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Regulatory mechanism of myeloid derived suppressor cell activity

Dissertation
Author: Cesar Alexander Corzo
Abstract:
Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network that develops during cancer. MDSC down-regulate immune surveillance and antitumor immunity and facilitate tumor growth. The ability of MDSC to suppress T cell responses has been documented; however the mechanisms regulating this suppression remain to be understood. This work proposes a biological dichotomy of MDSC regulated by the tumor microenvironment. In peripheral lymphoid organs MDSC cause T-cell non-responsiveness that is antigen-specific. These MDSC have increased expression of NOX2, enabling them to produce large amounts of reactive oxygen species. Since the transcription factor STAT3 is substantially activated in MDSC, its potential role in upregulation of NOX2 expression was investigated. Over-expression of a constitutively active form of STAT3 increases expression of NOX2 subunits, whereas attenuation of STAT3 activity leads to decreased expression of NOX2. The significance of NOX2 in ROS generation is demonstrated in mice devoid of NOX2 function; NOX2-deficient MDSC are unable to inhibit antigen-induced activation of T cells. In contrast, MDSC within the tumor microenvironment have a diminished potential to generate ROS but acquire expression of arginase and inducible nitric oxide synthase, enzymes implicated in T cell non-responsiveness. Upregulation of these enzymes results in MDSC ability to inhibit lymphocyte response in absence of antigen presentation. The tumor microenvironment also promotes the differentiation of MDSC to tumor associated macrophages. Hypoxia is an exclusive feature to the tumor microenvironment and we investigated its involvement in the properties of MDSC at the tumor site. Exposure of spleen MDSC to hypoxia converts MDSC to non-specific suppressors and induces a preferential differentiation to macrophages. Stabilization of HIF-1α, a transcription factor activated by hypoxia, induces similar changes in MDCS as hypoxic exposure. Finally, ablation of HIF-1α prevents MDSC from acquiring factors that enable the suppression of T cells in absence of antigen. These findings help to expand our understanding of the biology of MDSC and suggest a regulatory pathway of myeloid cell function exclusive to the tumor microenvironment. They may also open new opportunities for therapeutic regulation as we now should take into consideration how systemic location affects the function of MDSC.

TABLE OF CONTENTS

LIST OF FIGURES ................................ ................................ ................................ ........... iii

ABSTRACT……. ................................ ................................ ................................ ................ v

INTRODUCTION ................................ ................................ ................................ .............. 1

Cancer and the Immune System . ................................ ................................ ............. 1

Identification and Definition of MDSC…. ................................ .............................. 3

MDSC Expansion in Cancer.. ................................ ................................ .................. 5

Suppressive Mechanisms of MDSC:. ................................ ................................ ...... 8

Origin and F unctional P olarization o f TAM ................................ ......................... .15

Role of Hypoxia in A ccumulation of TAMs ................................ ......................... 17

Statement of Purpose ................................ ................................ ............................. 19

MATERIALS AND METHODS ................................ ................................ ....................... 21

RESULTS…… ................................ ................................ ................................ .................. 30

I. Suppressive Mechanism of MDSC in Peripheral Lymphoid Organs: Role of NADPH Oxidase (NOX2) and Signal Transducer and Activator of Tran scription 3 (STAT3) ................................ ........................... ..30

Hyper - production of ROS in splenic MDSC ................................ ............. 30

ROS generation by MDSC in peripheral blood samples of h ead and n eck cancer patients…………. ................................ ............................ 31

Increased Transcription of NAPDH Oxidase controls ROS upregulation and suppressive activit y of peripheral MDSC…… ........ 32

STAT3 recognizes promoter of NADPH Oxidase subunit ........................ 34

Stat3 a ctivity regulates e xpression of NADPH Oxidase s ubunits ............. 35

II. Suppressive Activity and Differentiation of MDSC in the Tumor Site: the Role of Tumor Hypoxia and HIF - 1 ! ................................ ......................... 45

