VHL Mutations and HIF-1alpha Protect Tumors by Regulating Antigen Presentation and

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1 VHL Mutations and HIF-1alpha Protect Tumors by Regulating Antigen Presentation and Generation of Extracellular Adenosine: Implications for Immunotherapies of Cancer by Shalini Sethumadhavan M.S. in Pharmacology, Massachusetts College of Pharmacy and Health Sciences A dissertation submitted to The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Doctor of Philosophy March 18, 2016 Dissertation directed by Michail Sitkovsky Professor of Biology and Pharmaceutical Sciences

2 Acknowledgements I would like to thank my advisor Dr. Michail Sitkovsky for giving me the opportunity to work with him. Your enthusiasm for research has been infectious and you have taught me important lessons in life as well. Thank you for the experimental independence you gave in projects, that has greatly helped me grow as a scientist. Dr. Akio Ohta, you have been a model scientist and your discipline and dedication to research has been inspirational. Thank you being such a great mentor. Your ideas and guidance over the years have played a big role in bringing this dissertation to fruition. I will never forget all that you have done for me, like driving me over in a snow storm to use the gamma counter! I am immensely thankful to my committee members Dr. Erin Cram and Dr. Veronica Godoy for not only being extremely resourceful in guiding the dissertation work but also being there to resolve any and all issues. No matter how busy, you always made time to meet with me and your advice over the years has been invaluable. Thank you Dr. Ban-An Khaw for your guidance and insights that has helped improve this dissertation. A special thanks to Akiko Ohta, whose dedication to work and family has been inspirational. I greatly appreciate all your guidance with molecular biology work early on. Thank you for always making time to help me out in spite of your own hectic experimental schedule. This work would have been impossible without the incredible support of my family, whose only goal was to see me succeed. Thank you doesn t seem enough for all the sacrifices my husband has made over the years and his unwavering faith in me. I would also like to thank my parents and my sister, who have always stood by me and have dedicated their lives to my happiness. ii

3 Thank you to Dr. Dimitry Lukhashev who taught me many techniques and aspects in molecular biology. I would also like to thank the members of the Sitkovsky lab members for making grad school fun and for all the help with the dissertation work including Dr. Robert Abbott, Phaethon Philbrook, Murillo Silva, Dr. Manasa Madasu, Ryan Cannici, Jasmine Labuda, Susan Ohman and Dr. Stephen Hatfield. iii

4 Abstract of Dissertation The tumor microenvironment (TME) plays a critical role in tumor initiation, progression and response to anti-tumor therapies including immunotherapy. One of the most pervasive TME stresses in solid tumors is hypoxia or low oxygen tension. The transcription factor hypoxia inducible factor-alpha (HIF-α) is known to be the master regulator of the cell s adaptive response to hypoxia. In co-ordination, hypoxia and HIF-1α promote an immunosuppressive TME but little is known about the mechanisms by which hypoxia affects immunogenicity, i.e., antigen presentation by MHC class I molecules on tumors. A significant fraction of human cancers are known to either downregulate or completely lose their expression MHC class I, thereby escaping recognition and rejection by anti-tumor T cells. Gene expression of MHC is controlled by complex regulatory signals, with the recruitment of distinct transcriptional machinery under different microenvironmental conditions. In Chapter 2, hypoxia in the TME is shown to serve as a local cue that negatively regulates antigen presentation by MHC class I molecules in a transcriptional, HIF-1α dependent manner. Corroborating this finding, hyperoxia (60% oxygen) upregulated MHC class I expression. In addition, oxygen mediated regulation of proteasome components (TAP1/2, LMP2 and LMP7) that play a dominant role in determining the surface density of MHC-peptide complexes is demonstrated. In this study, the importance of choosing the right in vitro culture platform to recreate the hypoxic TME-induced changes observed in vivo is underscored. In Chapter 3, extending my observations of oxygen-dependent regulation of antigen presentation, I hypothesized that by increasing the surface density of peptide-mhc complexes, hyperoxia could serve as a biotechnological tool to improve the efficacy of dendritic cell (DC)- based vaccines. Here I report that hyperoxia serves as an immunological co-adjuvant allowing DCs to attain a more activated state by significantly enhancing signal 1 (by upregulating MHC class I, MHC class II); signal 2 (by upregulating co-stimulatory molecules CD80 and CD86) and iv

5 signal 3 (increased proinflammatory cytokine profile post-activation by LPS). In addition to upregulating MHC class I expression, hyperoxia stabilized peptide-mhc complexes, increasing the half-life of the complex by 4x as compared with normoxic DCs. Extended display of peptide- MHC molecules augments potency of DCs by increasing the likelihood of contact between DCs and T cells. In Chapter 4, we turn our attention to yet another immune escape strategy used by hypoxic tumor cells. Through extensive and seminal work, Sitkovsky and colleagues have demonstrated that there is a metabolic shift in the hypoxic regions of the tumors toward production of extracellular adenosine in excess. This adenosine then binds to specific adenosine A2 receptors and inactivates infiltrating anti-tumor lymphocytes. Extracellular generation of adenosine from ATP is mediated by two ectoenzymes, CD39 and CD73, with CD73 being the rate limiting enzyme. Studies have shown that CD73 overexpression is associated with poor prognosis in several cancers such as ovarian and breast cancer and is also known to mediate chemoresistance, promote neovascularization and invasiveness of tumors. In endothelial cells, CD73 was shown to be induced by hypoxia in a HIF-1α-dependent manner. von Hippel-Lindau protein (VHL) tumor suppressor protein plays a key role in cellular oxygen sensing by ubiquitinating and targeting HIF-α to proteasomal degradation under normoxic conditions. Mutations in VHL is frequent in clear cell renal cell carcinomas (ccrcc), leading to constitutive stabilization of HIF-α irrespective of oxygen tension. Thus, in this study, we hypothesized ccrccs may have higher levels of CD73 due to mutated VHL and thus serve as a responder population for targeting the hypoxia-adenosinergic pathway. We found that VHL inactivation and HIF-1α upregulated CD73 expression transcriptionally. In contrast, HIF-2α did not affect CD73 expression. In addition, I describe a method to detect extracellular adenosine in in vitro cultures without the need to use labeled nucleotides. v

