reaction (24-28). The expression of IDO has immuno-modulatory effects on T cells as a result of the pen-cellular degradation of tryptophan, an amino acid required for T cell proliferation (29). Moreover, in recent studies, a clear association has been made between tryptophan catabolism and inhibition of inflammatory reactions in a broad range of physiologic and pathologic states. Indeed, IDO is important in the regulation of many different types of immune responses, such as the prevention of fetal rejection (29) and in pathological conditions including neoplasia (30,3 1), chronic infection (31), asthma (32) and autoimmune diseases (33). Previously, we confirmed the potential of IDO to protect engrafted xenogeneic fibroblasts propagated in collagen gel (21). Our group has found that IDO expression in keratinocytes down regulates their expression of cell-surface major histocompatibility complex (MHC) class I molecules (34). In addition, we have shown that skin cells including fibroblasts, keratinocytes and endothelial cells can survive and proliferate in IDO-induced tryptophan deficient environment for considerable period of time, but not the immune T cell (35). This phenomenon is consistent with what has been reported for IDO-expressing trophoblasts in the placenta in a mouse model, in which bystander infiltrated immune cells, but not trophoblast cells, were influenced by an IDO-induced  103  tryptophan-deficient environment (29). As a follow-up to our previous work, our aim in this study was to test the immunosuppressive function of IDO in skin substitutes containing keratinocytes, the main immunogenic cells of the skin. We hypothesized that modified human reconstructed skin composed of both dermal and epidermal components could be modified to suppress the infiltration of T cells and prevent or delay the rejection of non-autologus grafts in rats through enforced expression of IDO. Indeed, the non autologus skin substitute can serve not only as a temporary wound coverage but also as rich source of cytokines and growth factors to further prepare the wound bed for the completion of healing process by the host autologous cells.  Materials and Methods Fibroblasts and keratinocytes culture  All procedures were carried out following approval of the Ethics Committee of the University of British Columbia (UBC). Neonatal foreskin pieces were used as source of fibroblasts and keratinocytes, and cultures of human foreskin fibroblasts were established as described previously (36). Briefly, punch biopsy samples were prepared from human foreskins. Tissue was collected in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, GIBCO). Specimens were dissected free of fat and minced into small pieces of less than 0.5 mm in any dimension, washed with sterile medium 6 times, and distributed into 60x 15 mm Petri culture dishes (Corning Inc., Corning, NY, USA), at four pieces per dish. A sterile glass coverslip was attached to the dish with a drop of sterile silicone grease to immobilize the tissue fragment. DMEM containing antibiotics (penicillin G sodium, 100 U/mL; streptomycin  104  sulfate, 100 jig/mL and amphotericin B, 0.25 jig/mL; GIBCO) and 10% FBS was added to each dish and incubated at 37°C in a water-jacked humidified incubator in an atmosphere of 5% CO . The medium was replaced twice weekly. After 4 weeks of 2 incubation, cells were released from the dishes by brief (5-minute) treatment with 0.1% trypsin (Life technologies Inc., Gaithersburg, MD) and 0.02% EDTA (Sigma, MO, USA) in phosphate buffered saline solution (PBS; pH 7.4) and transferred to 75 cm 2 culture flasks (Coming Inc.). Once cells had grown to 90-100% confluence, the cells were subcultured 1:6 by trypsinization. Fibroblasts of between 4—7 passages were used for this study. To culture human foreskin keratinocytes, Keratinocyte Serum-Free Medium (KSFM, GIBCO) supplemented with Bovine Pituitary Extract (BPE, GIBCO) and recombinant Epidermal Growth Factor (rEGF, GIBCO) was used and procedure was done according to the manufacture protocol (GIBCO). In brief, foreskins were placed into a “rinse solution” of phosphate buffered saline (PBS) (without Ca 2 or Mg ) containing 2 Gentamicin (GIBCO) at a concentration of 20 ig/mL for approximately 2-3 minutes while the tissue was cut into 3 to 4 pieces of equal size. Each piece of tissue was then put into a 25.0 Caseinolytic Units per mL solution of dispase (GIBCO) and Gentamicin at 5 .tg/mL. The tissues were incubated for 18 h at 4°C. After incubation in dispase, the epidermal layer of human keratinocytes was lifted from the dermis and placed into a 15 mL sterile centrifuge tube containing 2 mL of Trypsin- EDTA (GIBCO). The tissue was incubated at 37°C for approximately 10-12 minutes during which time it was aspirated with a 2 mL pipette every 2-3 minutes to dissociate the cells. Following incubation, the action of the trypsin was stopped by adding 10-13 mL of Soybean Trypsin Inhibitor (GIBCO), at final concentration of 10 mg/mL dissolved in PBS and sterile filtered prior  105  to use. The cells were spun at 800 rpm for 8 minutes at room temperature. The cell pellet of human keratinocytes was gently resuspended in approximately 5 mL of complete medium (i.e., KSFM containing 25 jig/mL of BPE and 0.5 ng/mL of rEGF). The primary cells were seeded into T-75 flasks at a cell density of approximately 3 x 106 cells per flask in 10 mL complete medium. Primary cultured keratinocytes of between 3—5 passages were used for this study. Preparation of Engineered Skin Substitutes Engineered Skin Substitutes (ESS5) consisted of stratified layers of keratinocytes and fibroblasts embedded in a scaffold of type I bovine collagen and glycos-aminoglycan (GAG) were developed by the method previously described by Steven Boyce et a! (3740) from comminuted bovine hide collagen and chondroitin-6-sulfate, except without chemical cross-linking with glutaraldehyde. The pore size of the collagen-GAG substrates used in this study was optimized to promote better fibroblasts infiltration to the substrates. This would facilitate the migration of the cells from the wound bed to the scaffolds as well. Thickness of dermal substitutes was regulated by control of the volume and concentration of starting materials. For cell culture, the polymer substrates were rehydrated in hepes-buffered saline solution (HBSS), and changed into culture medium for inoculation of cells. For inoculation, substrates were placed on top of N-terface mesh, a cotton pad and a steel lifting platform. Non-treated, Ad-GFP or Ad-IDO-GFP treated human fibroblasts were inoculated within the collagen-GAG substrate at a concentration of 0.5 x 2 /cm and cultured at 37°C and 6 10 5% CO . Medium consisted of DMEM supplemented with 5% FBS and antibiotics was 2  106  used to culture human fibroblasts (41-42). On the following day, the collagen-human fibroblast substrates were rinsed and incubated overnight in the skin substitute serum-free culture medium composed of the equal volume of DMEM and KSFM supplemented with antibiotics. On the next day, incubation day 0, human keratinocytes (1.0  x  2 / 6 10 ) cm were  layered on top of the collagen- fibroblast substrates at the same side of the substrates that previously were inoculated by fibroblasts. The skin substitute culture medium was replaced daily until day 14 post-incubation. Biopsies for histology were collected on days 7 and 14 for microscopic evaluations. Adenoviral vector construction  The procedure for construction of the IDO-expressing adenoviral (Ad) vectors has been described previously (26). Briefly, to construct the adenovirus encoding human IDO, we cloned the PCR product encoding the full-length protein into a shuttle vector, which co expresses GFP as a reporter gene, by following the manufacturer’s instructions  (Q  Biognene, Carlsbad, CA, USA). The recombinant adenoviral plasmids were generated by electroporation of BJ5 1 83 E. coli the either the control shuttle vector or the vector expressing IDO. Recombinant adenoviral plasmids were then purified and transfected into 293 cells using Fugene-6 transfection reagent (Roche Applied Science, Laval, QC, Canada). Transfected cells were monitored for GFP expression and after three cycles of freezing in ethanol/dry ice bath and rapid thawing at 37 °C, the cell lysates were used to amplify viral particles in large scale to prepare the adenoviral stock. Then, viral titration was carried out with the 293 cell line as described previously (26).  107  Infection of fibroblasts with either Ad-GFP or Ad-IDO-GFP vector Recombinant adenoviruses were used to infect fibroblasts with a multiplicity of infection (MOT) of 100 as described previously (26,28). Free viral particles were removed from culture media 30 hours after infection. The success of infection was determined by fluorescence microscopy using a Motic inverted microscope (Motic Instruments, Richmond, BC, Canada) equipped with an FITC filter to view GFP-infected cells. The efficacy of infection and the expression of functional IDO in target cells were confirmed by analysing the presence of IDO protein by western immunoblotting and by measuring kynurenine levels in the conditioned media. Kynurenine measurement in the conditioned media We used the following assay to demonstrate IDO expression in fibroblasts that were engineered to express IDO with Ad-IDO-GFP. The biological activity of IDO was evaluated by measuring the levels of tryptophan degraded product, L-kynurenine, in the conditioned media from IDO expressing cells using a previously established method (43). Briefly, proteins in the conditioned media were precipitated by trichloroacetic acid and, after centrifugation at 10,000 rpm for 5 minutes at 4°C, 0.5 mL of supernatant was incubated with an equal volume of Ehrich’s reagent (Sigma) for 10 mm at room temperature. The absorption of the resultant solution at 490 nm was measured using a spectrophotometer within 2 hours. The amount of kynurenine in the conditioned media was calculated based on a standard curve. Protein isolation and western blot analysis Pieces of the NL-ESS, GFP-ESS, or IDO-ESS were weighed and cut into small pieces using a clean razor blade. 3 mL of ice cold Radio-lmmuno-Precipitation Assay (RIPA)  108  buffer (Sigma) was added per gram of tissue and, maintaining temperature at 4°C, tissues were disrupted using a homogenizer. Following this, 30 mL of 10 mg/mL of phenylmethylsulfonyl fluoride (PMSF) (Sigma) was added per gram of tissue and incubated on ice for 30 minutes. Following transfer to microcentrifuge tubes, samples were spun at 13,000 rpm for 20 minutes. Supernatant was retained and centrifuged again at 13,000 rpm for 20 minutes. The resulting supernatant was used as the total cell lysate. For detection of IDO expression, total proteins from cell lysates (20 ig per lane) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were probed with polyclonal rabbit anti-human IDO antibody (Washington Biotechnology Inc., Baltimore, MD, USA) at final dilution of 1:5000. Enhanced chemiluminescence detection system (ECL; Amersham Biosciences, UK) was used in all blots to detect the secondary HRP-linked antibody. In vitro proliferation assay for lymphocytes Lymphocytes were pulsed with [ H]-thymidine (1 iiCi/mL, Perkin Elmer, Boston, MA, 3 USA) on day 3 and harvested on day 4 for measurement of [ Hj-thymidine by f33 scintillation counting. Briefly, 74 KBq of [ H]-thymidine (Perkin-Elmer Life Sciences 3 Inc., Boston, MA) was added to each mL of the conditioned medium and the cells were incubated for 16 h After this period, lymphocytes were harvested, washed three times with PBS, dissolved in guanidium isothiocyanate, and added to scintillation fluid (Amersham Corp., Arlington Heights, IL). Radioactive counting was performed using a Beckman scintillation counter. All proliferation experiments were performed in 3 sets of experiments and reported as an average of counts per minute (CPM). In one control groups, we added concavaline A (10 ug/mL, Sigma) to stimulate the lymphocytes prior to  109  H]-thymidine incorporation assay as a positive control group for lymphocyte 3 [ proliferation. Grafting of ESSs-treated wounds in Sprague—Dawley rats Procedures on all animal studies were approved by the University of British Columbia (UBC) Animal Committee. Sprague-Dawley rats (10 weeks old) were anesthetised using isoflurane. The dorsal surface of animal was shaved and cleaned with 70% ethanol. Four full-thickness skin excision wounds were made on the dorsal surface of the rat using a 6 mm punch biopsy tool (Dormer Laboratories, Mississauga, ON, Canada). One wound was left non-treated as a control, while other wounds were treated with either NL-ESS, GFP-ESS, or IDO-ESS. In each set of experiment the order of treatment was changed to reduce the chance of bias in wound healing based on the place where the wounds were created on the animal. The wounds were then dressed with Tegaderm (St. Paul, MN, USA) and the grafted areas were bandaged. On day 7, the animals were killed and wound closure was measured and photographed. The entire wound, including a 2—4 mm margin of unwounded skin, was carefully excised. Each wound was divided in half and fixed in 4% paraformaldehyde (Fisher, Pittsburgh, PA, USA) in PBS solution and processed for  paraffin embedding. Immuno-histochemical staining  First, 5 tm thick sections were cut from paraffin-embedded wounds that had been treated with either NL-ESS, GFP-ESS, or IDO-ESS, and mounted on glass slides. Paraformaldehyde-fixed and paraffin-embedded sections (5 !Im) were de-paraffinized and hydrated by incubation in ethanol (Sigma) for 10 minutes. To retrieve cell surface antigens, a microwave oven heating pre-treatment was performed before blocking with  110  1% BSA (bovine serum albumin, Sigma) and 10% normal goat serum (Sigma) in TBS (tris-buffered saline). For CD3 staining, the sections were incubated with rabbit polyclonal anti-CD3 Ab (1:200, abcam, Cambridg, MA, USA) at 4°C for 16 hours. The secondary antibody used was biotinylated goat anti-rabbit IgG (1:5000, Vector Laboratories, Burlington, CA, USA). The signal detection was carried out using Vector VIP substrate kit (Vector Laboratories) according to manufacturer’s instructions. The slides were counterstained with methylene green for 3 minutes, dehydrated, mounted, and examined using microscopy. For HLA-I (human leukocyte antigen- type I) staining, the sections were incubated with mouse monoclonal anti-HLA-I Ab (1:10, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 16 hours. The secondary antibody used was biotinylated goat anti-mouse IgG (1:500, Vector Laboratories). The signal detection was carried out using Vector VIP substrate kit (Vector Laboratories) according to manufacturer’s instructions. The slides were counterstained with methylene green for 3 minutes, dehydrated, mounted, and examined using microscopy. Immuno-fluorescence staining First, 5 jim thick sections were cut from paraffin-embedded wounds that had been treated with either NL-ESS, GFP-ESS, or IDO-ESS, and mounted on glass slides. Paraformaldehyde-fixed and paraffin-embedded sections (5 jim) were de-paraffinized and hydrated by incubation in ethanol for 10 minutes. To retrieve cell surface antigens, a microwave oven heating pre-treatment was performed before blocking with TBS containing 5% BSA and 5% normal goat serum. For CD3 1 staining, pre-treated, blocked  111  sections were incubated with mouse monoclonal anti-PECAM-1 Ab (1:50, abcam) at 4°C for 16 hours. The secondary antibody used was Alexa-Fluor 488 goat anti-mouse (1:4000, Invitrogen, Carlsbad, CA, USA). The slides were then mounted with a DAPI (4’6-Diamidino-2-phenylindole) containing mounting medium (Vector Laboratories). DAPI is known to form fluorescent complexes with natural double-stranded DNA and so is a marker for nucleus. The fluorescence signal detection was carried out under fluorescent microscope. Statistical analysis Data were expressed as mean ± SD and analyzed with one-way ANOVA among different groups of each cell type where indicated. For post hoc testing, Student’s t-test with Bonferroni correction for multiple comparisons was used to compare groups. P-values of less than 0.05 are considered statistically significant in this study.  