Phenotype of MDSC in the tumor site ................................ ....................... 46

Function of MDSC in the tumor microenvironment.. ................................ 47

Manipulation of L - arginine metabolism is the main suppressive mechanism employed by Tumor - MDSC…. ................................ ........ 48

MDSC in Tumor Tissues of Cancer Patients….. ................................ ...... .49

Effect of Tumor - microenvironment on MDSC function and differentiation…. ................................ ................................ .................. 49

Effect of Hypoxia on MDSC function and differentiation….. .................. 51

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Effect of Hypoxia on MDSC function and differentiation….. .................. 51

Requirement of HIF - 1 ! for tumor microenvironment - and hypoxia - induced changes in MDSC……….. ................................ ..... 53

DISCUSSION……… ................................ ................................ ................................ ........ 73

RE FERENCES………….. ................................ ................................ ................................ 85

ABOUT THE AUTHOR ................................ ................................ ................... END PAGE

iii

LIST OF FIGURES

Figure 1: ROS level in MDSC from tumor - bearing mice and cancer patients ................. 38

Figure 2: ROS production in MDSC from patients with head and neck cancer ................ 39

Figure 3: Up - regulation of NADPH Oxidase in MDSC ................................ .................... 40

Figure 4: NADPH Oxidase is responsible for ROS production in splenic MDSC and the antigen - specific suppression of T cells ................................ ............... 41

Figure 5: STAT3 recognition of NADPH oxidase subunit promoter ................................ 42

Figure 6: STAT3 regulates expression of NADPH oxidase ................................ .............. 43

Figure 7: Effect of STAT3 inhibitor JSI - 124 on ROS level in MDSC .............................. 44

Figure 8: Phenotype of MDSC in tumor site ................................ ................................ ..... 57

Figure 9: Function of MDSC in tumor site ................................ ................................ ........ 58

Figure 10: Factors regulating MDSC suppressive activity ................................ .......... 59 - 60

Figure 11: MDSC i n peripheral blood and tumor tissues of cancer patients ..................... 61

Figure 12: Effect of the tumor microenvironment on MDSC function ....................... 62 - 63

Figure 13: Differentiation of MDSC in the tumor microenvironment .............................. 64

Figure 14: Regulation of MDSC function by hypoxia ................................ ....................... 65

Figur e 15: Regulation of MDSC differentiation by hypoxia ................................ ....... 66 - 67

Figure 16: Consequences of HIF - 1 !

stabilization in MDSC properties .......................... 68

Figure 17: Evaluation of HIF - 1 !

- deficient chimeric mice before and after tumor

iv

establishment. ................................ ................................ ............................. 69 - 70

Figure 18: Changes in MDSC function and differentiation induced by the tumor - microenvironment require HIF - 1 ! ................................ ................................ ... 71

Figure 19: Schematic of MDSC function and differentiation in tumor - bearing host ........ 72

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Regulatory Mechan ism of Myeloid Derived Suppressor Cell Activity

Cesar Alexander Corzo

ABSTRACT

Myeloid - derived suppressor cells (MDSC) are a major component of the immune suppressive network that develops during cancer. MDSC down - regulate immune surveillance and antitu mor immunity and facilitate tumor growth. The ability of MDSC to suppress T cell responses has been documented; however the mechanisms regulating this suppression remain to be understood. This work proposes a biological dichotomy of MDSC regulated by the t umor microenvironment. In peripheral lymphoid organs MDSC cause T - cell non - responsiveness that is antigen - specific. These MDSC have increased expression of NOX2, enabling them to produce large amounts of reactive oxygen species. Since the transcription fac tor STAT3 is substantially activated in MDSC, its potential role in upregulation of NOX2 expression was investigated. Over - expression of a constitutively active form of STAT3 increases expression of NOX2 subunits, whereas attenuation of STAT3 activity lead s to decreased expression of NOX2. The significance of NOX2 in ROS generation is demonstrated in mice devoid of NOX2 function; NOX2 - deficient MDSC are unable to inhibit antigen - induced activation of T cells. In contrast, MDSC within the tumor microenvironm ent have a diminished potential to generate ROS but acquire expression of arginase and inducible nitric oxide synthase, enzymes

vi

implicated in T cell non - responsiveness. Upregulation of these enzymes results in MDSC ability to inhibit lymphocyte response in absence of antigen presentation. The tumor microenvironment also promotes the differentiation of MDSC to tumor associated macrophages.