6 Table of Contents Acknowledgements ii Abstract iv List of Figures vii Abbreviations x Chapter Innate and adaptive immunity Antigen processing and presentation pathway Hypoxia, VHL and HIF transcription factors Anti-tumor immunity Significance 27 Chapter 2 30 Abstract 30 Introduction 31 Materials and methods 32 Results 36 Discussion and Future directions 41 Figures 45 Chapter 3 62 Abstract 62 vi

7 Introduction 63 Materials and methods 65 Results 67 Discussion and Future directions 71 Figures 78 Chapter 4 80 Abstract 80 Introduction 81 Materials and methods 82 Results 85 Figures 87 Discussion and Future directions 90 References and Notes 97 vii

8 List of Figures Figure 1.1: MHC class I antigen presentation pathway 9 Figure 1.2: MHC class II antigen presentation pathway 13 Figure 2.1: Hypoxia downregulates MHC class I expression on tumor cells in vivo 45 Figure 2.2: Hypoxia downregulates MHC class I expression in vitro in 3D but not in 2D culture systems 46 Figure 2.3: Deeper hypoxia achieved in 3D culture systems necessary to observe downregulating effects of hypoxia on MHC class I 48 Figure 2.4: Hypoxia-mediated downregulation of MHC class I expression impairs recognition and killing of tumor cells by CTLs 50 Figure 2.5: Hyperoxia upregulates MHC class I expression in vivo 51 Figure 2.6: Hyperoxia upregulates MHC class I expression in vivo and in both 2D and 3D cultures in vitro 52 Figure 2.7: 60% oxygen upregulates immunologically relevant peptide-mhc complexes and increases lethal hit delivery by cytotoxic T lymphocytes 53 Figure 2.8: Molecular oxygen regulates MHC class I expression transcriptionally 55 Figure 2.9: Hypoxia downregulates MHC class I expression via HIF transcription factors 56 Figure 2.10: Restoration of VHL and downregulation of HIF-1α upregulates MHC class I expression 58 Figure 2.11: Hypoxia downregulates and hyperoxia upregulates expression levels of TAPs and LMPs 59 viii

9 Figure 2.12: Hyperoxia upregulates presentation of ovalbumin peptide SIINFEKL 60 Figure 2.13: 60% oxygen induces reactive oxygen species in vitro 61 Figure 2.14: Hypoxia downregulates and hyperoxia upregulates HSP70 expression in tumors 62 Figure 3.1: Differentiation of monocytes into dendritic cells under 60% oxygen dramatically affects viability of the cells 74 Figure 3.2: 60% oxygen selectively upregulates antigen-presenting and co-stimulatory molecules on dendritic cells to acquire a more activated state 75 Figure 3.3: 60% oxygen increases density, stability and half-life of surface peptide-mhc complexes 76 Figure 3.4: Dendritic cells cultured under 60% oxygen acquire an increased ability to stimulate CD4+, CD8+ T cells and B cells 77 Figure 3.5: Culturing dendritic cells under 60% oxygen dramatically improves their response to stimulation by LPS 79 Figure 4.1: VHL-HIF axis regulates surface expression of CD73 90 Figure 4.2: Restoring VHL in HIF-2α expressing ccrccs does not affect surface expression of CD73 92 Figure 4.3: SiRNA-mediated knock down of HIF-1α but not HIF-2α downregulates CD73 expression 93 Figure 4.4: Overexpression of wild type (WT) but not functionally mutant HIF-1α upregulates CD73 expression 95 Figure 4.5: Mutant VHL-HIF-1α upregulates enzymatically active CD73 97 ix

10 Abbreviations MHC: Major Histocompatibility complex HIF: hypoxia inducible factor FIH: Factor inhibiting HIF TME: tumor microenvironment CTL: cytotoxic T lymphocytes APM: antigen processing machinery TAP: transporter associated protein LMP: low molecular weight protein VHL: von Hippel-Lindau ccrcc: clear cell renal carcinoma 2D: 2-dimensional 3D: 3-dimensional HP: hypoxyprobe-1 DC: dendritic cell MFI: mean fluorescence intensity sirna: small interfering RNA x

11 Chapter 1: Introduction 1.1. Innate and Adaptive Immunity (1) The innate immune system is the first line of defense against invading pathogens. The principal components of this system include physical and chemical barriers (e.g. epithelial layer), cells (natural killer cells, mast cells, eosinophils, basophil, macrophages, neutrophils, and dendritic cells) and the complement system. Components of the innate immunity are not antigen-specific, but rather recognize damage- associated or pathogen-associated molecules and do not demonstrate immunological memory. a) Neutrophils: They are the most abundant of the circulating white blood cells and mediate the earliest phase of inflammatory response and antimicrobial immunity. In addition to microbial phagocytosis and clearance, the role of neutrophils in cancer progression has recently gained momentum. Tumor associated neutrophils are known to be pro-tumorigenic (2). b) Mononuclear phagocytes: These include monocytes (in circulation) and macrophages (tissue-resident). Classical monocytes are identified by their expression of CD14 and lack of CD16. Macrophages play an essential role in ingesting and killing microbes and modulating the adaptive immune response through antigen processing and presentation. Macrophages also function to resolve inflammation and aid tissue repair. Depending on the cytokine milieu and local tissue microenvironment, monocytes can differentiate into the pro-inflammatory M1 phenotype or the anti-inflammatory M2 phenotype. Tumor-associated macrophages (TAMs) are known to be typically of the M2 phenotype and are involved in promoting cancer progression and metastasis. c) Mast cells, basophils and eosinophils: These cells have a role in elimination of helminthes and parasites but are better known for their role in mediating allergic reactions. 1