Results Histological characterization of the engineered skin substitutes In this study, we used an engineered skin substitute (ESS) that consisted of stratified layers of keratinocytes and fibroblasts embedded in a scaffold of type I bovine collagen and glycos-aminoglycan (GAG) as it has been developed before by the co-author, Steven Boyce, and his group (38-40). Briefly, a dermal skin substitute composing of collagen GAG and fibroblasts was used as a base to culture human keratinocytes on its surface. The primary fibroblasts and keratinocytes were extracted and cultured from neonatal foreskin pieces, as described in the methods, to be used for development of the skin substitute. As it is shown in figure 1, by using the optimized collagen-GAG scaffolds as  112  suitable substrates for the culture of human fibroblasts and keratinocytes, we have been able to develop a composite material that is histologically very similar to skin. Although this model cannot recapitulate all the delicate structure and appendages of the natural skin, it represents a good candidate to substitute the major barrier duties of the skin. Moreover, the alive cell component of this ESS can serve as a growth factor and cytokine factory to promote wound healing process.  Stratum Corneum  Epidermis  Dermis  { .  4  Stratum Basale  Fibroblasts populated in Collagen-GAG Matrix  Figure 4.1. Histological structure of the Engineered Skin Substitute (ESS). Fibroblasts were inoculated into collagen-GAG substrate at the concentration of 5 x 1 cells per 1 cm 2 of the substrate and cultured at 37°C and 5% CO . On the following day, 2 1.0 x 106 keratinocytes per 1 cm 2 of the substrate were inoculated on the lifted collagen fibroblast substrates in the skin substitute culture medium (incubation day 0), and the skin substitute culture medium was replaced daily until day 14 post-incubation. The hematoxylin and eosin stained ESS was viewed under light microscope. Scale bar equals to 50jim.  113  IDO-ESS has an immunosuppressive activity on co-cultured non-autologous immune cells As described in the methods, by using adenoviral vectors we developed genetically modified ESSs. Indeed, in our model of infecting fibroblasts by adenovirus containing green fluorescent protein gene (Ad-GFP) we made GFP expressing ESS (GFP-ESS) and in another one using adenovirus containing GFP and IDO genes (Ad-IDO-GFP) we developed IDO and GFP expressing ESS (IDO-ESS). In one set of the ESSs we left the fibroblasts without modification as a control group (NL-ESS). We then established whether adenoviral expression of IDO in IDO-ESS resulted in physiological levels of bioactive IDO enzyme. Levels of bioactive IDO in the IDO-ESS were compared with those in NL-ESS and GFP-ESS. For this purpose, we determined the levels of kynurenine, the main tryptophan metabolite of IDO activity, in the conditioned media from each of the different ESSs after 72 h of viral infection. As shown in figure 2A, the level of kynurenine in the conditioned medium from the IDO-ESS was significantly higher than that of either the NL-ESS or the GFP-ESS. To evaluate the total amount of IDO protein in the different ESSs, we subjected cell lysates of each skin substitute to western blot analysis, using 13-actin protein as a loading control to ensure equal loading in each lane. As shown in figure 2B, the results showed an intense band representing IDO protein in the cell lysate for IDO-ESS relative to that of controls. To investigate the potential of the inhibitory activity of IDO-expressing ESS to inhibit effector responses of non-autologous lymphocytes, lymphocytes isolated from peripheral lymph nodes of Sprague-Dawley rats were stimulated by exposing them with intact pieces of each of the different ESSs in co-culture system. Photomicrographs were taken  114  of these co-culture systems after 72 h which allowed visualization of the proliferation of lymphocytes in each co-culture. As shown in figure 2C, the proliferation of lymphocytes was suppressed in the IDO-ESS co-culture, but not in the other co-cultures, in which activated lymphocyte clusters were visible. The inhibitory effect of the IDO-ESS was markedly reversed in the presence of a specific IDO inhibitor, I -methyl-DL-tryptophan (1-MT). To confirm and quantifv these findings, proliferation of the co-cultured lymphocytes was measured using a [ H]-thymidine incorporation assay as described in 3 the methods. The results showed an almost five-fold reduction in the proliferation rates of the lymphocytes exposed to IDO-ESS compared to those exposed to GFP-ESS (from16329.33  ±  1785.786 for GFP-ESS to 2983.5  ±  903.72 for IDO-ESS, P<0.001,  figure 2D). The suppression of lymphocyte proliferation was specific to the IDO expression, as this inhibition was almost completely reversed with addition of 1-MT to this co-culture. Lymphocytes cultured for 72 h in the absence of ESS or with concavaline A were used as negative and positive controls, respectively.  115  ____  A  B 25  *  NL  GFP  IDO  IDO(42kD)  15 0  -actn  10  —  —  -  0  > 0’  NLESS  GFPESS  CNLESS  IDOESS  GFPESS  IDO ESS  GFP ESS  D  500 pm  40000 ._  35000  30000 26000  *  T 20000 Z  15000  I  0  () 10000 5000 0 Lymphocytes  +  Concavalin A  +  +  NLESS  -  OF? ESS  -  EDO £55 1MT  +  -  +  ‘I  -  + -  -  -  +  -  +  +  -  116  Figure 4.2. IDO expression and activity in ESSs. A) Measurement of kynurenine levels in the conditioned media ofNL-ESS, GFP-ESS, or IDO-ESS. Conditioned media was collected from the same number of infected and non-infected cells. B) Expression of IDO protein was evaluated in the different ESSs by western blot analysis. C) An equal size of each of the different ESSs was co-cultured with 2 x 106 cells/mL rat lymphocytes for 72h. Photographs were taken under light microscope. Scale bar equals to 500im. D) Lymphocytes were isolated from the co-culture and subjected to a [ H]-thymidine 3 incorporation assay to assess proliferative capacity. Data represent { H]-thymidine 3 incorporation of lymphocytes co-cultured with NL-ESS, GFP-ESS, or IDO-ESS. In one set of IDO-ESS, we added 1-MT at the final concentration of 800 jiM as a competitive inhibitor of IDO during the co-culture. Significant differences have been indicated by asterisks (* P value <0.001; n=3 per condition).  Expression of IDO improves the efficacy of ESS for wound healing of rat skin To test whether the IDO-ESS would also have an immunosuppressive effect in vivo, we tested the efficacy of the different ESSs in a rat model of wound healing. Four skin wounds (0.282 cm ) were made on back of each Sprague-Dawley rats (figure 3A). 2 Wounds were then covered with either NL-ESS, GFP-ESS, or IDO-ESS, and the fourth wound left non-treated as a control. After 7 days, photographs were taken to record the macroscopic aspect of the wounds (figure 3A). These results showed that the wound treated with IDO-ESS healed faster compared with non-treated wound or wounds that had been treated with control ESSs (figure 3A). The results of five sets of wounds showed that the wounds treated IDO-ESSs had an average of almost four-fold smaller surface area compared to those that received GFP-ESSs (2.9±1.7 mm 2 for IDO ESSs vs. 13.4±2.4 mm 2 for GFP-ESSs, P<0.05) 7 days after ESS engraftment (figure 3B). To evaluate the histological aspects of the wounds after treatment, they were harvested on day 7 post-engraftment, cross-sectioned, stained with hematoxylin and eosin, and analyzed by microscopy (figure 3C). The histology results revealed that those wounds treated with IDO-ESS were completely closed and were less infiltrated by cells compared  117  with control ESSs. We will characterize this cell infiltration in Fig.4. This finding indicates that wounds that were treated with IDO-ESS healed more quickly and with less inflammatory responses to the graft compared to the non-treated or control-treated wounds. Moreover, to confirm the engraftment process of the ESS cells, we stained the wound sections post- engraftment for HLA-1 (human leukocyte antigen-type I). As a result of this immuno-histochemistry staining, we have seen many positive cells in the wounds that received any of the three types of the ESS used in this study and no positive cells in the wounds that were left without any ESS engraftment (figure 3D). As HLA-I is specific to human cells and not the rat cells (39), any positive cells for HLA-I in these sections had to originate from the engrafted ESSs.  118  A  B **  Non-treated 20 E 15  NLESS 10  IDOESS  Non-treated  GFPESS  NLESS  GEPESS  100555  C Non-treated  NL ESS  GFPESS  100 ESS  fr  /  B Non-treated  Ni. ESS  GFP ESS  IDO ESS  —  119  Figure 4.3. Closure of 6mm circular wounds treated with different ESSs. Four wounds were made on the dorsal surface of Sprague-Dawley rats using 6 mm punch biopsy tool. Each wound was either left non-treated or treated with NL-ESS, GFP-ESS, or IDO-ESS. A) Wounds on day 0 before and after engraftment, and at day 7 after engraftment are shown. B) Quantification of wound closure 7 days after engraftment was determined by measuring wound surface area. Significant differences are indicated by asterisks (* P value < 0.05, ** P value <0.00 1, n=5 per condition). C) A representative hematoxylin and eosin stained wound sections on day 7 is shown. Scale bar equals to 500 im in the upper row and 100 jim in the lower row. D) Wound sections at day 7 after engraftment were stained for HLA-I to confirm the engraftment process of ESS cells. Scale bar equals to 200 jim in the upper row and 5Ojim in the lower row.  Treatment of wounds with IDO-ESS suppresses immune responses to the ESS at site of engraftment To evaluate the effects of the IDO expressed by the ESS on the rejection response of the rat immune cells to the non-autologous ESSs, skin wounds (0.282 cm ) were left non2 treated or were treated with the different ESSs, then harvested at day 7 post-engraftment. Sections of test wounds were then stained for detection of CD3 cells to analyze the degree of T cell infiltration. As shown in figure 4A, there were fewer infiltrating CD3 T cells in the grafted area of the IDO-ESS treated wounds compared with grafts of control treated wounds. In three experiments, the average number of infiltrating CD3 T cells in the wounds treated with IDO-ESSs was 2.5-fold lower than that of the wounds treated with GFP-ESS (32.6 +5.17 for IDO-ESS vs. 82.6 ± 18.9 for GFP-ESS group, P<0.001).  120  A  Non-treated  NL ESS  GFP ESS  IDO ESS  B 140 *  120 *  u.. 100  I  a .!!  80  1)  +  C,  60 40 20 0  Non-treated  NL ESS  GFP ESS  1DO ESS  Figure 4.4. Detection of CD3tiufiltrated lymphocytes in wound sections after engraftment. A) Immuno-histochemical staining of CD3 T cells in sections of wounds either left non-treated or treated with either NL-ESS, GFP-ESS, or IDO-ESS 7 days after engraftment. Scale bar equals to 200 jim in the upper row and 5Ojim in the lower row (CD3 T cells has been shown by yellow arrows) B) Statistical analysis of the number of infiltrating CD3 T cells per high-power field at magnification of 400x in each wound. The significant differences are indicated with asterisks (* P value < 0.001; n5 HPF/slide; n=3 slides/condition).  121  EDO expression in ESS promotes revascularization in treated wounds  It has been shown that slow revascularization is one of the causes of the rejection of non autologous skin substitute engraftment (44). Therefore, we wanted to assess whether IDO expression had a promoting effect on angiogenesis in wounds treated with ESSs. Six mm circular wounds (0.282 cm ) were treated with different ESSs or left non-treated, then 2 harvested at day 7 post-engraftment. The sections were then stained for CD3 I (platelet endothelial cell adhesion molecule-i, PECAM-1), a marker of the endothelial cells and vascular formation (44). As shown in figure 5A, there were a greater number ofCD3i cells and vessel-like structures in the wounds treated with IDO-ESS compared with nontreated wounds or wounds that had been treated with control ESSs. The quantification of the number of vessel-like structures per high power field (HPF, x400) for the three sets of the experiment has been given in figure 5B for each group. When three sets of experiments were averaged, the number of vessel-like structures per high power field in the wounds treated with IDO-ESS was more than four-fold higher than those treated with GFP-ESS (12.6  ±  1.1 /HPF in the IDO-ESS group vs. 3 ±1 /HPF, in the GFP-ESS group,  P<0.001).  122  A Non-treated  NL ESS  GFP ESS  IDO ESS  B 16•  * *  * *  14 12 U.. .  x  10•  0 0 0 0 C,  4-.  Non-treated NL ESS  GFP ESS  IDO ESS  Figure 4.5. Detection of vessel-like structures in wound sections after treatment with  ESSs. A) Immuno- fluorescence staining of CD3 1 cells in sections of wounds left nontreated or treated with either NL-ESS, GFP-ESS, or IDO-ESS 7 days after engraftment. Scale bar equals to lOOi.im in the upper row and 5011m in the lower row (vessel-like structures has been shown by red arrows) B) Statistical analysis of the number of vessellike structures per high-power field at magnification of 400x in each wounds is shown. The significant differences have been indicated with asterisks (* P value <0.001; n=5 HPF/slide; n=3 slides/condition).  