Hypoxia is an exclusive feature to the tumor microenvironment and we investigated its involvement in the properties of M DSC at the tumor site. Exposure of spleen MDSC to hypoxia converts MDSC to non - specific suppressors and induces a preferential differentiation to macrophages. Stabilization of HIF - 1 ! , a transcription factor activated by hypoxia, induces similar changes in MDCS as hypoxic exposure. Finally, ablation of HIF - 1 ! prevents MDSC from acquiring factors that enable the suppres sion of T cells in absence of antigen. These findings help to expand our understanding of the biology of MDSC and suggest a regulatory pathway of myeloid cell function exclusive to the tumor microenvironment. They may also open new opportunities for therap eutic regulation as we now should take into consideration how systemic location affects the function of MDSC.

1

INTRODUCTION

Cancer

and the Immune System

Cancer is among the most life - threatening diseases and has risen to become the second leading cause of death in the developed world. Most cancer patients are treated by a combination of surgery, radiation, and/ or chemotherapy. While these standard therapies are efficient at treating the primary tumor, cancer still causes 25% of mortalities in the industrialized world. The primary reason for the failure in mortality prevention is the ineffectiveness of traditiona l treatments in controlling metastatic spread of the disease.

Deficiencies in immune responses have been extensively described in cancer patients. The observation that tumor - infiltrating lymphocytes (TILs) and antigen presenting cells (APCs) are non - funct ional in tumor tissues exemplifies the defects in the immune system (1 - 5). Decreased numbers of mature dendritic cells (DCs) have been observed in the lymph nodes and spleen of tumor - bearing mice (1,2,3), and in peripheral blood of cancer patients (4). In addition to the DC defects, T cells are rendered tolerant to tumor antigens early during tumor progression (5) demonstrating that the T lymphocyte compartment also becomes systemically impaired .

The failure of the immune system to eradicate tumor cells is arguably due to its inability to recognize cancer cells in an immunogenic context. However, it was shown over a century ago that activation of the immune system using the highly immunogenic

2

Coley’s toxin induced a potent systemic inflammatory response tha t helped to control tumor growth and in some cases to eradicate solid tumors (6), demonstrating that if properly activated the immune system is capable of controlling and eliminating the disease. This concept helped establish the immunotherapy approach for cancer treatment. The purpose of cancer immunotherapy is to activate the immune system and to restore its functionality, hoping that it will be able to eliminate the primary tumor and prevent its metastatic spread.

Current therapies intended to boost the immune system involve administration of cytokines: interleukin - 2 (IL - 2) and interferon alpha (IFN - ! ) are FDA - approved for treatment of Kaposi’s sarcoma and multiple types of leukemia (7, 8). A second approach of immunotherapy involves antibody - based treat ment: Rituxan, Herceptin, Campath are example of commercially antibodies available for treatment of various leukemias, non - Hodgkin’s lymphoma, and colorectal cancer (9). Cancer vaccines are the latest strategy for prevention and treatment ofcancer. Cancer vaccines are intended to induce an endogenous, long - lasting tumor antigen - specific immune response. They involve the processing of tumor antigens by APCs and the accompanying presentation to T cells. Cancer vaccines include protein - containing vaccines, in which tumor - associated antigens (10) are usually combined with either adjuvants to induce a strong immune response, with irradiated autologous tumor cells, or with allogenic tumor cells lines transfected with cytokine genes (e.g. GM - CSF, IL2). Additionally , DC - based vaccines are currently under evaluation. Autologous DCs are activated in vitro , provided with the tumor - antigen (either as peptide, or as mRNA or cDNA encoding the antigen), and re - injected into the patient. DC - based vaccines have shown promisin g results in animal models and in the

3

clinical setting.