12 d) Natural Killer (NK) cells: NK cells are known to have effector functions against pathogeninfected and tumor cells. They are also known to reciprocally signal to T cells, dendritic cells and macrophages, acting as regulatory cells. NK cells do not display immunological memory and have a short life-span. Functions of NK cells are controlled by a balance between inhibitory and activating signals. Interaction between Major Histocompatibility Complex class I (MHC class I) on target cells and cognate inhibitory receptor (KIR) on NK cells typically dominate and lead to NK cells being maintained in a resting state. NK cell activating ligands are not typically expressed by healthy cells but are upregulated in response to cellular stress like microbial infection or neoplastic transformation. Activating ligands include NK group 2 member D ligand (NKG2DL, major histocompatibility complex I-related chain A and B (MICA and MICB) and UL16 binding protein 1 6 (ULBP1-6). These ligands interact with NKG2D receptors on NK cells and CD8+ T cells (3). e) Dendritic cells (DCs): DCs serve as an essential link between innate and adaptive immunity. DCs are the most potent antigen presenting cells (APCs), cells that capture antigens from microbes and other sources like tumors and present them to lymphocytes to stimulate their activation and proliferation, followed by macrophages and B cells. DCs are also capable of capturing antigens from live cells through a process called nibbling. DCs present captured antigens in context of MHC molecules to naïve T cells. Peptides presented by MHC class I molecules are recognized by CD8+T cells whereas peptides presented by MHC class II are recognized by CD4+T cells. The CD4+ T cells can then differentiate into T helper (Th1, Th2, or Th17 phenotype), T-follicular helper (Tfh) cells that regulate humoral immunity (i.e, B- cell/antibody mediated) or regulatory T cells (T-regs) that downregulate immune response. Lipid antigens are processed differently and are loaded onto non-classical MHC molecules of the CD1 family. 2

13 Typically DCs in the periphery are immature and lead to T cell tolerance due to lack of costimulatory signals and other cytokines. They have increased antigen capturing ability but reduced expression of MHC class II, CD80, CD86 and chemokine receptors. In response to environmental cues, DCs mature and home to lymph nodes where they present antigens to T cells. DC maturation is also dependent on CD40 ligation, interaction of CD27 on T cells with CD70 on DCs and different environmental cues lead to development of distinct DC phenotypes. Two subsets of DCs are known: myeloid and plasmacytoid DCs (pdc). Both are derived from hematopoietic progenitor cells but through different developmental pathways. pdcs are the first responders to viral infections. They secrete large amounts of IFN-γ in response to viruses, which in turn activates T cells and other DCs. They also allow differentiation of mature B cells into plasma cells secreting antibody. mdcs or the conventional/classical DCs have a role in innate immune responses against pathogens and in priming and cross-presentation to adaptive immune cells The adaptive immune system differs from innate immunity in its specificity, immunological memory, diversity of receptors and antibodies generated by somatic recombination of genes, clonal expansion and contraction. There are 2 arms of adaptive immunity, cell-mediated and humoral. Humoral immunity mainly comprises of B lymphocytes and the antibodies they produce. Cell-mediated immunity includes T lymphocytes; helper, cytotoxic, regulatory, NKT and γδ T cells. NKT cells or natural killer-t cells are T cells that express NK cell receptors like CD56. The TCR of NKT cells recognize lipid molecules presented by the CD1 molecules. They are known to produce cytokines in response to lipid antigens that in turn activate B cells to secrete antibodies to these lipid antigens. 3

14 γδ T cells, are T cells that are of distinct lineage from that of T cells expressing the αβ TCR. Signaling via the TCR is similar in both lineages but the γδ T cells do not recognize peptides presented by MHC complexes, i.e., they are not MHC restricted. Their exact physiological role is still unknown but they may have a role in microbial clearance at the epithelium until the more antigen-specific αβ T cells are activated. T cells can only recognize antigens presented by MHC class I or class II molecules and do not respond to soluble/free antigens. T cell receptor (TCR) recognition of peptide-mhc complexes constitute signal 1 for T cell activation, signal 2 comes from interactions of CD28 with corresponding co-stimulatory molecules (eg, B7.1 and B7.2 on APCs) and signal 3 includes cytokines. Negative regulators of T cell activation include: a) cytotoxic T-lymphocyte antigen-4 (CTLA4), which is expressed only on activated T cells and has 20x more affinity over CD28 to B7 molecules. b) Programmed death 1(PD1) receptors limit T cell effector functions within the tissue and bind to 2 different ligands, PDL1 and PDL2. C) Adenosine A2A receptors on T cells that are upregulated upon activation and bind to adenosine. Elevation in intracellular camp downregulates T cell activation. d) BtH-4 is expressed on activated T cells, B cells and monocytes. Its ligand is unknown. e) The lymphocyte activation gene 3 (LAG-3) is expressed only on activated T and NK cells. It binds to MHC class II and has higher affinity for it over CD4 and thus inhibits TCR/CD3 signaling. f) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is typically expressed by terminally differentiated Th-1 cells and binds to galectin Antigen presentation and processing machinery (1) MHC (in mice) or human leukocyte antigen class I (HLA, in humans) molecules are expressed on all nucleated cells. They are heterodimers of membrane bound heavy chain with three extracellular domains (α1, α2, α3) and a cytoplasmic domain with β2-microglobulin (β2m). β2m is essential for the stable expression of MHC class I. The α1 and α2 domains form a 4