123  Discussion The management of difficult-to-heal wounds remains among the most prevalent of medical problems and the use of appropriate skin substitutes is among the most promising way to overcome the difficulties in treating such wounds. Despite of the considerable advances in the development of skin substitutes over the past three decades, there are unfortunately almost no commercial products readily available that can permanently replace both dermis and epidermis layers of the skin in a single-stage application procedure (7). Considering many limitations in obtaining autologous skin substitutes, there is an urgent need to come up with a feasible model of skin substitutes from non-autologous sources to address the important to life as well as aesthetic needs of patients suffering from burn and non-healing wounds. However, using non-autologous keratinocytes without effective immunosuppressive strategy will cause immunologic rejection (45,46). Therefore, in this study we engineered a bilayer skin substitutes composing of main epidermal and dermal components of the skin which have been equipped with the ability to express IDO as an immuno-modulatory factor to protect itself from the host immune response. Indeed, transferring of the gene encoding IDO has been shown to be effective in prolonging the survival of pancreatic islet (24,28), lung (47) and corneal (48) allografts in animal models. Moreover, in our previous reports, we provided compelling evidence that both major subsets of human T cells, CD4 and CD8T cells, are sensitive to IDO induced low tryptophan environment and that the sensitivity of T cells to IDO mediated low tryptophan and high kynurenine environment is due, at least in part, to GCN2 kinase (general control non-derepressible-2 kinase) activation in T cells (25,35). We also confirmed that using IDO as a local immunosuppressive factor has no  124  significant adverse effect on non-immune cells including the keratinocytes and fibroblasts (35). Although in a preliminary in vivo study our group was able to show that the expression of IDO can be immuno-protective for the engrafted xenogeneic fibroblasts populated in a collagen matrix (21), in that study the keratinocytes, the main immuno stimulant component of the natural skin (7), were not included. Therefore, advancing our previous reports, in this study we proposed that locally expressed IDO by the non autologous skin substitute cells can be immuno-protective for this non-autologous skin graft model even in the presence of immunogenic keratinocytes and therefore eliminate the need for systemic immunosuppressive agents to protect the graft. To examine this hypothesis, first by measuring the proliferation rate of lymphocytes co-culture with non autologous IDO expressing (IDO-ESS) and non- IDO expressing ESSs (NL-ESS and GFP-ESS) we have found a significant efficacy for IDO expression in suppressing the proliferation of lymphocytes in this in vitro model. Then, applying these 3 types of ESSs in wounds created on totally immuno-competent Sprague Dawley rats, we confirmed the role of IDO expression in promoting wound healing in this system. Moreover, to investigate the positive role of IDO expression in revascularization in the wounds, an idea first proposed by our group based on a preliminary skin substitute model (21), we looked at the endothelial cell marker and interestingly we found promising evidence to further support this proposal. Interestingly, based on our previous study (21), revascularization due to IDO expression in skin substitute model happens not after kynurenine addition, but rather after incubation of endothelial cells in a collagen gel in tryptophan deficient media. This phenomenon as emphasized by other researchers (49) is analogous to the production of VEGF under conditions of hypoxia leading to revascularization. Indeed, in this study,  125  we found that IDO expression is not only effective to immuno-protect the non-autologous skin substitute grafts, but also it has a remarkable effect on revascularization in the wounds. Therefore, based on our findings, immuno-protection ability and angiogenetic effect of IDO expression can regulate an improvement in the efficacy of non-autologous ESS in promoting wound healing. In addition to previously described advantages of IDO-ESS, like many other skin substitutes composing of alive cellular components, living fibroblasts and keratinocytes in this composite can provide some important cytokines and growth factors for wound healing process and serve as an appropriate barrier and coverage for wounds which prevents heat and fluid loss from the wound as well as reducing the chance of infection (44). Another improvement in our skin substitute model in the current study is using the scaffold that has been approved for clinical use before (3) as the backbone structure of this new reconstructed human skin which brings our developed engineered skin substitute closer to the future clinical application. Our next goal in further investigating the engraftment mechanism of this genetically modified ESS model is to track the ESS cells and explore the interactions of these cells with the wound bed and the host cells in a more dynamic reciprocal approach. In addition, we are currently working to find a more clinically feasible mode of gene delivery with longstanding effect to substitute the adenoviral vectors in our current model. In summary, our results show that the engraftment of a non-autologous and genetically modified IDO-ESS may improve the wound healing process in rats due to suppression of graft rejection and promotion of the graft revascularization. However, we believe that more studies with longer time points, perhaps in bigger animal models, and further to the conclusion of wound healing need to be done to advance the result of this study.  126  Moreover, it is necessary to know the long-term effect of this approach on different phases of the wound healing process. Moreover, we found that the expression of IDO by IDO-ESSs modulates the immune response at the wound site. In fact, it has been shown before that waning of the inflammatory response seems to lessen the scar formation in remodelling phase of wound healing. Therefore, it would be interesting to know its potential effect in remodelling and scar formation phase of the wound healing in a study with longer follow up of the treated wounds. If proven so, this can be useful to prevent hypertrophic scar formations, especially in some burn related injuries. Furthermore, in such experiments with longer study duration, the concerns regarding to prolonged immunosuppression by the expression of IDO, such as the chance of infection or skin cancer development, can be addressed as well. Overall, based on the evidence presented here, we conclude that a non-autologous, IDO-expressing ESS is a promising strategy that may be developed for the treatment of those patients who suffer from wound healing difficulties such as burn and a variety of other non-healing wound conditions.  127  Conflict of Interests The authors state no conflict of interest.  Acknowledgments This study is supported by Canadian Institutes of Health Research (CIHR). Farshad Forouzandeh is holding the University of British Columbia Graduate Fellowship and the BC Innovation Council Scholarship. Reza B. Jalili is holding the University of British Columbia Graduate Fellowship. The authors are grateful to Dr. J.M. 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Topical nutrients promote engraftment and inhibit wound contraction of cultured skin substitutes in athymic mice. J.lnvest.Dermatol. 1995 Mar; I 04(3):345-349.  (45) Erdag G, Morgan JR. Allogeneic versus xenogeneic immune reaction to bloengineered skin grafts. Cell Transplant. 2004;! 3(6):70 1-712.  (46) Llames SG, Del Rio M, Larcher F, Garcia E, Garcia M, Escamez MJ, et al. Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin. Transplantation 2004 Feb 15;77(3):350-355.  (47) Liu H, Liu L, Fletcher BS, Visner GA. Novel action of indoleamine 2,3-dioxygenase attenuating acute lung allograft injury. Am.J.Respir.Crit.Care Med. 2006 Mar I ;173(5):566-572.  135  (48) Beutelspacher SC, Pillai R, Watson MP, Tan PH, Tsang J, Mcclure MO, et al. Function of indoleamine 2,3-dioxygenase in cornea! allograft rejection and prolongation of allograft survival by over-expression. Eur.J.Immunol. 2006;3 6(3):690-700.  (49) Penberthy WT. Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease. Curr.Drug Metab. 2007 Apr;8(3) :245-266.  136  CHAPTER 5 Conclusion The management of difficult-to-heal wounds remains as a common medical problem and the use of proper skin substitutes is among the most promising ways to overcome the difficulties in treating such wounds. In fact, in tissue engineering by making use of the principles of cellular biology with the fine teóhnological advances in engineering methods, the development of three-dimensional structures such as skin has become possible for the last three decades and skin was the first engineered tissue in the laboratory (1). However, despite the significant advances in the development of skin substitutes over time, there are unfortunately almost no commercial products readily available that can permanently replace both dermal and epidermal layers of the skin in a single-stage application procedure (2). Considering many limitations in obtaining autologous skin substitutes, there is an urgent need to come up with a feasible model of skin substitutes from non-autologous sources to address life saving strategy as well as aesthetic needs of patients suffering from burn and non-healing wounds. However, using non-autologous keratinocytes without effective immunosuppressive strategy will cause immunologic rejection (3,4). There is now convincing evidence that tryptophan metabolism through IDO dependent pathway plays an important role in immuno modulation in physiologic, paraphysiologic, and pathologic states in mammals (5-7). There are also several reports that cells expressing IDO can suppress T cell responses and promote tolerance (7,8). Indeed, transferring of the gene encoding IDO has been shown to be effective in prolonging the survival of pancreatic islet (9,10), lung (11) and cornea! (12) allografis in animal models. Moreover, Brandacher et al. showed that the expression  137  of IDO is a natural immuno-modulatory mechanism that the recipient immune system uses to counteract the immune-rejection process against allogeneic skin grafts. Indeed, based on their study, IDO was found to be expressed in high amount during the acute rejection process after engrafiment of a non-autologous skin which caused significant increase in serum kynurenine and decrease in tryptophan concentration (13,14). However, this overwhelming non-physiological expression of IDO sounds not to be strong enough, at least in most cases, to prevent the acute graft rejection process completely. Therefore, in this study we propose to engineer a bilayer skin substitutes composing of main epidermal and dermal components of the skin which have been equipped with the ability to express IDO as a local immuno-modulatory factor to protect the skin substitute from the host immune response. In fact, by inducing the overexpression of IDO in our skin substitute model we want to further increase the level of IDO expression more than the level that naturally over-expressed in response to non autologous engrafiment as described before. In order to systematically achieve the goal of developing a non-rejectable bilayer skin substitute using immonumodulatory effect of IDO, in the current study, first we have investigated whether this immunosuppression strategy could potentially harm the host or skin substitute associated primary skin cells or not. Based on the results of our experiments, we found that IDO expression has a selective effect on different primary human cell types compared to T cells examined. Indeed, 7-AAD staining, a very sensitive way to detect the loss of membrane integrity during apoptosis and caspase-3 activation assay, indicated that bystander T cells, but not fibroblasts and keratinocytes, will undergo apoptosis when they are co-cultured with IDO expressing cells (15). Therefore, the local immunosuppressive properties of IDO can  138  have a significant role in the development of a local immunosuppressive barrier for grafted skin cells and tissues without compromising the viability of the host’s primary skin cells. This phenomenon is absolutely crucial for the idea of using IDO as a local immunosuppressive factor in engineered grafts without compromising the survival of nonimmune cells. This will help us and other researchers in the field to make use of IDO immunosuppressive ability in development of non-autologous grafts such as a non rejectable skin substitute to be used as wound coverage. Further we have shown that this differential effect of IDO expression is at least in part due to the differences in activation of GCN2 kinase pathway. GCN2 kinase pathway is a stress response pathway which is usually activated as a result of nutrient deficiency in some cell types (16,17). Indeed, we have found that while GCN2 kinase is activated in bystander immune T cells in response to tryptophan deficiency caused by IDO expression, it remains inactivated in primary keratinocytes and fibroblasts under the same experimental condition. Also, we have found that the activation of GCN2 kinase pathway coincides with the activation of apoptotic cascade in T cells in response to IDO generated low tryptophan environment (15,18). Another interesting finding is that even there were some differences between different subpopulation of immune T cells, mainly CD4 and CD8 T cells, in response to the tryptophan deficient environment containing a high level of kynurenine produced by IDO activity. In fact, we demonstrated that IDO expression has a marked effect on viability and proliferation of both activated CD4 and CD8 T cells while this effect was greater in CD8 relative to CD4 T cells (18). This finding is consistent with those reported by Munn et al. using murine T cell populations (19), however, as reported by Mulley et al., the apoptotic effect of IDO was unknown (20) until we have reported in  139  this study. As T cells play the main role in the acute immune rejection process, this information would open a new insight in the field of transplantation. Although in a preliminary in vivo study our group was able to show that the expression of IDO can immuno-protect engrafied xenogeneic fibroblasts populated in a collagen matrix (21), keratinocytes as the main immuno-stimulant component of the normal skin (22) were not included. Therefore, in this study, we proposed that locally expressed IDO by the non-autologous skin substitute cells can be immuno-protective even for immunogenic keratinocytes and that would eliminate the need for systemic immunosuppressive agents to protect the graft. Indeed, based on the evidence found by other investigators and our previous and current studies on the immuno-modulatory role of IDO, as main part of the current project, we engineered a bilayered skin substitute composing of the non autologous keratinocytes and IDO expressing fibroblasts in a collagen matrix. In order to genetically modifS’ the skin substitute cells to express IDO, we used an adenoviral vector transfection method. In fact, despite the recent attention to non-viral vectors, viruses are so far the most common transduction method because they are easier to develop and are more effective transduction methods into eukaryotic cells compared to non-viral vectors (23). However, due to toxicity and inflammatory potentials of viruses used in some preclinical studies for gene transduction (23), we do agree that a non-viral method needs to be ultimately developed to make our skin substitute model possible to be used in clinical settings. The reason that we chose adenovirus but not other viruses which cause permanent gene over-expression in our non-autologous skin substitute model was because we desire to have transient expression of IDO. Indeed, adenoviruses do not integrate the gene of interest to the cellular genome and this makes it a safer approach for  140  further clinical use. In fact, as our skin substitute model has non-autologous origin, its cells supposed to be replaced by the host cells when the host body is capable of taking care of the wound itself. At that point of time, the expression of IDO neither in the remnants of the skin substitute nor in host cells is desired since it may create some concern such as developing a skin cancer as a result of permanent local immuno suppression (24). Moreover, adenovirus is very easy to produce and its amplification process is quite easy and can lead to have a very high viral yield. Therefore, engineering our bilayer IDO expressing skin substitute, we tested it in various sets of in vitro and in vivo experiments to evaluate the efficacy of this novel skin substitute based on different criteria. In brief, first we have confirmed the IDO expression ability of the genetically modified fibroblasts of our skin substitute by measuring the level of main metabolite of IDO, kynurenine, in the media of the skin substitute in culture. Also, we have confirmed the level of IDO expression at protein level by western blot analysis of the skin substitute lysate. Moreover, in a co-culture system we have tested the