The success of cancer vaccines is partly restrained by the accumulation of a group of myeloid cells with immune suppressive activity. These cells can take up antigen delivered by vaccination, present it to activated T cells and thereby inhibit the same antigen - specific T cells that the vaccination strategy is aiming to activate (11). This makes even the most effective antigen - delivery strategy ineffective because cancer patients or animals can have con siderable numbers of these myeloid - derived suppressor cells. Reducing the numbers of these suppressive cells and/or inhibiting their suppressive factors have been demonstrated to improve the efficacy of immunotherapy in both animal models and cancer patien ts (12). This inhibitory population presents one of the many roadblocks to the success of cancer immunotherapy and their elimination is a priority for cancer patients who are candidates for active immunotherapy.

Identification and Definition of MDSC

A su ppressive myeloid cell population associated with tumor development and immunosuppression was described three decades ago (13). The first reports demonstrated that administration of a Gr - 1 specific antibody slowed the growth of an experimental tumor. It wa s later found that the Gr - 1 antibody eliminated both polymorphonuclear and mononuclear cells in the blood. The Gr - 1 + cells were comprised of cells at different stages of maturation along the myeloid differentiation pathway (14). This suppressive population is referred to as myeloid - derived suppressor cells (MDSC). MDSCs are characterized in mice by the co - expression of the myeloid - cell lineage differentiation antigen Gr1 and CD11b, also known as ! M - integrin (14, 15). Gr - 1 + CD11b + cells are

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normally present i n the bone marrow of healthy mice and accumulate in the spleen and blood of tumor bearing mice (16 – 19). MDSCs lack the expression of cell - surface markers that are specifically expressed by monocytes, macrophages or DCs, and comprise a mixture of immature m yeloid cells (IMC) that have the morphology of granulocytes or monocytes and have been prevented from fully differentiating into mature cells (20). Gr - 1 + CD11b + IMC present in steady - state conditions are not able to induce suppression of stimulated T cells, and in healthy animals, they can quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). Normal mouse bone marrow contains 20 – 30% of cells with this phenotype, but these cells make up only a small proportion (2 – 4%) of spleen cells and are absent from the lymph nodes. In tumor - bearing animals, cells with this phenotype can make up 50 - 70% of all bone marrow cells and up to 40% of all splenocytes (these percentages fluctuate in tumor models). The human equivalents of mouse MDSC a re most commonly defined as CD14 – CD11b + CD33 + cells or, more narrowly, as cells that express the common myeloid marker CD33 but lack the expression of markers of mature myeloid and lymphoid cells, and of the MHC class II molecule HLA - DR (21,22). In healthy individuals, IMCs constitute ~0.5% of peripheral blood mononuclear cells (22); in the blood of patients with different types of cancer a tenfold increase in MDSC numbers has been detected (21 - 24).

Although initial observations of MDSC expansion were made in the field of cancer, an expansion of immunosuppressive myeloid cell population has been documented in multiple pathological inflammatory conditions. The importance of MDSC has transcended into other scientific fields and MDSC - related research has extend ed to areas involving bacterial infections (25 ,31 ), parasitic infections (26 - 30), traumatic stress,

5

tran splantation and autoimmunity (32 - 33)