15 groove, the peptide-binding cleft, which houses the antigenic peptide between 8-10 amino acids in length. The heavy chains are encoded by three genes (HLA-A,-B, and -C in humans) that are highly polymorphic. The allomorphs differ in their specificity for peptides. Consequently, individuals express a unique set of peptides on their cells depending on their class I genes (potentially six different allomorphs) and the peptides generated by the cells (4). Constitutive expression of MHC class I genes is regulated in a tissue-specific and developmental manner. For example, germline and neuronal tissues expresses about 2 fold lower MHC class I molecules as compared with lymphoid cells. In addition to the tissue-specific regulation of MHC, expression can also be modulated by extrinsic signals like cytokines and hormones (i.e., inducible expression). For example, IFN-γ upregulates MHC class I expression whereas thyroid hormone dowregulates MHC expression. The levels of MHC class I molecules expressed at the surface of a cell is a factor of both the transcriptional regulation of the MHC class I heavy chains, β2m and the efficiency of the antigen processing machinery (APM) that generate and load antigenic peptides for presentation to the immune cells (5, 6). Empty MHC class I molecules are unstable under physiological conditions and are rapidly internalized and degraded. The antigen processing machinery includes the constitutive and inducible forms of the proteasome, peptide transporters (TAP1 and TAP2), endoplasmic reticulum chaperones (calnexin, calreticulin, ERp57, and tapasin), and the Golgi apparatus. The proteasome is a multimeric protein complex that functions to cleave ubiquitinated proteins to generate short peptides and releases it into the cytosol. It has the 20S core and the 19S regulatory cap. The 19s region binds to ubiquitinated proteins, unfolds the protein and directs it into the 20S core (7). The proteins processed by the proteasome could be retirees (normal protein turn over) or defective ribosomal proteins (DRiPs). DRiPs are a subset of nascent proteins that are rapidly shunted to proteasomes for degradation because of either stochastic errors in gene expression (transcription, translation, protein folding or assembly) or a deliberate process of translating 5

16 standard and nonstandard mrnas for the purpose of immunosurveillance (8, 9). Under inflammatory conditions and by cytokines such as IFN-γ, the β-subunits of the proteasome are replaced by low molecular weight protein (LMP) 2, LMP7, and LMP10 to form the immunoproteosome, which processes proteins much more efficiently and generates more immunodominat epitopes. Peptides produced in the cytosol are transported into the endoplasmic reticulum (ER) by TAP1 and TAP2 in an ATP dependent fashion. These peptides are then further trimmed by the enzyme ERAP1 (ER aminopeptidase associated with Ag processing1) within the ER to allow the peptide to fit into the groove of the MHC class I molecules. Peptides that do not associate with MHC class I are recycled back to the cytosol by the translocon SEC61 (10).The newly synthesized MHC class I heavy chains translocate to the ER and 4 chaperones; calnexin, ERp57, calreticulin, and tapasin help assemble the heavy chains, β2m complex with the peptides. The completely assembled MHC class I molecule then disassociates from the chaperones and is targeted to the plasma membrane via the golgi complex (11). The half-life of peptide-mhc molecules is dependent on the cell type and stability of the complex. Free MHC class I heavy chains may appear at the plasma membrane but are quickly internalized and degraded. Peptide-MHC complexes are internalized by ubiquitination by MARCH proteins and are either recycled or degraded by the endosomal pathway (12). The process is summarized in Figure 1. Variations to the generation and loading of peptides onto MHC class I complex (13): 1) The peptides generated by the proteasomes may be either too small or too long to be transported by the TAP complex in which case the peptides are degraded and do not enter the MHC class I peptidome. 2) Although 6 different alleles of MHC class I can be expressed by the cell, in most cases, a maximum of only 4 different MHC alleles have been found. Thus, although a peptide may be of the right length for loading onto the MHC class I molecule, there may be sequence 6

17 incompatibility, in which case the peptide will dissociate from the loading complex and will have to move to another loading complex with different MHC allele. 3) Multiple alternative antigen processing and presentation pathways independent of TAP activity has also been found (14). For example, in TAP deficient cells, peptides with an ER signal sequence are preferentially loaded on to the MHC complex (12). An alternate repertoire of peptides presented by MHC class I molecules has been found in cells deficient in TAP, tapasin or the proteasome. These peptides have been termed the T cell epitopes associated with impaired peptide processing (TEIPP) and a subset of CD8+T cells have been identified that specifically recognize these TEIPPs (15, 16). The TEIPP peptides can also be from normal endogenous proteins but are not typically presented by the MHC class I molecules using the classical antigen processing pathway (17). Thus, these endogenous peptides now suddenly serve as neo-antigens tagging cells with impaired antigen processing pathways (18). For example, housekeeping protein Trh4 was shown to be processed by signal peptide peptidase, independently of TAP or the proteasome (19). 4) In TAP deficient cells, peptides may be highly hydrophobic and thus diffuse freely into the ER lumen to be loaded onto the MHC molecule (20). Alternatively, there is some evidence that chaperones such heat shock protein (HSP) 90 and protein disulfide isomerase (PDI) may serve to bind free peptides in the ER and chaperone it to the MHC class I peptide binding groove (21). 5) ERAP1 is not associated with the MHC loading complex and thus, before the peptides translocated by TAP can reach either ERAP1 or the loading complex, it may re-circulate back into the cytoplasm. 6) MHC class I molecules have also been shown to display peptides not coded by the genome by a process termed post-translational protein splicing (22). In this process, peptides generated by proteasomes can fuse/ligate to form neo-epitopes including tumor-specific antigens (7). 7