immunosuppression ability of the IDO expression by the skin substitute cells on the bystander lymphocytes. After confirming the immunosuppression effectiveness of IDO in all of these settings, we have applied our engineered IDO expressing skin substitute on full thickness wounds on Sprague-Dawley rats and compared the effectiveness of this skin substitute on promoting wound healing and suppressing the infiltrating immune cells with other control groups. All of the results of this part of the project have been presented in chapter 4 of this thesis and has been submitted for publication. In brief, first by measuring the proliferation rate of lymphocytes co-cultured with non-autologous IDO expressing and non-IDO expressing skin substitutes, we have found a significant efficacy for IDO expression in  141  suppressing the proliferation of lymphocytes in this in vitro model. Then, applying these different skin substitutes in wounds created on totally immuno-competent Sprague Dawley rats, we confirmed the role of IDO expression in promoting wound healing in this system. Moreover, to investigate the positive role of IDO expression in revascularization in the wounds, an idea first proposed by our group based on a preliminary skin substitute model (21), we looked at the endothelial cell marker and interestingly we found promising evidence to further support this proposal. Indeed, based on our previous study (21), revascularization due to IDO expression in skin substitute model happens not because of high level of kynurenine, but rather after incubation of endothelial cells in a collagen gel in tryptophan deficient media. This phenomenon as emphasized by other researchers (25) is analogous to the production of VEGF under conditions of hypoxia leading to revascularization. Indeed, in this study, we found that IDO expression is not only effective to immuno-protect the non-autologous skin substitute grafts, but also it has a remarkable effect on revascularization in the wounds. Therefore, based on our findings, immuno-protective ability and angiogenetic effect of IDO expression can regulate an improvement in the efficacy of non-autologous ESS in promoting wound healing. In summary, our findings indicate that an IDO-mediated environment differentially influences the biological functions of human T cells compared with either fibroblasts or keratinocytes. This differential effect seems to be, at least in part, due to difference in activation of the GCN2 kinase pathway. Also, our findings indicate that IDO-induced low tryptophan environment differently influences the biological functions of primary human CD4 and CD8 T cells which again this differential effect seems to be due to, at least in  142  part, differences in the activation of GCN2 kinase pathway in these T cell subpopulations. Moreover, our results show that the engraftment of a non-autologous, genetically modified IDO expressing skin substitute improves the wound healing process in wounds in rats due to suppression of graft rejection and promotion of revascularization. Overall, based on the evidence presented here, we conclude that a non autologous, IDO-expressing skin substitute is a promising strategy that may be developed for the treatment of those patients who suffer from wound healing difficulties such as burn and a variety of other non-healing wound conditions.  Significance and Potential Clinical Applications Over- and non-healing wounds are extreme conditions with far reaching clinical and economic implications. Indeed, skin lost due to burns or other non-healing wounds, is among the major causes of mortalities and morbidities in medicine. Utilizing a readily available skin substitute would be a logical way to overcome these problems, however, this skin substitute as a foreign body for the host patient immune system will be rejected after being grafted to the patient. Thus, in this project, we planned to make use of indoleamine 2, 3- dioxygenase (IDO) as a local immunosuppressive agent in our novel skin substitute model to improve its rate of take. Moreover, the feasibility of the skin substitute proposed by this project can benefit any patient suffering from difficult-to-heal wounds as well. The approach of using IDO as a local immunosuppressive factor in a non-autologous engineered graft can be a significant breakthrough not only in skin, but also for other organ grafting in future studies. In fact, the findings of this study suggest the selective effect of 1DO on some human immune cell subsets vs. some nonimmune  143  cell strains such as mesenchymal and epidermal cells and this differential effect support the proposal of using IDO as a local immunosuppressive factor for engraftment of a non autologous graft, in our case skin substitute, without compromising the viability of nonimmune cells. Moreover, in addition to previously described advantages of IDO expressing skin substitute, like many other skin substitutes composing of alive cellular components, living fibroblasts and keratinocytes in this composite can provide some important cytokines and growth factors for wound healing process and serve as an appropriate barrier and coverage for wounds that prevents heat and fluid loss from the wound as well as reducing the chance of infection. Another improvement in our skin substitute model is the use of a scaffold that has been approved for clinical use before (26). This would function as the backbone structure of the new reconstructed human skin substitute and that would bring the use of a non-rejectable wound coverage one step closer to its potential application in clinical settings.  Limitations and Suggestions Although, our hypothesis has been substantiated by our preliminary and recently published data and the research plan has been outlined and executed according to our previous experiments in all aspects of this project, some modification in the proposed experiments may be necessary should we aim for a more clinically acceptable skin substitute. Indeed, the ideal properties of a skin substitute are: adherence, control of evaporative water loss, safety (sterile, hypoallergic, non-toxic, non-pyrogenic), flexibility, durability and stability on various wound surfaces, bacterial barrier, ease of application and removal, availability and ease of storage, cost-effectiveness, and  144  haemostatic efficiency (27-29). Although reaching all these standards was far beyond the scope of the current project, we have to declare that our developed skin substitute needs to undergo some optimization steps before it can be considered in any clinical settings to help patients. Moreover, further investigation are needed to explore the engrafiment mechanism of our genetically modified skin substitute model including a tracking procedure to evaluate the skin substitute cells and their interactions with the wound bed and the host cells in a more dynamic reciprocal approach. In addition, we are currently working to find a more clinically feasible mode of gene delivery with longstanding effect to substitute the adenoviral vectors in our current model. Also, we believe that the angiogenetic effect of IDO expression needs further systematically investigation. Furthermore, there are still several important questions that need to be addressed regarding to the immuno-modulatory role of IDO before making the use of it as a local immunosuppressive strategy in a clinical setting, including the application of IDO expressing skin substitute.  145  References  (1) Andreadis ST. Gene-modified tissue-engineered skin: the next generation of skin substitutes. Adv.Biochem.Eng.Biotechnol. 2007; 103:241-274.  (2) Boyce ST, Foreman TJ, English KB, Stayner N, Cooper ML, Sakabu S, et a!. Skin wound closure in athymic mice with cultured human cells, biopolymers, and growth factors. Surgery 1991 Nov;1 10(5):866-876.  (3) Swope VB, Supp AP, Boyce ST. Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice. Wound Repair Regen. 2002 Nov-Dec;10(6):378-386.  (4) Tredget EE, Shankowsky HA, Pannu R, Nedelec B, Iwashina T, Ghahary A, et al. Transforming growth factor-beta in thermally injured patients with hypertrophic scars: effects of interferon alpha-2b. Plast.Reconstr.Surg. 1998 Oct; 102(5):! 317-28; discussion 1329-30.  (5) Munn DH, Mellor AL. IDO and tolerance to tumors. Trends Mol.Med. 2004 01;10(1):15-18.  (6) Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB, et a!. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 2002 09/13;297(5588):1867-l870.  (7) Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat.Rev.Immunol. 2004 1 0;4( 1 0):762-774.  146  (8) Mellor A. Indoleamine 2,3 dioxygenase and regulation of T cell immunity. Biochem.Biophys.Res.Commun. 2005 1 2/09;338( I ):20-24.  (9) Hayashi T, Beck L, Rossetto C, Gong X, Takikawa 0, Takabayashi K, et a!. Inhibition of experimental asthma by indoleamine 2,3-dioxygenase. J.Clin.Invest. 2004 07;1 14(2):270-279.  (10) Jalili RB, Rayat GR, Rajotte RV, Ghahary A. Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts. J.Cell.Physiol. 2007 10;213(0021-9541; 1):137-143.  (11) Uyttenhove C, Pilotte L, Theate I, Stroobant V, Colau D, Parmentier N, et al. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat.Med. 2003 l0;9(10):1269-1274.  (12) Wood FM, Kolybaba ML, Allen P. The use of cultured epithelial autograft in the treatment of major burn injuries: a critical review of the literature. Burns 2006 Jun;32(4):3 95-40 1.  (13) Brandacher G, Margreiter R, Fuchs D. Clinical relevance of indoleamine 2,3dioxygenase for alloimmunity and transplantation. Curr.Opin.Organ.Transplant. 2008 Feb;13(l):I0-15.  (14) Huurman VA, Hilbrands R, Pinkse GG, Gillard P, Duinkerken G, van de Linde P, et al. Cellular islet autoimmunity associates with clinical outcome of islet cell transplantation. PLoS ONE 2008 Jun 1 8;3(6):e2435.  147  (15) Forouzandeh F, Jalili RB, Germain M, Duronio V, Ghahary A. Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts. Wound Repair Regen. 2008 May-Jun;16(3):379-387.  (16) Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, et al. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 2005 05 ;22(5):633-642.  (17) Kimball S, Anthony T, Cavener D, Jefferson L. 5 Nutrient signaling through mammalian GCN2. ; 2004. p. 113-130. (18) Forouzandeh F, Jalili RB, Germain M, Duronio V, Ghahary A. Differential immunosuppressive effect of indoleamine 2,3-dioxygenase (IDO) on primary human CD4 and CD8 T cells. Mol.Cell.Biochem. 2008 Feb;309(1-2): 1-7.  (19) Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J.Exp.Med. 1999 05/03;1 89(9): 1363-1372.  (20) Mulley WR, Nikolic-Paterson DJ. Indoleamine 2,3-dioxygenase in transplantation. Nephrology (Carlton) 2008 Jun; 1 3(3):204-2 11.  (21) Li Y, Tredget EE, Ghaffari A, Lin X, Kilani RT, Ghahary A. Local expression of indoleamine 2,3-dioxygenase protects engrafiment of xenogeneic skin substitute. J.Invest.Dermatol. 2006 01 ;126(1): 128-136.  148  (22) Atiyeh BS, Costagliola M. Cultured epithelial autograft (CEA) in burn treatment: three decades later. Burns 2007 Jun;33(4):405-413.  (23) Tomanin R, Scarpa M. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction. Curr.Gene Ther. 2004 Dec;4(4) :357-372.  (24) Muller AJ, Sharma MD, Chandler PR, Duhadaway JB, Everhart ME, Johnson BA,3rd, et al. Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase. Proc.Natl.Acad.Sci.U.S.A. 2008 Nov 4; 105(44): 17073-17078.  (25) Penberthy WT. Pharmacological targeting of IDO-mediated tolerance for treating  autoimmune disease. Curr.Drug Metab. 2007 Apr;8(3):245-266.  (26) Supp DM, Boyce ST. Engineered skin substitutes: practices and potentials. Clin.Dermatol. 2005 Jul-Aug;23(4):403-4 12.  (27) Horch RE, Kopp J, Kneser U, Beier J, Bach AD. Tissue engineering of cultured skin substitutes. J.Cel l.Mol.Med. 2005 Jul-Sep;9(3):592-608.  (28) Nangia A, Gambhir R, Maibach H. Factors influencing the performance of temporary skin substitutes. Clin.Mater. 1991 ;7( 1 ):3- 13.  (29) Clark RA, FAU Ghosh K, Ghosh K, FAU Tonnesen,Marcia G., Tonnesen MG. -  -  Tissue engineering for cutaneous wounds. J Invest Dermatol.2007 May;127(5):l018-29. -  (1523-1747 (Electronic)).  149  A.1 REPRINTS OF PUBLISHED PAPERS Woindtkpth MdR.qM1azbm  —  Skin cells, but not Tcells, are resistantto indoleamine2, 3-dioxygenase (lDO expressed byallogeneic fibroblasts Earthad Farouzandeh, MD Rem BJaIIL MIY; Marc Gerrnain. PhD ; Vincert Dururiic, FThD Add 6hahary PhD t 1 SCt¼bso’,4 Fm F*ituW &rn rd Ward 4aatnq Rascxcn Lanerfl’y. tiopnmvil otSuoy, tkwo tt&tti Crtrb4 Vrcou.  tie. t1c1Z4 wd  2 Ora,td 4a3dr*AJrivuatyaf tiflsh ecArnUa. bncarar. tiCSnd  Rqflit pa  Aá Grrj. Pt4 2 On Stwaat Zi Jactti& tian$ Canfl tcaua-, til. a  Ta. (dzl Bita slat rnst ni,sSntetwçafla  Mnimrpl rwava± Aupst2. ZO1 t.ccptadi, lira taint ctarya 2Q 1  l4irz44m)tmcarnz  ADsrRadrr We have previoutly demonstrated that indoleamine 2, 3-dioxygeixase (100) espresmd by dennal fibroblasts generated a tryptoplarn defrient environment in whkh intmietse cells, hut not skin cell, underge apoptosis Howese; the much anrism by which primary skin cells such as flbroblasts and keratinteytan are re-As tarn to this culture environment, is not elucidated. Here, we asked the quastionof whether the activity of the general cannel nonckrepraraing-2 (GCW2) khnse pathway in prmnnr-y inenune and skin cells a differently regulated in reqoaieto lD0-indnced tryptoçilnn deficient environment. Before aditresi ng this question, the expression ofiDOin IDO-adenoviral infected libroblasts, aaa seurc’eoflDO expraion, twa ‘isslidated. We then demonstrated a signifuant inamuneQspprs the effect of 100 expression in primary human I cell co-cultured with 100 a presaing fibrabhsts in the prance of ailogeneic places ofeitherepideridsorfiall thicknetiskit Evahndngthemschainmbvwhichskmncells. butnot Tcells. are reistan t to 11)0 induce! low tryptophen enviromiient,we then c cultured 100exprewing fibroblasta with bystander human T cells, the flbrobbista,or keratin ocytes for 3thys. The results showed a tignificant activation of apoptotic path way as analyzed by caase-3 induction s welt as the expression of CHOP, a downstream effuctorof (XTh2 kinase pathway mT cell, but not in skin cells.  Indoleamine 2 34ioaygetnse (10(0, a hane-containhig rate-limiting citzynie talant conveision of tryptophan to kynwenina as the main tiptopltan metaboliteY ‘p initial and rate-limiting raction of the kynuienine pathway is the oxidation of tryptoØian to N-fcnnykkycnsie itire, cntalydd b hçpatic tryptophan 3’dioxygeinm Jfl(j2 (900) or the ubiquitous, enrahepa&, DX) has besn found it nonhepatic cells mainly in tiojzhthlaa dendritk celk, monceytes, and macttplns The a pression of IDQ ins inmiunomodulatory efihcte on T cels that are related to the perketlular degzstatioa of tryptoplnit Tryptoplan is the lea ansihNe eatentini amino add and is zequi tart by all forms oflilk fbr protainsynthe sit and abe risnportant metabolic functions Raind on this infonnatiot we hypothedse tint an ailogeanic skin sub stitute whceecelhibr components cetopically cap rats 100 would ha nonrejertable. As 100 degrades the esemtial amino acid tryptoplnn, it might affat pathwaysknown respond to amino add anbolint One preibillty it the activation of nutricnt-ntniti mammalian target of rap. amycin (mTOR) lint pathway Howievar it has hem shown tint inhibitors of mTOR such as rapan’iytindo not i-txapitulate the profound pn-ilferative arrest in inmurt cells men with IOU-re diated suppratsionfrA anondami no atid-scinitive pathway is mediated by the geanral con troi no:n&arepresthw-2 (OCN2) kitnm. (3042 nontaire a regtilatory domain that hats the uncharged form of tRNA. Amino aidinsuffl&ncyeaiemsasiseinuxt)nrgai tRNA, which activates the (3042 kinse domain and mi tiatac dawnstresin tiginling. Recently, it has heen shown W&w.dfle Aegi  i  iena- ®  SOSb%.t. wataasoae  tint the CCN2 kirase pathway is invoked in the itwna nesuppresive effect of I DO espresnonY In an attempt to ate 100 expression as a local menu nreupprestive factor to r.oprotect anallogeneic &kin substitute made of an epideran) podion of primary cut tiered keratintryles arid adenniti portion of deralfibro— blasts populated in a ccllagcn—GAG *1. a serits of experiments were cc nthrtal; prevromrly, ue thowed tInt difettypesofimmune cell such as aW Ju&at edit, THP-l nionocytes, and huaun psñpheral blood Iymflo cytes (PIlL) heroine appptotic within 3 days whencetiered with IOQ expressing &UsYr Moreover, under a iminlar experimental condition, a signi&aat down-regula tion of cell nnrnhrame associated MKCrthssI antinin 100 geriati&IIy modified keratinceytes relative to that of indict noninfe2ted err infected cell with adenoviras with out lOt) was shownYe In an in vivo audy. l)er press ing human flhrohlasts embedded within bovine collagen placed on rat wounds accelerated wound healing bypn> mating neovarcularization during the early stages of )i meg and by providing protection of x.enogertic fibrobla.” Tle findings collectiwly suast that IIX) expression may function as a local imniunosuppresive  tAAb C-IC? COlT 1 Mt  ?