MDSC Expansion in C ancer

The past decade of research has failed to identify a single factor responsible for expa nsion of MDSC. The expansion of MDSC is predominantly viewed as the result of the combined effort of many different factors, including pro - inflammatory mediators. The contribution of inflammation to tumor initiation and progression is an old concept. It wa s proposed

by pathologist Rudolf Virchow over 140 years ago (34). Evidence linking inflammation and cancer comes from studies demonstrating that long - term users of nonsteroidal anti - inflammatory drugs, including aspirin, are at a significantly lower risk o f developing colorectal (35), lung, stomach, esophageal (36), and breast (37) cancers. In addition, the block of inflammatory mediators or signaling pathways regulating inflammation reduces tumor incidence and delays tumor growth, whereas heightened levels of proinflammatory mediators or adoptive transfer of inflammatory cells increases tumor development (38). These observations support the notion of a causative relationship between chronic inflammation and cancer onset and progression. The list of inflamma tory mediators implicated in MDSC expansion includes the complement protein C5a, prostaglandins PGE2, and the family of calcium binding proteins S100A8/A9.

The anaphylatoxin C5a is a complement component and a potent chemoattractant and inflammatory media tor. Studies with C5aR - deficient mice demonstrate the contribution of C5a to tumor progression, as after tumor challenge C5aR - / - had lower tumor volumes than littermate controls (38). C5a promotes the accumulation of MDSC not only in tumor tissues, but in peripheral lymphoid organs as well. The ability to

6

suppress T cells of MDSC from tumor - bearing C5aR - deficient was impaired and consequently, tumor - bearing C5aR - / - had higher numbers of infiltrating CTLs in the tumor tissue than wild type counterparts (38). A second set of potent inflammatory mediators produced by many tumors and implicated in MDSC expansion are the PGE2 molecules. PGE2 synthesis begins with the COX - 2 catalyzation of arachidonic

acid to prostaglandin G2 (PGG2), which is subsequently modified

by PGE synthase to PGE2. Mouse MDSC were shown to express all four PGE receptors and coculture of bone marrow progenitors with receptor agonists induced the differentiation of precursors cells into suppressive MDSC. Blocking the PGE2 pathway with COX - 2 in hibitors in tumor - bearing mice decreases the numbers of MDSC and delays progression of spontaneous mammary carcinomas (39).

Myeloid progenitors express receptors for the S100 family members and accumulating evidence confirms their role in MDSC expansion d uring infection and inflammation. The S100 calcium - binding protein family comprises of 12 proteins that serve as inflammatory mediators released by cells of myeloid origin. S100A8 and S100A9 have been implicated in MDSC expansion. These proteins are releas ed in response to cell damage, infection, or inflammation, and function as pro - inflammatory danger signals. When wild type mice were injected with complete Freud’s adjuvant (CFA), a fivefold increase in the proportion of circulating MDCS was observed betwe en days 6 and 9 post - injection. In contrast, in S100A9 deficient mice, the number of circulating MDSCs did not increase after the treatment and the proportion of MDSC in the spleens of S100A9 - / - mice after CFA challenge was threefold lower than challenged wild type animals (40).

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S100 proteins - binding to their receptors activates nuclear factor kappa - light - chain - enhancer of activated B cells (NF - kB) in MDSC (41), and a potential function of NF - KB in MDSC development has been proposed in microbial infections , during which accumulation of MDSC appears to result from Toll - like receptor (TLR) signaling. A recent study focused on polymicrobial sepsis in mice, induced by ligation of the cecum and a double enterotomy, found dramatic MDSC accumulating in spleens and peripheral lymph nodes after the procedure. MDSC accumulation did not occur in mice deficient for MyD88, an adapter protein operating downstream of TLRs (except TLR 3) that transmits signaling from the receptors. The ultimate target of MyD88 is the activa tion of NF - kB, suggesting a possible involvement of NF - kB in the accumulation of MDSC, at least during infection and tissue damage ( 31 ).