18 Role of cytosolic peptidases in generation of antigen peptides for MHC class I presentation: Cytosolic peptidases play an important role in determining the peptide display by MHC class I molecules as these enzymes can either destroy peptides generated by the proteasome before they bind to TAP or trim peptides to the right size for translocation by TAP (23, 24). Tripeptidyl peptidase II (TPP II) and Insulin-degrading enzyme (IDE) have been shown to replace proteosomal need for peptide generation in some cases (24, 25). The main functions of MHC class I molecules include presentation of antigenic peptides for recognition by CD8+ T cells, development and selection of CD8+ T cells in the thymus, as well as tolerance of NK cells. Other non-classical functions of MHC have been reported. For example, reverse signaling via MHC class I molecules after ligation or cross-linking has been shown to affect endothelial cell proliferation and migration and attenuate inflammation by downregulating signaling via Toll-like receptors (26). In addition, MHC class I molecules were shown to act as a tumor suppressor gene by controlling cell cycle genes and invasiveness of melanomas, thus regulating the intrinsic oncogenic potential of the cells (27). MHC class I has a role in synaptic pruning, i.e., a process by which synapses are formed/degraded and thus has implications in neurological disorders like Parkinson s and Alzheimer s (28). 8

19 A B Figure 1.1: MHC class I antigen presentation pathway (Adapted from (12)) (A) Retiree proteins or products of Defective ribosomal products (DRiPs) can serve as substrates for proteosomal degradation and generation of peptides. Peptides maybe re-ligated by post-proteosomal processing. The peptides are then transported into the lumen of endoplasmic reticulum (ER) by the transporter associated proteins (TAP). Here the peptides may further be trimmed by the ER-associated aminopeptidase (ERAAP). The peptide is then loaded on MHC class I molecules by the peptide loading complex (PLC) consisting of calcineurin, calcireticulin, ERp57, tapasin. The peptide-loaded MHC complex then dissociates from the PLC and is translocated to plasma membrane via the golgi complex. (B) Internalization of the peptide-mhc complex can occur by ubiquitination of the MHC by MARCH proteins and can be targeted for degradation by lysosomes. A fraction of the MHC molecules is recycled for exchange with endosomal peptides. 9

20 1.2.2 MHC class II (in mice) or HLA- DR/DQ/DP (in humans) present peptides to CD4+ T helper cells. In contrast to class I molecules that are present on all nucleated cells, Class II molecules are only present on immune cells constitutively and can be induced in few other non-immune cells like endothelial cells by cytokines. Cytokines like IFN-γ can also enhance expression of class II molecules on immune cells, there by amplifying the adaptive immune response. Some non-antigen presenting cells are known to induce expression of MHC class II molecules without the expression of co-stimulatory molecules to induce peripheral tolerance (29). The rate of transcription of the MHC chains is the major determinant of MHC molecule synthesis and expression on the cell surface. Class II transcription activator (CIITA): It is considered as the master regulator of class II gene expression (12). The transcription factor CIITA itself is subject to tight transcriptional regulation and its transcription can be influenced by cytokines. In addition, there are 3 different isoforms of CIITA; I, III and IV that have differential expression in various cell types (30). The promoter of the MHC class I genes have a W-X-Y box that is bound by RFX, CREB, and NF-Y transcription factors to form the MHC class II enhanceosome. The enhanceosome then allows for binding of CIITA, which in turn binds to other transcriptional co-activators to control the transcription of the MHC class II gene. Furthermore, CIITA activity can be modulated by post-translational mechanisms (31). Histone deacetylase HDAC1 has been shown to negatively affect the interaction of CIITA with the enhanceosome complex whereas HDAC2 can deacetylate CIITA and target it for degradation (32). Acetylation of CIITA has been shown to increase nuclear translocation. Phosphorylation followed by monoubiquitylation of CIITA can stabilize the interaction between CIITA and MHC class II promoter (33). In addition to CIITA, remodeling of nucleosomes via acetylation and deacetylation events seem to play a critical role in controlling the transcription of the MHC class II gene (34). 10

21 Antigen presentation by MHC class II: The MHC class II molecules consist of 2 polymorphic chains, α and β. The α1 and β1 regions form the peptide binding cleft that houses a peptide between the lengths of amino acids. Only the fully assembled trimer (α, β chains and peptide) are expressed at the cell surface. Most of the MHC class II peptides are derived from internalization of extracellular protein antigens into endocytic compartments. The endosomes then communicate with lysozymes and the internalized protein antigen is degraded in the late endosomes and lysosomes. Class II molecules (α, β chains) are synthesized in the ER. The peptide binding cleft of the newly synthesized MHC molecules is blocked with an invariant chain (Ii or CD74) that prevents the class II molecules from accepting peptides before it reaches the endosome-lysosome. It also aids in proper folding of class II molecules and directing it to the right compartments. The MHC class II-Ii complex is then transported to the late endosome and lysosome compartment termed MHC class II compartment (MIIC). In the endosomal-lysosomal vesicles, the Ii is cleaved to a shorter peptide termed class II-associated Ii peptide (CLIP) by proteases. In the MIIC, HLA-DM is required to facilitate the exchange of CLIP with the newly generated peptides from the endosomal pathway. The fully assembled class II trimer is then shuttled for surface expression. Transport of MHC class II-Ii complex to the MIIC (35): In addition to blocking the loading of endogenous peptides onto the MHC class I peptide-binding groove, the cytoplasmic tail of the Ii has motifs that direct trafficking of the MHCII-Ii complex to MIIC via the sorting adaptor AP1. In cases where the MHCII-Ii complex have reached the plasma membrane via the trans-golgi complex, the sorting AP2 interacts with the cytoplasmic tail of Ii and allows for endocytosis and trafficking to the MIIC. Transport of MHC class II-peptide complex from MIIC to the plasma membrane (12): The MIIC has been shown to release MHC class II-peptide complexes after specific periods of time and this in turn has been shown to be controlled by several factors such as cholesterol, GTPases, 11