-amr-o&n’r-ytn .  CCAI Tlonncntrnrq IThnn rn’rooqas rfl Gewt cart-a ncrde-cpsan-rq 2 I  otrptixre,  Sm  150  ma tencT can sEr eels  fator to protect an aDogeiic skin snintititte in which keatinocytesand librobleats serve as the cellular coinpo meats of thle wound coverage. Nowever wint is not known is why priinaty tkin &lls but not inn cElk, are reistant to 100 indired low t.yptoplnn environment Here, by conducting a ser of exporisnentawe proided nindence tInt (1) small pi of buena skin are highly antigenic for primary inn T celk and om be sup pressed by byaeder 100 expressing llbroNas%, ( Cnspaw3 and CHOP levels $5 two indicatois of adiva boa ofapoptotic and (3042 kinase pathways, raectiie ly, either slightly increased or remninned andetectaNe in fibrohlasts and keratinocytes relative to these of T cells grown in an environment geirerated by oo-cuitured 100 expensing ñhrohlaets.  MATERIALS AND METHODS Aejenovfr& vsda eansiucwon The procedure efoonetrection of 100 expressing adeno vital vectors has In daiibed hfora re Brolly, to con stnict the adent*inn encodhighuman IDQ,we cinnal the polynierase chain rattion product encoibag the full length protein into a shuttle vector, which ooexp teases OF? as a reporter ne following the mactutor’s in stnections (Q- Rio jtene,Catklnd, CA). The recombinant adunoviral plasnids were nerated by elect.roporalion of BJS 183 Eetherihia cdi naing tIre shuttle sector either with or without 100. Recombinant a4enoriral plasnids were then purified and transfected to 33 rielk using Fugene-6 traasfiretion reagent (Roche Apphed Stienos, Laval, QC. CaInds). Infected cells were monitored for (IEP expres sion and after that eyelet of freatheg in ethan4idty ice bath and rapid thawing atIl C the cell lyaste were ured to ampulr viral partiies inlarge scale to piejte adenovi ral sto&. Then, viral titration was done asing 293-cell litre aaderrihedpmvioxasly.  Neonatal fo reskirt pie were used as mince of tibroblasts and keratinecytes and the proctiure was done bared en the appro’al of Ethim (bntnittee ofthe University crf British Columbia (UBQ. Cultures of human forsikin llbroblesis were established as descrihed preiotnly: 5 Icr brief, punch biopsy nntples were prepared from human foreski ret. The there was collected in :Dulbasob Modified Eagle Media (OMEM) with :I(4 fetal bovine rennel (FBS QIRCO, Grand Island, Nt crincedintosmailpimesof c 0.Sn in any dirwi.nsion,, washed with sterile medium six timer, and dittributed into 6O,r ISn 1%tIi culture &hrs (Coming Inc., Corning. NYJ, four pieces per dish. A ster il.e glass coveralip was attached to the dish with a drop ofsterlle silicorea grense to ininohilise the tissue fragment OMEMtantibiotics (panidUin (1 satin IOQU?nti, streptomycin sulfate tfljtgjletL, and arnphotedcin B 11.25 peJniL SmL, (118(20) with 10% FBS was edded to eech dish and incubated at 37C(2 in a wata.’eckai humid illed incuintor in an atmeqhere of 5% CO. The medium was replaced twire weekly. After 4 weeks of incubation, cells were releaaid from dishes by brief (5 minute)  ago  c,o.cai.ion cia  -  tntmentwithêl % typein (Life technologisslire.,Oaith ersburg, MQ) and 0.02% EDTA (Sigma, St Louis, MO) in phoqihata-buffered sabre rolution (PBS; p11 IA) and trasferred to ?Scnr culture flasks (Corning Inc.). Theta afteç, orte visual confluence was renched, tire cells were subcultered t 6 by trwsinization. Fibroblasts from pea rages .4—1 were nazi for this study. In order to culture hu iran foreskin keratinocytes, keratinocyte sent ni free nredinni 1CSFM, 018(X)) supplr’.rnentcd with bovine p3— teitary enr-nit (2SjnrL) and spite anal growth &tor ((kSnWmL) were art Winary cultured keratinccytes at passages 3—$ were used for this study -  hrtec$on ml %eoNa*a with ltJQad.nevfral vecbr Reconthimnnt adenovi ruts were med to inibet fibroblasts atthemuhgilicityofithctionof10O(M0I IFreevi’ ral jnrticb were removed from culture rrtediunr 30 hours after infection. The sass of inthetion was determined by flow cytonretmy measuring the OF? protein expression and fluorea,ent microaiopy using a MotIc inve rind micreecope equipped with an teorescein iroshiocyanate ((ITO filter to view OF? (Motic Frastrurnents, Richmond, BC, Cainda). hinges were captured using a digital cam eta. Moreover, the efficacy of infection and expression of functional 100 were assewed by iasnunohistocheretistry for 100 expression, and by Western blot using anti-ha ann 11K) Mi, and ntasuring the levels of kynurenine.  C-wksnssdIDO a fi&thIans Mid hynredet eels thing 30mm Mlllioell Sterihand Culture Plate Inserts (Millipote. Bedford, MA). we set ups co-culture system in whmdh IDO-exprestiag fibroblasts were grown on the upper chamber- of a ax-well plate, while bystander tiit her kcralirsocytes. fibro blasts or T celk -were cultured en the lower chamber. Therefore, there was no direct contams be tween 100 expr’a’ing fibro blasts and bysrandcr cells. lt should ako be rnentiorred tint adenos inn or IF’N-y—trcs ed fibrobtasts were washed with PBS before covculturirtg them with other colts, to remove arty escectaderi cvi rasand IFN-y, rerrctiely.  100 expreation was detected byintracdllmtlar staining Fi broblasts remained untreated, treated with WNdf or in focted with Ad-lOO-OFP for 3 days and fixed by peraformaldebyde 4%.. Cells were then incubated with pdl3clonal anti-human [DO antibody, caired he rabbits by Washington Bioteclerroiogylnc.(Raltimore, MD), at final cotntration oft t,000at4CCovernight.Theprooedure vets followed by incubating cells with biotinytatat-goat anti-rabbit imntunoglobulin (Vector Laberatorirs, The ruin game, Ok). The signal detection was carried out using 3. -diani nobeaddi ire-enhanced hquid subarame system t (Sigma). The slides were counterstained with hematorcylin forS mconds. and then soctiomn were dehydrated, mount at, andexaminedundermicreacope. WndAc  pemj rean-a  as’s  sar4wrv 5ttbI  151  ft denmi T an a* a  CI  fluonneencs aetetaded sedhigd human pedphara( bleed monanuchar talk (PENt) tacT eta Total PBMC were 101 hydutsitygiadient atdimenta tion on ILstojnqae-1077 (Sigma) axeiding to the mann fxture?spmtocoi. Brlfly, whole blood teas layered on am equal volume of Hieopaque and centrifugal at Z000 :t..in. for 20 minuta at mont temperature and stopped without any brake. PEMC were biased and s suspended in RPM! 1640+10% FES and pelleted hycen teifugation at 2,00G r.pnt for 10 ninuta and were farther washed twice in P85+1% FBS. IJmig PE.onnjutal monat anti-human CDSmAh(ED, Oakiille,ON), FflG conjusated ntou* anti-human (1)4 saAb (ED), arid all cphyc*xyathi (APC)—oonjuptal eanse anti-h reman CbS nt4b (ED) at the concentration of 20 jsl4I It cells, an stained the P8M The calls tale then cubated at room temperature for 30 aura ibereafter, ealis were washed twice and rarnpended at l0’cell*rtL in P85+1% FBS far fluorement atth’ated cell ending (FACS). For paeparatien of a pure popuition of CDI CD4 4 and CD3CI*’ T celk, we gated on CDrCD4’ and CDI 4 CW Tcelk after excluding thedead cells and cell debt-b basal on FSC and SSC parameters and sorted th tus, cell populatiora into aepxatie tubas.  After being sated front Noor, T calls were propagated in RPMI 1440 (Hydone,tJT)splemented with I 0¾ FES, 0.1 U pers n/eeL, and 0.1mg streptomyci&rnL at WC in ahumidifled 3% CO. 1 atmosphere to be used forfurther t teatmeaL  Cdl aredud evduatlen  The surtival of two different sernitive (034 and CDt I cells) and two difibrent redstarrt (&rrnal fibroblasts and keratinecytes) cell types in an Iflaindsted tryptophan deficient environment was conipaind by ?-AAD staining. 1-AAD intercalates into double-stranded nucleic acids. It is excluded by vinbie cells but run penetrate cell mer brar of dying or dead cells. After each treatment, cells were Isariested, washed in PBS, stained far 7-AAD and tin exanaraid eating FACS aaslysia, as per the manufsa turer’sprotecol (SD).  ED). 11 Ia ta were stripped and reprobed for Ji-actin as equal loading control. For detection of IDO espraion, Ohm htssts were lnrverterlafter48 hours of WN-y treatment or 12 hours pan viral infection and washed with PBS. Gills were then lysed in lyas buffer (50mM Tria—NCI, pH 7A :10 triM EDTA 5mM EGTA 0.5% NP4O: 1% Triton X 100, and proteam inhibitor cocktad, Sipia). Equal amounts of total protein from each individual fibroblast crifture were sperated by 10% SDS-PAtiEJtroteirss were then translbrred to a PYDF rnenebrane (Mililpore) and irnmunoblocted with polycloreal anti-human 100 anti body saiend in rabbits by Washington otechnologyIrsc. at final dilrrtion of 1:5,000. Enlrarted dulwnirtwence detection system (ECL Amersiram Eioaierwa little Chalfont, UK) was reed in all blots to detat the secaid  arAb.  na-ne rusearrement In the cendoned niedkern The biologiral ectivityof 11)0 wasevaluated by manning the la-nb of tryptoplan degraded product, r.-kynurerirrm, pxeatnt in the conditioned medium derived from 11)0 and control vectorvinlhcted cells. The tenount of t-kynurenine was mastered by a previously established metho4Y Briefly, proteins in the conditioned niedium were precipi tated by trichioreacetic acid, and after centrifugation, tkSntL of supenratant was incubated with an equal miume & Ehricht reagent (Sigma) for tOmitautes at morn temperature. Atairption of the resultant ambition was treasured at 49Onrn by a spectrophotonreter within 2 hours. The n.bar of kynurenine in the conditioned medium were cakulated by a starerhrd curve with the rInfrrvad kynurenine (0-100mM) corntrstion. In this Study, we have used this nay as an evideece of 100 expression wherever we hew induced the fibroblasts to express AX) either by Ad- ID( FP infection orbylFwq treatment.  [asia were expremal as Mean t SD and arralyand with ore-wayA NOVA among diffcre nt groupe of each cell twe where indicated. p-valrrra <01)5 are conridera! stat IS eally gnthcantin ties study. RESULTS  In id*relrdiesan nay tarT tea Tce!Iswere perked with [ H]4hymidi e(ljitJ7nit, Perkire 1 fleer, Boston MA) on dey. and harvester! on dey 41cr measurement of cell lyaste-assothtad tritierreby -scinti1lation counting. All prnlilbration experiments were penformal in tripltate and reported stats] of cell count per minute (c.p.nt).  Weasra blot artalyds Total proteins from cell lysate (20 jag) were separated by S1EPAGE and transferred to PVDF membrane (hfiJli pore). The hiatt were probed for CHOP using anti-GAD DIflCHQP1OAb(l :25ldllution, Sigma) and foractise caspase.3 with anti-actiw coajxaat-3 Ab (I: t000 dilution, W&tdRc Aç(m 15579-SW  (b  Zr*brtheWatd4einaSaej  3O expraalen Inhths Tat pedltealan Inducadhy en-tinned a&oenelc piaces d spider miter Ulintara  Before evaluating the irresrunrisupprisive eftntt of 100 expression on immune cells in seaports to allogericjia of epidermis or frail thikts skin, the ex.ptaskmn of 100 was tahlated in I’lbroblasts infected with an Ad-IDO-GFP etttor(MOI 7 I00)ortratedw ( l0001J/mLi mthfFW a potent indurrar of 11)0 eapradon. Indeed, the effi 4 ratyoflDO etpresion hythacetihroblastswastetted by different rret beds. The results of inunumohbtechenttal experiments showed a strong 100 protein staining in fihroblsts either infected with Ad-IDO-GFP sector or treated with IFN-y (flgure 1A). Ths finding seas Sal  152  to atcit  A  Vt iflt  c_s  Non-inti-ctal  Ad4EP intattd  p,44pc3-GFF trifectoti  IFP4’f  AI4FP  tratlet  Ad.iDOFP lnfec%d  ii  C  Non  teciBti  Ri IDO spesweo at tst Stha tinted veift M4Ua Gfou Vfld wdl lAd7 100 eqtes a flbrobbets atha minted -sMh M-41J0-GFP lM0ltWl or united wits WN7 41OOOtvrlj wni stoves by Ennerer tntton*ety at treat eat tot 1130 pttn after of oiturng Itsem vs tots &arrter nitini are) corned wtt tie sneitsi fl fwei A). Ate, tM GFP eqenistas ot AIGIPoc Ad-tOO-GE? wetted fibre time vnwteJ irder Itoessnit ni is  5*50  Wi paid K  Meseo.,a, tie aonrwmasoo of 1130 eqwenon by tie it cets tat tateir: ailOWn by WStSSI biOt atlaffle ti fIX) p.eten by utrrg lOG poi doe5 at Ab at axtesisalest ci t:5,WO t&t4 0. tOO, itddnitrvne 2, 3-db*  confirniat under INc resteace n±nniccpy for GEP expresr son at well (Figure 18). To fierther confIrm thi fInding noastrete thet the itumunostain ing of 11)0 apres& and lag celk is not due to nomai& staining, cells were then harvested and the totalpnoteira were subjected to Weeern blot analynia. As shown in Figure IC, the results showed an inteacelDO protein handinthc& fibróbiasts that were either infected with M-1004.FP wctor or treated with IFN-y. Under a thnllar as nlalcondition,&lls treat ed with tector without ItO poe or untreated cellsthowed little orno 100 expresien. This diffesac was not due to  variation in protein loattingasthe inteestyofjoctinpto  tam, a loading control, vat relatively the sane in nil tam  pias(flgnhe ta As out ultimate pal is to snake use of the 11)0 inn nesuppressive effect to develop a nontejectable hila3ered skin substitute, we were intetested in looking at the eflèct of 100 xpreson on human T cells aiinulated with the intact pheesofeitherallogeseic epidermiror fbli ihickts 382  ikinin an eavivoco-cultnrentodek. For this purpose, hu nan T ealk wee co-cultured with allogetnet pkees of ei  ther epidermis or full thirknms kin in the preatnce of fibrobiasts infected with M—100-CIFP senor or control llbroblasts. otorniuographe were taken from this cc culture sstan after 72 hours. As shown in Figure IA, hu stan I cdt proliferated in nspnnm to stimuinlion by all oteic epiderais or fuLthicknea skin when co-cultu red with either nontrented fibroblasts (first row) or M-GFP treated 11 broblasts (second row). However, there was a se srwrkable reduction in the number of lymphocytes when co-cultured with &flsn&c epldernisor full thtkneat skin in the preaire of preticAfly modified 100-expiating libro biases (third row). De supproaive effect of 100 was nnrk ally seduced in the puesesre of 100 inhibitor, i-MT (fourth sew). To cssnthm and quantify thee flndin peoli&ration of nphocytes neasusert by flfl-th3enidiat iucorpora ion any. The nulls showed an almost sixfold seduction in the proliferation rain oft talk when cocultured with Wcs’dAe. As  iOta) lSS79—2&  0 SaMy @ atta tq&rs Ward HaS,  ) 53  acnn t €a  ccwouardwt at  Wilt —I cats  A  Fiji tT ii.lbs  m  S  i  kat  tin  Nq.re2. Prd*ratai rate of liuna That stamIalal %4thabrneic pea it ci ether epaent a tot tãMth tepreettoect atzeiceot 100 eipreshg ca. Hienat tihabbas were t1eted web AdtO-GPP lMOhlco ad tin wathed will PRS. tasaw4 ad ao-aitad web  Tatrn andenet pecee  Ad.IDO.GFP + 1MT  B  ci eItiecepdeent 4!) at tat ttItht tt4. To anepcqaratha citest a flp 100 ttttat 1-Mt acbdet tetirairettenta tat ci 8000.4. toosiRra ci (twa ties n4th I cat se were ds hc*ut a atitier 00141* saaci A). Mta attep. Tce were that stkØfl at to fl4•titn4n irta00eatori 4atd 8). Data rqtraent rHF tt$VLthtte iiotiprrstiat IcnIsIt fatl insure cp.m) sscbNA&bystactac I cats aadurah will enter untie) & ttis&MMs bold tasJ. Ad-GEP jMctk-A4 Etaled lttsthfaets klpet lstkt400GFPwtecwd ftrctn fatitiltas) or1D0-apeat Thao bats i tie resWict d 1-MT leia*ed fried heaL The dkat ‘p % <0fl0l tiltratces have taut tn&4 fry ataSs 4t. 1-MT. 14naD-wpt4ist tact. trictia z 74o4eIase. —  4O0O  20Xt Ii  scco  FIb’T ceia4rp. aIotwec skin in the presels of 100-cariSin! fibrohint compan$ with thaw of lymphocytat co-as Itated with all cith skin and a 4DO-expaeitg tihrcbbsts in aneti ally znodiôed pn paratiota (p< (1001. r 4! 