In cancer, the expansion of MDSC has been primarily attributed to the numerous cytokines and growth factors produced by tumor cells. Primary evidence supporting this conclusion derives from studies revealing a decline of circulating MDSC after surgical resection of tumors, and by early experiments that showed that conditioned medium from tumor cells cultured in vitro pr evented the differentiation of hematopoietic progenitor cells (HPC) into mature APCs (42, 43) . The cytokines and growth factors implicated to MDSC expansion include stem cell factor (SCF), IL - 1 ! , macrophage colony - stimulating factor (M - CSF), IL - 6,

granuloc yte - macrophage colony - stimulating factor (GM - CSF), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF - ! ), IL - 10, IL - 12, and IL - 13 (44). Most of these cytokines trigger signaling cascades that converge in a common signaling path way, the Janus tyrosine kinase (JAK) protein family members and signal transducer and activator of transcription 3 (STAT3) , which are

8

signaling molecules that are involved in cell survival, proliferation, differentiation and apoptosis (45).

STAT3 is a memb er of the STAT family of transcription factors which consists of seven members: STAT - 1, - 2, - 3, - 4, - 6, and the closely related STAT5A and STAT5B (46 - 48). Engagement of cytokine receptors activates JAKs which subsequently recruit and phosphorylate STAT mem bers. STATs undergo homo - or hetero -

dimerization with other STAT proteins followed by translocation to the nucleus. STATs modulate the expression of genes involved in cell growth, survival and differentiation.

Abnormal activation of STAT3 during tumor pr ogression is well documented. STAT3 is constitutively activated in tumor cells (49) and in diverse tumor - infiltrating immune cells (50), leading to inhibition of proinflammatory cytokine, reduced chemokine production, and to the release of factors that dow nregulate the immune response. Hyper activation of STAT3 is also observed in MDSCs from tumor - bearing mice (51), and its persistent activation preventing myeloid progenitors from differentiating. An in vitro

study showed that exposure of hematopoietic prog enitor cells (HPCs) to supernatants from tumor - cell cultures results in the accumulation of Gr - 1 + CD11b + MDSC and diminution of mature DCs. Blocking of STAT3 activity in the HPCs restored their ability to differentiate into mature DCs. These findings were f urther confirmed in vivo (40). Thus, hyper - activation of STAT3 in MDSC promotes their expansion in tumor - bearing animals; however other potential outcomes stemming from STAT3 signaling in MDSC remain to be elucidated.

Suppressive M echanisms of MDSC

MDSC - mediated suppression of T cell activation has been extensively studied and

9

proven by many research groups. These immunosuppressive activities appear to require direct contact with the target cell, suggesting that these suppressive activities function throu gh cell - surface receptors and/or through the release of short - lived soluble mediators. Factors implicated in suppression of T - cell function include reactive oxygen species (ROS), regulation of L - arginine metabolism, production of TGF - ! , depletion of cystei ne, induction of T - regulatory cells (Treg), down regulation of L - selectin surface proteins on T cells and others. (18,82 - 85,94 )

1. ROS production

Increased production of ROS is one of the main characteristics of MDSC (18,20,52 - 57). ROS are crucial immunos uppressive mediators; inhibition of ROS generation abrogates the suppressive function of MDSC in vitro (18,52,54). ROS are highly reactive molecules due to the presence of unpaired valence shell electrons. Generation of ROS occurs as the normal byproduct o f oxygen metabolism and include such species as super oxide (O 2 - ), hydrogen peroxide (H2O2), hydroxyl radical (OH - ), hypochlorous acid (HOCl - ), and peroxynitrite (ONOO - ). Traditionally ROS are known for their propensity to cause oxidative damage to nucleic acids, proteins and lipids; this property is exploited by phagocytes to destroy invading pathogens. ROS also play a regulatory role in signal transduction and gene expression.

The primary stimuli promoting ROS production in MDSC may be contact with other cells. One study demonstrated that cell - cell interactions mediated by the integrins CD11b, CD18 and CD29 significantly increased ROS production by MDSC (18). Thus, adhesion molecules may contribute

to MDSC - generation of ROS.