22 and ph. The transport of MIIC from and to the plasma membrane is controlled by microtubulebased motor proteins. Fast MIIC transport is enabled by dynein (from plasma membrane) and kinesin (to plasma membrane). Slow transport is facilitated by the actin-myosin system. MIICs have been shown to express different receptors essential for binding of the motor proteins. Many different signals and the maturation state of the antigen-presenting cells control the expression of both the expression of the motor proteins and their respective vesicle receptors. For example, lipopolysaccharide (LPS) that induces maturation of dendritic cells signals to increase the surface density of MHC class II-peptide complexes at the surface by signaling to increase the microtubule network and transport of MIICs to the plasma membrane. (36) Half-life of peptide-mhc II complexes at the plasma membrane: The half-life of the complexes is determined in a cell-type specific manner and in relation to the maturation status of the cells. For example, mature dendritic cells have significantly longer half-life than immature dendritic cells. Ubiquitination of the cytoplasmic tail by MARCH proteins is one of the mechanisms by which MHCII molecules are internalized and targeted for degradation or recycling (37). Other mechanisms of MHC class II internalization are yet to be defined since inducing mutations in the ubiquitination sites of the MHC cytoplasmic tail did not eliminate MHC II uptake and recycling (38). Alternate functions of MHC class II: In addition to antigen presentation, MHC class II molecules have been shown to behave as signaling molecules. For example, signaling via MHCII was shown to induce caspase-independent cell death in B cells (39). Conversely, MHCII expressed on melanoma cells when ligated by lymphocyte activation gene-3 (LAG3) expressed on infiltrating T cells, induced apoptosis resistance in the melanoma cells (40). Intracellular MHC II has been shown to act as an adaptor molecule in macrophages and dendritic cells to amplify signaling via the toll like receptors. TLR signaling lead to association of intracellular MHCII with 12

23 CD40, which in turn activated the tyrosine kinase Btk and resulted in an augmented inflammatory innate immune response (41). Figure 1.2: MHC class II antigen presentation pathway (Adapted from (12)) The α and β chains of the MHC class II molecule associate with the Invariant chain (Ii) that prevents association with endogenously generated peptides. It also has the signaling sequence that targets the MHCII-Ii complex to late endosomal compartment (MIIC). The MHCII-Ii complex may also be indirectly shuttled to MIIC by transport to plasma membrane via the golgi complex and then endocytosis. In the MIIC, the Ii chain is cleaved by proteases leaving the class IIassociated Ii peptide (CLIP) in the peptide binding grove. HLA-DM exchanges the CLIP for peptide and the assembled peptide-mhc complex is shuttled to the plasma membrane by either the dynein-kinesin or the actin-myosin system. 13

24 Cross-presentation: Cross-priming or cross-presentation refers to the ability of dendritic cells to present exogenous protein antigens on MHC class I molecules for presentation to CD8+T cells. Thus the DCs need not be infected to be able to present foreign peptides for CD8+ T cell activation. Internalized antigens in the endocytic compartments fuse with the ER, the proteins are then exported to the cytosol where they are degraded by the proteasome, transported back into ER by TAP and assembled onto MHC class I molecules. Three modes of cross-presentation have been identified so far (10). - Extracellular proteins taken up by DCs into the early endosomes are processed into peptides by proteosomes. The endosome can also have MHC class I molecules that are internalized from the surface. Thus, exogenous peptides can get loaded onto the MHC class I molecules and is then recycled to the cell surface. - The phagosome contains several ER components (for example, TAP and Sec61). Exogenous protein internalized by a phagosome can be translocated into the cytosol by Sec61, where it is processed into peptides by proteasomes. The peptides then re-enter the phagosome through TAP and are loaded onto internalized MHC class I molecules. In cases where the phagosome is attached to the ER, the peptides that re-enter the phagosome can be loaded onto newly synthesized MHC class I molecules. - A certain proportion of the newly synthesized MHC class I molecules can associate with the invariant chain (Ii), which has a similar function as in MHC class II antigen presentation pathway. This prevents the association of endogenous proteins and directs the MHC class I complex to MIIC for peptide loading (42). 1.3 Hypoxia, VHL and HIF transcription factors The ability to maintain oxygen homeostasis is central to all metabolic processes. The human body thus has a complex co-ordination between circulatory, respiratory, and neuroendocrine 14

25 systems to sustain precise oxygen levels within cells and tissues. In addition, all nucleated cells in the body can sense and adapt to changing microenvironmental oxygen tensions (43). Hypoxia or a state of inadequate or reduced oxygen supply can occur due to several reasons: 1) high cellular or local oxygen consumption with supply deficit and 2) increased distance from vasculature (diffusion range of oxygen is up to 200 μm depending on blood oxygen levels) (44). Within the body, there exists steep oxygen gradients both among different organs and within tissues. For example, the pulmonary vasculature typically has an oxygen tension of 13-15% and as the oxygen gets delivered to tissues, the venous blood oxygen tension drops to 5-7%, with the levels of oxygen within tissues and cells being even lower (45). Hypoxia is a common feature of solid tumors (46). Hypoxia in tumors initially develops as the vasculature is unable to keep pace with the rapidly dividing neoplastic cells, causing a diffusion-limited low oxygen tension. The induced hypoxia in turn leads to the development of abnormal and inefficient tumor microvasculature that fails to rectify the oxygen deficit (46-48). In response to hypoxia, the transcription factor hypoxia inducible factor-α (HIFα), known as the master regulator of the hypoxic response, is stabilized. HIF belongs to the Per-ARNT-Sim (PAS) domain family of basic helix-loop-helix factors and forms a heterodimer with the constitutively expressed HIF-1β, also known as aryl hydrocarbon nuclear translocator (ARNT). This dimer then regulates gene transcription post translocation to the nucleus. Under normoxic conditions, a set of non-heme Fe(II)- and 2-oxoglutarate dependent dioxygenases (prolyl hydroxylase domain; PHD, also known as Egln) inactivate HIF by posttranslational hydroxylation of specific amino acid residues in the α subunits. Prolyl hydroxylation enables interaction with the von Hippel Lindau protein (pvhl) E3 ubiquitin ligase complex, which then targets HIF to proteosomal degradation. Asparaginyl hydroxylation is known to block coactivator recruitment (49). For example, Factor-inhibiting HIF-1 (FIH) blocks HIF transactivation by hydroxylating an asparaginyl residue and blocking association of HIFα with the p300 coactivator protein (50). 15