2. The suppraioan of iimnune cell picliferation s spxifc to fiX) exprtwton, krossa liii effezt wee resertihb upon adduce of 1 MW an 1DO athihnor Further coutml  hrennn T celk cc-cultarat with aIlcmak tibathists alone showed no signi&ant prolifeadon (Figure 1k and IlL Xynwflne n let. ci althlky Hit cUb. banal Hi fifrthbsts and ka-aNneefla inonderto etalimtetlwpostihletatcffasofkynurenitw,  the main Inainthsced tryptoplna devadiative prod act, iitsa,w ® rca&wt*wdtraesv6er  flb+T ciSlil,  on human T cells, fibrobletisated keiatinocyta, net aniat eh of these caR types with various coutentraticas of kyt. nurenina. The cosrentrations of kpeaienine were thoatn based on panbable coiaaatratioia tint em atafly an t froinourlTiOsx:pretängftbrohlaea and alto ourpsevioan Cells were then Itanested after 4 days and evahiat edict their surtival tate bred cm 7AAD staining exam med byffowcytonterryTherewltsshowed tint iarrawing concentiation ofkynuienine *iwn a dgnificnnt reduction (fmmfl* (7% to tlt± i$% p r flit) unteell vnhil ity at concentration as low as t2SjtM of kynnreniae seed. 14&never, under the nine esperhental conditioan, the tiability of fibroblasu and keratinaytae temained an clnngai ewe at higher cosrent ration of kynuienine (Figure 1). This finding indiDates that T cells, but not skin czefls,nrese,sitive tokynurenine. Sn  154  mc tatnnt cain iS’  ian  rcn reaL  -IL  -  i—a  -100  to so  ‘5iii  VU a  ire  ‘5-  so  %LPS  0  ti  20  25€  MuF’  t-2  tcnnin,rereflm ciir,i;  flgins. The died ci kyriiraseie trite sanind rate of hu man alto cSs and t a. Three *ffetnrr ceO tpns tdecfrq librcbasts tflne, frnttra4es lrsde* and? eels jtn ØerJ  ID  recStrjrst in The teaerte of oiaeimng uxicifentatedi  of kyusenne for 4 days Ax thy $ tie cs were harvested to  heir asw.al rate based n 7-AM) flwtq with flow cytcniesy. the grri1ceri 4iirtht <OW1)&tereiees frays  bear n&ste) by attrdas Pt  -  4O  -  lit’  £ Li  m. snvlvdrasoftralê,butnetfbmMas*sand  S  kaa-ftoci.s. nsra&rce’d by nlhsbrg shear vAt 100 expednqeais AsUeviáNlityoftlsdntrellsiscnal to tuake anyalki— genim ski ii saiwtinste to be used as wound coverage with iUO-expreiaing cells, it & important to demonstrate whether 10 -inducedeniAronrnent differencialty infin es the viability of skin and immune cells. For this rsen, fibsobiacts were infected with Ad-100-GFP or titual with IFN-y and co—cukured with CD4 4 T cells. CDt’ T celk, keratimocyter or another strain of fib rob bsts for 4 thyt lii there experirrents, cells cultured either alons, co-culnired with non infected cells or co-cultured with Ad-OF? infected fibrol,hslri were inchded as rxrptive controht AL day 4, the bystander cells were Tnt-sorted and evaluated for their survival rates based en 7LAAD  j  staining with flew cytr4netry. Asdanorrstrated in Figure the control groajt irecliedirig cells co-cultured with norrtreated librobiasts and co-cultured with Ad—GFP  treater! fibrobiasts showed no slgnnlcant differences in sur’ that raze oomjnred with non-cc,-cu km red cells, whereas, as shown in Figure #F1, there were signi&antramuctions in survival rates of both typer of T cells (p ‘c RLIOI) on cultured with Ad-IDO-OF? treated fibroblan In Cf)4 T cells, the survisirl rate decreased by about 14% and in CUP Teells t9%.Inaddition, asdemortrtratedin Figure  40, in IFN-i treated groups the sursival rate reductions ofTedils were even greater (p -sc QMOL). Ta CD4 t T cells. the reduction was 27°’ and in CT Tcelk utwaslá% These inhibitory effects were set red rearkedly by aiding the 100 inhibitor, I-hiT, to the preparations. On the other mud, as shown in Figure 4K-C, no sirifi cant decrease in the suriial rates of fibroblasts and keratinocytes in any of the treatount groups was obeerved compared with their control cells, which were  cultured akise.  “e ,,  -  -  en-  sorn  iruiw  Olin4. Sleet of IIX) eqsrven on ontO savael rare ci Pin nest *irr cats and? a. Abrottssts were titar bit it irstted on  seated with yflous athnosssas IP.€OI:iOO) rate then washed arid rsr-arttired  ii AlCOWrnL). (list  with tie irdated cdl i’ppe in two drariter co-coterie systems 5cr 4 days, in the tharrre or prarercu ci rOw :fl)Q fltrrcy i-Mt (X)LM). teWs*&4riy von Then anatyzedby FPC$srarrgi—AAD tnts A—U im ctwis care*xirrcffte to tIre ssnvet ot of ascIi bystanrhr rpr it escir irentrieni eoop was one. psad to mar of S-re ridma rios—et-edrured eat that coat tirat 1 (X)% nsravd rate icr eat eel twa The ret-irs of rIte cuapnscrt aresbrwn kit Itsetiset acid bask iraratarceytes lepentad, pstilal Q)4’ Ta (haidrat ttersl. and pisted t a 4rte&ad bead bat TOw srqreiiart n Sue <OSD1) diltereices hirer linen indcet& ti t astadas f’). 1IX 1 tddearirte 2-dórrrase:FAa. lirewarsea actretat cell —-  S’S1Sr3  Caepne4 activation ks bysandot T ceib but irrot ftrrcbints and kednocyle oo-cubwadisrtth lUG  The results presented en far indicate that 100 espression  cea a kiss of vinhiuity specillexily in T edIt To &ter. in whether this was the ooiwquerre of increased ap optcsis. we analyxal tin cells for caspsse.3 activation. Osçail is an effector cuase ubiquitously activated through proteolytic deascage following induction of ap optonis As shown in Figure 5k there was a sigeifrant Wv-sdAc tri9i3omiss3W ft Z byT..W.ad ifaiir Sre  155  _______  loG aaY ca  WJ  3L[>. an an.  CI’Q  ft  IN-lIST  ccncra  ciii  cut  P  !PJ4  .t  #  i  .r  p  .flt  4rfl—t  -  cs;pav  f12K.i  RbJiii  4W]  —t  tiC 7  W  T  P  CU—i- 11  Pluse5. Hoe c( G042 trese oath way n sniective ripeobte Sleet of IDQ apeseon &r bsats I cats vs. atli ceft. ftro&ae wdv oft eater untreated or rested witir I FN flOWtJRnU. Cas were stan wasted wrdcOc4slurstt with peorart Cb4CTce*s 1C04. psvrliat coa I cabs CtS. teflocytes 4I. a a ctterenataitotfitsmbtasssjff)tosa days It a t%ve &srbee cocatsire tryatren. o the Stuerice a aesoa ci mac*s anpØerrerstert watt 14(1’ IGp1M. Cats tysates were prenred and ansged toe tIre expresnia of Ct$OP ann deseed easp.se-3 Weawn ttt. Sois were srrrpp.d and rerebad foe Jiacrat as it$rd lig eeed. A repeseertawe e* peknars re Stews lprrel Al. More ewe. tlweaast eshe raterrsed writs *6GF? 4Mcek-Art) a Ad-ItiO-Gffi  w  V.  cHOP(a’KO1  Cseawcd  sn ca  17540: —  Ua%pare-a i2 kfl  1)001 wee  ajttjsid wriji: tssn&i  Teds (ii. ttsrobast l cue tseratIrr otytes (It) tr 3 days vs a tWO thso bet ot-auture syatets. wi tie S.-acttn(43PW) — thee-tee or preaceree of spectre 100 Istibiter, HAT lax)fls). fiysts’dee cabs were nswssth ant cdi lysate wore geoseal and awtyzort true The erasai of CIOP and crewdeaspaae-3 byvarasem na. ots wee xsrrpedard rieotect for Wnctirr as eo.S Scabsocesirrd. A flrttSrneeoednnrts sioweirei G04-Z .$mwS oarolnletbrearsa,2:IDQ. tndtesnne 2, a4icawe are’ 1-Mt. .  actisation in easpos&3 in human T cells cecamed with TFNIt treated fibmbhsls, asiudiorted by the appearatxa of the 17 and 12 KU subunit of active caase-3. Mtiva  titrated fibroMsets we lnrscstod and evaluated for the expression of downstram effector of CCNI, CHOPY As shown in :F’iguresA. therew sasii&ant istrease in  tion ofcaspaw$ was sjgni&antiy inhiNted byan aldition of 1-MT. Moretwer no caspase-3 actisation was seen in any of Lbs fibrobalts or kerstinocytesin different prepa rations. To further can&m this 11ndini in another set of experiments, A440043F? infected llbrOblasts, asackan 54)0102 of 100 exproriot, was used and the ioels of csepose4 in bystander cells were evthnted (Figure SB). Asanti sisistent with tint obtainod ted, the result wa from the 1FN-y induced 11)0 expression. These findings collectively conlirtead tint tegatdleis of the strategksumd tointhacu 100 the level ofcsepose4i:n byanderTceth, but not fibroblasts and keratinceytes, co-cultural with 100 expressing cells will ha iecreased.  CNOPexprasaioninTcell suhsetsandfhiswas :reaomedby skiing 1-MT in cultures. Moreover. the expresion of (310? was not sean in any oft he examined fibroblasms or kerati.nocytes co-cultured with 100 expressingeells. Sim Ilarly, when Ad-lOG-OF? infected fibroblasts were used sea clean seusoe of 100 expression, the trunk of CHOP induction in bwander T cells was the nese tint found in IFN-y experiments (Fsns SB). As shown for caspase 3, regaidlass of stratees used to induce 100, the level of (310? in bystanderT cells, hut not fibrohkns and hera thbocytea, cercultural with 100 expressing cells will he in cased.  a  DiSCUSSION O hi ease pal lwway Isinvoindin sebcjve apo$*MJc affect diDOen human Tce&n skh aedh It lnsbeen suggested tlntactivation ofOOl2kitnseinT cells by um2hrged LRNM sne&atesprolderatsve artest in T cells and aucrr inductiost 5 Nerein, we propose tint the selective apoptoha effect of 100 expresion on human T evils vs. skis fibroblasts and keratinocyta can be due to their different responses to tryptophan deficiency via the 0012 kistase pathway. To examine this point, imnmum and flO:flflUtUtSS cells co’cultu red for 3 cLays with lFNy Wo.ridR  €bzer  1I59-3 L xcebytaWatdRa&mgSEt.v  Several recant audia demonstratat that 100 rioght Inve a role In inun4ueodulatrolt Our group her nit ready shi wa tint 100 expression fusrtions ass local ml sinosisp9reeds’e factor to pro teat alit,- or sunogeneic skin cuBs.” However, the sneche.nian(s) by whi2h EDO affects Tceflsispoodydeflrssd. Two nalnnionslnve hare sag gested for the ire nunosuppresaive effect of 11)0: first, the production of toxic metabolites of tryptophan (La. ky meressisse and 3-hydroxas.ihra]uinc atid) results in Tcell deathS and sent, the local tryptophan depletion causes a decrease in Tee]] proliferation and apopcosa In this tab  156  too a.ct 7 cdi, n ti. a  study, seven] experimental arateger were med to esasn inc these two posiNbties Ta the current study, we Ibund that TOO erprassionhesa setive effort on different pri mary human cull types. Indeed, MAD aainitrg a very sensitive way to detect the hit ofmembrune integrity dat ing pqnosis and pese-3 autivtoa away dkrated tInt bystander T celk, but not librobtasts and keratin ocyt will undergo apoptasit when they are ce-cultated with IOU ep resting elk Moreover we also showed tint an immense in proliferation of human TcelIsstimulat ed bya high ininnanogeniceflhct efpkeei of beth epider’ mat and full skin cue hrisuppacsedinthe presence ofiTY) eiçpressing cells. This Anding may pave the way to practi cally me mU espresien as a lore) inmrnnouepprsise factor to prevent the rejection of allogeneic grafts, esh as aJloneis skin sulastitute to be ured not only ass wound coverage hut also as source of wound haling promoting  ractor.  Muon et aVlnve reentIy shown tlatfCNlserveses a molecukr sensor in murineT cells that allows them to de tort and respond to the immunoregulSor eigrni tserat eel by 100, and It has also been shown that overexpression of 0-lOP cancas apoptosk in keratinocyta in cultureY’ Based on these data, to e&borate the niechanlast by wiekh immune cub hut not skin ceU are winltiws to the 100 med latest asvircnrsent, we stied the activation of pro apoptotic factor CHOP as a dowrntrsam gnal for the 0CN2 lcinase pathwa9 in tle different cell types The findingsshowed a signi&ant imrase in OfOPexpression in slinuriated human 1’ cell in responas to co-culturing with 11)0 expressing cek however, there was no detect able exprewion of CHOP In ttbrobiasts and keratinocytas under thallar conditions. Tie other woids, we were abls to donorestrate that there is a ditThrence in the actiurtion of downstream pathways in reqtortse to 100 between p rims ry human I ciells and skincells. In nmenary, our 1lndln indicate that an 100-mediat ed ens ironnient differentially inituemes the biologtal furections or human T cells rxtnipamed with dther febro blasts orkeratinocytes. This differentialeffort seems to be, at leastinprrt,d difference in activation of the tICN2 icirnsepathway. The Ilndinr ofthksstndy therefore, gest the selective effort of 100 on some iranian immune cell sul*ets vs. sane nonirresiune cell subsets and well sup port the proposal of ining IOU as a local intmunomppres sise factor for engraftrntnt of an alioneic: grail, in our case skin sulietitute, wit bout compromising the viability of nonirrenune cells.  Fniro ataL.  RE$SENCES 1. Teniee P, &tci IN, Rot L, DuRrr, Wafrhk A, Stawit H, Opeir a [ublintiati of aflouinc T cell pnthfentinn l’ tixtoleamine 2mttxpicadi dardtitt ceTh: xne &aticmofsupjrendouby titojthen. masboifles. S &p Md t2 l%:W-5 a StfrrAJ,T*nckrntCTC [nth eZliraytum in minim naimi at aura.. Or Canter Dnq Thrgtm XKt7; 7: 3- Allrni 38, Woig ilL, Can OL, eik€wski Mi, WaN SN. &pknofnrmocyte fimcion and dilTetniatal m,guhtiaa cttL-l emit tt4n by .[b4c4rttibuLatotolutieudeqta’ maim] arthxttii. Jinnanni I93- IS i: 4344-,5 L 4. tgnrwx RA, Maaague I. Transf’irming .gwth fnr-beu nthnuhien The expw.iim f fiiraiedin ant diarc and thdirhratjxnmim toni the afrntdlalannfliz.J Thai Ciwan t9S6 261:4337-45. 5. 5iorn;Mfl,RdiertaA8, Wakc±IdctLM, dieCB, &nttnrtt &h’anms in lit cbeniehy ant biolo y S train fmining 8 .arowthfrtor-irta. iCe)? Dial icr; i 0± 1039—45. 6. ti 7, TretFt EEL, (llnhry A. Cell ewbat ajietoan of MHC tiers I aniin is ep-pirad in m&niuunine Z3cbnzynate geudically nioditted Ira inicytec tmtariorsi in aDoniec slim; enhatitate nssçafttneit Their imwiuesni 4 6± 1 14-fl 7. Munr DII, Slufirmiteb 5 Atinarad FL, &mdamv I, Pashine A, Melkir AL tuhilition: ofT cdl prolifention by niro iagetzyptqr.han nirab brni)Lrp.Me51999 ill9 1363— 72. E Ssrtheir a, ThdFr tn, ti Y, atri UT, TThxL II, (Irahary A. Patitfention at perIpheral bloat moixtiirrar ate is erqnnnad by the indaleainirr Z3-dtorvsarare a’ penion of intafr*n-nnra-twured situ cell’r in a cars)  lure rrysteit WsrnrrdRepEr.W 2€Xt3; 1 1: 337-45. MO, Bahan B, Ilardog It?, Ziranj Y, Ron 0, Meikir AL CCN2ktnnin T cdknnitires 2 rdif’ aathrr anrert and anay tndrrticrn in reaponie lu tutolea nine 2,3-dinaygenat. ieenraurOy C5; 22. 633-42. 10. (lrairary:A, Li 7, budget EEl, Kilani UT; twashiren T, Ear’ anitA, LinK. ram Se2,3tiioxyarein U Mum 033,  &nnal ithatlasrar tindirms at a tacit tnanc,nriw thetor. .1 ba-ar .&nriafaiX)4; 122:953-6.  11. tiY,Tr&Lgett!E,cmalTartA,UnX,KtbriRT,OluuraiyA. local aprcaikn of itetoknmtme 2,34inyrase fl aigraitment Sxaincpeieit situ srrhrlibtte. I hinter .Deoeirsi Xr6; 126:126-34. 