Our lab has demonstrated the s ignificance of ROS generation by MDSC; the ROS molecule

10

peroxynitrite produced during direct contact with T cells resulted in nitration of the T - cell receptor and CD8 molecules, altering specific peptide binding and rendering T cells unresponsive to antige n - specific stimulation (12). Peroxynitrite is produced by the chemical reaction between nitric oxide (NO) and O 2 - ; it is one of the most powerful oxidants that are produced in the body capable of inducing the nitration and nitrosylation of the amino acids cysteine, methionine, tryptophan and tyrosine (63).

Although studies have suggested that ROS molecules are important factors in tumor - mediated immune suppression, the mechanism leading to generation of ROS by MDSC remains to be elucidated. The mitochondri a and various oxidative enzymes can generate ROS. The main source of ROS in leukocytes is a multi - subunit enzyme called NADPH oxidase (NOX2), a complex that generates

O 2 - in the one - electron reduction of O 2 using electrons supplied

by NADPH after activatio n of its various components (58). The oxidase complex consists of two membrane - bound proteins,

gp91 phox and p22 phox , cytosolic components

p47 phox , p67 phox , p40 phox , and a small GTPase protein

Rac1 or Rac2 (58).

The phagocyte NOX2 plays a key role in inna te immune responses against microbial pathogens by generating ROS that act as powerful microbicidal agents (59). Sustained NOX2 activity requires continuous renewal of the enzyme complex; without it rapid deactivation occurs (60). The activation of the com plex, which is essential for its full functionality, requires phosphorylation of the cytosolic components and their translocation to the plasma membrane where the generation of

O 2 - takes place (61). The assembly and activation of the NOX2 complex can be in duced by pro - inflammatory cytokines, such as GM - CSF and TNF - alpha (62). Cell adhesion molecules and integrin engagement are also capable stimulants of NOX2 - dependent ROS generation (160)

11

2. Metabolism of amino acid L - arginine

Metabolism of the amino acid L - arginine has been implicated in the suppressive activity of MDSC. Metabolic manipulation of L - arginine is a survival strategy conserved in lower organisms (64). This strategy is exploited by MDSC to limit the expansion and function of T cells. L - arginine serves as a substrate for two distinct but related enzymes arginase and inducible nitric oxide synthase.

A) Arginase (ARG) : The importance of this enzyme in tumor progression is reflected by the observation that its inhibition slows the growth of a lung ca rcinoma in a dose - dependent manner (65). Two distinct isoforms, Arg1 and Arg2, have been identified in mammals; they are encoded by different genes and are located in the cytoplasm and mitochondria, respectively. Arg1 is primarily located in the cytosol of hepatocytes and is an important component of the urea cycle. Arg1 expression is induced in myeloid cells by exposure to the Th2 cytokines IL - 4 or IL - 13 ( 66, 67 ) , TGF - ! (68), and GM - CSF (69).

Arg2, also known as kidney - type arginase, is constitutively expr essed in the mitochondria of various cell types, including renal cells, neurons, macrophages and enterocytes .

Arginases hydrolyse the amino acid L - arginine to L - ornithine and urea. L - Ornithine is a precursor for the synthesis of polyamines by the ornithine decarboxylase (ODC) pathway; polyamines have an anti - inflammatory role and inhibit the release of pro - inflammatory cytokines from monocytes (70).

The primary consequence of arginase upregulation in MDSC is the depletion of L - arginine from their surroundi ngs. In the absence of L - arginine, T cells cultured in vitro fail to proliferate upon stimulation and fail to produce interferon gamma (IFN - ! ) (71). L - arginine deprivation triggers several negative effects on T cell activation. First, T cells

12

deprived of L - arginine are deficient for CD3 !

chain and become arrested in the Go – G1 phase of the cell cycle (72) . Second, the expression of cell cycle regulators cyclin D3 and cyclin - dependent kinase 4 is also compromised (73). Finally, L - arginine starvation can resul t in phosphorylation of eukaryotic translation initiation factor 2 (EIF2a), halting the initiation of translation and repressing protein synthesis (74).