26 PHD and FIH have different affinities for oxygen, with FIH having lower Km for oxygen than PHD. Thus, FIH is active at intermediate levels of hypoxia where HIF may be stabilized and thus contribute to the hypoxic gene signature (51). Although a large focus has been placed on posttranslational, oxygen mediated regulation of HIF-α, it can also be regulated transcriptionally and translationally (52). HIF is stabilized by decreasing oxygen concentrations in a graded fashion. HIF can also be stabilized under non-hypoxic conditions by mutations in VHL, hormones like angiotensin, cytokines such as TGF-β and upon TCR activation (44). In addition, the PHD enzymes can be inhibited by elevated levels of succinate or fumarate, thereby stabilizing HIF (44). Similarly, iron chelators can inhibit PHDs and stabilize HIF (44). Hundreds of genes have been shown to be positively and negatively regulated by HIF. Interestingly, only 40% of the genes have a HIF binding site within 2.5kb of the transcriptional start site (50). Mole et al. (53) showed that HIF binding sites could even be 100kb away from the transcription start site, however, it is not known if additional transcriptional factors are needed to mediate HIF response or if HIF binding can affect gene expression over long distances. In addition, the authors also showed that positive regulation of genes by HIF in >20% of genes was mediated by direct HIF binding to the promoter whereas negative regulation by HIF was almost always indirect. Indirect mechanisms may include activation of other transcriptional repressors, micrornas or epigenetic modulation through control of DNA methylases and histone acetylases. Direct down regulation of genes by HIF may occur in rare instances where HIF is able to dislodge a transcriptional activator or if a HIF binding site is present, HIF may recruit other negative regulators (53). There are 3 known isoforms of HIF-α, HIF-1α, HIF-2α and HIF-3α. Of these, very little is known about the HIF-3α isoform with the 1α and 2α isoforms being better studied. Initially thought to be redundant form of 1α, it is becoming more evident that the HIF-1α and HIF-2α can have unique 16

27 or even opposing roles in gene regulation (49). Hif-1α is ubiquitously expressed whereas HIF-2α has restricted expression (51). In renal cell carcinomas, a number of studies have pointed toward HIF-1α being the tumor suppressor while HIF-2α is the oncogene (54). In addition to having opposing effects in ccrcc, the HIF-α isoforms have also been shown to be mutually suppressive (52). The 2 isoforms can also be differentially regulated at level of transcription, translation and protein stability (55). For example, TH1 cytokines increased 1α transcription whereas TH2 cytokines increased 2α transcription. Similarly, translation of 1α mrna was controlled by mtorc1 and mtorc2 kinases whereas 2α translation was only dependent on mtorc2. Only 2α translation was inhibited by iron response element binding protein 1 (IREBP1) although 1α has an identical iron response element (IRE). mrna stability also varies between the isoforms. HIF-associated factor (HAF) was shown to selectively destabilize 1α mrna and not 2α mrna. Instead, HAF association with 2α increased its transcriptional activities (55) Anti-tumor Immunity Cancer immunosurveillance, the theory that the immune system can recognize and eliminate primary tumors without any external therapeutic intervention was conceptualized over 100 years ago (56). However, the idea was more rigorously tested and began to be accepted only in the 1990s when two landmark studies showed that endogenous Interferon gamma (IFN-γ) protected the host from both transplanted and spontaneous tumor growth (57). Similarly, it was demonstrated that mice lacking perforin, an important lytic component of the cytotoxic granules from cytotoxic T cells (CTL) and natural killer (NK) cells, were more prone to carcinogen induced tumor formation (58). Evidence for cancer immunosurveillance in humans came from retrospective epidemiological studies that noted a significantly higher incidence of cancer in immunosuppressed and immunocompromised individuals (59, 60). Correspondingly, a positive 17

28 correlation was seen in melanoma patients between the number of tumor infiltrating lymphocytes and patient survival (61, 62). It is now well appreciated that the immune system not only works to protect the host from the tumor but also has an important role in shaping the tumor progression. Thus the term cancer immunosurveillance was replaced with the term cancer immunoediting to reflect these dual, opposing functions of immunity. Cancer immunoediting is an extrinsic tumor suppression mechanism that deploys only after the cells have undergone neoplastic transformation and all the intrinsic tumor suppression mechanisms have failed. It encompasses three different phases, Elimination, Equilibrium and Escape (63-66). In the elimination phase (67), the immune system is able to effectively fight neoplastic cells even before the cancer becomes clinically apparent. Cells of both branches of immunity, innate and adaptive, have an important role in tumor elimination. There are several mechanisms by which the immune system is alerted to the presence of transformed cells. As the tumors start to grow invasively and remodel the stroma, several pro-inflammatory cytokines (danger signals) are secreted by the tumors attracting cells of innate immunity including NK cells, natural killer- T (NKT) cells, γδ T cells, macrophages and dendritic cells (DCs) to the site. In addition to these cytokines, damage-associated molecular pattern molecules (DAMPs) such as high mobility group box 1 (HMGB1) and hyaluronan fragments are secreted by the dying tumor and surrounding tissue that activate DCs. A third possible mechanism includes upregulation of stress ligands that activate cells of innate immunity which in turn secrete pro-inflammatory cytokines that create a microenvironment conducive to tumor-specific action of the adaptive immune cells. Dendritic cells (68) play a key role in anti-tumor immunity by controlling both humoral and cellular arms of adaptive immunity. This has led to exploration of use of DCs as prophylactic and therapeutic cancer vaccines. Presentation of antigens by immature DCs results in T cell 18