17. Cirahary A, TiStg& Slit, Shim Q, Eilant UT, Scat ThCI, Steak V. Marniom6jrlraqthettjtCit.Tt lrrcqrrtom rnedtsre  ACKNQWUEDCM[NS The Carndhn Institutes of Health Raseamth (CINR) and Britith Columbia Profewiorni Fire Fighters’ Burn Fund supported this study. Farsind Foroazandeh and Ran ft JieJIIi are holding University Graduate Fdllowthre from the University of British (kthsmbia. Mare Gemanin is the rotipient of a Postdoctoral Fellowship from the Michael Smith Foundation for Health Romarek The authors ate grateful to Dr. f.M. Chrin (Department of Micrtbioloti, MlaasiUnivertity,Oxfotd, OH) for hisgift of IOQ cONk Conflict qisuen The authors stete no oonfltt of iota mat.  rite cinch of [OF’) ifrttrmd htntlanafcrming growth fan’ brireta I on.apmnrion Stypetcdlnrgerr and nellagemarin &nnal Ilbimbin. Grnth Farsrrn2O&t 17: 167-16. If. Srlthnnh K, TT&Iget FIt, Karaini A, Uiudagit, twarblus T, Sllani RT, Ohahar A. Irnuime cell pmlt&nticwr is tarp jrernreit by lie itneiferampmma’itnducelt trnloieainine 2,3thirygeneic expiation, of ilbeoblasta popidatat in cdhgcrr (FPCC3. 102 Thathrwr 200 90: 206—li. 14. Seilitesh a, Tredga tIE, fluting H, Kilani UT, Karaurt 4, ii Y, Iwarhina T, Ohahary A.. Teiqtenlmr—resuitiit pdy’ rn-cnijial*t WNsnvna inthicar the eapianini of TOO rnELNA and ecttviry by ;fiintitts pnpnriatd in cdlageu gd (FPCCfl. I Ccli PJrysk’i O& 201:146-54.  age  157  IDO tcOJT:t  15. Thitng M, knniegs R,Sian M, Desnsnaeth .% antorA, &tnenmn N; Meflr AL, Maen 011, AntotiaSi. .1minba nine 2$’exygnen onettlenten to tar call osnelan ofT calI’rnedisted etoit hifl OwerlO2 101: 151-1 I IL Ihyath5i T, Nak I ReeLt, C, 0on K, Taktkawa 0, Taka’ luyaibiK. &d&DN OarsonflA, RazE Iniib’ticm. of en’ pettnein4 abma by inthidaneine 2)4ioxyaia. .1 Cit hvar04; 114:270—9. 13. Mint 0H Thou N; Atwood iT, Eondxav 1., Conway $1 ManIla, Ezuwn C, Motor AL. Prevention of atcnet ltd eajeclion by irypbptan catabdIn. S&nre 19; 281: 1191—i, Ii. Mime DI!, Slemnn MD, Ibis 0,Eaban 3 lao3R, Anienis $1, Monies IL, Cbaidfr F, KAni PA, Mdlor AL. Erpan’ on of ind.damier Zh’ ygeia by plnmxytäd skit’ th&ca’l&intandiJyenphneda IOu hwn200I 11* 280-90. 19. Uyttnbene C, PiloLte 1, ‘nra!, Ezooba V. Cohn 0. Patnentiw K, lain T; Van dot Een& El a’kbace In a tenon) inennnr rokiaiia neabankin based on tqthan  4Zot1 lISfl.W @  .  21. 22.  23.  24.  vs sAte  degrade isen byinddnentire2,3’dioxynar. War Med2001 9:1289-74. Faflezints F, Crdnnann U, Va,raC, Biandi 3, 0.abona C, L%nataA, flevati MC Pnantti 1. T call apoptnis:by fle” Inplesncatabobent Cell Death D#e R102; 9: i069’77. &binit 1, Uitloebcaait CII, Okwti IV. Soudiw nrtbod trneeasttinqoj*oá antI cdl pnotype in Iuner.n tyinoce: by ibwc3loindfl. Cvt’nrtq I 994 15:1 3-al. Sdiniid 1, briuaitrt CII. Meld B. Ctirgi IV. A rapid nrthod for nanning optds aid dna].colozimund1m aeneenca by single ItT flow c1oendsy. Jbewiwnl Ifet&t 199 170: 145—53. Amand 5, (leababarti 5, Kawannna 1!, TayIct CR, Maim LV. rnnimoht flgbt (liVE and UVA.) indncese it thma’ nepnntha tanrciption hdcn CI:IOPfgadd).5i in nanrina at lntenan ejacknniis evidence For a inadeaniene ajacific to imad skin. IThw# &neeatati\’lOS; 125: 123-1 Wok IC, hang flY, Anthony TO. Ccping with steis dPI Imnaira and mnendabonal control. .Dthsfrnr 5cc Threw 2006; M(nl)7-1 1.  387  158  Mel CdT Biacfaem C2(XIS) 3:I.$ LOT tftl 001/si iOlO.&t7:.9635v  Differential immunosuppressive effect of indoleamine 2+3dioxygenase (11)0) on primary human cDe and CDS t T cells Fardiaci Foroumah Ron I jMtIt Mare Gemda Viral Durodo Aria Chahary -  Raeive l July I0Y IAccqt& 18 Octb )U11Pthiidid cm8at: Ili Ne:venter fl C fqringrx &iesna&áias Mtha, LIC 2007  We have wtvi0tly dcmonasatat tha indok amine 23-dloxyge nate (100) caprestion by skin cells geitsata a ta*oçhan de&ia awhoiuna in wbith THP-t, India cellsn well as human P2MCam wsthle to smvivt Howeva, fr ssthsds of dmsyhnman T cells am tenslive to typtçhan deplalon have not beae i&nti&d. In this study. ist asked wtwfrr the proliferation aid viability of byasn&s CD4 and COW’ T cells are 1j1asM lnnm to10Q 4iieed tryxcçhan deficient etwiionnt and if so, wIndier their zespome is diffarent. To ahbess ttwne questions, we cot dl 3-cxprcsing fibrobiasts with bystander htrnun C04 T cells 4 and Ahant  -  coa  fit 4 days and then the asuvival and prolifesthon istra as  cells are suppressed In response t tsyjtoçhan deficient environment caused by 100: expression and it is mom so for at T titan C04 T cells.  Keywords Indoleaminc 2,3-dioxygemase (100)GCN2 kinase pathway Kymuenhte  Abfrevlafioas CHOP CCAATioehsncer-binding proein homologous paseeke (MT l-Mzthyl-D-TryptqAialL 7-AAD 7-Ainino-Aesinomycin  well as downstream ntttholie psthway of tiyptophan  degrmlatkm in these cells wean cvaluatd. The wants showed a maciced inunanoiatppressive &ect of 100 erpression on both subsets of prinuty haitian T eell& bnerestingl dices was also a snificnt difiircntx in rho suppresiave effest of 100on prolibaion of CDII’ onespared to the of (304’ T cells. ‘lice result of submqncnt eaex4anents showed tin this disenipaney is diet to diflW sires in GOt! kinase pathway netivinion &fleae these two sott of inunane cells, In aendusion, die finding of Ills study tevenlial that die probfaatkne of C06 and CD4 T  V. Thzierai&i, R. ft bib A. (Ththwj C1) BCPrefe’v&unl FeVn’ mci WcnstTlsJiis itaasch Tab., Dqwtuaa of Spey, tkiseaiiy c1Bi Coto’dis 26&) Oat Snet, IS] .h& BeD Rneath Vests, Vanccan’a, BC, Camds V6H Z6 saD: a4nlniyflnthrg&tsa  M.Gnsnah’ V. Th,onia Depflua of Wdcace. twzáy of Bñtsti Cdrba, Vaarcsiva, BC, Cam& V&1 3211  Introduction Indolcainine Z3’diosygaam (100) is a heane-coataining rate-intaing enzyme, which catalyzes the convedon of teyptophan to kysesratine, the main teyptophan madiolite (1]. 100 has heen found in nonlaepaie cells mainly in trqphoblnazs, dendrifle cells monoeyes, and macreptuges [2-5]. The expression of TOG has i.mnntno-modulaory effects on T cells tta arc tthtal to the pttellular deg tadotion of typtophan [4, 61. Tryptophan Is fr least availuble essential aniswi acid and teqmaed by aU ficenu of life for protein synthesis and othes impoitant nmttholle flutctions, liXi-genersted low tzyptoçtian enviroasnent may case the activstion of stress pathways such as nutrientsensitive mammalian target of rapamycin (mTOR) kina.se pathway in byslanda celL Howewt ithastceen showatha inhihias of mTOR such as eapamydn do not sttaplttelte the profound proliferative arrest seen with UK) expresalon [fl A second amino add-sensitive pahway is medisted by  Sflg  159  2  )&il Cell Thochuf8)Xl91$  the 0CM kinase. GcNleoniains a rgitIaPiy domain Ga binds fr uncharged foim of tRNA& Amino atd hssnffi ciancy mutt a rise in uncharged IRNAs, ntich nctisatcs the 0012 kiiate dentain and inSet downtrcarn sit aaling. Recently, it has been shown Ga 0042 brase pahway is irvolsed in the in mauppicesive effect of 11)Q expression on ammite irnnune cells [7). As the inurimo-nnhilanry effect of ltX) is being invcstigaed for Ia usage an a laid ininumosipprasive suaegy for llogenek grafts, it is linpixtant to piedsely demonsuate is effects on thffaent subsets ofimmune cells important in graft sectin Hat we, therefore, asked wiaether the prolifnion and viability of byaandms (1W aid CDt T cells are matulatd in response t trwtophan de&knt cov*murbu and ifso. wiicther their response Is differeit hi this stialy, for the first tint, we provided compelling evidence that both sularts of hurnanT cells are senslive to TOO iiahteal low tryptophan environ mart and iris nnesofora)VrdaIvewthaofa)tcell& Furthermore we found Ga this diffoential response is chic, at kast in part to the differeite In tlt:leveTofGCNZ kinase nctlvaion betwust these twn atbscts of T cells.  Matetak  — n*thnth  Adenoviai wetor constnicflon The procedure ofconstruction of 11)0 expressing akooviral veetmex ban been described before JSJ Briefly, t construct the adeniovirus encoding hunun IIX), we elated & P434 prodsartencoding the fal 1-length protein iut ashnuk or, which co-csprnsa GFP an a reporter gaic following the manufflra’s instructions (Q-Biognene. Carlebril, CA). The recombinant adenoviral plaids were gcneiatd by ekdroporntion of hIS 1*3 £ cdi using the dauttle sector ether with or without 100. Recornhimnr adenoviral pits niids nan then purified and transfrted to 293 celLs uirsg Fugene-6 naresfection ruagart 4Roe& Applied Sdence, Laval, Q: Canada)Transfected ruin wore monitored tin GEPexpression and afta thror cycles of freezing in ethandl dry ice bath and rapid thawing at 37CC, the &ll l.yrate wore used to amplify vial pasticks in large scale to prepare Adenovial tnt Then, viral tiranion was done using 293cdl line andescrhe’dpreviously f 8).  Transfection of ltthlasts with TOO aGe novfral vector Reconbinam* adcaovinmses were used to infect Iluoblarts at a Mukiplicity of Infection of 100 QvICIL 100) an deribaipreviosny 19. &ee viral particles were removesi fran cuknre medium 30 h after transfection. The suoreex  of transfection wan daernined by flow cytometry mm suong the GE? pretein cspsesioa and fluorescent micrercopy using a Nikon inverted nrictorope. equipped with an FlIt tiler to slew GPP, Images were cupared using a dital camera. Moronver, the efticrey of traits fection and erprossion of flamtalored 100 nat confirmed by dctotuin of 100 protein by Western blot analysis arid kynercAine level in the corulitionul medium wn mcasrred according to a procedure described before [9.  Fhaoreacaiee-aetivated cell sorting (FACS) of human ?BMC forT cells  Total PBMC were isoloted by denalty gralient redirin stan at Hlstopaque- 1077 (Sigma) axordng to the masadnre’s protocol Briefly whok blood was layered on an equal. volume of Thstopapae and csrnfl’ugert a 2,000 rpm for 20 miii at ronm ternperannre and stopped without any brake. PBW were irroluted and added to RPMI 1640 + 10% PBS and prlleted by cattmifsation a ZOOt) rpm for 10 nib and wan further washed twice iii PBS ±1% PBS. We used 20 jsl/iO’celk of?E-coa)ugaed matte anti-human (203 mAli (ED, Qakville 0N) P11(2conjtgatal mouse antI-burma (204 rnAb (BE)), and alice pbycoeyanin (AWy.conjugatcd mona: anti-human (208 arAb (ED). The cells were unabated at mom teirperatore for 30 rein. Mn stoin% cells were washed twice 1 resuspended a 10’ cdlslni in PBS + 1% PBS for FACS. For preparatIon of a pure popirlanion of C03t04 and (203-CUr T eulk we grted on CDVCD4 4 and C03C011T cells after excluding the deal cells and cell debris based on P3(2 and 55(2 parameters and sorted &se rwcecell population into separate ashes.  T-cdll culture and survival evalualon Afsrr being sorted from blaxt T cells were propagrted in RPMI 1640 (Hyclone, Uuh, Uf) srmppknierstesl with 10% FBS. 0.1 U penidlliwiul, and 0_i mg slrepkrmytimimni a 3?(2 in a Immidilled 5% (20.. amo* to be sired for fiuthmr treatna The survival of two different sensitive (cfl44 and err T cells) and two different reskiant (dorsal flbmblants and kerainocytes) cdl types in an TOO-induced tptophan dctktnt environment was ec’mwai by 7-MD staining. 7-AAD tinercaintes into double-stranded nucleic acids. k ls esduded by viable cells but can peaictrute cell membranes of dying or deal celia. Afte each tratnent, cells were ham’t-csted, washed In PBS, stained for 7-AAD and then examined sting FACS analyis, an per tIre nunufadure?s protocol (80).  160  kIc*l Cdi llkc CO) YS:i7  T-cell. stimuhticn and in vito proliferaflon assay T cells woe stimelaed wit sail-CE 3(1 isgtnt RU); The cells woe poked with {HJ-thynaldine (1 jCltnt Pcskin Elmer. Boston 1 MA) on day 3 and harvested on day4 for xneasuzenfl of cell lysat-asaxiacd Uttiun by &sebntii Iton counting. All preliferaidon oxpiments were pafonned in tiiplke mid reported as Count Per Minute. (CPM)  Western blot analysis Total proteins front cell lysac (20 jig) woe arparaed by SDS-PAOE and tsnsferred te WE)? rneinbzane. (Mliii ore, Bedfcad,. MA). The blots west pithed for t310P using antiGADDi53/CN0Ttl0 Ab produced in rab bit (h250 dilutio Signn). We have aced gent anti- rattit Ab (i:2$(X) dilution, Bie-Rad) as the secondary Ak Enhanced elarmi,Auninescezwc dettetion systm 4EC1; Amesshani Biosdatees. UK) was aced in all Not todetct the• seinted secondary Ab. Blots were then flped and reprthed for flardn and med as a osatid of protein loakg.  ?rqasring Llwfl solution RH (Sigma) was pr. as n 20 ruM stcek athrtion in (It N NaGH and adjusted to p11 lA. The reagent was storat rn 4°C and protected fitin light.  Statistical analysis User were expresard as mean ± SI) and aaalyzoi wish nm-way .ANOVA among different groups of cash cell type where indicated. ?-vshses -cURS are ccnsklercd tatisti tally significant in this study.  Remus  3  sub was more than 99%. In parallel, fbroNast population were cultured and infectd list 72 h wish recomhinant adenovimi vertors expressing eidmr OF? (Ad-Gfl) or 100 plus OF? (Ad-OFP-I0O), as desaibed in Methods. Based on OF? capression, infection rnte was ostimrned te he around 1)40% in cads art of espoirnents. Moreover, 100 expression was cordlrtned by western hint analysis (dan not shown). We darn setup liar co-culture. system in wtnldm 100-expressing cells we grown in the upper chsmlaras of ti-wall plant, stalk T cells were arltunrd in the lower chanil as bystander cells (Fig. la) Therefore, there was rio direct centact between fibroblasts aid T cells. It shoild also be mentioned tha Adenovints or 1R4-flred fibre’ blasts, as descrilarstirefeire {9, were washed wih PBS prior to co-eulturing them with other cells, to remove can adunovirus and IFN-y. We. used anti.-C03 (1 pg/nil) fOr activaing liar resting T cells. Bystander T cells were has’ verted on day 4 and evshared for 7-AAD staining by FA(S analysis. The histograms of one set of experiment, out of three arrs liar E4 and CDT cells ate shown in Fig 3 and c respectively. The india of FACS analysis representing the result of three set of experiments are shown In FIg. 2d and a As shown in panel 24 stlmakerd (2W T cells were sensitive t DCNnduced low trypto phan environment as their cell airvival rate dropped from 91+ 15% to 77±73% and M ± 41% when to-al tinted with now-tend flbrobkst. A40,nLB)0 or IFN-y eternal fibrohlsst respectively. However, resting (DtT cells dd not show any significant sensitivity so the sante 100 Induetti environment On the otlan hand, both resting ar-el stimsnlatdCl3WT cells woe araxisise to lEN) induced low tryptophan environnxsnt (panel 2e; P < 0001). liar viability of resting CDt T cells was reduced from 92±2.9% in co-culture wit non-treated fibntlazas to 74±45% and 67 ± 35% in co-cultured with Ad-OW100 and IFN-’y ge respectively. These decswses woe also evident In stimulated COB 4 T cells whose viability of % ± 16% dropped to 72±2% and 67±14%, respec tively (POAX)i). Moreover, addition of a competitive 100 inhibiar, IMT (8(1) pM) f 10], reversal the cffcct.of Ifl0errpresslonon CD4 andCDVT cells signiflsnnlly for 4 both Ad-OW-TOO and IFN-y treated flbroblasss (F ld and &j.  Sensitivity of (1W and (It to iDO-Üuhtced low tryptophan environment In trW to tin orttgte the tmrnunosuppstssive effects of 100 espression on human T cells, we set up a cocukunr. system of either CIW or CDV cells wish IDO-expresing dermal fibroblarns. Total human PBMC (Fig. la) as well as Cl3t(g. lb)aadCDR’{Pj. Ie)Tcellsweresartedby FAQI and analyand for panty. The resale shown in Fig. I revealed that tim parity of both (1W (Id) and CDt (Ic)  Inhibition of Tcell proliferation by IDQ-intheed low tryptophan environment In order to clarify the immunosuppressive effea of TOO expression on T-cdl suhssss, we looked at cell proliferation ofT cells inlOO-indnced low tryptophan environment. On the third day of co-culturing stirnriatd (D4 and CDS T 4 cells with fibtoblasts receiving different treatments, 1 p(1  161  4  Mel 1l lodien (IO8) 3U9:i7  . I Fmviie 1 Fl Cfll &ñe -E tmtaii ?BMC fw T celk Tc1 PBMC we 1 ty dey gri g m itIe 1L17? Pñmzy gate hwA ,ycl pime (fcmd d a  ITGIPMCI  ssc  —  ee±d&ad clk crdth The CDY4I.i4’ (1 imi  -  we ecrd  LTkilkiwin frC1I is) 8 the Iy of ioi1ztke wianel ow xne md fci1 to EThCWC t 99% (a aiid 4 Sit  0  1FC-A  Total PRMC  I  1 Total PRMC  C  I  §  ia  ir  io  ia  1L;o  to  PEori 3 p  rcc4t-ceibttersorth1  io  t  PEae1 CD  I  E  CD 7-veils fte sorting  it? 10  it? Pioli rnJ .LHI-thymidin wat nd&d to Tcd ctfturci and aftf 16 h, in poitbit of JH]-thyrnklin to clla1ar DNA wat As thown in B. 3. wa an almoit S-fãld rhktien in th prolifion ofT d1s w&n t-cil twcd wth alloi’ek -xpting fiboNattn onrnparxi to th d T calls eoc*iitwd ith mon4DOcxpnsang  fihnb1ats (P <Oi)O1).. Th supprtsaion & iirnnnn call pliktnwat spni& to IDO zpnssIon, bccaac aUiion & .IMT sigai&antiy wctscd thc cffc of 100 on CD4 nisd C08 4 T lls for both AdGW  ‘  Cr)3  E  t?  