B) Nitric Oxide Synthase (NOS) . L - arginine is also the substrate for a family of enzymes known as NOS. These enzymes catalyse the reaction between oxygen and L - arginine, generating L - citrulline and NO. Three distinct isoforms of NOS are the products of different genes: NOS1 is primarily found in neuronal tissue; NOS2 is the inducible isoform (also known as iNOS) and is found in various cells of the immune system, including several types of myeloid cell. NOS3 is found in endothelial cells (75 - 77).

NOS inhibitors reverse immune suppression demonstrating immunoregulatory properties of NO (78). NO operates thro ugh various mechanisms to suppress T cell function. It interferes with the IL - 2R - signalling pathway by blocking the phosphorylation of signal - transducing pathways coupled to IL - 2R and by altering the stability of IL - 2 mRNA (79). Exposure to NO can also lea d to cellular apoptosis (80). NO also interferes with the cytotoxic effector phase (81). NO causes mRNA instability of Ras, a critical molecule in the signal transduction cascade from TCR activation to cytolytic granule release, resulting in inefficient ex ocytosis of the cytotoxic granules. Through this mechanism, NO prevents activated lymphocytes from killing target cells.

3. Production of TGF - !

The immunosuppressive molecule TGF - " has also been implicated to MDSC function. TGF - " is a cytokine with multiple immunosuppressive properties ( 82 ).

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Inhibition of TGF - ! by antibody or soluble receptor inhibits tumor growth in vivo ( 83 ) and T cells that d o not respond to TGF - ! were resistant to implanted tumor cell lines B16 and EL4 (84). A separate study suggested that MDSC are the main source of TGF - ! in tumor - bearing animals. In this study, MDSC induced by a mouse fibrosarcoma or colon carcinoma when st imulated with IL - 13 through the IL - 13R "

Full document contains 112 pages
Abstract: Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network that develops during cancer. MDSC down-regulate immune surveillance and antitumor immunity and facilitate tumor growth. The ability of MDSC to suppress T cell responses has been documented; however the mechanisms regulating this suppression remain to be understood. This work proposes a biological dichotomy of MDSC regulated by the tumor microenvironment. In peripheral lymphoid organs MDSC cause T-cell non-responsiveness that is antigen-specific. These MDSC have increased expression of NOX2, enabling them to produce large amounts of reactive oxygen species. Since the transcription factor STAT3 is substantially activated in MDSC, its potential role in upregulation of NOX2 expression was investigated. Over-expression of a constitutively active form of STAT3 increases expression of NOX2 subunits, whereas attenuation of STAT3 activity leads to decreased expression of NOX2. The significance of NOX2 in ROS generation is demonstrated in mice devoid of NOX2 function; NOX2-deficient MDSC are unable to inhibit antigen-induced activation of T cells. In contrast, MDSC within the tumor microenvironment have a diminished potential to generate ROS but acquire expression of arginase and inducible nitric oxide synthase, enzymes implicated in T cell non-responsiveness. Upregulation of these enzymes results in MDSC ability to inhibit lymphocyte response in absence of antigen presentation. The tumor microenvironment also promotes the differentiation of MDSC to tumor associated macrophages. Hypoxia is an exclusive feature to the tumor microenvironment and we investigated its involvement in the properties of MDSC at the tumor site. Exposure of spleen MDSC to hypoxia converts MDSC to non-specific suppressors and induces a preferential differentiation to macrophages. Stabilization of HIF-1α, a transcription factor activated by hypoxia, induces similar changes in MDCS as hypoxic exposure. Finally, ablation of HIF-1α prevents MDSC from acquiring factors that enable the suppression of T cells in absence of antigen. These findings help to expand our understanding of the biology of MDSC and suggest a regulatory pathway of myeloid cell function exclusive to the tumor microenvironment. They may also open new opportunities for therapeutic regulation as we now should take into consideration how systemic location affects the function of MDSC.