29 tolerance due to lack of co-stimulatory signals and other cytokines. Thus adjuvants in DC-based vaccines are designed to activate the DCs to a mature phenotype to elicit an effective T and B cell response. The recent FDA approval of the first dendritic cell based vaccine, Provenge, has provided a great stimulus to the field. Provenge involves isolating lymphocytes from the blood of patients with prostate cancer, purifying dendritic cells and pulsing with prostate cancer specific protein, prostatic acid phosphatase (PAP) and returning the cells to the patient via I.V injections. This therapy has been shown to induce tumor-specific CTLs and extend the life-span by several months. Prostvac, currently in phase 3 clinical trials, uses pox-virus based vector system to infect DCs and deliver prostate specific antigen. The infected DCs then elicit a robust tumor specific CTL response. Thus, dendritic cells and cytotoxic T lymphocytes have an essential role in anti-tumor immunity during the elimination phase. Only the transformed cells that survive elimination move on to the equilibrium phase (69). In this phase, the cells of the immune system are no longer able to effectively kill the tumors but are still able to prevent its rapid outgrowth. The neoplastic cells under a constant immunological pressure go through further transformation, resulting in tumor cell variants that are able to effectively evade an immune attack. This leads to the third phase of cancer immunoediting Escape. It is in this escape phase (69) that the cancer becomes clinically apparent. Immune evasion strategies include downregulation of the tumor immunogenicity, induction of an immunosuppressive state within the tumor microenvironment, insensitivity to immune effector mechanisms, and aberrant expression of immunomodulatory molecules, among others (70). Thus the success of an immunotherapy regime relies heavily on identifying and bringing down the immuno-evasive barriers around the tumors (71, 72). Tumor immunogenicity or the ability to induce adaptive immune responses depends on the antigen expression and antigen presentation by the tumors. CD8+ T cells, considered as the main effector arm of the adaptive anti-tumor immune response, recognize tumor specific 19

30 peptides presented by Human Leukocyte Antigen (HLA) class I in humans (or MHC in mice) (73-75). Downregulation of HLA class I molecules reduces the levels of tumor-associated antigens expressed by tumor cells. This in turn negatively impacts their recognition and subsequently their destruction by CTLs (76-78). Although downregulation of HLA molecules could activate NK cells, which recognize and lyse HLA negative cells, several tumors are known to expresses coinhibitory receptors for NK cells, shed ligands for the NK cell activating receptor NKG2D or maintain the HLA molecules at levels high enough to inhibit NK cells but too low to effectively activate CTLs (79). In addition to downregulating their own HLA molecules, tumors can also disable DC-based antigen presentation to T cells. They do so by either producing antigens that cannot be efficiently processed by DCs (like mucin 1 and other glycoproteins) or by directly interfering with maturation of DCs by producing cytokines like IL-10 that induce DC anergy. Tumors are also known to produce cytokines that result in dendritic cells that activate Th2 subset of CD4+T cells that in turn secrete cytokines (e.g IL-4 and Il-13) that prevent tumor cell apoptosis and induce secretion of epidermal growth factor (EGF) by tumor-associated macrophages (80, 81). Two different types of HLA lesions have been identified in tumors, soft (or reversible) and hard (or irreversible). Soft lesions result from reversible changes in either the expression of HLA class I heavy chains, β2 microglobulin (β2m) or components of the antigen presentation machinery. Dysregulation of HLA expression can occur at the transcriptional, posttranscriptional or at the epigenetic level. Hard lesions result from HLA or β2m haplotype loss or mutations in the genes (82, 83). In addition to immunogenicity, the tumor microenvironment has been identified as a critical factor in controlling the progression and metastasis of the cancer, along with dictating response to conventional anti-tumor therapies (84, 85). In particular, hypoxia is considered as one of the 20

31 most pervasive tumor microenvironmental stresses. Our lab has previously shown that in hypoxic regions of the tumor, there is a metabolic shift toward production of extracellular adenosine (86-88). Immunosuppressive properties of Adenosine (89-93) Adenosine is a ubiquitous purine nucleoside that is present at extremely low levels in interstitial fluids of unstressed tissues (~ nm) but can rapidly rise (>30µM) in response to pathophysiologic conditions like hypoxia, ischaemia, inflammation or trauma. Thus adenosine is considered as a danger signal that aims to restore tissue homeostasis and prevent collateral damage from overactive immune cells and inflammatory response. The persistence of increased adenosine levels beyond the acute injury phase is detrimental to tissues as it activates pathways to maintain a continuous wound-healing process leading to fibrotic remodeling or neoplastic changes. Levels of extracellular adenosine is dependent on the activities of ectoenzymes (CD39 and CD73) that generate adenosine from ATP, nucleoside transporters (ENT1 and 2) that shuttle intracellular adenosine to the extracellular space, enzyme activities of adenosine deaminase that converts adenosine to inosine, adenosine kinase that phosphorylates adenosine to AMP, and S-adenosyl-homocysteine hydrolase that hydrolyses S-adenosyl homocysteine to adenosine. Hypoxia is known to affect the expression or activity of the molecules involved in adenosine metabolism such that there is a dramatic increase in the levels of extracellular adenosine. The majority of the signaling action of extracellular adenosine occurs via G-protein coupled receptors (GPCRs); A1, A2A, A2B and A3. A1 and A3 are Gi linked, A2A Gs linked and A2B can either be coupled to Gs or Gq. Adenosine signaling through A2AR is known to suppress the functions of T cells, NK cells, NKT cells, macrophages, dendritic cells, and 21