PE-h CD3  IDO and IFNi iiord tthla. En in this xpnmcnt. T cells sho*l ight1y more seatitvity to TDO conazed to CD4 4 T ceU.  crW  OCN2 kinase pathway is tvolvcd in thi effect of TOO on htwian T calls It hat beco tggcstcd that thc adivation of OCN2 kiaa% by wscharged tRNAn in nirinc T calls medias T cdl  Springi  162  ____  M Cd  Bxiem  _____  2X) 3O7  S  C  I.FP  NL F H  A4FP  IiL.Fi  I  “.•.  I—I  1—4—-i  •‘i”’I: ‘...n’iii  (flHrnIn.fl  .iP_4.2i  (  F  I  ....Lir—iiiic:t.:  ———  I  rl  t  fl_-H 11N  IT I  ir.  ?L1 F{  I  -  ,Fi, 2i  IFN.1M  1? 1D  IC  ..  .  t ?‘  1)  is  ,,  ..‘  d  c  *.t  i’ , VL ll.H-.l  I.:— II  1W ti  II’)  I[[ NI Fl  4J-FP  jjt][  d-FP-IOG .I-GFp.4OQ  IFN  II  .F  *  * 1M1  E  JI..f.[. ra.  AFP  ?4GFP4Dc MGFFfl:  Theffe of 11 T1’*  FN  coonkire eyn f& 4  Ii IFN.ThT  c.),  the cr ree  of  id CD T 1k i4 kft & wb eifrT dXIII NOt 1) c W (i,EO CX% waied as ccu1ed ith pified ri • ind iinwy mn C04 mI CT)f T c n two cbnth  asid CD (jueik e T cd1 w &ien7id hyFMD .iuiy%ie ig AAD The iufret (Pvahie OO&1 ffere h eeei ndId y —  pctNfcran ant and enrjr i dion fr]. To v&fy di poim in puma V human T-cD sths, C1W and CI)?.T cdh c-ctilturc*1 ith pwg obhit e  karvtd and wnd fnh eiofa&wnrnni effeioof GCN2, CRC? 11 Wc, hfoc, aluatnd t1 1c1 of QPcipiuon in human T-U iuibtc ud  UXI in1ilIcr  1MT(Lfl i1A The .iiiy of  4ei h arid d  163  Cal) Thcdwnn aXis 3(99:14 45000 áirti, SJDOD  EE:  —  I ILill I NLflb  M4IFP  M4WNDO  I.) Wtrct of iDGix1M low om tan red bi kynentan 1 enviragneiicm jwobfaatrmaf TeeD *ta Onte tart thy&ce etc. I udhn) [ tyniditewas al&d a> ninilamed CD’S 4 Teeli (ccilid Ones) or utinarlad CDt T edit (rçan tnes) ailurn Mrs 16 Ii, irapcatai of [ tJ-tynidiie to celbahe DNA was nte,aced.  we euliusat itt cilt tryptophai-fxee mall en in the presence of kyntrenine (128 ISMI. hi anothersa of apu intents, t tested also ndwth r the addlion of an cxs  anriunt of tsyj*oçhan could pevent the effect of EDt) epacdon at T cells. As shown in Fig. 4, thcs’e ns a ssgn)flntindtace in CEO? espiesslon in CDV, but not in CDt T cells in tDC)-inductd low tayptophan environntnt This &cet was preventable to some tint by adding can annunts of typtaphan in the environment. 1trawer ad swing the human (DW 4 T cells in the alsenee of tryptnpdsan ox in h presence of kyinueni.ne can induce OCN2 kinase aetivntion in them cells, howevex, not at the level of EDO induced kw iryptophan and high kynwenine avixonnunt. buexe-ingly, (NO? expiesdon remained low in CD4 uncta all conditions tsrd (Fig. 4).  NDR-treat€ct  POt  hlOFPIOO tibli  9994  :WNWT  The bin skmw ipngIncyt troliferaic rats, Can Pa ir5m (CRbt) The elgilficat (lkvahe tI(99)) differatais in trnwb. gte robfeniat cia ccrind. a> tat ci tan cement ktfltc enlteed wit crud qattaed) ttlrthIaus hare been in±atd by  astai*c (*) Ø,, 3)  In this study, we densusstrated fre the first the tla the proliferation of CIt and CDt’ T cells is siçpasal in response to rsyptophan deficient awironniem canal by  100 expresion. This was even more so for COP 4 T rein tive to that of (i)4 T cells. Tins flnshug is eonsisent wilt those reported by h4um et at using rrcrine ‘f-cell popu Intions [7[ However, we founds differential senaitiviy in cell proliferation beswten two suisecs of CIW Tand eTC T cefis utica grown in sit same 100-induced low trypto  phan deficient enviromn’ient Ahhough some rqsn suggasied tat T cells are api cificully aenitiveto EDO espresion [6), the nia2ttanisni(s) by utich 100 affccs primary huunn T cells is poorly  Wilt tip  Trpfra  kynurenlne  (‘HOP (29KD  $-nctin 445Klfl  4 QCNI kinan w*way is in,olved ii sswendian eaff&t of t anion at bunar T-cefl str, Ptitds)as ‘an left eithe e tramed with WN-y (lsxm: 5JAn19. (kIn were thai wanbed at ar’cnltrth wi .nifia1 CISC TceJa (CTh4, tnrnlnl nifled  mc)  CISC T edit t2) firS &vs ii a tat chants co’culne sfln it the alwrnce a imae cinmti .5ppknned wit ca arncn of  f4  nyjtjdna In n)dikn edit wee sqtzat)y cobsed in etc ayj*ptnn fare naalii cr in the prna of kynurair, Cell titi  were jwqard and analyred frar the ezpnkc of CHOP by wetn Na lltr were ntijp&1 ansi reinled ktr &4ciin an egret hang caret A rqrranatw apeñnait it öoat tN — 3)  Sprtngn  164  Cdl  xtiin 2O) Y:]7  7  dcthind. Two in ‘ism,s have 1 gcatnd for the imiaupaive effect of DO tra, th p tuctioa of toxic metabolles of uyØephaa (Le, kyimreniiic and 3-hydrnxyanthialiwic acid rotolt in T cdls death (1 ij.  Scnd. la1 tiyprohan &jetht cansea a decreata n Tcell prndfation and açeØá [121 in this study. wn donomtmtcd that TDO cpreasion haa mkad cffot on vithility and fimtkn ofboth tiatcd (1W and CDR T ceik Uowever, this nifect wan patar n CD8 calk relative to CT)4’ T cdis. The findings alan showed that saremtec atnsithe 1DO ghwnancIr m induced low tiypthan environmEnt than (1)4k T cells. This diffcrene wan a]ao re1cted in ItDQ exprranion induced cell death of ii celk Mwrn et L. hat reeently shown (2tS) that GC?12 svea as a imleuilar sensor in nanrinc T cells, ithidi a1ow them to datet and respond to the inunanemiju laLoty inxi n(raLed by JOCk We studind the activation of the pn-apeptic for CHOP an a downstream signL for OCN2 kinma pathway [13J. 1 ñndiags showed a sigaitatit ineremE in (J(JP espies aim in s,tnnulatotI himma Ci)8 T cells and al;tanst no :rnorot, ha stimiated human CD4* T cells in response  to TOO expression. inannunary, ow’ fimdng indicate. that DO-induced low tryptophazi iiOannt diffcrentially inlhiencns the blo logknl funtions of pnmay himian CD4 and CD T celk This dWfcreatial effect scems part, to  be due, at leant in  diffearoces in the retivation of GCN2 idnase  pathway. ‘fln Qaa izc of tlri and ihitid nthia Prnfdmd PIa. fhura’ Ba F rntoi t1a ondy. Th-stod Friuzzmli and Rza B. Jalib ar ‘s Uvr1y (a±a Ftfl aliipc fm th Undaiiy of BTtict CI?m Mrc t7enn,n is die5 cf a PXaI FtTlwibip frcm tha Mithaei nith Fcdaticai fE 1 ‘  griefI to O. JM Cretin tDesin  of Mkretto1cigy ani Uüvand’, ttX) cDNk  OH)  of  I. Tenura P,Ba &TMJL,i árC,W1k ASiuiLidz ((2) Tilldncsi aFaflogi& Tfl rcbkra byini*a1Tii 2gemweaãt hyis.JEMd t%M7451 2. Ailmi iB, Wr IlL GL,kiMJ, WallSM(i993) &iWrecd irnc*cy f*mc ami .ntto&t gik of 1L1 d LI by IL-4 ccwil nindm of 15 bt3444i5i 1 Tgiatz RA, Maacagre J (196 Traatkrmii 1re11s ia indcii of and cdhi d rafr ircpoa&m to 5 enl1a  I Bici Orasn  26i:4i3743t5 4. Jahh RB &IGALVTE 200 h1TÜ 2)ayIem (1tO katon aula,tiy rde in ydakra’ ant thnl. o of tl boty. Keraia. tndit Rereudi gm 5.. S,oi MR. RObn AB, Waknte1 LM, di CR (t9571 Sa in iJ c1aiiar and bilc af arfndn cin J Cr’B Bid iQ5:10 1045 3toui mi, Stofirediti B, Anvot J1. Bntv t, iadmn A, )&flrr AL (1999) Jthitrm of T cdl rdIfÉadcm by mann ax canboBim. I Rq Md 19i B3.-I. 372 7. )&.in DII, Binrea MD, Bii it. ILidog HP, 7Jg Y, RaD, )rI1cw AL (20(s) (II32 toT caRcmsdis aIifrorAe sod reoy kidi in to indiinomi Z3dtoay,en. tmoy 22li3t2 S. (ndsyA, Li Y,TedetIBt, KSItTtT, Iwsddi T,KsmTñ A, Lin X Of) 1moiin 2ay,emar in 20 .onici of I &ma1 Bib1acts forriisra rea irai toio i’a fticx JTxDn.al I25396t 9. I.IJIII RB, Rayst (R,R4sERV, (Rmhay A CJi7) SWead of 41 aflonre& noiio xnie by intin.UTire 2,5. diazy 5eei5 tIt.n. I Cdin1 Vbyinlasy 2Lk i57. 143 J0.L1 Y, Tredgt EB, (Ruffi A, tin X, 3th itt, Guba A i6) Lcal areiii of indi1reyi 23xyn px anraar of xeoek i ndiot2fl I i2 H. Falirino F, Gbmazm U, Vatca C, Bisixhi it, (cn1 C A, Ficroai 1C, PondP (2002) 7 1l apçacins by csanbliun C11 £d DIf4 9I0 1077 12 )doio DII, StuMD, :H D, T5fCS 13 L lit, Azinda J, )dininaJL, QTcwd L,t4dtyK AL (2t .Rttrntiw of in sn Z5dkrzvti by nmscd di*tiiiic rJk in trixdinir iym ooto. I Oin Tovs 11 WRC 1ian WY,Ackmy 20tilCqio iOnc nIF2 tire d xanilsu1 coatrsl. Biocbin S Trxa 3&741  165  A.2 UBC RESEARCH ETHICS BOARD CERTIFICATES  USC  t  The &n.wsê of&itictt Coksrnbia Offlue ofResearch Services Ckvcsi Research Ethics Boaid- Room 210 828 West 10Th Avenue Vancouver BC VSZ ILS  ETHICS CERTIFICATE OF EXPEDITED APPROVAL: AMENDMENT RINCIPAL INVESTiGATOR: EPARTMENT: ISIS Chahay IJBC*tedicine. Faculty of/Surgery NSTJTIJTION(S1 WHERE RESEARCH WLL BE CARRIED 01ff:  JBC CREB NUMBER:  )-405-70537 at  (ancouvar Coastal HEalth (VCHRINCHA) Is. jnnrah w  115ev taudlcre ater.  Vancoirver General Hospital  be eoraiahe:  AlA :o-nJvEsTIGAToR(s tiiagizlIani ihad Forouzandeh liiU bGhaffa  iliresa Rezakhartou tezaBsadsJahli  thetardo Medena P0NSORING AGENCIES: lanadian Institutes ci’ HeaRit Researdi (CIHR) t The Molecular Role of Keratinocytes and Fibroiblasts in Wound fealtkigw FTherapeutio Evaluation of Keratkiccyte Derived AnSFibrogenic Factors (KDAF) fr the Improvement nd!or P antler of Fibrobo Condtbon’ ROSECT TITLE: I )Development arid Application otNon-Rejeotable Aflogenic Skin Substitute as a Wound Coverage -  -  ) Functional Roles dKeratinoovte Derived Mti-fibropenic Factors in Wound Healing REMiNDER: The current UBC CREB approval for this study expires: January 18 2009 IrMENOMENT(S): Dale  IAMENDMEP4T APPROVAL DATE: ebruaryS, 2008  Addtkinof Co-Investdlrirs Change ii Project Title 0031 Jamy 2008 ERTIFlCATiC*1: n respect of clinical thals: 1 The membership of This Research Ethics Board conoiles with The mernbersh4i, requiremenb for Research Ethics Boards defined hi Dk4tiion 5 of the Food and Onig Regulations 2 The Research Ethics Board carries cilia *snothins hr a manner consistent w*h Good Ctktical Pncficea This Research Ethics Board has reviewed and approved the clinical blat protocol and inkirmed cons ent form o,.:the trial which iv te be conducted by the qualilicd irwestigator named above at the specified clinical blat site This approval and The vie s of this Reseanth E1IaIcSBOWd have bees docrnnentedin wrtbbg  The amendment(s) for the above-named project has been reviewed by the Ch.ir of the :uvamity of British Columbia CRnical Research Ethics Board and the accompanying docsnentation was found to be acceptable on ethical grounds for research involving human subjects.  On James McConnad  Associate Chair  https:Ifnse.ubccalns&DocMrFG3MR6VKIJKV400LSH32G9RCE/fromstrinfltml  2/23/2009  166  The Uniwrsiiy of flritish COIL.rnbia  Biohazard Approval Certificate PROTOCC_  runin I 1*5—0103  irvrr GATCR OR CCJHSz LNRI-C:Tofl; (liuhury .iiis 31 pArTw rNT:  Surg?ry  i OH cousr TrL:  Dvvelopnient at iwtii—rejectaLIe slut ststnditIL(e  APROVAL DAlE. 08-12-31 AFpaovEDcown’JNPtNI 1.. ‘F : I LJNI) MG .srr’cy;  2  Canadian Tiistitutes of health  RcsejiriJ  (CIIIR)  TM Priicirici linLgaLiir!trso Oirc:o’ is resporibl Ic eisui tot ;II IoscrLth or course work i’wc ;irq bin nqicsl bac’c. sco’idjctod iriiccorcice;vilti t -le;i1h Cairln, I c4boialay P rafety Guidel no, (21d LuiLicii 1 J90. Copieii cit te Ci.ineliws (1 J9’5) uo .wJlab [rough ho FYosafe: Office. Deprirm&iroIHi>aI:l, &lcly and rwirorirerr. Room !3C 207!I We.hrook WI 1JFC WicIxivor. DC. V6T 12’. 622-7690. Fax: S27-6fl66 -  Approvs of tie UBC Borozacs Coirrrilleeby nni of: Chor. ftrJ3y Committee rinaçer, Biosafety Ethics L)lrec:o’. CI!icu ol Riseaith Sar;ir.e lii s carl f oa:e  val d for oe year frcr thu bcju appru’al dare pro:ioad there is i’o cI’CiiQc in [ha axpr’irrintal poedrves tnnuaI [cDwlcw is raqu rni. A ocy or ihie UIthIUaLe ,,iutb dipInycd  ii  your tIIly  r- oI1Jievt IDZ, 692 A;rurt)rI. i.yir VSrrn!?v  ‘lonti: O5 2? 1 1  Ix  ‘?IH l!  .i;-im  167  Th9 UnIvar.iIyof Bitish Columbia  Biohazard Approval Certificate PRCTL)CO_ NUWBER:  1105-0103  IwrsTicATr)R Ofl C’) LiYH I MbF’.  .  .Azir  9r rjinrrroR. Cliahan’.  Surgery  PRO.EC OR CCJRSE TITLE: APPROVAl 9Ar:  Developrnvn( of noIL-rLlnlaIJIc  sitlil sub( ía uie  U7- 12-14  APPROVEC CG1TAiM4rIT i,rvri 2 l-t)NVIN(J AGbNDY  Canadian Tntitutes cii He3lth Reseireli (C[JIR  The Princ:pal Investicatc4Course Director resparsib 1cr osi.ing :hw si rcstairC or w ro work invoNlrig biological razarc& corated in accoreJ3nce with t.e Health Caracs Lsbcrrnory Sicmefety Suicp ices.. (2nd Ecition i9fd3. Cujies o the Z3uidslirias (‘096) iru wa:Ilo LIoi .iiu Jiuu dy O1iiu L)opaiVcIt of Hcolth. Safely and Frjirorrnen;, Pram 511- 2075 Weshrmk Vail, tJBC, Vancointar. BC. ‘JOT it, 622-7596, Fn: 322-8550.  Rppxvai ot the iJOC BioiiarJs Chair, Biosafet’?  Curni  ito by oiio c?1:  Ccnrr’;itee  Miiasr, BiosseLy _lii.c Direrrnr. Office n Research Se’yices This txrLiislo is vat U Fur uru wuir ixiiii thu ibuvu lar o’ opp’eiai o:o vA-lci-ever is iar’i r;ided tie’e 3 no c aine ir 11-e epe’i tie itsi prc.eiirea. Annual re.ie is recuired A eupy of tlh  tGricL riiuL  Oh ce  l dipIvbrJ  eerc  1r2, Gic&l Açr::,.airv Rad, Phçrc II-4.P.>f  ‘.1  -  iii  yOuV FBCiIiL4  seroi  ‘?nti;.’m,  :r,x.  VST 1Z  1iJ4 12 1!t  168  The tJn:vcrsity of BriUsh Columbia  Biohazard Approval Certificate plc C2COL ‘4U ‘4SER:  crinr-r  OR EIEPARTMFNT  1105-0103 DInLO :Oc  Gh;ikary,  Atlz  Surgery  pRo.l[(: I () ::OLNSE TrTLE: APPliA’AL JWE:  ikvciopiiieiit  of non—rej?chibk sldn &iilistitute  06-12—14  APPRCW[ I) (()N IAIh MEW LEVEL:  rusjr.Nn AGrNc’Y  Canadian  2  or Ilealrb Reseai-clt  1nsihiws  I l P1rcipol IIWPS!iqalor:Couse LNrocbr $ ASonS Me for errriiiy LHU all r.sear& or ccjrs work irwo iirg L uIouita hazajds s cor&tec in radai’oe wih rhe He’al;h CiimIu. Lcboratory buafeLy Guile.liqs, (2nd EU Liui iSE?b. Copes of the Guidoliiios i9E3U) are available :hrout [he Bnsat% tilipu. LJLp r mnr of Health Sa stj ii U En., rorre[ Rnom 55 2U7t Wi. P ok Mall, ¶1G. Vnonuver. BC. ‘167 it 322 (:U6. Fmc: 822-6050.  UUC iloPazarc Gwrmil:ea El osMesy Cwomittee  Approval of he  (ihu  by su of.  r’Aaraqer, 3iosu IciLy LLP Go  LI ccLor Off ra of esearch Surviucs This cenilicaLe is vliu ‘or one year from the xnu riwL cit approval date whithever i laLar) prird0U Lhere is ro rharie ir thu c4vflhlIOnlGl procediras. ?siriul rcniOw IS required.  4  of Lhl sI$  1c.  niin4bc  dipiaed  ChoFOeccespth .3zn?no; R.nU, V9n.1ur,  4gwniiy  sit. ss,14’  iii  r  iii ur tacIII1y  viii 17  &J4.-82...-5 93  169  The University of British Coltimbia  Biohazard Approval Certificate PROTOCOL NUMBER  1105-0103  INVSTIGAI OH OR CDUSE DIRECTOR. (‘hahnry, Miz DEPARTMENT:  Surgery  PRQJ;rCT r)I{ COO NSE ARPHO VAt. DATE:  TILE:  Dcirelopriicnt at nrni-rejectthLe skin stslstitiitt  0$- 12-05  APPRDVED ONiAINMhNT LEVEL: FUis 0! NG AGENCY: Caiiaiii an  2  Tris lii ales of Uta Ill) Research  The Princ!pal lnes1igstor:Ccu-sa Director is responsible trensurirq The: all reaeaith ir course wa-k inssilving bialoqica hazards is conducted in nccordancewiLh tho Health Carada. I ahcrrrory Ricsak4y ULudelVes. (?ri Erliton 1996). CopIes oftheGjideliries (996) are avthbIe through the Biosafely Office Dupaitirwrtof Health Safetg and Enwirorment Room 50- ?075 Wesbroak Mall, LBC. VarD:uv&r, AC. VOl Z1, 622-759,l Ox. 2266iO  ApprDal zt iha USC Uichaza’ds Comnitteey one ci: Chair 8icsoer Corn mittee Manager. Sics-alety Ltflics Director, Office or Research Seivices  This qertfica:e a iIkJ ía- oi’e year frcr’ the abovE atartar apprcal date (whichever is lat2r:P providec there s no chnrlQe in the exprarirricntal proceoures Annual reviev is required. A jiopy ot mis rifIab must be dispisytd  iii  yvur i’o1II4q  Ofrrr 1 kns.2rci SerQioim  02. EflOA.jitnoy Hwf. /an;I;.x!r. V8T 1ZS raii3 Phone. 6M,327-5’ II F$jt 504  170  

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